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| J Biol Chem, 2000 Jun 30, 275(26), 19933 - 41 Syntenin-syndecan binding requires syndecan-synteny and the co-operation of both PDZ domains of syntenin; Grootjans JJ et al.; Syntenin is an adaptor-like molecule that binds to the cytoplasmic domains of all four vertebrate syndecans . Syntenin-syndecan binding involves the C-terminal part of syntenin that contains a tandem of PDZ domains . Here we provide evidence that each PDZ domain of syntenin can interact with a syndecan . Isolated or combined mutations of the carboxylate binding lysines in the inter-betaAbetaB loops and of the alphaB1 residues in either one or both the PDZ domains of syntenin all reduce syntenin-syndecan binding in yeast two-hybrid, blot-overlay, and surface plasmon resonance assays . PDZ2 mutations have more pronounced effects on binding than PDZ1 mutations, but complete abrogation of syntenin-syndecan binding requires the combination of both the lysine and the alphaB1 mutations in both the PDZ domains of syntenin . Isothermal calorimetric titration of syntenin with syndecan peptide reveals the presence of two binding sites in syntenin . Yet, unlike a tandem of two PDZ2 domains and a reconstituted PDZ1+PDZ2 tandem, a tandem of two PDZ1 domains and isolated PDZ1 or PDZ2 domains do not interact with syndecan bait . We conclude to a co-operative binding mode whereby neither of these two PDZ domains is sufficient by itself but where PDZ2 functions as a "major" or "high affinity" syndecan binding domain, and PDZ1 functions as an "accessory" or "low affinity" syndecan binding domain . The paired, but not the isolated PDZ domains of syntenin bind also strongly to the immobilized cytoplasmic domains of neurexin and B-class ephrins . By inference, these data suggest a model whereby recruitment of syntenin to membrane surfaces requires two compatible types of bait that are in "synteny" (occurring together in location) and engages both PDZ domains of syntenin . The synteny of compatible bait may result from the assemblies and co-assemblies of syndecans and other similarly suited partners in larger supramolecular complexes . In general, an intramolecular combination of PDZ domains that are weak, taken individually, would appear to be designed to detect rather than drive the formation of specific molecular assemblies. J Biol Chem, 2000 Jul 14, 275(28), 21678 - 87 Maize cap1 encodes a novel SERCA-type calcium-ATPase with a calmodulin-binding domain; Subbaiah CC et al.; A cDNA (CAP1) isolated from maize roots shares sequence identity with genes encoding P-type Ca(2+)-ATPases and restores the growth phenotype of yeast mutants defective in Ca(2+)-pumps . CAP1 was transcribed and translated in the yeast mutant . Furthermore, the membrane-integrated product formed a Ca(2+)-dependent phosphorylated intermediate and supported Ca(2+) transport . Although CAP1 shares greater sequence identity with mammalian "endoplasmic reticulum-type" Ca(2+)-pumps, it differs from these genes by having features of calmodulin (CaM)-regulated Ca(2+)-pumps . CAP1 from yeast microsomes bound CaM, and the CAP1-dependent Ca(2+) transport in yeast was stimulated by CaM . Peptides from the C terminus of CAP1 bound CaM . Anti-CAP1 antibodies specifically recognized a maize microsomal polypeptide that also bound CaM . A similar polypeptide also formed a Ca(2+)-dependent phosphoenzyme . Our results suggest that cap1 encodes a novel form of CaM-regulated Ca(2+)-ATPase in maize . CAP1 appears to be encoded by one or two genes in maize . CAP1 RNA is induced only during early anoxia, indicating that the Ca(2+)-pump may play an important role in O(2)-deprived maize cells. J Exp Med, 2000 Apr 17, 191(8), 1365 - 80 Evidence for class-specific factors in immunoglobulin isotype switching; Shanmugam A et al.; Immunoglobulin class switch recombination (SR) occurs by a B cell-specific, intrachromosomal deletional process between switch regions . We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities . The plasmids are novel in that they lack a eukaryotic origin of DNA replication . The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells . The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes . These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity . Finally, we provide evidence for two distinct switching activities which independently mediate mu-->alpha and mu-->gamma3 SR. Cancer Gene Ther, 2000 Feb, 7(2), 177 - 86 Enhanced antitumoral effect of adenovirus-mediated cytosine deaminase gene therapy by induction of antigen-presenting cells through stem cell factor/granulocyte-macrophage colony-stimulating factor gene transfer; Cao X et al.; Suicide gene therapy has been studied intensively for the treatment of cancer . A limited antitumoral effect was obtained by intratumoral injection of adenovirus harboring Escherichia coli cytosine deaminase gene (AdCD) in tumor-bearing mice followed by continuous administration of 5-fluorocytosine (5FC) . To address the drawbacks of the limited potential for the induction of antitumoral immunity by CD suicide gene therapy, we hypothesized that antigen-presenting cells (APCs) might contribute to the efficient induction of an antitumoral immune response in tumor-bearing mice undergoing suicide gene therapy . We preinjected the mice with murine stem cell factor (SCF)-encoding adenovirus (AdSCF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-encoding adenovirus (AdGM-CSF); after 7 days, the mice were inoculated with CT26 colon adenocarcinoma . AdCD was injected intratumorally into tumor-bearing mice followed by 5FC administration . The results showed that AdSCF/AdGM-CSF treatment could increase the number, surface molecule expression, and function of APCs efficiently . A more significant growth inhibition of established tumors and a prolongation of the survival period were observed in tumor-bearing mice after AdSCF/AdGM-CSF pretreatment in combination with AdCD/5FC therapy when compared with mice treated with AdSCF or AdGM-CSF in combination with AdCD/5FC, or AdCD/5FC alone (P < .01) . Cytotoxic T-lymphocyte activity was induced efficiently after the combined therapy, and mRNA of tumor necrosis factor-alpha, interleukin-4, interferon-gamma, and interleukin-2 was present in the tumor mass after combined therapy, suggesting that a more potent antitumoral response was induced by enhanced APCs . Our results demonstrated that AdSCF/AdGM-CSF pretreatment could activate APCs, and that these APCs could present the tumor antigens released from AdCD/5FC-killed tumor cells and activate the antitumoral response of the host, thus increasing the therapeutic efficiency of suicide gene therapy. Biotechniques, 2000 Apr, 28(4), 789 - 93 Multiple epitope tagging of expressed proteins for enhanced detection; Hernan R et al.; Three FLAG epitopes have been incorporated into the mammalian expression vector pCMV-5 to create a transient expression vector, p3XFLAG-CMV-7 . The vector was designed to express FLAG fusion proteins that can be detected at tenfold lower expression levels than the current FLAG fusion protein expression system . The usefulness of this expression and detection system was demonstrated by expression of bacterial alkaline phosphatase in COS-7 cells . In addition, 3XFLAG bacterial alkaline phosphatase was expressed in Escherichia coli, purified on anti-FLAG M2 affinity gel, and detection of 500 pg of purified protein by Western blot analysis is demonstrated. Biotechniques, 2000 Apr, 28(4), 784 - 8 New positive selection system based on the parD (kis/kid) system of the R1 plasmid; Gabant P et al.; The use of vectors that are designed to allow positive selection of recombinants facilitates cloning experiments in E . coli . Using kid, a lethal gene of the R1 plasmid parD locus, we generated pKID vectors leading to high selective efficiency of recombinants (greater than 90%) . The E . coli bacterial host used to propagate these vectors produces the Kis protein, the natural antagonist of Kid . This new positive-selection system exhibits the same efficiency as the original ccdB-based selection vectors, pKIL (4) . We also show that the ccdB and kid systems are independent . This property increases the potential of plasmidic poison-antidote systems for genetic applications and opens the door to a generation of new vectors containing the two selection systems. Biotechniques, 2000 Apr, 28(4), 660 - 2, 664, 666 passim Efficient DNA subcloning through selective restriction endonuclease digestion; Spear MA; Described here is a selective restriction endonuclease digestion method that eliminates the electrophoresis step that is usually used during the subcloning of new DNA sequences into typical E . coli-based plasmids . The method increases yield while decreasing laboratory resource and time utilization . By using donor and acceptor sequences that contain unique restriction sites found only outside of the intended recombination sequences, the initial digestion products can be directly combined without electrophoresis if the ligation step is followed by a selective digestion using the unique restriction enzymes before transformation . This system is based on the several order of magnitude decrease in transformation efficiency of linearized compared to circular plasmids . As an example, this method was used to obtain recombinants between a 3.6 kb acceptor plasmid and 3.0 kb insert following one ligation reaction after the failure of nine standard reactions using similar amounts of input DNA . It is particularly applicable to situations in which low subcloning efficiencies are expected . The technique can be extended to a large percentage of planned recombinations by using nonidentical compatible cohesive or blunt-ended fragments, or site-directed mutagenesis. Biochem J, 2000 May 1, 347 Pt 3, 881 - 6 Heteroduplex DNA and ATP induced conformational changes of a MutS mismatch repair protein from Thermus aquaticus; Biswas I et al.; ATP hydrolysis by MutS homologues is required for the function of these proteins in mismatch repair . However, the function of ATP hydrolysis in the repair reaction is not very clear . We have examined the role of ATP hydrolysis in oligomerization of Thermus aquaticus (Taq) MutS protein in solution . Analytical gel filtration and cross-linking of MutS protein with disuccinimidyl suburate suggest that TaqMutS is a dimer in the presence of ATP . ATP binding and hydrolysis by TaqMutS reduces the heteroduplex-DNA binding by the protein . Using limited proteolysis we detected extensive conformational changes of the TaqMutS protein in the presence of ATP and heteroduplex DNA . Heteroduplex-DNA binding is necessary for the observed conformational changes since F39A mutant protein defective in DNA binding does not display ATP-induced conformational changes . The implications of the observed conformational changes in the MutS protein are discussed with respect to two different models proposed for the role of ATP hydrolysis by MutS in DNA mismatch repair. Biochem J, 2000 May 1, 347 Pt 3, 797 - 805 Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site; Kuo PH et al.; Subunit a of the ATP synthase F(o) sector contains a transmembrane helix that interacts with subunit c and is critical for H(+) transport activity . From a cysteine scan in the region around the essential subunit a residue, Arg-210, we found that the replacement of aGly-213 greatly attenuated ATP hydrolysis, ATP-dependent proton pumping and Delta mu(H)+-dependent ATP synthesis . Various amino acid substitutions caused similar effects, suggesting that functional perturbations were caused by altering the environment or conformation of aArg-210 . aG213N, which was particularly severe in effect, was suppressed by two second-site mutations, aL251V and cD61E . These mutations restored efficient coupling; the latter also increased ATP-dependent proton transport rates . These results were consistent with the proposed functional interaction between aArg-210 and cAsp-61, the likely carrier of the transported proton . From Arrhenius analysis of steady-state ATP hydrolytic activity, the transport mutants had large increases in the transition-state enthalpic and entropic parameters . Linear isokinetic relationships demonstrate that the transport mechanism is coupled to the rate-limiting catalytic transition-state step, which we have previously shown to involve the rotation of the gamma subunit in multi-site, co-operative catalysis. Biochem J, 2000 May 1, 347 Pt 3, 601 - 12 Role of lipids in the translocation of proteins across membranes; Van Voorst F et al.; The architecture of cells, with various membrane-bound compartments and with the protein synthesizing machinery confined to one location, dictates that many proteins have to be transported through one or more membranes during their biogenesis . A lot of progress has been made on the identification of protein translocation machineries and their sorting signals in various organelles and organisms . Biochemical characterization has revealed the functions of several individual protein components . Interestingly, lipid components were also found to be essential for the correct functioning of these translocases . This led to the idea that there is a very intimate relationship between the lipid and protein components that enables them to fulfil their intriguing task of transporting large biopolymers through a lipid bilayer without leaking their contents . In this review we focus on the Sec translocases in the endoplasmic reticulum and the bacterial inner membrane . We also highlight the interactions of lipids and proteins during the process of translocation and integrate this into a model that enables us to understand the role of membrane lipid composition in translocase function. Biochemistry, 2000 Apr 25, 39(16), 4915 - 23 The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin; Yang X et al.; Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity . Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E . coli bacterioferritin . Consistent with previous UV-visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined . The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe(2+) ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe(2+) + P(Z) --> {Fe(2)-P}(Z) + 4H(+), where P(Z) is the apoprotein of net charge Z and {Fe(2)-P}(Z) represents a diferrous ferroxidase complex . The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: {Fe(2)-P}(Z) + (1)/(2)O(2) --> {Fe(2)O-P}(Z) where {Fe(2)O-P}(Z) represents an oxidized diferric ferroxidase complex, probably a mu-oxo-bridged species as suggested by UV-visible and EPR spectrometric titration data . The third phase corresponds to mineral core formation according to the net reaction: 4Fe(2+) + O(2) + 6H(2)O --> 4FeO(OH)((core)) + 8H(+) . Iron oxidation is inhibited by the presence of Zn(2+) ions . The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn(2+)per 24mer, i.e., 2 Zn(2+) per ferroxidase center. Biochemistry, 2000 Apr 25, 39(16), 4846 - 54 Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport; Dumas F et al.; The structural and functional consequences of a mismatch between the hydrophobic thickness d(P) of a transmembrane protein and that d(L) of the supporting lipid bilayer were investigated using melibiose permease (MelB) from Escherichia coli reconstituted in a set of bis saturated and monounsaturated phosphatidylcholine species differing in acyl-chain length . Influence of MelB on the midpoint gel-to-liquid-phase transition temperature, T(m), of the saturated lipids was investigated through fluorescence polarization experiments, with 1,6-diphenyl-1,3,5-hexatriene as the probe, for varying protein/lipid molar ratio . Diagrams in temperature versus MelB concentration showed positive or negative shifts in T(m) with the short-chain lipids DiC12:0-PC and DiC14:0-PC or the long-chain lipids DiC16:0-PC and DiC18:0-PC, respectively . Theoretical analysis of the data yielded a d(L) value of 3.0 +/- 0.1 nm for the protein, similar to the 3.02 nm estimated from hydropathy profiles . Influence of the acyl chain length on the carrier activity of MelB was investigated in the liquid phase, using the monounsaturated PCs . Binding of the sugar to the transporter showed no dependence on the acyl chain length . In contrast, counterflow and Deltapsi-driven experiments revealed strong dependence of melibiose transport on the lipid acyl chain length . Similar bell-shaped transport versus acyl chain length profiles were obtained, optimal activity being supported by diC16:1-PC . On account of a d(P) value of 2.65 nm for the lipid and of various local constraints which would all tend to elongate the acyl chains in contact with the protein, one can conclude that maximal activity was obtained when the hydrophobic thickness of the bilayer matched that of the protein. Biochemistry, 2000 Apr 25, 39(16), 4831 - 7 Spin labeling analysis of structure and dynamics of the Na(+)/proline transporter of Escherichia coli; Wegener C et al.; With respect to the functional importance attributed to the N-terminal part of the Na(+)/proline transporter of Escherichia coli (PutP), we report here on the structural arrangement and functional dynamics of transmembrane domains (TMs) II and III and the adjoining loop regions . Information on membrane topography was obtained by analyzing the residual mobility of site-specifically-attached nitroxide spin label and by determination of collision frequencies of the nitroxide with oxygen and a polar metal ion complex using electron paramagnetic resonance (EPR) spectroscopy . The studies suggest that amino acids Phe45, Ser50, Ser54, Trp59, and Met62 are part of TM II while Gly39 and Arg40 are located at a membrane-water interface probably forming the cytoplasmic cap of the TM . Also Ala67 and Glu75 are at a membrane-water interface, suggesting a location close to the periplasmic ends of TMs II and III, respectively . Ser71 between these residues is clearly in a water-exposed loop (periplasmic loop 3) . Spin labels attached to positions 80, 86, and 91 show EPR properties typical for a TM location (TM III) . Leu97 may be part of a structured loop region while Ala107 is clearly located in a water-exposed loop (cytoplasmic loop 4) . Finally, spin labels attached to the positions of Asp33 and Leu37 are clearly on the surface of the transporter and are directed into an apolar environment . These findings strongly support the recently proposed 13-helix model of PutP {Jung, H., Rubenhagen, R., Tebbe, S., Leifker, K., Tholema, N., Quick, M., and Schmid, R . (1998) J . Biol . Chem . 273, 26400-26407} and suggest that TMs II and III of the transporter are formed by amino acids Ser41 to Gly66 and Ser76 to Gly95, respectively . In addition to the topology analysis, it is shown that binding of Na(+) and/or proline to the transporter alters the mobility of the nitroxide group at the positions of Leu37 and Phe45 . From these findings, it is concluded that binding of the ligands induces conformational alterations of PutP that involve at least parts of TM II and the preceding cytoplasmic loop. Biochemistry, 2000 Apr 25, 39(16), 4821 - 30 Role of metal ions in the reaction catalyzed by L-ribulose-5-phosphate 4-epimerase; Lee LV et al.; H97N, H95N, and Y229F mutants of L-ribulose-5-phosphate 4-epimerase had 10, 1, and 0.1%, respectively, of the activity of the wild-type (WT) enzyme when activated by Zn(2+), the physiological activator . Co(2+) and Mn(2+) replaced Zn(2+) in Y229F and WT enzymes, although less effectively with the His mutants, while Mg(2+) was a poorly bound, weak activator . None of the other eight tyrosines mutated to phenylalanine caused a major loss of activity . The near-UV CD spectra of all enzymes were nearly identical in the absence of metal ions and substrate, and addition of substrate without metal ion showed no effect . When both substrate and Zn(2+) were present, however, the positive band at 266 nm increased while the negative one at 290 nm decreased in ellipticity . The changes for the WT and Y229F enzymes were greater than for the two His mutants . With Co(2+) as the metal ion, the CD and absorption spectra in the visible region were different, showing little ellipticity in the absence of substrate and a weak absorption band at 508 nm . With substrate present, however, an intense absorption band at 555 nm (epsilon = 150-175) with a negative molar ellipticity approaching 2000 deg cm(2) dmol(-1) appears with WT and Y229F enzymes . With the His mutants, the changes induced by substrate were smaller, with negative ellipticity only half as great . The WT, Y229F, H95N, and H97N enzymes all catalyze a slow aldol condensation of dihydroxyacetone and glycolaldehyde phosphate with an initial k(cat) of 1.6 x 10(-3) s(-1) . The initial rate slowed most rapidly with WT and H97N enzymes, which have the highest affinity for the ketopentose phosphates formed in the condensation . The EPR spectrum of enzyme with Mn(2+) exhibited a drastic decrease upon substrate addition, and by using H(2)(17)O, it was determined that there were three waters in the coordination sphere of Mn(2+) in the absence of substrate . These data suggest that (1) the substrate coordinates to the enzyme-bound metal ion, (2) His95 and His97 are likely metal ion ligands, and (3) Tyr229 is not a metal ion ligand, but may play another role in catalysis, possibly as an acid-base catalyst. Biochemistry, 2000 Apr 25, 39(16), 4808 - 20 13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase; Lee LV et al.; On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4 . With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively . H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2 . 69 and 1.99% at C-4, respectively . Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N . At C-4, the corresponding values were 9.6, 14, and 19% . These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6 . The Y229 mutant shows only the internal commitment of 0.6 . The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket . The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24 . The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm. Biochemistry, 2000 Apr 25, 39(16), 4722 - 8 Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain; Zhang S et al.; The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe . To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E . coli strain NK6024, and studied for their effect on PDT activity . Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy . Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry . In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L . PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe . Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis . The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein . The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein . These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism . To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala . None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis . However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding . For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca . 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower. Biochemistry, 2000 Apr 25, 39(16), 4704 - 10 The role of enzyme isomerization in the native catalytic cycle of the ATP sulfurylase-GTPase system; Wei J et al.; ATP sulfurylase, from E . coli Kappa-12, is a GTPase.target complex that conformationally couples the free energies of GTP hydrolysis and activated sulfate (adenosine 5'-phosphosulfate, or APS) synthesis . Energy coupling is achieved by an allosterically driven isomerization that switches on and off chemistry at specific points in the catalytic cycle . This coupling mechanism is derived from the results of model studies using analogue complexes that mimic different stages of the native catalytic cycle . The current investigation extends the analogue studies to the native catalytic cycle . Isomerization is monitored using the fluorescent, guanine nucleotide analogues mGMPPNP (3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-{beta, gamma-imido}triphosphate) and mGTP {3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-triphosphate} . The isomerization is shown to be initiated by an allosteric interaction that requires the simultaneous occupancy of all three substrate-binding sites . Stopped-flow fluorescence and single-turnover studies were used to define and quantitate the isomerization mechanism, and to show that the isomerization precedes and rate-limits both GTP hydrolysis and APS synthesis . These findings are incorporated into a model of the energy-coupling mechanism. Biochemistry, 2000 Apr 25, 39(16), 4640 - 8 Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit; Guittet O et al.; Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase . The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity . This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues . In this report, nitrated residues in the E . coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing . Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit . The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da . Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein . A mean of two nitrotyrosines per E . coli protein R2 dimer were obtained at 400 microM peroxynitrite . Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122 . Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility . Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed . However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E . coli protein R2. Biochemistry, 2000 Apr 25, 39(16), 4630 - 9 New reactions in the crotonase superfamily: structure of methylmalonyl CoA decarboxylase from Escherichia coli; Benning MM et al.; The molecular structure of methylmalonyl CoA decarboxylase (MMCD), a newly defined member of the crotonase superfamily encoded by the Escherichia coli genome, has been solved by X-ray crystallographic analyses to a resolution of 1.85 A for the unliganded form and to a resolution of 2.7 A for a complex with an inert thioether analogue of methylmalonyl CoA . Like two other structurally characterized members of the crotonase superfamily (crotonase and dienoyl CoA isomerase), MMCD is a hexamer (dimer of trimers) with each polypeptide chain composed of two structural motifs . The larger N-terminal domain contains the active site while the smaller C-terminal motif is alpha-helical and involved primarily in trimerization . Unlike the other members of the crotonase superfamily, however, the C-terminal motif is folded back onto the N-terminal domain such that each active site is wholly contained within a single subunit . The carboxylate group of the thioether analogue of methylmalonyl CoA is hydrogen bonded to the peptidic NH group of Gly 110 and the imidazole ring of His 66 . From modeling studies, it appears that Tyr 140 is positioned within the active site to participate in the decarboxylation reaction by orienting the carboxylate group of methylmalonyl CoA so that it is orthogonal to the plane of the thioester carbonyl group . Surprisingly, while the active site of MMCD contains Glu 113, which is homologous to the general acid/base Glu 144 in the active site of crotonase, its carboxylate side chain is hydrogen bonded to Arg 86, suggesting that it is not directly involved in catalysis . The new constellation of putative functional groups observed in the active site of MMCD underscores the diversity of function in this superfamily. Biochemistry, 2000 Apr 25, 39(16), 4622 - 9 Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli; Haller T et al.; The Escherichia coli genome encodes seven paralogues of the crotonase (enoyl CoA hydratase) superfamily . Four of these have unknown or uncertain functions; their existence was unknown prior to the completion of the E . coli genome sequencing project . The gene encoding one of these, YgfG, is located in a four-gene operon that encodes homologues of methylmalonyl CoA mutases (Sbm) and acyl CoA transferases (YgfH) as well as a putative protein kinase (YgfD/ArgK) . We have determined that YgfG is methylmalonyl CoA decarboxylase, YgfH is propionyl CoA:succinate CoA transferase, and Sbm is methylmalonyl CoA mutase . These reactions are sufficient to form a metabolic cycle by which E . coli can catalyze the decarboxylation of succinate to propionate, although the metabolic context of this cycle is unknown . The identification of YgfG as methylmalonyl CoA decarboxylase expands the range of reactions catalyzed by members of the crotonase superfamily. Biochemistry, 2000 Apr 25, 39(16), 4590 - 602 Evolution of enzymatic activities in the enolase superfamily: crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli; Gulick AM et al.; D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydration of both D-glucarate and L-idarate as well as their interconversion via epimerization . GlucD is a member of the mandelate racemase (MR) subgroup of the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion substrate . Alignment of the sequence of GlucD with that of MR reveals a conserved Lys-X-Lys motif and a His-Asp dyad homologous to the S- and R-specific bases in the active site of MR . Crystals of GlucD have been obtained into which the substrate D-glucarate and two competitive inhibitors, 4-deoxy-D-glucarate and xylarohydroxamate, could be diffused; D-glucarate is converted to the dehydration product, 5-keto-4-deoxy-D-glucarate (KDG) . The structures of these complexes have been determined and reveal the identities of the ligands for the required Mg(2+) (Asp(235), Glu(266), and Asn(289)) as well as confirm the expected presence of Lys(207) and His(339), the catalytic bases that are properly positioned to abstract the proton from C5 of L-idarate and D-glucarate, respectively . Surprisingly, the C6 carboxylate group of KDG is a bidentate ligand to the Mg(2+), with the resulting geometry of the bound KDG suggesting that stereochemical roles of Lys(207) and His(339) are reversed from the predictions made on the basis of the established structure-function relationships for the MR-catalyzed reaction . The catalytic roles of these residues have been examined by characterization of mutant enzymes, although we were unable to use these to demonstrate the catalytic independence of Lys(207) and His(339) as was possible for the homologous Lys(166) and His(297) in the MR-catalyzed reaction. J Gen Virol, 2000 May, 81(Pt 5), 1335 - 45 Enzymatic properties of hepatitis C virus NS3-associated helicase; Paolini C et al.; The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion . In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed in Escherichia coli and purified to homogeneity by conventional chromatography . Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates . On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3' to 5' direction . In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA . Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem-loop RNA structure (SL I) within the 3'-terminal 46 bases of the viral genome . Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule. J Histochem Cytochem, 2000 May, 48(5), 685 - 93 Immunohistochemical localization of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in rat tissues; Kotera J et al.; We raised a polyclonal antibody against maltose binding protein fusion human cGMP-binding, cGMP-specific phosphodiesterase (PDE5) produced in E . coli . This antibody immunoreacted specifically with recombinant human and rat PDE5 proteins expressed in transfected COS-7 cells and with a native form of PDE5 in extracts of rat platelets, lung, and cerebellum . Immunohistochemical analysis showed that the anti-PDE5 antibody detected immunoactive materials in Purkinje cell layers of the cerebellum, proximal renal tubules, collecting renal ducts, and epithelial cells of pancreatic ducts in rats . Reverse transcriptase-polymerase chain reaction analysis demonstrated that PDE5 transcripts are also present in rat cerebellum, kidney, and pancreas . Here we described a cell-specific localization of PDE5 in various rat tissues, suggesting the possibility of the presence of a cGMP/PDE5 pathway in these tissues. Infect Immun, 2000 May, 68(5), 2954 - 61 Characterization of heat, oxidative, and acid stress responses in Brucella melitensis; Teixeira-Gomes AP et al.; Brucella melitensis is a facultative intracellular pathogen which is able to survive and replicate within phagocytic cells . Therefore, it has to adapt to a range of different hostile environments . In order to understand the mechanisms of intracellular survival employed by virulent B . melitensis 16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses by two-dimensional (2-D) polyacrylamide gel electrophoresis was used . Depending on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis . On the basis of N-terminal sequence analysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified . Some of them were previously reported for Brucella, such as BvrR, DnaK, GroEL, and Cu-Zn superoxide dismutase (SOD) . Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydrogenase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 component beta subunit . Results indicated that B . melitensis could bring specific regulatory mechanisms into play in response to stress conditions . For example, the ribosome releasing factor in B . melitensis appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for Escherichia coli, was strongly down-regulated in B . melitensis in response to heat stress . Some of the identified proteins and their potential specific regulation could be required for the adaptation of B . melitensis to environmental stresses encountered in phagocytic cells and possibly for bacterial virulence. Infect Immun, 2000 May, 68(5), 2791 - 6 mkp-1 encoding mitogen-activated protein kinase phosphatase 1, a verotoxin 1 responsive gene, detected by differential display reverse transcription-PCR in Caco-2 cells; Kojima S et al.; The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing Escherichia coli is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release . Four differentially expressed genes, including mkp-1 (encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells . Northern blot analysis showed the induction of mkp-1 mRNA 6 h after VT1 stimulation . Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced mkp-1 mRNA, but mkp-1 mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor . Therefore, we concluded that the A subunit of VT1 was essential for mkp-1 induction . Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin . Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1 . This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins. Infect Immun, 2000 May, 68(5), 2775 - 82 Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection; Weltzin R et al.; The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice . We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H . pylori urease antigen via the parenteral route . Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H . pylori . Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H . pylori gastric infection . Parenteral immunization using either LT or LTB as adjuvant protected mice against H . pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant . LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization . A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum . Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used . Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a . Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice . These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H . pylori infection. Infect Immun, 2000 May, 68(5), 2766 - 74 Identification of a gene within a pathogenicity island of enterotoxigenic Escherichia coli H10407 required for maximal secretion of the heat-labile enterotoxin; Fleckenstein JM et al.; Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens . ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease . These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis . Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E . coli pathogenicity islands . Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia . The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses . An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection . Although previous studies have suggested that E . coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island. J Physiol Pharmacol, 2000 Mar, 51(1), 85 - 102 Protective role of endogenous nitric oxide (NO) in lipopolysaccharide--induced pancreatic damage (a new experimental model of acute pancreatitis); Jaworek J et al.; Lipopolysaccharide (LPS) derived from the bacterial cell wall activates the inflammatory response in the tissue but the role of LPS in the pathogenesis of pancreatic damage and in the activation of NO system in the pancreas has not been fully explained . The aim of this study was to investigate the effect of repeated administration of LPS to the rats on the integrity of the pancreas, on the ability of isolated pancreatic acini to secrete the amylase and on the plasma level of tumor necrosis factor alpha (TNFalpha) . The role of NO in the pancreatic resistance to the damage was assessed in animals subjected to repeated administration of LPS . To induce pancreatic damage one group of rats received intraperitoneal (i.p.) injection of LPS (from E . coli) every day during 5 consecutive days (10 mg/kg--day) . Another groups of animals were given N(G)-nitro-L-arginine (L-NNA), an inhibitor of NO synthase (NOS) (20 mg/kg i.p.) alone or in combination with L-arginine (100 mg/kg i.p.), 30 min prior to each LPS injection . Plasma level of TNFalpha was determined by ELISA kit . Repeated administration of LPS produced mild pancreatic inflammation that was most pronounced at day 5 of LPS treatment and manifested as edema, neutrophil infiltration and hemorrhage of the pancreas . The survival rate after 5 days treatment with LPS was 87.5% . Pancreatic weight, plasma levels of TNFalpha and amylase, pancreatic blood flow (PBF) and NO generation by pancreatic acini were markedly increased in rats subjected to repeated administration of LPS whereas the amylase response of isolated pancreatic acini to pancreatic secretagogues was significantly attenuated . Suppression of NOS by L-NNA resulted in a dramatic increase in the mortality of the animals reaching 50% and significantly increased inflammatory changes in the pancreatic tissue, decreased PBF, abolished the ability of pancreatic acini to release NO and to secrete amylase . Pancreatic weight and plasma levels of amylase and TNFalpha significantly increased in the group of rats treated with combination of LPS+L-NNA as compared to the animals received LPS alone . Addition of L-arginine to L-NNA+LPS administration reversed all harmful effects produced by L-NNA in the pancreas . We conclude that repeated administration of high doses of bacterial LPS to the rats could induce pancreatic tissue damage by itself, however, it is not able to produce severe pancreatitis . Suppression of NO generation significantly aggravates the pancreatic lesion produced by LPS leading to the dramatic mortality in treated rats . The rise of plasma level of TNFalpha corresponds to the severity of pancreatic inflammation. Hybridoma, 2000 Feb, 19(1), 1 - 13 Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus; Long MC et al.; A novel recombinant single-chain fragment variable (scFv) antibody against Western equine encephalitis virus (WEE) was constructed and characterized . Using antibody phage display technology, a scFv was generated from the WEE specific hybridoma, 10B5 E7E2 . The scFv was fused to a human heavy chain IgG1 constant region (CH1-CH3) and contained an intact 6 His tag and enterokinase recognition site (RS10B5huFc) . The RS10B5huFc antibody was expressed in E . coli and purified by affinity chromatography as a 70-kDa protein . The RS10B5huFc antibody was functional in binding to WEE antigen in indirect enzyme-linked immunosorbent assays (ELISAs) . Furthermore, the RS10B5huFc antibody was purified in proper conformation and formed multimers . The addition of the human heavy chain to the scFv replaced effector functions of the mouse antibody . The Fc domain was capable of binding to protein G and human complement . The above properties of the RS10B5huFc antibody make it an excellent candidate for immunodetection and immunotherapy studies. Electrophoresis, 2000 Mar, 21(5), 993 - 1000 A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell; Choe LH et al.; We tested and compared three different commercially available instruments for isoelectric focusing for proteome analysis by two-dimensional protein electrophoresis . These instruments, the Multiphor, the IPGphor, and the Protean IEF cell, were used with 18 cm immobilized pH gradient strips and run under various conditions . The total number of spots and features was obtained by Melanie software (Bio-Rad Laboratories) and separately by visual inspection . The Multiphor consistently resulted in the highest number of spots detected per gel independent of sample type, immobilized pH gradient (IPG) and method to calculate the number of spots . The Protean IEF cell had the next highest number of spots detected per gel . In the experiments performed, the IPGphor afforded good reproducibility in the total number of Melanie-detected spots from gel to gel while the Protean IEF cell offered better reproducibility in the total number of manually detected spots from gel to gel . Among gels run with the different instruments, differences in the quality of the ammoniacal silver stain were also observed . A measure of quantitative reproducibility suggests that the Protean IEF cell, which was the easiest instrument to use, performs better than the other instruments, although all three instruments had demonstrated good quantitative reproducibility in the experiments performed. J Chromatogr A, 2000 Mar 31, 874(1), 27 - 43 Immobilised metal affinity chromatography of beta-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption; Clemmitt RH et al.; The development of an expanded bed process for the direct extraction and partial purification of beta-galactosidase from unclarified Escherichia coli homogenates using its natural affinity for metal loaded STREAMLINE Chelating is described . Small packed beds were used to determine the effect of chelated metal ion (Cu2+, Ni2+, Co2+ or Zn2+), loading pH and ionic strength on the selective binding capacity, and recovery of beta-galactosidase from clarified homogenates . An elution protocol was developed using the competitive displacer, imidazole, to recover beta-galactosidase in 87% yield and 3.4-fold purification . These results were then used to develop a separation for the recovery of beta-galactosidase from unclarified homogenates in a 2.5-cm diameter expanded bed . Although Ni2+ loaded STREAMLINE Chelating had a 5% dynamic capacity for beta-galactosidase of just 118 U ml(-1) (0.39 mg ml(-1)), the low capacity was thought to be due to the large size of the target (464,000) relative to the exclusion limit of the macroporous adsorbent . Despite this low capacity, Ni2 STREAMLINE Chelating was used successfully to recover beta-galactosidase from an unclarified homogenate in 86.4% yield and at 5.95-fold purification . The degree of purification relative to a commercial standard, as assessed using the purification factor and sodium dodecyl sulphate-polyacrylamide gel electrophoresis was high suggesting that this pseudo-affinity procedure compared favourably with alternative methods. Mutat Res, 2000 Apr, 462(2-3), 71 - 82 Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicity of methylating agents; Bignami M et al.; Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells . Their effects can be ascribed to an ability to modify DNA covalently . Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O(6)-methylguanine (O(6)meG) as a potentially important DNA lesion . Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells - including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli - have defined the contributions of O(6)meG and other methylated DNA bases to the biological effects of these chemicals . More recently, the role of O(6)meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O(6)meG and the mismatch repair pathway . Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O(6)meG . We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent . Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive . Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens. Gene, 2000 Apr 4, 246(1-2), 321 - 30 PCR-mediated gene replacement in Escherichia coli; Murphy KC et al.; The hyper-recombinogenic properties of an E . coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli . Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCDDelta::red substitution . This technique allows for gene replacement in E . coli without prior cloning of the gene of interest . In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates . In other experiments, electroporation of recBCDDelta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation . The placement of lambda red and gam at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyper-rec as one containing the lambda red for recBCD substitution . The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E . coli . Three of these replacements were performed without prior cloning of the genes. Gene, 2000 Apr 4, 246(1-2), 255 - 64 Identification and recombinant expression of glyceraldehyde-3-phosphate dehydrogenase of Plasmodium falciparum; Daubenberger CA et al.; The gene coding for the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was isolated from Plasmodium falciparum . The gene contains 1 intron and the A+T content is characteristic for the codon usage of P . falciparum . The predicted open reading frame codes for 337 amino acids (36651Da) and is 63.5% identical to the human erythrocytic GAPDH . GAPDH sequences from several field isolates of P . falciparum displayed 100% conservation . Phylogenetic analysis supports the hypothesis that dinoflagellates and Plasmodium are closely related . The protein encoded by the pfGAPDH was expressed recombinantly in Escherichia coli and exhibited enzymatic activity with NAD(+) but not with NADP(+) as cofactor . Antiserum raised against the recombinantly expressed enzyme detected specifically all developmental stages of cultured P . falciparum blood-stage parasites. Gene, 2000 Apr 4, 246(1-2), 151 - 5 Candida glabrata shuttle vectors suitable for translational fusions to lacZ and use of beta-galactosidase as a reporter of gene expression; El Barkani A et al.; The functionality of beta-galactosidase encoded by the E . coli lacZ gene as a reporter of gene expression in C . glabrata was investigated . C . glabrata/E . coli shuttle vectors were constructed, containing both a C . glabrata CEN-ARS cassette, to allow regular segregation and episomal replication of the plasmids, and the lacZ coding sequence of E . coli . The functionality of beta-galactosidase in C . glabrata was verified by inserting the promoter and the 5' coding region of the HIS3 gene from C . glabrata directionally upstream of the lacZ gene . By fusing the promoter of the copper-controlled MTII gene to the lacZ reporter, we showed that beta-galactosidase activity can be differentially induced in C . glabrata . beta-galactosidase reporter activities were detected qualitatively by an indirect filter assay and quantitatively from permeabilized cells. FEBS Lett, 2000 Apr 14, 471(2-3), 211 - 4 GroES co-chaperonin small-angle X-ray scattering study shows ring orifice increase in solution; Timchenko AA et al.; GroES consists of seven identical 10 kDa subunits and is involved in assisting protein folding as the partner of another oligomeric protein, the GroEL chaperonin . Here we studied the GroES structure in solution using small-angle X-ray scattering (SAXS) . The SAXS pattern, calculated for the GroES crystal structure, was found to be different from the experimental one measured in solution . The synchronic shift in the radial direction and some turning of the protein subunits eliminate the difference and result in the increase of the hole diameter in the GroES ring-like structure from 8 A in the crystal to 21 A in solution. FEBS Lett, 2000 Apr 14, 471(2-3), 173 - 6 Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration; Simon H et al.; The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated . The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration . As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site . The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents. J Exp Zool, 2000 May 1, 286(6), 656 - 65 cDNA cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle; Kimura A et al.; A cDNA for rat prolyl oligopeptidase was cloned which contained an open reading frame of 2,130 nucleotides encoding a protein of 710 amino acids . The deduced amino acid sequence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% to bacterial prolyl oligopeptidases . The recombinant prolyl oligopeptidase generated in E . coli was purified and its properties were examined . The substrate specificity and the susceptibility to proteinase inhibitors were similar to those of the native enzyme . Northern blot analysis showed wide expression of the prolyl oligopeptidase gene . Using ovaries from hormone-treated rats, it was found that both the mRNA expression and enzyme activity increased in the luteal phase . These findings suggest the involvement of prolyl oligopeptidase in events associated with corpus luteum formation and/or luteal regression. J Biol Chem, 2000 Apr 21, 275(16), 12261 - 5 Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme; Arnold DA et al.; Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination . Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion . Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme . However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele . Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo. J Biol Chem, 2000 Apr 21, 275(16), 12214 - 22 Transcriptional regulation of the divergent paa catabolic operons for phenylacetic acid degradation in Escherichia coli; Ferrandez A et al.; The expression of the divergently transcribed paaZ and paaABCDEFGHIJK catabolic operons, which are responsible for phenylacetic acid (PA) degradation in Escherichia coli, is driven by the Pz and Pa promoters, respectively . To study the transcriptional regulation of the inducible paa catabolic genes, genetic and biochemical approaches were used . Gel retardation assays showing that the PaaX regulator binds specifically to the Pa and Pz promoters were complemented with in vivo experiments that indicated a PaaX-mediated repression effect on the expression of Pa-lacZ and Pz-lacZ reporter fusions . The region within the Pa and Pz promoters that is protected by the PaaX repressor in DNase I footprinting assays contains a conserved 15-base pair imperfect palindromic sequence motif that was shown, through mutational analysis, to be indispensable for PaaX binding and repression . PA-coenzyme A (PA-CoA), but not PA, specifically inhibited binding of PaaX to the target sequences, thus confirming the first intermediate of the pathway as the true inducer and PaaX as the only bacterial regulatory protein described so far that responds to an aryl-CoA compound . Superimposed in the specific PaaX-mediated regulation is transcriptional activation by the cAMP receptor protein and the integration host factor protein . These global regulators may adjust the transcriptional output from Pa and Pz promoters to the overall growth status of the cell. J Biol Chem, 2000 Apr 21, 275(16), 12009 - 16 The yeast mitochondrial citrate transport protein . Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway; Kaplan RS et al.; The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine . A cysteine-less CTP retaining wild-type functional properties served as the starting template . The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system . The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function . These rate constants varied by seven orders of magnitude . With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193 . Based on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix . Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192 . We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. J Biol Chem, 2000 Apr 21, 275(16), 11951 - 6 Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression; Meibom KL et al.; At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes . Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes . Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP is responsible for the transcriptional inactivity of the complexes. J Biol Chem, 2000 Apr 21, 275(16), 11915 - 20 Filling the gap in vitamin A research . Molecular identification of an enzyme cleaving beta-carotene to retinal; von Lintig J et al.; Vitamin A and its derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation with important clinical implications . It has been known for 50 years that the key step in the formation of vitamin A is the oxidative cleavage of beta-carotene; however, this enzymatic step has resisted molecular analysis . A novel approach enabled us to clone and identify a beta-carotene dioxygenase from Drosophila melanogaster, expressing it into the background of a beta-carotene (provitamin A)-synthesizing and -accumulating Escherichia coli strain . The carotene-cleaving enzyme, identified here for the first time on the molecular level, is the basis of the numerous branches of vitamin A action and links plant and animal carotene metabolism. J Biol Chem, 2000 Apr 21, 275(16), 11865 - 73 Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins; David-Cordonnier MH et al.; Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity . Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage . These proteins were tested for their efficiency to induce a single-strand break on a (32)P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2'deoxyguanine, or 7, 8-dihydro-8-oxo-2'deoxyadenine at positions 1, 3, or 5 base pairs 5' or 3' to either an AP site or DHT on the labeled strand . DHT excision is much more affected than cleavage of an AP site by the presence of other damage . The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg . Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages. J Biol Chem, 2000 Apr 21, 275(16), 11758 - 64 Identification of the pore-forming region of the outer chloroplast envelope protein OEP16; Steinkamp T et al.; The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids . The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants . Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli . The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein . Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel . The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel . Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93. J Biol Chem, 2000 Apr 21, 275(16), 11672 - 7 A distinct seven-residue trigger sequence is indispensable for proper coiled-coil formation of the human macrophage scavenger receptor oligomerization domain; Frank S et al.; We have recently identified a distinct 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils but not in trimers, tetramers, or pentamers . This coiled-coil trigger pattern was demonstrated to be indispensable for the assembly of the oligomerization domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum and the leucine zipper domain of the yeast transcriptional activator GCN4 . With the aim to extend our knowledge on trigger sequences we have investigated the human macrophage scavenger receptor type A oligomerization domain as a representative of three-stranded coiled coils . We prepared a variety of recombinant N- and C-terminal deletion mutants from the full-length oligomerization domain by heterologous gene expression in Escherichia coli and assessed their ability to form trimeric coiled-coil structures by circular dichroism spectroscopy and analytical ultracentrifugation . Deletion mapping identified a distinct seven-residue sequence that was absolutely required for proper coiled-coil formation, supporting our previous results that heptad repeats alone are not sufficient for oligomerization . The finding that all fragments containing this particular sequence exhibited similar thermal stabilities indicates primarily a stabilizing function of the coiled-coil trigger . Based on sequence similarity, we suggest that functionally related sites are present in other three-stranded coiled-coil proteins. J Biol Chem, 2000 Apr 21, 275(16), 11610 - 7 Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids; Powell KA et al.; Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC) . Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization . An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid . Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM . Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone . Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin . Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase . However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain . Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals. J Biol Chem, 2000 Apr 21, 275(16), 11591 - 6 The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli; Stanley NR et al.; In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif . The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI . Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue . Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair . The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain . Unexpectedly, the consensus lysine residue was found to retard Tat transport . These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues . Tat signal peptides characteristically have positively charged carboxyl-terminal regions . However, changing the sign of this charge does not affect export of SufI. J Biol Chem, 2000 Apr 21, 275(16), 11576 - 84 Sequence-specific interaction between the disintegrin domain of mouse ADAM 2 (fertilin beta) and murine eggs . Role of the alpha(6) integrin subunit; Bigler D et al.; Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins . Mouse ADAM 2 (mADAM 2; fertilin beta) is a sperm surface protein involved in murine fertilization . We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria . The protein produced in insect cells (baculo D+C) contained a signal sequence followed by the disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells . A bacterial construct containing the disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera . Baculo D+C, as well as the D domain of the bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs . Using concentrations in the range of 1 to 5 microM, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (approximately 75 versus approximately 55% maximal inhibition) . Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine . Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-alpha(6) monoclonal antibody, GoH3, but not by a nonfunction-blocking anti-alpha(6) monoclonal antibody . Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2. J Biol Chem, 2000 Apr 21, 275(16), 11541 - 4 Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein; Jhee KH et al.; Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease) . We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase . The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase . The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism . The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm . The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction . Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function . The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes . The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase. J Biol Chem, 2000 Jun 30, 275(26), 19482 - 9 Butadiene-induced intrastrand DNA cross-links: a possible role in deletion mutagenesis; Carmical JR et al.; To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon . The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli . Both stereoisomeric forms (R, R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic . Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E . coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links . The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E . coli UvrABC complex . UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system. J Biol Chem, 2000 Jul 14, 275(28), 21349 - 54 X-ray scattering studies of Methylophilus methylotrophus (sp . W3A1) electron-transferring flavoprotein . Evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase; Jones M et al.; Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp . W(3)A(1)) . Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states . Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution . These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible "hinge" sequences that are unique to M . methylotrophus ETF . The x-ray scattering data are consistent with previous models concerning the interaction of M . methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase . Our data reveal that an "induced fit" mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process. J Biol Inorg Chem, 2000 Feb, 5(1), 67 - 74 Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry; Cowan JA et al.; Crystallographic studies of the Mn(2+)-doped RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have revealed two bound Mn2+ separated by approximately 4A and surrounded by a cluster of four conserved carboxylates . Escherichia coli RNase H is structurally similar to the RNase H domain of HIV-1 RT, but requires one divalent metal cation for its activity, implying either that the HIV-1 RT RNase H domain contrasts in its ability to bind two divalent metal ions, or that the crystallographic data reflect specific use of Mn2+ and/ or the doping technique employed . Metal binding stoichiometry has been determined for Mn2+ and the biologically more relevant Mg2+ cation by solution calorimetric studies of native and recombinant p66/p51 HIV-1 RT . Three Mn2+ ions bind to HIV-1 RT apo-enzyme: one at the DNA polymerase and two at the RNase H catalytic center, the latter being consistent with crystallographic results . However, only one Mg2+ ion is bound in the RNase H catalytic center . Several mechanistic implications arise from these results, including the possibility of mutually exclusive Mg2+ binding sites that might be occupied according to the specific reaction being catalyzed by the multifunctional RNase H domain . The occurrence of distinct binding stoichiometries for Mg2+ and Mn2+ to multifunctional enzymes has previously been reported. Anticancer Drug Des, 1999 Oct, 14(5), 411 - 20 A similarity model for the human angiogenic factor, thymidine phosphorylase/platelet derived-endothelial cell growth factor; Cole C et al.; Thymidine phosphorylase (EC 2.4.2.4), identical to the angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), is up-regulated in several tumour types . A similarity model of human thymidine phosphorylase was built, based on the crystal structure of the Escherichia coli enzyme . The high residue conservation between the two enzyme sources (39% sequence identity and 53% sequence similarity) aided model building . The human model was very similar to the E . coli enzyme's crystal structure, with the main tertiary structure difference being the destruction of helix 15 in E . coli by the presence of a loop in the human model . The model was used to rationalize the nature of the binding of the substrates thymine and thymidine, and of known inhibitors using a quantitative docking algorithm . Ab initio calculations on the nM inhibitor 5-chloro-6-(1-(2-iminopyrrolidinyl)methyl)uracil hydrochloride gave its conformation and distribution of charge . Subsequent quantitative docking studies have led to the suggestion, for the first time, that this inhibitor behaves as an oxycarbenium ion transition-state analogue, explaining its strong reported inhibition. Vestn Ross Akad Med Nauk, 2000, (3), 49 - 53 {Carbohydrate transport systems in Escherichia coli and regulation of catabolism}; Gershanovich VN; The mechanism of carbohydrate uptake in E . coli with involvement of the phosphoenol pyruvate-dependent phosphotransferase system (PTS) is dealt with . The genetic structure of the glucose transport system and fructose operon is given in detail . How the products of these systems can affect the total bacterial cellular catabolism dependent of complex of cyclic 3'5'-monophosphate + protein regulator of the above nucleotide is shown . Some section is devoted to the bacterial transport of beta-glycosides with the participation of Po-independent terminator and to the role of PTS as a highly sensitive sensory system associated with carbohydrate catabolism. Domest Anim Endocrinol, 2000 Feb, 18(2), 187 - 97 Alterations of growth hormone, cortisol, luteinizing hormone, and insulin concentrations in early-postnatal calves affected with diarrhea; Bruckmann A et al.; The aim of the study was to investigate the influence of diarrheic infections during the early postnatal phase of calves on the concentrations of hormones controlling reproduction and metabolism . Blood samples were collected from 20 male and female calves via jugular vein catheters every 15 min for 6 hr at Days 3, 9, and 21 of life . The animals were classified into three groups . Group 1 (controls): healthy calves (n = 9) . Group 2: calves affected with diarrhea at Day 9 (n = 7) . Group 3: calves with diarrhea at Days 3 and 9 (n = 4) . Infections occurred spontaneously and were mainly due to E . coli infections . All affected calves had recovered at Day 21 . Mean GH concentrations in the calves in Groups 2 and 3 compared to control calves had increased by Day 3 (P<0.01; P<0.001) . Cortisol levels of calves in all groups were highest at Day 3 and decreased thereafter (P<0.001) . Cortisol concentrations were lower at Day 3 in animals in Groups 2 (P<0.001) and 3 (P<0.05) than in controls . Pulsatile LH release was detectable at Days 9 and 21 only in healthy calves . Insulin increased at Day 9 during diarrhea . The results indicate that cortisol concentrations decreased whereas GH concentrations were increased before diarrhea was observed . The onset of pulsatile LH release was delayed in diarrheic calves . It is concluded that diarrhea exerts effects upon the release of reproductive and metabolic hormones in early postnatal calves. J Biol Chem, 2000 Jun 23, 275(25), 19098 - 105 Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain; Cruz MA et al.; The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury . It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb) . The GPIb-binding site within vWF has been localized to the vWF-A1 domain . Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R . (1998) J . Biol . Chem . 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site . We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations . The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination . Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site . One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here. J Biol Chem, 2000 Jun 16, 275(24), 18557 - 65 cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome; McCutchen-Maloney SL et al.; The primary structure of PI31, a protein inhibitor of the 20 S proteasome, was deduced by cDNA cloning and sequencing . The human protein has a calculated molecular weight of 29,792, a value in excellent accord with 31,000, as estimated by SDS-polyacrylamide gel electrophoresis for purified bovine PI31, and is not similar to any other protein in current data bases . PI31 is a proline-rich protein, particularly within its carboxyl-terminal half where 26% of the amino acids are proline . Wild-type PI31 and various truncation mutants were expressed in Escherichia coli and purified to homogeneity . Recombinant wild-type PI31 displayed structural and functional properties similar to those of PI31 purified from bovine red blood cells and inhibited the hydrolysis of protein and peptide substrates by the 20 S proteasome . Analysis of truncation mutants demonstrated that proteasome inhibition was conferred by the carboxyl-terminal proline-rich domain of PI31, which appears to have an extended secondary structure . Inhibition of the 20 S proteasome by PI31 involved formation a proteasome-PI31 complex . In addition to its direct inhibition of the 20 S proteasome, PI31 inhibited the activation of the proteasome by each of two proteasome regulatory proteins, PA700 and PA28 . These results suggest that PI31 plays an important role in control of proteasome function, including that in ubiquitin-dependent pathways of protein degradation. J Biol Chem, 2000 Jun 30, 275(26), 19461 - 8 Functional mapping of the GAGA factor assigns its transcriptional activity to the C-terminal glutamine-rich domain; Vaquero A et al.; GAGA is a nuclear protein encoded by the Trithorax-like gene in Drosophila that is expressed in at least two isoforms generated by alternative splicing . By means of its specific interaction with DNA, GAGA has been involved in several nuclear transactions including regulation of gene expression . Here we have studied the GAGA(519) isoform as a transcription factor . In vitro, the transactivation domain has been assigned to the 93 C-terminal residues that correspond to a glutamine-rich domain (Q-domain) . It presents an internal modular structure and acts independently of the rest of the protein . In vivo, in Drosophila SL2 cells, Q-domain can transactivate reporter genes either in the form of GAGA or Gal4BD-Q fusions, whereas a GAGA mutant deleted of the Q-domain cannot . Our results give support to the notion that GAGA can function as a transcription activating factor. J Biol Chem, 2000 Jun 23, 275(25), 18818 - 23 Ligand binding and structural properties of segments of GABAA receptor alpha 1 subunit overexpressed in Escherichia coli; Hang J et al.; The gamma-aminobutyric acid, type A (GABA(A)), receptor is the target for numerous therapeutic compounds . In the present study, the Gln(28)-Leu(296), Gln(28)-Arg(276), Gln(28)-Arg(248), and Gln(28)-Glu(165) (numbering of bovine precursor protein) segments of its alpha(1) subunit were overexpressed in Escherichia coli, along with Cys(166)-Leu(296) produced previously, for structural analysis by circular dichroism and ligand binding studies by fluorescence spectroscopy . Results showed that the protein segments were rich in beta-sheet structures . Binding of the fluorescent benzodiazepine Bodipy-FL Ro-1986 was evident from fluorescence resonance energy transfer and fluorescence anisotropy measurements . The binding affinity was in the micromolar range . The binding was attributable more to Cys(166)-Leu(296) than to Gln(28)-Glu(165) and was inhibited by known central benzodiazepine site ligands . Three point mutations, Y187A, T234A, and Y237A, were found to perturb protein secondary structures . Studies with the single Trp mutants W198Y and W273Y indicated that Trp(273) was closer to the binding site than Trp(198). J Biol Chem, 2000 Jun 2, 275(22), 16401 - 3 A common interface on histidine-containing phosphocarrier protein for interaction with its partner proteins; Wang G et al.; The bacterial phosphoenolpyruvate:sugar phosphotransferase system accomplishes both the transport and phosphorylation of sugars as well as the regulation of some cellular processes . An important component of this system is the histidine-containing phosphocarrier protein, HPr, which accepts a phosphoryl group from enzyme I, transfers a phosphoryl group to IIA proteins, and is an allosteric regulator of glycogen phosphorylase . Because the nature of the surface on HPr that interacts with this multiplicity of proteins from Escherichia coli was previously undefined, we investigated these interactions by nuclear magnetic resonance spectroscopy . The chemical shift changes of the backbone and side-chain amide (1)H and (15)N nuclei of uniformly (15)N-labeled HPr in the absence and presence of natural abundance glycogen phosphorylase, glucose-specific enzyme IIA, or the N-terminal domain of enzyme I have been determined . Mapping these chemical shift perturbations onto the three-dimensional structure of HPr permitted us to identify the binding surface(s) of HPr for interaction with these proteins . Here we show that the mapped interfaces on HPr are remarkably similar, indicating that HPr employs a similar surface in binding to its partners. J Mol Biol, 2000 Apr 28, 298(2), 273 - 82 High-resolution structure of the OmpA membrane domain; Pautsch A et al.; The membrane domain of OmpA consists of an eight-stranded all-next-neighbor antiparallel beta-barrel with short turns at the periplasmic barrel end and long flexible loops at the external end . The structure analysis has been extended from medium resolution to 1 . 65 A (1 A=0.1 nm), and the molecular model has been refined anisotropically to show oriented mobilities of the structural elements . The improved data allowed us to locate five further detergent molecules and 11 more water molecules . Moreover, the two large non-polar packing contacts have now been defined in detail . The analysis indicates that the beta-barrel constitutes a solid scaffold such that the long external loops need not contribute to stability . These loops are highly mobile and thus cause a major problem during the crystallization process . The beta-barrel was related to those of lipocalins . Two further crystal forms with exceptionally dense packing arrangements were established at medium resolution . J Mol Biol, 2000 Apr 28, 298(2), 195 - 209 Quadruplet codons: implications for code expansion and the specification of translation step size; Moore B et al.; One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid . The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined . With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 % . The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations . The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA . When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced . Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet . J Mol Biol, 2000 Apr 14, 297(5), 1129 - 43 The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit; Lieberman KR et al.; Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix . We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing . We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA . A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding . Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits . Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains . There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit . J Mol Biol, 2000 Apr 14, 297(5), 1105 - 20 A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin; Schlehuber S et al.; We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules . 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis . From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation . DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a K(D) of 30.2(+/-3.6) nM . The derivative compound digitoxigenin is bound even more tightly, with a K(D) of 2.0(+/-0.52) nM, whereas the steroid glycoside ouabain is not recognized at all . Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant . Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called "anticalins", which provide a promising alternative to recombinant antibody fragments . J Mol Biol, 2000 Apr 14, 297(5), 1037 - 44 A thermodynamic coupling mechanism can explain the GroEL-mediated acceleration of the folding of barstar; Bhutani N et al.; Despite extensive structural and kinetic studies, the mechanism by which the Escherichia coli chaperonin GroEL assists protein folding has remained somewhat elusive . It appears that GroEL might play an active role in facilitating folding, in addition to its role in restricting protein aggregation by secluding folding intermediates . We have investigated the kinetic mechanism of GroEL-mediated refolding of the small protein barstar . GroEL accelerates the observed fast (millisecond) refolding rate, but it does not affect the slow refolding kinetics . A thermodynamic coupling mechanism, in which the concentration of exchange-competent states is increased by the law of mass action, can explain the enhancement of the fast refolding rates . It is not necessary to invoke a catalytic role for GroEL, whereby either the intrinsic refolding rate of a productive folding transition or the unfolding rate of a kinetically trapped off-pathway intermediate is increased by the chaperonin . Am J Respir Crit Care Med, 2000 Apr, 161(4 Pt 1), 1087 - 93 Cardiac contractility is not depressed in early canine endotoxic shock; Pinsky MR et al.; We investigated effects of acute endotoxemia (Escherichia coli endotoxin, 1 mg/kg, intravenously) on left ventricular (LV) function in the first 4 h after induction of endotoxic shock in anesthetized canine preparations (n = 7 each, endotoxin and control groups) . LV pressure and conductance (volume) catheters were used to construct pressure-volume loops . Transient inferior vena cava occlusion was used to rapidly and reversibly alter LV end-diastolic volume . LV contractility was assessed from the slope of the LV end-systolic pressure-volume relationship (Ees) and from preload-recruitable stroke work (PRSW), and from their change (DeltaEes and DeltaPRSW, respectively, measured at 2 and 4 h only), in response to a dobutamine infusion (5 microg/kg/min) . Diastolic function and arterial tone were assessed as the maximal negative change in filling pressure versus time (max -dP/dt), filling rate, and arterial elastance (Ea), respectively . Ees, PRSW, Ea, diastolic function, and hemodynamics were measured hourly . Endotoxemia induced an immediate hypotensive, hyperdynamic, tachycardic state with progressive lactic acidosis . By 2.5 h after endotoxin infusion, heart rate returned to preendotoxin and control levels, but the other changes remained . However, no change occurred in LV Ees, DeltaEes, PRSW, DeltaPRSW, diastolic function, or Ea during the 4-h measurement interval . The cardiovascular collapse seen during the first 4 h of endotoxemia is therefore not due even partly to alterations in LV contractility. Anal Chem, 2000 Apr 1, 72(7), 1611 - 7 Amperometric detection of Escherichia coli heat-labile enterotoxin by redox diacetylenic vesicles on a sol-gel thin-film electrode; Peng T et al.; Supramolecular assemblies (bilayer vesicles) prepared from ferrocenic diacetylene lipid and the cell surface receptor ganglioside GM1 are utilized to construct an amperometric biosensor for Escherichia coli heat-labile enterotoxin on a sol-gel thin-film electrode . The bilayer vesicles adsorbed on the sol-gel film provide an open platform for molecular recognition, while the electrochemical communication between the incorporated redox lipids and the electrode is influenced by the binding of the toxin . Cyclic voltammetric studies suggest a facile redox reaction for the adsorbed supramolecular assembly, which allows the sensor to detect enterotoxin up to 3 ppm (3.6 x 10(-8) M) concentration . The apparent diffusion coefficients for the redox lipids in the assembly were observed to be in the range of 4.73 x 10(-8) -2.30 x 10(-8) cm/s2 . A mechanism of lateral electron transport of redox lipids controlled by biomolecular recognition is proposed. Anal Chem, 2000 Apr 1, 72(7), 1462 - 8 Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry; Zhang CX et al.; A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS) . Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries . Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary . Protein zones are stepwise eluted from the capillary by changing the mobilization conditions . Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary . We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses . The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E . coli lysate proteins using CIEF-ESI-MS . These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS. Clin Lab Haematol, 2000 Feb, 22(1), 49 - 53 Simultaneous occurrence of the 5q- syndrome and multiple myeloma; Rios R et al.; We report a case of the 5q- syndrome with simultaneous occurrence of multiple myeloma, characterized by a very complicated course . To the best of our knowledge, this is the first report of such an association. J Bacteriol, 2000 May, 182(9), 2672 - 4 A common regulator for the operons encoding the enzymes involved in D-galactarate, D-glucarate, and D-glycerate utilization in Escherichia coli; Monterrubio R et al.; Genes for D-galactarate (gar) and D-glucarate (gud) metabolism in Escherichia coli are organized in three transcriptional units: garD, garPLRK, and gudPD . Two observations suggested a common regulator for the three operons . (i) Their expression was triggered by D-galactarate, D-glucarate, and D-glycerate . (ii) Metabolism of the three compounds was impaired by a single Tn5 insertion mapped in the yaeG gene (proposed name, sdaR), outside the D-galactarate and D-glucarate systems . Expression of the sdaR gene is autogenously regulated. J Bacteriol, 2000 May, 182(9), 2619 - 23 Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase; He M et al.; A three-dimensional model of the Streptomyces coelicolor actinorhodin beta-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes . The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site . Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production . (3)H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile. J Bacteriol, 2000 May, 182(9), 2559 - 66 Purification and characterization of the alanine aminotransferase from the hyperthermophilic Archaeon pyrococcus furiosus and its role in alanine production; Ward DE et al.; Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography . The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence . The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor . Optimal activity was found in the pH range of 6 . 5 to 7.8 and at a temperature of over 95 degrees C . The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P . furiosus genome database . The gene was expressed in Escherichia coli, and the recombinant enzyme was purified . The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P . furiosus . The k(cat)/K(m) values for alanine and pyruvate formation were 41 and 33 s(-1) mM(-1), respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine . Northern analysis identified a single 1.2-kb transcript for the aat gene . In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate . The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P . furiosus. J Bacteriol, 2000 May, 182(9), 2498 - 506 Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili; Anantha RP et al.; Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells . A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E . coli . We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA . Here we report the construction and analysis of nonpolar mutations in six genes of the bfp cluster, bfpG, bfpB, bfpC, bfpD, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein . We found that mutations in bfpB, which encodes an outer membrane protein; bfpD, which encodes a putative nucleotide-binding protein; and bfpG and bfpC, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA . The mutation in bfpP, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA . The mutation in bfpH, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA . For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes . We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins . These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly. J Bacteriol, 2000 May, 182(9), 2468 - 75 Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis; Jiang X et al.; RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli . Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end . Here we show that the preference of E . coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region . This property is shared by the related E . coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species . Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E . coli . Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E . coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels. J Bacteriol, 2000 May, 182(9), 2370 - 5 Functional expression in Escherichia coli and membrane topology of porin HopE, a member of a large family of conserved proteins in Helicobacter pylori; Bina J et al.; HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin . In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies . HopE purified from E . coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H . pylori . A model of the membrane topology of HopE was constructed and indicated that this protein formed a beta-barrel with 16 transmembrane amphipathic beta-strands . The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane beta-strands but were tolerated in the adjoining loop regions . Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model . The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane. Int J Biochem Cell Biol, 2000 Apr, 32(4), 405 - 16 Molecular organization, catalytic mechanism and function of serine hydroxymethyltransferase--a potential target for cancer chemotherapy; Rao NA et al.; Serine hydroxymethyltransferase, a pyridoxal-5'-phosphate dependent enzyme, catalyzes the retro-aldol cleavage of serine to yield glycine and the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate to generate 5,10-methylene-H4-folate . The enzyme plays a pivotal role in channeling metabolites between amino acid and nucleotide metabolism . Dihydrofolate reductase and thymidylate synthase have been favorite targets for the development of anticancer drugs . However, development of resistance to drugs, due to a variety of reasons, has necessitated the identification of alternate targets for cancer chemotherapy and serine hydroxymethyltransferase is one such potential target . A detailed study of the kinetics of interaction of serine and folate analogs with this enzyme revealed several unique features that can be exploited for the design of new chemotherapeutic agents . The pathways for the reversible unfolding of the dimeric Escherichia coli and the tetrameric sheep liver enzyme, although different, revealed a requirement for the cofactor in the final step for generating an active enzyme . The gly A gene of Escherichia coli has been shown to code for this enzyme . Analysis of available gene sequences indicate that serine hydroxymethyltransferase is one of the most highly conserved proteins . The isolation of the cDNA clones for the enzyme and their overexpression in heterologous systems has enabled the probing of the molecular mechanisms of catalysis and the role of lysine, arginine and histidine in cofactor, substrate(s) binding and in maintaining the structure of the protein . Recently, the three-dimensional structure of the human liver serine hydroxymethyltransferase has been published . This, along with the information already available, provides a framework for the rational design of drugs targeted specifically towards this enzyme. Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 263 - 6 Characterization of the Aspergillus parasiticus major nitrogen regulatory gene, areA; Chang PK et al.; The major nitrogen regulatory gene, areA, was cloned from Aspergillus parasiticus . It encoded a polypeptide of 864 amino acids which contained a nuclear localization signal (NLS), a highly acidic region from positions 497 to 542, a Cys-X(2)-Cys-X(17)-Cys-X(2)-Cys DNA-binding motif and a conserved carboxy-terminus . Electrophoretic mobility shift assays suggested that the A . parasiticus AREA DNA-binding domain fusion protein bound cooperatively to single GATA elements in the A . parasiticus niaD-niiA intergenic region . AREA also bound to the aflR-aflJ intergenic region of the aflatoxin biosynthesis gene cluster . Regions of areA were fused to a yeast GAL4 DNA-binding domain coding region to localize putative transcription activation domain(s) of AREA based on activation of the GAL1(p)::lacZ reporter gene expression . The portion between NLS and the acidic domain demonstrated 16-20-fold higher activation activities than other portions of AREA, which suggests that the transcription activation domain is located in this region. Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 185 - 95 Reiterative transcription initiation from galP2 promoter of Escherichia coli; Rostoks N et al.; The expression of gal operon in Escherichia coli is driven by two promoters, P1 and P2 separated by 5 bp . The transcription initiated from the P2 generates a large amount of abortive transcripts to produce a comparable amount of full-length transcript as P1 in vitro . In this study, we investigated the source of the abortive transcripts by employing a quantitative potassium permanganate footprinting method that determines the extent of open promoter complex formation . The extents of open promoter complex formation at the two gal promoters were about the same during the given reaction time while the amount of transcription initiation determined by in vitro transcription assay showed a considerable difference: several hundred-fold more transcription initiation from the P2 than the P1, most of which was abortive . Thus, it was concluded that the abortive transcripts are generated reiteratively by a small fraction of RNA polymerase . An in vitro transcription assay using an immobilized DNA template revealed that the fraction of RNA polymerase generating abortive transcripts never produces the full-length transcript and it remains bound to the promoter . We concluded that there are two kinds of RNA polymerase-promoter complexes formed at galP2, at least in vitro, productive complex and nonproductive complex; and, the nonproductive complex is responsible for generating large amount of abortive transcripts from the P2. Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 143 - 60 Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv; Kaydamov C et al.; We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv . at early developmental stages . The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits . Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N . plumbaginifolia genome . Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin . Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins . The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences . This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development. Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 1 - 6 Growth-phase regulation of the Escherichia coli thioredoxin gene; Lim CJ et al.; The two promoters of Escherichia coli trxA gene were separately cloned into pKO100 as well as pJEL170 . Galactokinase expression in cells containing the pKO100 derivatives was found to be negatively correlated with growth rate and was 6- to 20-fold higher in stationary cultures than in exponential cultures . The expression of trxA-galK was induced by amino acid starvation in a RelA(+) strain but not in an isogenic Rel(-) strain indicating that the control involves guanosine 3',5'-bispyrophosphate (ppGpp) . RpoS, which appears to be essential for expression of most stationary phase expressed genes, is not required for trxA expression . Increased expression of relA, which increases ppGpp concentration, increases trxA expression. FEBS Lett, 2000 Apr 7, 471(1), 99 - 102 Soluble P-type ATPase from an archaeon, Methanococcus jannaschii; Ogawa H et al.; MJ0968 has been proposed to be an ancestor of P-type ATPase, because its primary structure is highly homologous to that of the core catalytic domain of P-type ATPase . However it completely lacks amino acid sequences that possibly constitute transmembrane domains . To examine if MJ0968 is indeed a P-type ATPase, it was overexpressed in Escherichia coli and purified . It did show ATPase activity, autophosphorylation and inhibition by vanadate . All these properties support the idea that MJ0968 is indeed a soluble P-type ATPase. Vet Immunol Immunopathol, 2000 Apr 19, 74(1-2), 137 - 44 Production of antibodies against chicken interferon-gamma: demonstration of neutralizing activity and development of a quantitative ELISA; Lambrecht B et al.; Four monoclonal antibodies (mAbs) specific for chicken interferon-gamma (ChIFN-gamma) were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-gamma . Each mAb reacted specifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-gamma in ELISA and Western Blot analysis or natural ChIFN-gamma in immunofluorescence experiments . As determined by competition ELISAs, mAbs 3D5, 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-gamma molecule, whereas mAb 1E12 recognized a distant epitope . Moreover, this latter mAb was able to highly neutralize the biological activities of both recombinant and natural ChIFN-gamma as measured by inhibition of viral replication and macrophage activation . To improve the detection of ChIFN-gamma, a capture ELISA was developed using mAb 1E12 as capture antibody and biotinylated mAb 4C6 as detection antibody . In addition to being more rapid and easier to perform than classical cell-mediated immunity tests, this ELISA has excellent sensitivity and improved specificity . The use of a specific rabbit polyclonal serum as revealing antibody further increased the sensitivity of the detection down to 0.5ng/ml of ChIFN-gamma . This ELISA would provide a sensitive tool to measure the in vitro release of ChIFN-gamma by T-cells in response to specific recall antigen. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 4221 - 6 Conversion of alpha-lactalbumin to a protein inducing apoptosis; Svensson M et al.; In this study alpha-lactalbumin was converted from the regular, native state to a folding variant with altered biological function . The folding variant was shown to induce apoptosis in tumor cells and immature cells, but healthy cells were resistant to this effect . Conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells) required partial unfolding of the protein and a specific fatty acid, C18:1, as a necessary cofactor . Conversion was achieved with alpha-lactalbumin derived from human milk whey and with recombinant protein expressed in Escherichia coli . We thus have identified the folding change and the fatty acid as two key elements that define HAMLET, the apoptosis-inducing functional state of alpha-lactalbumin . Although the environment in the mammary gland favors the native conformation of alpha-lactalbumin that serves as a specifier in the lactose synthase complex, the conditions under which HAMLET was formed resemble those in the stomach of the nursing child . Low pH is known to release Ca(2+) from the high-affinity Ca(2+)-binding site and to activate lipases that hydrolyze free fatty acids from milk triglycerides . We propose that this single amino acid polypeptide chain may perform vastly different biological functions depending on its folding state and the in vivo environment . It may be speculated that molecules like HAMLET can aid in lowering the incidence of cancer in breast-fed children by purging of tumor cells from the gut of the neonate. Proc Natl Acad Sci U S A, 2000 Apr 11, 97(8), 3838 - 43 The human DINB1 gene encodes the DNA polymerase Poltheta; Johnson RE et al.; The human DINB1 gene shares a high degree of homology with the Escherichia coli dinB gene . Here, we purify the hDINB1-encoded protein and show that it is a DNA polymerase . Because hDinB1 is the eighth eukaryotic DNA polymerase to be described, we have named it DNA polymerase (Pol) theta . hPoltheta is unable to bypass a cis-syn thymine-thymine dimer, nor does it bypass a (6-4) photoproduct or an abasic site . We also examine the fidelity of hPoltheta on nondamaged DNA templates by steady-state kinetic analyses and find that hPoltheta misincorporates deoxynucleotides with a frequency of about 10(-3) to 10(-4) . We discuss the relationship between the fidelity of hPoltheta and its inability to bypass DNA damage. Int J Biochem Cell Biol, 2000 Apr, 32(4), 465 - 73 Rhodanese as a thioredoxin oxidase; Nandi DL et al.; A major catalytic difference between the two most common isoforms of bovine liver mitochondrial rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been observed . Both isoforms were shown to be capable of using reduced thioredoxin as a sulfur-acceptor substrate . However, only the less negative form in common with the recombinant mammalian rhodanese expressed in E . coli, can also catalyze the direct oxidation of reduced thioredoxin evidently by reactive oxygen species . These activities are understood in terms of the established persulfide structure (R-S-SH) of the covalently substituted rhodanese in the sulfurtransferase reaction and an analogous sulfenic acid structure (R-S-OH) when the enzyme acts as a thioredoxin oxidase . The observations suggest a role for one rhodanese isoform in the detoxication of intramitochondrial oxygen free radicals. Int J Biochem Cell Biol, 2000 Apr, 32(4), 455 - 64 An endogenous proteinacious inhibitor in porcine liver for S-adenosyl-L-methionine dependent methylation reactions: identification as oligosaccharide-linked acyl carrier protein; Seo DW et al.; A proteinacious inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent transmethylation reactions was purified to homogeneity from porcine liver by size exclusion chromatography and FPLC . The molecular weight of the inhibitor was 12,222 Da . A 7400 Da polypeptide fragment of the purified inhibitor was sequenced by matrix-associated laser desorption ionization; time-of-flight MS, and was found to be identical with the known sequence of spinach acyl carrier protein (ACP) . Although the remainder of the molecule was not clearly defined, 1H and H-H correlation of spectroscopy (COSY) NMR analysis revealed the presence of an oligosaccharide with alpha-glycosidic linkage . The purified oligosaccharide-linked ACP inhibited several AdoMet-dependent transmethylation reactions such as protein methylase I and II . S-farnesylcysteine O-methyltransferase, DNA methyltransferase and phospholipid methyltransferase . Protein methylase II was inhibited with a Ki value of 2.4 x 10(-3) M in a mixed inhibition pattern, whereas a well-known competitive product inhibitor S-adenosyl-L-homocysteine (AdoHcy) had Ki value of 6.3 x 10(-6) M . Commercially available active ACP fragments (65-74) and ACP from Escherichia coli had less inhibitory activity toward S-farnesylcysteine O-methyltransferase than the purified inhibitor . The biological significance of this oligosaccharide-linked ACP which has two seemingly unrelated functions (inhibitor for transmethylation and fatty acid biosynthesis) remains to be elucidated. Mol Microbiol, 2000 Apr, 36(1), 223 - 9 Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop; Wade JT et al.; The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter . MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point . In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region . Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression . Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter . The two MelR binding sites required for this optimal repression are separated by 177 bp . We suggest that, in the absence of melibiose, MelR forms a loop between these two sites . We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated . However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region . Parallels with the Escherichia coli araC-araBAD regulatory region are discussed. Mol Microbiol, 2000 Apr, 36(1), 211 - 22 Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein; Belyaeva TA et al.; MelR is a melibiose-triggered transcription activator that belongs to the AraC family of transcription factors . Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter . This activation is dependent on MelR and melibiose . These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR-dependent transcription activation . DNAase I footprinting has been exploited to investigate the location of MelR-and CRP-binding sites at the melAB promoter . We showed previously that MelR binds to two identical 18 bp target sequences centred at position -100.5 (Site 1) and position -62.5 (Site 2) . In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1', centred at position -120.5, located immediately upstream of Site 1, and Site R, at position -238.5, which overlaps the transcription start site of the divergent melR promoter . MelR can bind to Site 1', Site 1, Site 2 and Site R, in both the absence and the presence of melibiose . However, in the presence of melibiose, MelR also binds to a fifth site (Site 2', centred at position -42.5) located immediately downstream of Site 2, and overlapping the -35 region of the melAB promoter . Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding Site 1 and Site 2 . Thus, tandem-bound MelR recruits CRP to the MelR . We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2' . Optimal expression of the melAB promoter requires Sites 1', Site 1, Site 2 and Site 2'; CRP acts as a 'bridge' between MelR bound at Sites 1' and 1 and at Sites 2 and 2', increasing expression from the melAB promoter . In support of this model, we show that improvement of the base sequence of Site 2' removes the requirement for Site 1' and Site 1, and short circuits the effects of CRP. Mol Microbiol, 2000 Apr, 36(1), 174 - 82 Identification of Escherichia coli K1 genes contributing to human brain microvascular endothelial cell invasion by differential fluorescence induction; Badger JL et al.; Most cases of Escherichia coli K1 meningitis arise as a result of haematogenous spread, however there is a limited understanding of the mechanisms by which circulating E . coli K1 cross the blood-brain barrier . We have previously shown that environmental growth conditions both positively and negatively influence the capabilities of E . coli K1 to invade brain microvascular endothelial cells (BMEC), for example growth in media supplemented with 50% newborn bovine serum (NBS) increased BMEC invasion, whereas growth in media supplemented with 0.2 M NaCl repressed invasion in vitro and in vivo . In this study, differential fluorescence induction (DFI) was used to identify E . coli K1 genes involved in this differentially expressed invasion phenotype . E . coli K1 promoter libraries were constructed and screened for gfp expression in a manner analogous to the above growth conditions . Twenty-four clones were isolated that showed fluorescence induction when grown under the invasion-enhancing condition (i.e . NBS) . Four of these clones also demonstrated repression or no induction of fluorescence when grown under the invasion-repressing condition (i.e . 0.2 M NaCl) . One such clone, containing a ygdP promoter and an open reading frame (ORF), showed significant homology to Bartonella bacilliformis IalA (invasion associated locus) . Among the other NBS-inducing loci, finPtraJ was identified as well as several clones with no homology to other known genes . When ygdP, finPtraJ and several of the unique loci were disrupted in E . coli K1, there was a significant decrease in human BMEC (HBMEC) invasion . RNA transcript analysis determined that these newly identified invasion loci were differentially regulated at the transcriptional level . This is the first demonstration of using DFI to identify E . coli K1 genes contributing to HBMEC invasion. Mol Microbiol, 2000 Apr, 36(1), 132 - 40 The aspartate chemoreceptor Tar is effectively methylated by binding to the methyltransferase mainly through hydrophobic interaction; Shiomi D et al.; In the chemotaxis of Escherichia coli, adaptation requires the methylation and demethylation of transmembrane receptors, which are catalysed by the methyltransferase CheR and the methylesterase CheB respectively . CheR binds to major chemoreceptors through their C-terminal motif NWETF, which is distinct from the methylation sites . In this study, we carried out a systematic mutagenesis of the pentapeptide sequence of Tar . Receptor methylation and adaptation were severely impaired by the alanine substitution of residue W550 and, to a lesser extent, by that of F553 . Substitution of residues N549, E551 and T552 had only a slight or little effect . The defects of the W550A and F553A mutations were suppressed by high- and low-level overproduction of CheR respectively . Expression of a fusion protein containing the NWETF sequence, but not its W550A and F553A versions, inhibited chemotaxis of the Che+ strain . In an in vitro assay, CheR bound to the wild-type version but not to the mutant versions . These results and further mutagenesis suggest that the hydrophobicity and the size of residues W550 and F553 are critical in the interaction with CheR, a conclusion that is consistent with the crystal structure of a CheR-NWETF complex . On the other hand, the negatively charged side chain of E551 and the polar side chains of N549 and T552 may not be strictly required, although the presence of a salt bridge and hydrogen bonds between these residues and residues from CheR has been noted in the co-crystal. Mol Microbiol, 2000 Apr, 36(1), 85 - 92 Antagonistic control of the Escherichia coli bgl promoter by FIS and CAP in vitro; Caramel A et al.; The wild-type Escherichia coli bgl promoter is silent in vivo but active in vitro . Silencing in vivo is directed by silencer sequences that flank the promoter, and requires nucleoid-associated protein H-NS and other unidentified cellular factors . Here we show that the DNA bending protein FIS is a repressor of the bgl promoter . Two FIS binding sites, centred at positions -52 and -27, overlap the CAP binding site and the -35 box respectively . FIS efficiently competes with CAP for binding to the wild-type promoter . However, FIS does not prevent binding of RNA polymerase . It interferes with the formation of a heparin-resistant complex and represses transcription initiation up to 40-fold . The presence of CAP has very little effect on the FIS-mediated repression of the wild-type bgl promoter in vitro . However, when a bgl promoter allele was tested that carries an improved CAP binding site (which leads to activation in vivo) CAP effectively counteracted repression by FIS in vitro . These results suggest that FIS contributes to silencing of the wild-type bgl promoter in vivo, presumably in the early exponential phase when FIS is predominantly expressed. Mol Microbiol, 2000 Apr, 36(1), 33 - 43 Functional polarization of the Escherichia coli chromosome terminus: the dif site acts in chromosome dimer resolution only when located between long stretches of opposite polarity; Perals K et al.; In Escherichia coli, chromosome dimers are generated by recombination between circular sister chromosomes . Dimers are lethal unless resolved by a system that involves the XerC, XerD and FtsK proteins acting at a site (dif) in the terminus region . Resolution fails if dif is moved from its normal position . To analyse this positional requirement, dif was transplaced to a variety of positions, and deletions and inversions of portions of the dif region were constructed . Resolution occurs only when dif is located at the convergence of multiple, oppositely polarized DNA sequence elements, inferred to lie in the terminus region . These polar elements may position dif at the cell septum and be general features of chromosome organization with a role in nucleoid dynamics. Mol Microbiol, 2000 Apr, 36(1), 24 - 32 A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor; Qin L et al.; Histidine kinases function as dimers . The kinase domain of the osmosensing histidine kinase EnvZ of Escherichia coli consists of two domains: domain A (67 residues) responsible for histidine phosphotransfer and dimerization, and domain B (161 residues) responsible for the catalytic and ATP-binding function . The individual structures of these two domains have been recently solved by NMR spectroscopy . Here, we demonstrate that an enzymatically functional monomeric histidine kinase can be constructed by fusing in tandem two domains A and one domain B to produce a single polypeptide (A-A-B) . We show that this protein, EnvZc{AAB}, is soluble and exists as a stable monomer . The autophosphorylation and OmpR kinase activities of the monomeric EnvZc{AAB} are similar to that of the wild-type EnvZ, while OmpR-binding and phosphatase functions are reduced . V8 protease digestion and mutational analyses indicate that His-243 of only the amino proximal domain A is phosphorylated . Based on these results, molecular models are proposed for the structures of EnvZc{AAB} and the kinase domain of EnvZ . The present results demonstrate for the first time the construction of a functional, monomeric histidine kinase, further structural studies of which may provide important insights into the structure-function relationships of histidine kinases. Mol Microbiol, 2000 Mar, 35(6), 1560 - 72 Identification of additional genes belonging to the LexA regulon in Escherichia coli; Fernandez De Henestrosa AR et al.; Exposure of Escherichia coli to a variety of DNA-damaging agents results in the induction of the global 'SOS response' . Expression of many of the genes in the SOS regulon are controlled by the LexA protein . LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA-regulated gene . Alignment of 20 LexA binding sites found in the E . coli chromosome reveals a consensus of 5'-TACTG(TA)5CAGTA-3' . DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA . By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E . coli genome to identify novel LexA-regulated genes . These searches identified a total of 69 potential LexA-regulated genes/operons with a heterology index of <15 and included all previously characterized LexA-regulated genes . Probes were made to the remaining genes, and these were screened by Northern analysis for damage-inducible gene expression in a wild-type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non-inducible lexA(Ind-) cell . These experiments have allowed us to identify seven new LexA-regulated genes, thus bringing the present number of genes in the E . coli LexA regulon to 31 . The potential function of each newly identified LexA-regulated gene is discussed. Mol Microbiol, 2000 Mar, 35(6), 1506 - 17 The ChiA (YheB) protein of Escherichia coli K-12 is an endochitinase whose gene is negatively controlled by the nucleoid-structuring protein H-NS; Francetic O et al.; The chromosome of Escherichia coli K-12 contains a putative gene, yheB (chiA), at centisome 74.7, whose product shows sequence similarity with chitinases of bacterial and viral origin . We cloned the chiA (yheB) gene and demonstrated that it codes for a 94.5 kDa periplasmic protein with endochitinase/lysozyme activity . Under standard laboratory growth conditions, chiA expression is very low, as shown by the Lac- phenotype of a chiA transcriptional fusion to a promoterless lacZ reporter . To identify factors that control chitinase gene expression, we generated random Tn10 insertions in the chromosome of the fusion-containing strain, selecting for a Lac+ phenotype . The majority of the mutations that caused a Lac+ phenotype mapped to the hns gene, encoding the nucleoid-structuring protein H-NS . Transcription of chiA in vivo is driven by a single sigma70 promoter and is derepressed in an hns mutant . Using a competitive gel retardation assay, we demonstrated that H-NS binds directly and with high affinity to the chiA promoter region . In addition to hns, other E . coli mutations causing defects in global regulatory proteins, such as fis, crp or stpA in combination with hns, increased chiA expression to different extents, as did decreasing the growth temperature from 37 degrees C to 30 degrees C . A possible physiological function of ChiA (YheB) endochitinase in E . coli K-12 is discussed. Mol Microbiol, 2000 Mar, 35(6), 1443 - 53 Specific amino acid changes enhance the anti-recombination activity of the UmuD'C complex; Sommer S et al.; In addition to being an essential component of trans-lesion synthesis, the UmuD'C complex is an antagonist of RecA-mediated homologous recombination . When constitutively expressed at an elevated concentration, the UmuD'C complex sensitizes recA+ bacteria to DNA damage, whereas it has no effect on bacteria expressing a RecA {UmuR} protein that overcomes recombination inhibition . Using as a genetic screen enhanced cell killing on mitomycin plates, we isolated novel umuD' and umuC mutations that restored mitomycin sensitivity to recA D112G {UmuR} bacteria overproducing the UmuD'C complex . The mutations were named {Rin++} because a characterization in a recA+ as well in a recA D112G background showed that they enhanced UmuD'C-promoted recombination inhibition in two assays, conjugational recombination and recombinational repair of palindrome-containing DNA . The {Rin++} mutations affect five amino acids, G25D, S28T, P29L, E35K, and T95R, in UmuD' and seven, F10L, Y270C, K277E, F287L, F287S, K342Q and F351I, in UmuC . These amino acids might play a key role in the UmuD'C anti-recombination activity . None of the {Rin++} mutations enhanced UmuD'C-promoted mutagenic bypass of UV lesions, in contrast, several lead to a defect in this process . In this study, we discuss a few molecular mechanisms that could account for the recombination and mutagenesis phenotypes of a mutant UmuD'C {Rin++} complex. Mol Microbiol, 2000 Mar, 35(6), 1413 - 20 Escherichia coli response to hydrogen peroxide: a role for DNA supercoiling, topoisomerase I and Fis; Weinstein-Fischer D et al.; Bacterial cells respond to the deleterious effects of reactive oxygen species by inducing the expression of antioxidant defence genes . Here we show that treatment with hydrogen peroxide leads to a transient decrease in DNA negative supercoiling . We also report that hydrogen peroxide activates topA P1 promoter expression . The peroxide-dependent topA P1 activation is independent of oxyR, but is mediated by Fis . This nucleoid-associated protein binds to the promoter region of topA . We also show that a fis deficient mutant strain is extremely sensitive to hydrogen peroxide . Our results suggest that topA activation by Fis is an important component of the Escherichia coli response to oxidative stress. Mol Microbiol, 2000 Mar, 35(6), 1360 - 74 Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function; Gordon EH et al.; DipZ is a bacterial cytoplasmic membrane protein that transfers reducing power from the cytoplasm to the periplasm so as to facilitate the formation of correct disulphide bonds and c-type cytochromes in the latter compartment . Topological analysis using gene fusions between the Escherichia coli dipZ and either E . coli phoA or lacZ shows that DipZ has a highly hydrophobic central domain comprising eight transmembrane alpha-helices plus periplasmic globular N-terminal and C-terminal domains . The previously assigned translational start codon for the E . coli DipZ was shown to be incorrect and the protein to be larger than previously thought . The experimentally determined translational start position indicates that an additional alpha-helix at the N-terminus acts as a cleavable signal peptide so that the N-terminus of the mature protein is located in the periplasm . The newly assigned 5' end of the dipZ gene was shown to be preceded by a functional ribosome-binding site . The hydrophobic central domain and both of the periplasmic globular domains each have a pair of highly conserved cysteine residues, and it was shown by site directed mutagenesis that all six conserved cysteine residues contribute to DipZ function. Mol Microbiol, 2000 Mar, 35(6), 1348 - 59 The transfer region of IncI1 plasmid R64: similarities between R64 tra and legionella icm/dot genes; Komano T et al.; The entire nucleotide sequence of the transfer region of IncI1 plasmid R64 was determined together with previously reported sequences . Twenty-two transfer genes, traE-Y and nuc, were newly identified in the present study . The protein products of 17 genes were detected by maxicell experiments or by the T7 RNA polymerase expression system . Mutagenesis experiments indicated that 16 genes were indispensable for R64 transfer both in liquid and on surfaces . In summary, the R64 transfer region located within an approximately 54 kb DNA segment was shown to encode the most complex transfer system so far studied . It contains at least 49 genes and may produce 58 different proteins as a result of shufflon DNA rearrangement and overlapping genes . Among the 49 genes, 23 tra, trb and nik genes have been shown to be indispensable for R64 conjugal transfer in liquid and on surfaces . Twelve additional pil genes are required only for liquid matings . The amino acid sequences of 10 R64 tra/trb products share similarity with those of the icm/dot products of Legionella pneumophila that are responsible for its virulence, suggesting that the R64 transfer and L . pneumophila icm/dot systems have evolved from a common ancestral genetic system. Mol Microbiol, 2000 Mar, 35(6), 1312 - 25 The role of tandem IS dimers in IS911 transposition; Turlan C et al.; Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer . When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other . These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR-IRL junction, to generate simple insertions . In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination . This also creates an active IRR-IRL junction by generating a head-to-tail IS tandem dimer ({IS}2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction . Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule . Inter-IS figure 8 molecules generated in vitro could be resolved into the {IS}2 form following introduction into a host strain by transformation . Resolution did not require IS911 transposase . The {IS}2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro . Previous studies had demonstrated that the IRR-IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro . Integration of the {IS}2 derivative would result in a product that resembles a co-integrate structure . It is also shown here that the IRR-IRL junction of the {IS}2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro . These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements . This behaviour is probably characteristic of a large number of IS elements. Eur J Biochem, 2000 Apr, 267(8), 2260 - 7 An anti-idiotypic antibody with an internal image of human interferon-gamma and human interferon-gamma-like antiviral activity; Depraetere H et al.; D9D10, a monoclonal antibody that inhibits the biological activity of human interferon-gamma (IFN-gamma), was used to generate monoclonal anti-idiotypic antibodies . After a first selection, the monoclonal anti-idiotypic antibody AA1E5 was chosen to be fully characterized . To the best of our knowledge this is the first description of a monoclonal antibody with an IFN-gamma-like antiviral activity; AA1E5 competed with IFN-gamma for binding to D9D10 indicating its anti-idiotypic character . However, AA1E5 also fully mimics HuIFN-gamma as it not only binds to the HuIFN-gamma-receptor, where it competes with HuIFN-gamma, but more importantly AA1E5 and its Fv fragment, cloned and expressed in Escherichia coli, mimic the antiviral activity of HuIFN-gamma . Indeed, 15 microg of AA1E5 and 2.5 microg of its Fv fragment had an effect comparable to that of 10 IU of HuIFN-gamma in an antiviral assay on A549 cells . Sequence comparison between the complementarity determination regions of the antibody and the sequence of HuIFN-gamma revealed that both the heavy chain variable domain, VH, and the kappa light chain variable domain, Vkappa, have epitopes of 3-4 amino acids that are present in the HuIFN-gamma sequence, some of which contribute to receptor binding, as identified by Walter et al . {M . R . Walter, W . T . Windsor, T . L . Nagabhushan, D . J . Lundell, C . A . Lunn, P . J . Zauodny & S . K . Narula (1995) Nature 376, 230-235}. Eur J Biochem, 2000 Apr, 267(8), 2242 - 51 The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition; Carcamo JG et al.; To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis . The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme . The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme . The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes . The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity . In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive . In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity . These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP. Clin Exp Immunol, 2000 Apr, 120(1), 218 - 23 Autoepitopes on autoantigen centromere protein-A (CENP-A) are restricted to the N-terminal region, which has no homology with histone H3; Muro Y et al.; Anti-centromere autoantibodies (ACA) are commonly found in the serum of patients with a limited type of scleroderma and other systemic autoimmune diseases . CENP-A is one of the major antigens against ACA and a histone H3-like protein . To analyse the autoantigenic epitopes of CENP-A, a series of truncated peptides of human CENP-A were expressed in Escherichia coli and immunoblotting analysis was performed with 91 ACA+ sera . Eighty sera (88%) with the ACA reacted to the 52-amino acids N-terminal region which is not homologous to H3, while no sera reacted to the C-terminus which has a sequence similarity with H3 . Moreover, ELISA was also employed in this study using two synthetic peptides corresponding to the amino acid sequences 3-17 (peptide A) and 25-38 (peptide B) . Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively . Core antigens of hepatitis B virus (HBV) and hepatitis C virus (HCV) have similar sequences to peptide A and/or peptide B, but three sera containing HBV without ACA and five sera containing HCV without ACA were found to be reactive to neither peptide . Centromere localization of CENP-A is dependent on the H3-like C-terminal domain which is not autoantigenic, while the antigenic N-terminal domain, which might play unidentified functional roles, should be an important region for the induction of ACA. Br J Surg, 2000 Apr, 87(4), 448 - 53 Aminoguanidine attenuates endotoxin-induced mesenteric vascular hyporeactivity; Kavuklu B et al.; BACKGROUND: The aim of this study was to investigate the effects of inducible nitric oxide synthase inhibition by aminoguanidine on endotoxin-induced reduction in mesenteric blood flow . METHODS: Twenty Sprague-Dawley rats (180-230 g) allocated into four groups were administered either Escherichia coli endotoxin 1 mg/kg intraperitoneally or its solvent saline and were pretreated with either aminoguanidine (15 mg/kg intraperitoneally 20 min before and 2 h after endotoxin injection) or saline . Some 4 h after endotoxin injection, animals were anaesthetized, arterial blood pressure and mesenteric blood flow were measured and the resistance in the mesenteric vascular beds was then calculated . The effect of phenylephrine (1-30 microg/kg intravenously) on these parameters was also investigated . RESULTS: Endotoxin did not significantly modify the mean arterial blood pressure but decreased mesenteric blood flow by increasing the vascular resistance (mean(s.e.m.) 7.8(1.0) versus 13.7(1.2) mmHg per min per ml for control versus endotoxin groups; n = 5, P = 0.0099) . Aminoguanidine alone had no effect on either the mean arterial blood pressure or mesenteric blood flow, but it completely blocked the effects of endotoxin . On the other hand, endotoxin significantly attenuated the responsiveness to phenylephrine which was restored by aminoguanidine . CONCLUSION: The present results indicate that endotoxin decreases the mesenteric vascular blood flow by increasing vascular resistance and decreases responsiveness to phenylephrine . The effects of endotoxin were inhibited by aminoguanidine . The mesenteric vasoconstriction in response to endotoxin might not be explained by the overproduction of nitric oxide; other actions of aminoguanidine may explain its inhibitory effect . Presented in part to the 10th Annual Meeting of the Surgical Infection Society - Europe, Istanbul, Turkey, May 1997 Plant Physiol, 2000 Apr, 122(4), 1311 - 21 Cloning and expression of cytochrome P450 enzymes catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of cyanogenic glucosides in Triglochin maritima; Nielsen JS et al.; Two cDNA clones encoding cytochrome P450 enzymes belonging to the CYP79 family have been isolated from Triglochin maritima . The two proteins show 94% sequence identity and have been designated CYP79E1 and CYP79E2 . Heterologous expression of the native and the truncated forms of the two clones in Escherichia coli demonstrated that both encode multifunctional N-hydroxylases catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the two cyanogenic glucosides taxiphyllin and triglochinin in T . maritima . This renders CYP79E functionally identical to CYP79A1 from Sorghum bicolor, and unambiguously demonstrates that cyanogenic glucoside biosynthesis in T . maritima and S . bicolor is catalyzed by analogous enzyme systems with p-hydroxyphenylacetaldoxime as a free intermediate . This is in contrast to earlier reports stipulating p-hydroxyphenylacetonitrile as the only free intermediate in T . maritima . L-3,4-Dihydroxyphenyl{3-(14)C}Ala (DOPA) was not metabolized by CYP79E1, indicating that hydroxylation of the phenol ring at the meta position, as required for triglochinin formation, takes place at a later stage . In S . bicolor, CYP71E1 catalyzes the subsequent conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile . When CYP79E1 from T . maritima was reconstituted with CYP71E1 and NADPH-cytochrome P450 oxidoreductase from S . bicolor, efficient conversion of tyrosine to p-hydroxymandelonitrile was observed. Plant Physiol, 2000 Apr, 122(4), 1289 - 99 Identification of a Hsp70 recognition domain within the rubisco small subunit transit peptide; Ivey RA 3rd et al.; The interaction between SStp, the transit peptide of the precursor protein to the small subunit of Rubisco (prSSU) and two Hsp70 molecular chaperones, Escherichia coli DnaK and pea (Pisum sativum) CSS1, was investigated in detail . Two statistical analyses were developed and used to investigate and predict regions of SStp recognized by DnaK . Both algorithms suggested that DnaK would have high affinity for the N terminus of SStp, moderate affinity for the central region, and low affinity for the C terminus . Furthermore, both algorithms predicted this affinity pattern for >75% of the transit peptides analyzed in the chloroplast transit peptide (CHLPEP) database . In vitro association between SStp and these Hsp70s was confirmed by three independent assays: limited trypsin resistance, ATPase stimulation, and native gel shift . Finally, synthetic peptides scanning the length of SStp and C-terminal deletion mutants of SStp were used to experimentally map the region of greatest DnaK affinity to the N terminus . CSS1 displayed a similar affinity for the N terminus of SStp . The major stromal Hsp70s affinity for the N terminus of SStp and other transit peptides supports a molecular motor model in which the chaperone functions as an ATP-dependent translocase, committing chloroplast precursor proteins to unidirectional movement across the envelope. Plant Physiol, 2000 Apr, 122(4), 1193 - 9 The plastidic phosphoglucomutase from Arabidopsis . A reversible enzyme reaction with an important role in metabolic control; Periappuram C et al.; An Arabidopsis cDNA (AtPGMp) encoding the plastidic phosphoglucomutase (PGM) predicted a 623-amino acid protein with an N-terminal sequence typical of a plastid signal peptide . Expression of a recombinant protein in Escherichia coli confirmed its enzyme activity . The recombinant enzyme had an apparent K(m) value of 98.5 microM and a V(max) of 4.48 micromol min(-1) (mg protein)(-1) . The Calvin cycle intermediates fructose-1,6-bisphosphate and ribulose-1, 5-bisphosphate exerted an inhibitory effect on PGM activity, supporting its proposed involvement in controlling photosynthetic carbon flow . A point mutation was identified in the AtPGMp gene of the Arabidopsis pgm-1 mutant . The mutation in the mutant transcript generated a stop codon at about one third of the wild-type open reading frame, and thus rendered the polypeptide nonfunctional . Storage lipid analysis of the pgm-1 mutant seeds showed a 40% reduction in oil content compared with that of wild type . Our results indicate that plastidic PGM is an important factor affecting carbon flux in triacylglycerol accumulation in oilseed plants, most likely through its essential role in starch synthesis. Plant J, 2000 Mar, 21(5), 445 - 54 A bifunctional epimerase-reductase acts downstream of the MUR1 gene product and completes the de novo synthesis of GDP-L-fucose in Arabidopsis; Bonin CP et al.; L-Fucose is a monosaccharide found as a component of glycoproteins and cell wall polysaccharides in higher plants . The MUR1 gene of Arabidopsis thaliana encodes a GDP-D-mannose 4,6-dehydratase catalyzing the first step in the de novo synthesis of GDP-L-fucose from GDP-D-mannose (Bonin et al . 1997, Proc . Natl Acad . Sci . USA, 94, 2085-2090) . Plant genes encoding the subsequent steps in L-fucose synthesis (3,5-epimerization and 4-reduction) have not been described previously . Based on sequence similarities to a bacterial gene involved in capsule synthesis we have cloned a gene from Arabidopsis, now designated GER1, which encodes a bifunctional 3, 5-epimerase-4-reductase in L-fucose synthesis . The combined action of the MUR1 and GER1 gene products converts GDP-D-mannose to GDP-L-fucose in vitro demonstrating that this entire nucleotide-sugar interconversion pathway could be reconstituted using plant genes expressed in Escherichia coli . In vitro assays indicated that the GER1 protein does not act as a GDP-D-mannose 3, 5-epimerase, an enzymatic activity involved in the de novo synthesis of GDP-L-galactose and L-ascorbic acid . Similarly, L-ascorbate levels in GER1 antisense plants were unchanged indicating that GDP-D-mannose 3,5-epimerase is encoded by a separate gene. Plant J, 2000 Feb, 21(3), 305 - 10 Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana; Oster U et al.; Chlorophyll (Chl) biosynthesis and degradation are the only biochemical processes on Earth that can be directly observed from satellites or other planets . The bulk of the Chls is found in the light-harvesting antenna complexes of photosynthetic organisms . Surprisingly little is known about the biosynthesis of Chl b, which is the second most abundant Chl pigment after Chl a . We describe here the expression and properties of the chlorophyllide a oxygenase gene (CAO) from Arabidopsis thaliana, which is apparently the key enzyme in Chl b biosynthesis . The recombinant enzyme produced in Escherichia coli catalyses an unusual two-step oxygenase reaction that is the 'missing link' in the chlorophyll cycle of higher plants. J Immunol Methods, 2000 Apr 21, 238(1-2), 69 - 80 Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents; Devito C et al.; An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens . Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively . Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA . No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens . Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 . The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E . coli recombinant antigen had the most restricted linear epitope response . The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G . Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations. J Membr Biol, 2000 Apr 1, 174(3), 199 - 205 Mutants of the lactose carrier of Escherichia coli which show altered sugar recognition plus a severe defect in sugar accumulation; Varela MF et al.; Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force . Mutants of the E . coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog beta-thio-o-nitrophenylgalactoside (TONPG) . TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked . These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked . Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation . The mutants had a poor apparent K(m) for both lactose and melibiose in transport . One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V(max)) of normal . All of the mutants accumulated methyl-alpha-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate . Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane . Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V . Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition . We conclude that the amino acids M23, W151, G257, A295 and G377 of the E . coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation. Biochemistry, 2000 Apr 18, 39(15), 4493 - 9 Evidence for a role of helix IV in connecting cation- and sugar-binding sites of Escherichia coli melibiose permease; Cordat E et al.; To improve the structural organization model of melibiose permease, we assessed the individual contributions of the N-terminal tryptophans to the transporter fluorescence variations induced by the binding of cations and beta-configured sugars, by replacement of the six N-terminal tryptophans by phenylalanines and the study of the signal changes . Only two mutations, W116F located in helix IV and W128F located in the cytoplasmic loop 4-5, impair permease activity . The intrinsic fluorescence spectroscopy analysis of the other mutants suggests that W54, located in helix II, W116, and W128 are mostly responsible for the cation-induced fluorescence variations . These tryptophans, W116 and W128, would also be responsible for the beta-galactoside-induced fluorescence changes observed in the N-terminal domain of the transporter . The implication of W116 and W128 in both the cation- and beta-galactoside-induced fluorescence variations led us to investigate in detail the effects of their mutations on the functional properties of the permease . The results obtained suggest that the domains harboring the two tryptophans, or the residues themselves, play a critical role in the mechanism of Na(+)/sugar symport . Taken together, the results presented in this paper and previous results are consistent with a fundamental role of helix IV in connecting cation- and sugar-binding sites of the melibiose permease. Biochemistry, 2000 Apr 18, 39(15), 4455 - 71 Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro; Courtenay ES et al.; To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required . To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes . These include Escherichia coli cytoplasmic osmolytes {potassium glutamate (K(+)Glu(-)), trehalose}, E . coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO) . For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal) . Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations . Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)) . Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine >> proline >TMAO > trehalose approximately K(+)Glu(-) > glycerol . The order of exclusion of E . coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E . coli at high external osmolality . For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O) . Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes . More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account. Biochemistry, 2000 Apr 18, 39(15), 4443 - 54 Energetics of S-adenosylmethionine synthetase catalysis; McQueney MS et al.; S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase) catalyzes the only known route of biosynthesis of the primary biological alkylating agent . The internal thermodynamics of the Escherichia coli S-adenosylmethionine (AdoMet) synthetase catalyzed formation of AdoMet, pyrophosphate (PP(i)), and phosphate (P(i)) from ATP, methionine, and water have been determined by a combination of pre-steady-state kinetics, solvent isotope incorporation, and equilibrium binding measurements in conjunction with computer modeling . These studies provided the rate constants for substrate binding, the two chemical interconversion steps {AdoMet formation and subsequent tripolyphosphate (PPP(i)) hydrolysis}, and product release . The data demonstrate the presence of a kinetically significant isomerization of the E.AdoMet.PP(i).P(i) complex before product release . The free energy profile for the enzyme-catalyzed reaction under physiological conditions has been constructed using these experimental values and in vivo concentrations of substrates and products . The free energy profile reveals that the AdoMet formation reaction, which has an equilibrium constant of 10(4), does not have well-balanced transition state and ground state energies . In contrast, the subsequent PPP(i) hydrolytic reaction is energetically better balanced . The thermodynamic profile indicates the use of binding energies for catalysis of AdoMet formation and the necessity for subsequent PPP(i) hydrolysis to allow enzyme turnover . Crystallographic studies have shown that a mobile protein loop gates access to the active site . The present kinetic studies indicate that this loop movement is rapid with respect to k(cat) and with respect to substrate binding at physiological concentrations . The uniformly slow binding rates of 10(4)-10(5) M(-)(1) s(-)(1) for ligands with different structures suggest that loop movement may be an intrinsic property of the protein rather than being ligand induced. Biochemistry, 2000 Apr 18, 39(15), 4358 - 65 Phosphoaminoglycosides inhibit SWI2/SNF2 family DNA-dependent molecular motor domains; Muthuswami R et al.; Members of the SWI2/SNF2 family of proteins participate in an array of nucleic acid metabolic functions, including chromatin remodeling and transcription . The present studies identify a novel strategy to specifically inhibit the functional DNA-dependent adenosinetriphosphatase (ATPase) motor domain common to SWI2/SNF2 family members . We have identified preparations of phosphoaminoglycosides, which are natural products of aminoglycoside-resistant bacteria, as inhibitors of the in vitro activities of three SWI2/SNF2 family members . These compounds inhibit the ATPase activity of the active DNA-dependent ATPase A domain (ADAAD) by competing with respect to DNA and thus have no effect on DNA-independent ATPases or on RNA-dependent ATPases . Within the superfamily of DNA-dependent ATPases, these compounds are most potent toward SWI2/SNF2 family members and less potent toward other DNA-dependent ATPases . We demonstrate that it is feasible to target DNA-dependent ATPases of a particular type without affecting the function of other ATPases . As the SWI2/SNF2 proteins have been proposed to function in all aspects of DNA metabolism, this paper provides an archetype for development of DNA metabolic inhibitors. Biochemistry, 2000 Apr 18, 39(15), 4276 - 87 Mechanism-based inactivation of cytochrome P450 3A4 by L-754,394; Lightning LK et al.; Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4 . Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein . The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively . L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion . In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion . The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached . However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394. Biochemistry, 2000 Apr 18, 39(15), 4250 - 8 Unfolding and disassembly of the chaperonin GroEL occurs via a tetradecameric intermediate with a folded equatorial domain; Chen J et al.; The chaperonin GroEL is a homotetradecamer in which the subunits (M(r) 57 000) are joined through noncovalent forces . This study reports on the unfolding and disassembly of GroEL in guanidine hydrochloride and urea . Kinetic and equilibrium measurements were made using amide hydrogen exchange/mass spectrometry, light scattering, and size-exclusion chromatography . Hydrogen exchange in GroEL destabilized in 1.8 M GdHCl (the unfolding midpoint is 1.2 M GdHCl) shows that the apical and intermediate domains unfold 3.1 times faster than the equatorial domain . Light scattering measurements made under the same conditions show that disassembly of the native GroEL tetradecamer occurs at the same rate as unfolding of the equatorial domain . This study of the kinetics of GroEL unfolding and disassembly demonstrates the existence of an intermediate that was identified as a tetradecamer with the apical and intermediate domains unfolded . Although this intermediate was easily detected in dynamic unfolding measurements, its population in equilibrium measurements at the midpoint for GroEL unfolding was too small to be detected . This study of GroEL unfolding and disassembly points to features that may be important in the folding and assembly of the GroEL macroassembly. Biochemistry, 2000 Apr 18, 39(15), 4237 - 42 Proteomics on full-length membrane proteins using mass spectrometry; le Coutre J et al.; A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins {Whitelegge, J . P., le Coutre, J., et al . (1999) Proc . Natl . Acad . Sci . U.S.A . 96, 10695-10698} . Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized . Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented . In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences . Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification. Biochemistry, 2000 Apr 18, 39(15), 4217 - 24 Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5'-monophosphate decarboxylase; Harris P et al.; Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5'-monophosphate, the last step in the de novo synthesis of uridine 5'-monophosphate . ODCase is a very proficient enzyme {Radzicka, A., and Wolfenden, R . (1995) Science 267, 90-93}, enhancing the reaction rate by a factor of 10(17) . This proficiency has been enigmatic, since it is achieved without metal ions or cofactors . Here we present a 2.5 A resolution structure of ODCase complexed with the inhibitor 1-(5'-phospho-beta-D-ribofuranosyl)barbituric acid . It shows a closely packed dimer composed of two alpha/beta-barrels with two shared active sites . The orientation of the orotate moiety of the substrate is unambiguously deduced from the structure, and previously proposed catalytic mechanisms involving protonation of O2 or O4 can be ruled out . The proximity of the OMP carboxylate group with Asp71 appears to be instrumental for the decarboxylation of OMP, either through charge repulsion or through the formation of a very short O.H.O hydrogen bond between the two carboxylate groups. Genetics, 2000 Mar, 154(3), 1291 - 300 Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice; Stuart GR et al.; Mutation frequency and specificity were determined as a function of age in nuclear DNA from liver, bladder, and brain of Big Blue lacI transgenic mice aged 1.5-25 months . Mutations accumulated with age in liver and accumulated more rapidly in bladder . In the brain a small initial increase in mutation frequency was observed in young animals; however, no further increase was observed in adult mice . To investigate the origin of mutations, the mutational spectra for each tissue and age were determined . DNA sequence analysis of mutant lacI transgenes revealed no significant changes in mutational specificity in any tissue at any age . The spectra of mutations found in aging animals were identical to those in younger animals, suggesting that they originated from a common set of DNA lesions manifested during DNA replication . The data also indicated that there were no significant age-related mutational changes due to oxidative damage, or errors resulting from either changes in the fidelity of DNA polymerase or the efficiency of DNA repair . Hence, no evidence was found to support hypotheses that predict that oxidative damage or accumulation of errors in nuclear DNA contributes significantly to the aging process, at least in these three somatic tissues. Genetics, 2000 Mar, 154(3), 1239 - 53 The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway; O'Donnell AF et al.; Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-{(N-succinylamino)carbonyl}-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively . In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC . We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E . coli purC purine auxotroph . This cDNA encodes AIRc yet is unable to complement an E . coli purE mutant, suggesting functional differences between Drosophila and E . coli AIRc . In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis . In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies . Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated . Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated . Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs. Genetics, 2000 Mar, 154(3), 959 - 70 The consequences of growth of a mutator strain of Escherichia coli as measured by loss of function among multiple gene targets and loss of fitness; Funchain P et al.; We have examined the composition of members of mutator populations of Escherichia coli by employing an extensive set of phenotypic screens that allow us to monitor the function of >700 genes, constituting approximately 15% of the genome . We looked at mismatch repair deficient cells after repeated cycles of single colony isolation on rich medium to generate lineages that are forced through severe bottlenecks, and compared the results to those for wild-type strains . The mutator lineages continued to accumulate mutations rapidly with each increasing cycle of colony isolation . By the end of the 40th cycle, after approximately 1000 generations, most of the lineages had reduced colony size, 4% had died out, 55% had auxotrophic requirements (increasing to 80% after 60 cycles), and 70% had defects in at least one sugar or catabolic pathway . In addition, 33% had a defect in cell motility, and 26% were either temperature-sensitive or cold-sensitive lethals . On the other hand, only 3% of the wild-type lineages had detectable mutations of any type after 40 cycles . By the 60th cycle, the typical mutator cell carried 4-5 inactive genes among the 15% of the genome being monitored, indicating that the average cell carried at least 24-30 inactivated genes distributed throughout the genome . Remarkably, 30% of the lineages had lost the ability to utilize xylose as a carbon source . DNA sequencing revealed that most of the Xyl(-) mutants had a frameshift in a run of eight G's (GGGGGGGG) in the xylB gene, either adding or deleting one -G- . Further analysis indicated that rendering E . coli deficient in mismatch repair unmasks hypermutable sites in certain genes or intergenic regions . Growth curves and competition tests on lineages that passed through 90 cycles of single colony isolation showed that all lineages suffered reduced fitness . We discuss these results in terms of the value of mutators in cellular evolution. Connect Tissue Res, 1999, 40(4), 251 - 8 Recombinant expression and characterization of dentin matrix protein 1; Srinivasan R et al.; Dentin matrix protein 1 (DMP1) is an extracellular matrix noncollagenous protein (NCP) initially isolated from dentin and now found to be present in calcified tissues like calvaria and long bone . The characteristic feature of DMP1 is that it contains a large number of acidic domains and has properties which implicate it as a key participant in regulating matrix mineralization . The level of DMP1 in the tissue is sparse and it is not easily isolated from dentin because it copurifies with other dentin NCPs . The exact function of DMP1 is not known and this is due to the inherent difficulty of obtaining enough protein from the mineralized tissues . In order to understand the physiologic role for DMP1 during the formation of mineralized tissues we have produced milligram quantities of recombinant DMP1 in E . coli . The objective of this work was: (1) to prepare unmodified apoprotein so that it could be used for studying the function of DMP1; and (2) to prepare polyclonal antibody against the recombinant DMP1 antigen . The DMP1 polyclonal antibody did not cross-react with other NCPs present in dentin or with bone acidic glycoprotein-75 (BAG-75) present in the bone matrix, confirming the specificity of this antibody and thus making it a valuable tool to determine the in vivo function of DMP1. Microbios, 2000, 101(400), 157 - 68 Biosynthesis and secretion of several enzymes in Escherichia coli dnaK and dnaJ mutants; Wolska KI et al.; Escherichia coli null dnaJ and dnaKdnaJ mutants were defective in the biosynthesis and secretion of several enzymes . The synthesis of beta-galactosidase induced in delta dnaJ and delta dnaKdnaJ mutants was abolished at 42 degrees C and significantly decreased at 30 and 37 degrees C . The activity of alkaline phosphatase in the periplasm in both mutant strains at high temperature was lower than in the wild-type strain . The synthesis of b-type cytochromes was defective in two deletion mutants while the synthesis of nitrate reductase-A at 42 degrees C was influenced by dnaK mutation only . The lack of DnaK and DnaJ does not impair the activity of catechol 2,3-dioxygenase irrespective of growth temperature. Haematologica, 2000 Apr, 85(4), 346 - 51 Immunohistochemistry of HFE in the duodenum of C282Y homozygotes with antisera for recombinant HFE protein; Zuccon L et al.; BACKGROUND AND OBJECTIVE: HFE is a class-I MHC related protein which carries the C282Y mutation in most patients with hereditary hemochromatosis, an iron overload disease . HFE protein is expected to have a relevant role in the regulation of duodenal iron absorption, and HFE protein was immunohistochemically identified in the crypt cells . The aim of the work was to analyze whether the C282Y mutation affects HFE accumulation in the duodenum . DESIGN AND METHODS: We developed antisera for the extracellular portion of recombinant human HFE protein expressed in E . coli . The antisera were specific for HFE protein and the C282Y mutant in immunoblotting, immunoprecipitation and immunocytochemistry experiments of transfected cells, and they did not cross react with HLA antigens in various analyses . The antisera gave positive results in the staining of paraffin-fixed sections of duodenal slices of subjects with hemochromatosis . RESULTS: The antisera stained evident supranuclear granules in all enterocytes of 7 C282Y homozygous subjects, and a dark area in the same region in 3 other C282Y homozygotes . Granular bodies were absent from the duodenal sections of 8 C282Y negative subjects, from 2 C282Y heterozygotes and 3 C282Y homozygotes, with or without hemochromatosis . INTERPRETATION AND CONCLUSIONS: The detection of HFE-protein in granular bodies in the enterocytes of the large majority (77%) of C282Y homozygotes and not in other subjects suggests that the mutation facilitates protein accumulation in the duodenum. J Pharm Sci, 2000 May, 89(5), 664 - 73 A novel heparin/protamine-based pro-drug type delivery system for protease drugs; Liang JF et al.; Previously we proposed a heparin/protamine-based system for delivery of protease drugs such as tissue-specific plasminogen activator (tPA) . To demonstrate the feasibility of this approach as well as its pro-drug and triggered release features, positively charged peptides {(Arg)(7)Cys} were successfully linked to tissue-specific plasminogen activator (tPA) using the crosslinking agent N-succinimidyl-3-(2-pyridyldithio)- propionate . This cation-modified tPA showed much stronger heparin affinity than the parent tPA . The complex formed by mtPA and heparin was stable in human plasma, and the activity of mtPA in such a complex was inhibited by the appended heparin . Similarly, the activity of mtPA could also be inhibited by a heparin-antifibrin IgG conjugate in which heparin was linked, via endpoint attachment, to the sugar moieties in the F(c) region of anti-fibrin IgG . Aside from this pro-drug feature exhibited by the binding of the macromolecule heparin to mtPA, results from chromogenic and in vitro clot lysis assay demonstrated that the heparin-induced inhibition of the mtPA activity could be easily reversed by the addition of an adequate amount of protamine . These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA . In addition to the chemical conjugation method, modified tPA could also be produced by the recombinant DNA method . The expressed modified tPA (EmtPA) thus prepared retained the full catalytic activity of the parent tPA, and this activity could also be inhibited by heparin, and the heparin-induced inhibition could be reversed following the addition of protamine . J Allergy Clin Immunol, 2000 Apr, 105(4), 827 - 33 Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3); Shen HD et al.; BACKGROUND: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area . It is important to understand the allergenic composition of this ubiquitous fungal species . OBJECTIVE: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein . mAbs were prepared with the recombinant protein as antigen . The corresponding natural allergen in the fungal extracts was identified with the mAbs . METHODS: A Uni-Zap XR P citrinum cDNA library was screened with sera from asthmatic patients . An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein . The frequency of IgE binding to the expressed protein was analyzed by immunoblotting . Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation . RESULTS: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients . An IgE-binding cDNA clone was obtained and designated as PCE2 . PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame . The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3) . PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3 . Sera from 13 (46%) of the 28 Penicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3 . In addition, 11 of the 13 Pen c 3-positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3 . The presence of IgE cross-reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition . Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3 . The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients . CONCLUSION: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum . PCE2 is a full-length cDNA clone encoding this allergen . In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts. Mol Vis, 2000 Apr 07, 6, 51 - 62 G239T mutation in Repeat 1 of human IRBP: possible implications for more than one binding site in a single repeat; Gross EA et al.; PURPOSE: Interphotoreceptor retinoid-binding protein(IRBP) is a four-repeat protein found in the interphotoreceptor space . Each repeat can bind retinoids and fatty acids . The purpose of this study was to examine the effects of the single amino acid substitution, G239T, versus the wild type sequence of human IRBP Repeat 1, on ligand binding at equilibrium, ligand off rates, and protection of retinol from degradation . METHODS: G239T was created by site-specific mutagenesis, expressed in E . coli, and purified . E . coli expressed wild type Repeat 1 (EcR1) and G239T were subjected to thermal denaturation and analyzed by circular dichroism spectroscopy . We compared the ligand binding properties by fluorescence enhancement of retinol and 16-anthroyloxy-palmitate, tryptophan quenching of the proteins by different ligands, binding competition assays, protection of retinol from degradation, and stopped-flow kinetics to measure transfer of ligands to and from model membranes . RESULTS: Circular dichroism, fluorescence, and absorbance spectroscopy of G239T and EcR1 showed similar wavelength scans . G239T exhibited about three-fold less fluorescence of bound all-trans-retinol or 13-cis-retinol versus EcR1 . Retinol quenching of intrinsic protein fluorescence was reduced by 37% in G239T versus EcR1 . Other retinoids used as quenchers produced no difference between intrinsic protein fluorescence of either G239T or EcR1; all exhibited saturable high affinity binding to each protein . Docosahexaenoic acid (DHA) served as a competitive inhibitor of retinol fluorescence enhancement with EcR1 . However, DHA did not alter retinol fluorescence with G239T . 16-anthroyloxy-palmitate (16-AP) exhibited about 30% higher levels of fluorescence enhancement when bound to G239T versus EcR1 . EcR1 prevented oxidative damage of all-trans-retinol, whereas G239T provided much less protection . Each protein could accept 9-cis-retinal from small unilamellar vesicles (SUVs) as measured by stopped flow kinetics . Off rates were the same in comparing G239T and EcR1 as acceptors . CONCLUSIONS: Despite the general similarity in shape between G239T and EcR1 and the nearly identical binding behavior with some ligands, distinct differences exist in the ligand binding properties of G239T and EcR1 . Fluorescence enhancement/quenching and retinol protection experiments suggest that retinol binding is reduced by about 50% in G239T versus EcR1 . The data suggest that either: (1) EcR1 contains two binding sites for retinol and G239T has lost one site or (2) EcR1 has a single binding site that is altered in G239T to reduce retinol binding . Results of all the experiments were consistent with the first model while some of the data were not consistent with the second model . Thus, it is possible that position 239, found in Domain B2 of IRBP Repeat 1, is located in or near one of two retinol binding sites. Mol Vis, 2000 Apr 07, 6, 40 - 50 Effects of dispersed point substitutions in Repeat 1 of human interphotoreceptor retinoid binding protein (IRBP); Gross EA et al.; PURPOSE: The purpose of this study was to measure the effects of mutations on the retinol binding capability of human Repeat 1 of interphotoreceptor retinoid-binding protein (IRBP) . First, we predicted important functional amino acids by several computer programs . We also noted the lack of shared functions between Tail-specific protease (Tsp) and IRBP, which bear sequence similarity, and this aided in predicting functional residues . We analyzed the effects of point substitutions on the retinol and fatty acid binding properties of Repeat 1 of human IRBP at 25 and 50 degrees C . METHODS: To find residues critical to retinol binding that might affect function, a series of thirteen mutations were created by site-specific mutagenesis between positions 140 and 280 in Repeat 1 of human IRBP . These mutants were expressed, purified, and tested for binding properties . The conformations of the proteins were examined by circular dichroism (CD) scans . RESULTS: Seven of the mutations exhibited reduced binding capacity, and five were not expressed at high enough levels to assess binding activity . Four of the mutants were purified, and their CD scans were very similar to those of Repeat 1 . Only one of the mutations did not affect binding, folding, or expression when compare to wild type Repeat 1 . CONCLUSIONS: Several IRBP mutants containing point mutations retained native structure but lost retinol binding function . The data suggest that retinol binding is affected by many different amino acid substitutions in or near a binding pocket . That even a single point substitution can profoundly affect binding without affecting overall conformation suggests that much of Domain B (from amino acid positions 80 to 300) is involved with ligand binding . This excludes three previously proposed IRBP-retinol binding mechanisms: (1) retinol binds to a small portion of the protein repeat, (2) retinol can bind to any hydrophobic patch in the protein, and (3) native conformation is not required for retinol binding to the repeat. Res Vet Sci, 2000 Apr, 68(2), 109 - 14 Canine leptin: cDNA cloning, expression and activity of recombinant protein; Iwase M et al.; Leptin, the product of the ob gene, is one of the key molecules for the regulation of appetite and whole-body energy balance, and thereby for the pathogenesis of obesity . In an attempt to clarify the roles of leptin in obesity and/or related diseases in companion animals, canine leptin c DNA was cloned by amplifying reverse-transcriptase products of RNA extracted from the adipose tissue of the beagle . A c DNA clone of about 3 kbp contained a 501 bp open reading frame coding a 167-amino acid protein with a 21-amino acid signal peptide . The sequence of a 146-amino acid mature leptin was more than 79 per cent identical to those of other mammals . Northern blot analysis revealed abundant expression of leptin m RNA in adipose tissue, but not in other tissues, in adult beagles . When Chinese hamster ovary cells expressing the rat leptin receptor were stimulated with recombinant canine leptin produced by E . coli, some intracellular signal transduction proteins were phosphorylated, indicating that the recombinant leptin was biologically active . The data reported herein will be helpful for further studies of leptin of the dog in health and disease . J Mol Biol, 2000 Apr 21, 298(1), 35 - 59 The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5 A resolution; Mueller F et al.; The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an unfiltered version of the recently published 50 S reconstruction at 7.5 A resolution . At this resolution, the EM density shows a well-defined network of fine structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations . The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots . RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy . The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density . The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A or 20 A . Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA . Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit . The post-transcriptionally modified bases in the 23 S rRNA form a cluster close to the peptidyl transferase area . The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure . Nucleic Acids Res . 2000 May 1;28(9):E38. Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy; Tanigawa M et al.; Attempts were made to apply atomic force microscopy (AFM) imaging to the detection and mapping of the sites of base substitutions in DNA molecules . In essence, DNA fragments to be examined for possible base substitutions were mixed with an equal amount of a corresponding DNA standard and subjected to heat denaturation and subsequent annealing . The reassociated DNA was incubated with MutS protein, a protein that recognizes and binds to mismatched base pairs in duplex DNA . Bound MutS protein molecules were then detected by AFM and their positions along the DNA molecules were determined by calculating the distance from one of the DNA termini, which had been tagged with a biotin-avidin complex . Base substitutions present in DNA molecules >1 kb were effectively detected by this procedure, and the positions determined were in good agreement with the actual mutation sites . This method is quite simple, has virtually no limitations on the size of DNA fragments to be examined and requires only a very small amount of DNA sample. Nucleic Acids Res, 2000 May 1, 28(9), 1906 - 12 Structural basis for uracil DNA glycosylase interaction with uracil: NMR study; Ghosh M et al.; Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop . The(1)H resonances of this hairpin have been assigned almost completely . NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin . This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T(12)and T(15)nucleotides stack upon 3' and 5' ends of the stem, respectively . Further, T(14)stacks upon both T(12)and T(15) . Though U(13)partially stacks upon T(14), no stacking interaction is observed between U(13)and T(12) . All the individual nucleotide bases belonging to the stem and T(12)and T(15)of the loop adopt ' anti ' conformation with respect to their sugar moiety, while the U(13)and T(14)of the loop are in ' syn ' conformation . The turning phosphate in the loop is located between T(13)and T(14) . This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA . The epsilon of T(12)and alpha, beta, gamma, epsilon and zeta of U(13)and gamma of T(14), which partially influence the local conformation of U(13)in U2-hairpin are all locked in ' trans ' conformation . Such stretched out backbone conformation in the vicinity of U(13)could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney . This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases. Nucleic Acids Res, 2000 May 1, 28(9), 1879 - 84 Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli; Nishigaki K et al.; The sequence of an unknown PCR product generated by random (and conventional) PCR could be determined without sequencing when it is provided with the template DNA sequence . Theoretically, this was based on formerly established ideas which assert that the amount of random PCR product mainly depends on the stability of the primer-binding structures and that the dynamic solution structure of DNA is essentially governed by the Watson-Crick base pairing . However, it has not been clear whether this holds true for larger genomes of mega- to gigabase size, beside the lambda phage genome (of 50 kb) used previously, nor has it been ascertained to uniquely specify the sequence of a random PCR product . Here, we jointly use two computer programs together with experimental data from Genome Profiling (i.e . TGGE analysis of random PCR products) . The first procedure carried out by a newly remodeled computer program (PCRAna-A1) was shown to be competent to calculate a set of random PCR products from Escherichia coli genome DNA (4.7 Mb) . The other procedure performed with another program (Poland-H) played a critical role in determining the final candidate sequence by theoretically offering the initial melting temperature and the melting pattern of unspecified candidate sequences . The success attained here not only proved our method to be useful for sequence prediction but also confirmed the above-mentioned ideas as rational . We believe that this is the first case to computer-utilize a genome sequence as a whole. Nucleic Acids Res, 2000 May 1, 28(9), 1864 - 70 DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study; Burr T et al.; We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the -10 hexamer . Starting with an activator-independent promoter, with a 17 bp spacing between the -10 and -35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions -15 and -14 . Promoter activity is greatest when the 'non-template' strand carries T and G at positions -15 and -14, respectively . Promoter activity can be further enhanced by a second T and G at positions -17 and -16, respectively, immediately upstream of the first 'TG motif' . Our results show that the base sequence of the DNA segment upstream of the -10 hexamer can make a significant contribution to promoter strength . Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of 'TG motifs' upstream of the promoters' -10 elements . We conclude that correctly placed 'TG motifs' are found at over 20% of E.coli promoters. Nucleic Acids Res, 2000 May 1, 28(9), 1849 - 58 RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog; Costas C et al.; A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase . This analog, 8-{(4-azidophenacyl)thio}adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring . This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization . Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA . Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3' end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3' end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3' end binding site (beta, beta') . Both 8-APAS-AMP and the related 8-azido-AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA-RNA hybrids . Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated . Although the azide and linker together are larger in 8-APAS-ATP than in 8-N(3)-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase. Mol Plant Microbe Interact, 2000 Apr, 13(4), 456 - 64 Heterologous expression of functional Ptr ToxA; Tuori RP et al.; Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP) . NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E . coli at approximately 10 to 16 mg per liter of culture . Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA . A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA . These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity . A C domain construct with a cysteine residue mutated to glycine is inactive . This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity . The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance. Trends Biochem Sci, 2000 Apr, 25(4), 189 - 95 Coping with replication 'train wrecks' in Escherichia coli using Pol V, Pol II and RecA proteins; Goodman MF; DNA replication machineries tend to stall when confronted with damaged DNA template sites, causing the biochemical equivalent of a major 'train wreck' . A newly discovered bacterial DNA polymerase, Escherichia coli Pol V, acting in conjunction with the RecA protein, can exchange places with the stalled replicative Pol III core and catalyse 'error-prone' translesion synthesis . In contrast to Pol V-catalysed 'brute-force, sloppier copying', another SOS-induced DNA polymerase, Pol II, plays a pivotal role in an 'error-free', replication-restart DNA repair pathway and probably involves RecA-mediated homologous recombination. Trends Biochem Sci, 2000 Apr, 25(4), 185 - 9 PriA-directed replication fork restart in Escherichia coli; Marians KJ; The encounter of a replication fork with either a damaged DNA template, a nick in the template strand or a 'frozen' protein-DNA complex can stall the replisome and cause it to fall apart . Such an event generates a requirement for replication fork restart if the cell is going to survive . Recent evidence shows that replication fork restart is effected by the action of the recombination proteins generating a substrate for PriA-directed replication fork assembly. Trends Biochem Sci, 2000 Apr, 25(4), 156 - 65 Initiation of genetic recombination and recombination-dependent replication; Kowalczykowski SC; Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps . These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein . The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication . In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases) . The initial steps common to these recombination and recombination-dependent replication processes are reviewed. Pharmacology, 2000 Apr, 60(3), 128 - 35 In vitro pharmacological profile of SK-896, a new human motilin analogue; Tsukamoto K et al.; SK-896 ({Leu(13)}motilin-Hse) is a new human motilin analogue synthesized by Escherichia coli using a biotechnological method . We investigated the binding of SK-896 to motilin receptors and the contractile effect of SK-896 on smooth muscle preparations isolated from the gastrointestinal tract and various regional organs in order to clarify its in vitro pharmacological profile . SK-896 inhibited the binding of (125)I-human motilin to rabbit gastroduodenal motilin receptors with the same potency as unlabeled human motilin . The IC(50) values of SK-896 and human motilin were 3.5 +/- 1.5 and 3.1 +/- 1.8 nmol/l, respectively . The K(d) of human motilin was 3.0 +/- 1.5 nmol/l, and the Ki of SK-896 was 3.4 +/- 1.5 nmol/l . SK-896 induced contraction of smooth muscle preparations isolated from rabbit duodenum in a concentration-dependent manner . However, there was no effect of SK-896 on duodenal preparations isolated from the dog and the rat . SK-896 thus exhibited species specificity in its contractile effect . We next investigated the effect of SK-896 on various smooth muscle preparations isolated from rabbit gastrointestinal tract, trachea, bladder, gallbladder, uterus, vas deferens and artery . Results showed that SK-896 induced contraction of smooth muscle preparations isolated from gastrointestinal tract, with potencies in the order duodenum > gastric pylorus = jejunum = descending colon > ascending colon >/= ileum . However, there was no effect of SK-896 on smooth muscle preparations from gastric fundus and other regional organs . SK-896 thus exhibited regional specificity in its contractile effect . Moreover, the effects of SK-896 on smooth muscle preparations from rabbit duodenum were the same as those of human motilin, and were not inhibited by pretreatment with tetrodotoxin and atropine but were inhibited by verapamil . These findings indicate that SK-896 has the same pharmacological profile as human motilin . They suggest that SK-896 acts on gastrointestinal smooth muscle isolated from rabbit directly and specifically . Free Radic Biol Med, 2000 Mar 1, 28(5), 767 - 72 Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi; Lopez JA et al.; Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol . Chem . 378, 827-836, 1997) . The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry . Epimastigotes of T . cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C . fasciculata . In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T . cruzi (Acc . Nr . AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity . With TXN, TXNPx, trypanothione and TR, T . cruzi possesses all components constituting the crithidial peroxidase system . It is concluded that the antioxidant defense of T . cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism. FEMS Microbiol Lett, 2000 Apr 15, 185(2), 117 - 21 Homocysteine thiolactone is a positive effector of sigma(S) levels in Escherichia coli; Goodrich-Blair H et al.; sigma(S) is a regulator of the stationary phase response in Escherichia coli . Multi-copy suppressors were sought in a strain with decreased levels of sigma(S) and one such suppressor was found to encode HsrA, a putative efflux pump . Multi-copy expression of hsrA was shown to lead to accumulation of homocysteine, which is predicted to cause an increase in homocysteine thiolactone (HCTL) levels . A direct correlation between HCTL levels and sigma(S) accumulation was observed both in mutants and during normal cell growth, leading to the hypothesis that HCTL is a physiologically relevant positive effector of sigma(S) levels in vivo. Mamm Genome, 2000 Apr, 11(4), 275 - 80 Characterization of mouse Clpp protease cDNA, gene, and protein; Andresen BS et al.; Mutations that cause accumulation or rapid degradation owing to protein misfolding are a frequent cause of inherited disease in humans . In Escherichia coli, Clpp protease is one of the components of the protein quality control system that handles misfolded proteins . In the present study, we have characterized the mouse Clpp cDNA sequence, the organization of the mouse gene, the chromosomal localization, and the tissue-specific expression pattern . Moreover . the cellular localization and processing of mouse Clpp was studied by overexpression in transfected eukaryotic cells . Our results indicate that mouse and human Clpp have similar roles, and they provide the molecular basis for establishing a Clpp knockout mouse and to study its phenotype, thereby shedding light on a possible role of Clpp in human disease. Curr Opin Struct Biol, 2000 Apr, 10(2), 259 - 64 Unveiling ribosomal structures: the final phases; van Heel M; Unprecedented insights into the structure of the ribosome have been gained recently: X-ray crystallographic studies have yielded 5-9 A resolution structures and cryo-electron microscopy has elucidated the structure of the Escherichia coli ribosome in different functional states . A 7.5 A cryo-electron microscopy structure of the large subunit indicates that this technique is still in the race to determine the ribosome structure. Curr Opin Genet Dev, 2000 Apr, 10(2), 162 - 8 Sloppier copier DNA polymerases involved in genome repair; Goodman MF et al.; When chromosomal replication is impeded in the presence of DNA damage, members of a newly discovered UmuC/DinB/Rev1/Rad30 superfamily of procaryotic and eucaryotic DNA polymerases catalyze translesion synthesis at blocked replication forks . Although these polymerases share sequence elements essentially unrelated to the standard replication and repair enzymes, some of them (such as the SOS-induced Escherichia coli pol V) catalyze 'error-prone' translesion synthesis leading to large increases in mutation, whereas others (an example being the Xeroderma pigmentosum variant gene product XPV pol eta) carry out aberrant, yet nonmutagenic translesion synthesis . Ongoing studies of these low fidelity polymerases could provide new insights into the mechanism of somatic hypermutation, a key element in the immune response. Biotechnol Prog, 2000 Mar-Apr, 16(2), 278 - 86 Gene expression profiling by DNA microarrays and metabolic fluxes in Escherichia coli; Oh MK et al.; DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome . A total of 111 E . coli genes, including those in central metabolism, key biosyntheses, and some regulatory functions, were cloned, amplified, and used as probes for detecting the level of transcripts . An E . coli strain was grown in glucose, acetate, and glycerol media, and the transcript levels of the selected genes were detected . Despite extensive studies on E . coli physiology, many new features were found in the regulation of these genes . For example, several genes were unexpectedly up-regulated, such as pps, ilvG, aroF, secA, and dsbA in acetate and asnA and asnB in glycerol, or down-regulated, such as ackA, pta, and adhE in acetate . These genes were regulated with no apparent reasons by unknown mechanisms . Meanwhile, many genes were regulated for apparent purposes but by unknown mechanisms . For example, the glucose transport genes (ptsHI, ptsG, crr) in both acetate and glycerol media were down-regulated, and the ppc, glycolytic, and biosynthetic genes in acetate were also down-regulated because of the reduced fluxes . However, their molecular mechanisms remain to be elucidated . Furthermore, a group of genes were regulated by known mechanisms, but the physiological roles of such regulation remain unclear . This group includes pckA and aspA, which are up-regulated in glycerol, and gnd and aspA, which are down- and up-regulated, respectively, in acetate . The DNA microarray technology demonstrated here is a powerful yet economical tool for characterizing gene regulation and will prove to be useful for strain improvement and bioprocess development. Carcinogenesis, 2000 Apr, 21(4), 715 - 25 LacI mutation spectra following benzo{a}pyrene treatment of Big Blue mice; Shane BS et al.; The mutation spectrum of the lacI gene from the liver of C57Bl6 Big Blue transgenic mice treated with benzo{a}pyrene (B{a}P) has been compared with the spectrum of spontaneous mutations observed in the liver of untreated Big Blue mice . Mice were treated with B{a}P for 3 days followed by a partial hepatectomy one day after the last injection . Liver tissue was removed for analysis at hepatectomy and, again, 3 days later at the time of sacrifice . Earlier, we reported that the lacI mutant frequency in these B{a}P-treated mice was elevated in the liver both at the time of hepatectomy and at sacrifice; however, a statistically significant increase in the mutant frequency was observed only at sacrifice . In this study, the DNA sequence spectra of lacI mutations observed in the liver of B{a}P-treated Big Blue mice at hepatectomy and at time of sacrifice were compared with each other and with the spectrum of spontaneous liver mutations . No differences were observed between the two B{a}P-treatment spectra . However, mutation frequencies of both GC-->TA and GC-->CG at the time of hepatectomy and at sacrifice were significantly elevated compared with the spontaneous frequency of these same transversions . Also, the frequency of AT-->TA transversions was significantly higher than the spontaneous frequency at the time of hepatectomy but not at sacrifice . The frequency of all other classes of mutations scored was not significantly different from the frequency of these same events in the spontaneous spectra . These data support the view that B{a}P treatment results in the induction of GC-->TA and GC-->CG transversions within 1 day of the last injection and they provide insights regarding the relative roles of benzo{a}pyrene-7,8-diol-9, 10-epoxide and radical cations of B{a}P in B{a}P-induced mutagenesis in vivo . Finally, these data provide evidence for B{a}P-induced mutagenesis under conditions where no statistical increase in mutant frequency could be shown. Protein Sci, 2000 Mar, 9(3), 587 - 97 Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp . PCC 6803; Scott NL et al.; The genome of the unicellular cyanobacterium Synechocystis sp . PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins . Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands . To study the structural and functional properties of Synechocystis sp . PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli . Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate . Recombinant, heme-reconstituted ferric Synechocystis sp . PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM . At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm . The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron . Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form . Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm . This reduced form appeared capable of carbon monoxide binding . Optical data also suggested that cyanide ions could bind to the heme in the ferric state . The spectral properties of the putative Synechocystis sp . PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure. Protein Sci, 2000 Mar, 9(3), 440 - 51 The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein; Schlumpberger M et al.; The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the {URE3} phenotype . The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for {URE3} (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD) . We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble . Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence . The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy . Fiber formation proceeded in an autocatalytic manner . In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology . Aggregation of Ure2p could be seeded by UPD fibrils . Our results provide biochemical support for the proposal that the {URE3} state is caused by a self-propagating inactive form of Ure2p . We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation . The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2000 Apr, 17(2), 125 - 8 {Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro}; Lu Y et al.; OBJECTIVE: To establish a suitable model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells . METHODS: The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome-mediated transfection, and grew in the presence of G418 . One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT-PCR analyses on its genomic DNAs . RESULTS: (1) Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone . (2) One of lac I target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not . (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/microg DNA . CONCLUSION: The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established . The two lacI target genes in the cell line could imitate the functional states of expressed and non-expressed genes in mammalian cells respectively . The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. J Biol Chem, 2000 Jun 30, 275(26), 19897 - 905 Tobacco transcription factor TGA2.2 is the main component of as-1-binding factor ASF-1 and is involved in salicylic acid- and auxin-inducible expression of as-1-containing target promoters; Niggeweg R et al.; In higher plants, activating sequence-1 (as-1) of the cauliflower mosaic virus 35 S promoter mediates both salicylic acid (SA)- and auxin-inducible transcriptional activation . Originally found in promoters of several viral and bacterial plant pathogens, as-1-like elements are also functional elements of plant promoters activated in the course of a defense response upon pathogen attack . Nuclear as-1-binding factor (ASF-1) and cellular salicylic acid response protein (SARP) bind specifically to as-1 . Four different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and TGA2.2) are potential components of either ASF-1 or SARP . Here we show that ASF-1 and SARP are very similar in their composition . TGA2.2 is a major component of either complex, as shown by supershift analysis and Western blot analysis of DNA affinity-purified SARP . Minor amounts of a protein immunologically related to TGA2.1 were detected, whereas TGA1a was not detectable . Overexpression of either TGA2.2 or a dominant negative TGA2.2 mutant affected both SA and auxin (2, 4D) inducibility of various target promoters encoding as-1-like elements, albeit to different extents . This indicates that TGA2.2 is a component of the enhancosome assembling on these target promoters, both under elevated SA and 2,4D concentrations . However, the effect of altered TGA2.2 levels on gene expression was more pronounced upon SA treatment than upon 2,4D treatment. J Biol Chem, 2000 Jun 16, 275(24), 18145 - 52 The allosteric regulation of pyruvate kinase; Valentini G et al.; Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway . The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)) . Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit . The R271L and K413Q mutant enzymes exhibit altered kinetic properties . In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium . In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge . The mutants R292D and D297R are totally inactive . The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure . However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins . Furthermore, in the R292D structure, two loops that are part of the active site are disordered . The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator . R431E exhibits only slight changes in the regulatory properties . Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism . Taken together, these results support the notion that domain interfaces are critical for the allosteric transition . They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites . These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene. J Biol Chem, 2000 Jun 16, 275(24), 17968 - 73 The F420H2 dehydrogenase from Methanosarcina mazei is a Redox-driven proton pump closely related to NADH dehydrogenases; Baumer S et al.; The F(420)H(2) dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei Go1 . Here it is shown that cofactor F(420)H(2)-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H(+) translocated per two electrons transferred . The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + P(i) . The gene cluster encoding the F(420)H(2) dehydrogenase of M . mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D, H, I, J, K, L, M, N, and O . Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I) . Like the NADH-dependent enzymes, the F(420)H(2) dehydrogenase is composed of three subcomplexes . The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1 . FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1 . Homologues to the hydrophilic NADH-oxidizing input module are not present in M . mazei Go1 . Instead, the gene product FpoF may be responsible for F(420)H(2) oxidation and may function as the electron input part . Thus, the F(420)H(2) dehydrogenase from M . mazei Go1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways . The enzyme from the methanogenic archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F(420) and a modified output module adopted to the reduction of methanophenazine. Planta, 2000 Feb, 210(3), 407 - 15 Developmental regulation of the maize Zm-p60.1 gene encoding a beta-glucosidase located to plastids; Kristoffersen P et al.; A beta-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene . The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in-situ hybridization . It was found that the expression levels of the Zm-p60.1-specific mRNA changed after pollination of carpellate inflorescences . The Zm-p60.1 cDNA was expressed in E . coli and antibodies were raised against this protein . An antibody was used to determine the tissue-specific localization of this protein . By in situ immunolocalization experiments, this protein was found to be located in cell layers below the epidermis and around the vascular bundles of the coleoptile . In the primary leaf, the Zm-p60.1 protein was detected in cells of the outermost cell layer and around the vascular tissue . In floral tissue, Zm-p60.1 was present in the glumes, the carpels and in the outer cell layer of the style . In coleoptiles, as determined by immuno-electronmicroscopy, the Zmp60.1 protein was located exclusively in the plastids. Plant Cell Physiol, 2000 Jan, 41(1), 119 - 23 Identification of multi-gene families encoding isopentenyl diphosphate isomerase in plants by heterologous complementation in Escherichia coli; Cunningham FX Jr et al.; Two cDNAs encoding isopentenyl diphosphate isomerase (IPI) in Adonis aestivalis, Arabidopsis thaliana, and Lactuca sativa, and single examples from Oryza sativa and Tagetes erecta were identified . An analysis of these and other ipi leads us to suggest a separate origin for green algal and plant genes and propose that a single gene encodes plastid and cytosolic IPI in plants. J Dairy Sci, 2000 Mar, 83(3), 488 - 98 A quantitative approach to classifying Holstein cows based on antibody responsiveness and its relationship to peripartum mastitis occurrence; Wagter LC et al.; A quantitative approach was developed to classify Holstein cows and heifers based on phenotypic variation of serum antibody response and to determine associations with peripartum mastitis . Using an index, 136 cows and heifers were classified into high (Group 1), average (Group 2), or low (Group 3) antibody groups following immunization with ovalbumin at wk -8, -3, and 0 relative to parturition . The ranking of groups based on the quantitative index of serum antibody response to ovalbumin were similar for sera and whey antibody such that Group 1 > Group 2 > Group 3 . Animals were also vaccinated with Escherichia coli J5 (Rhone Merieux, Lenexa, KS) at wk -8 and -3 relative to parturition . The ranking of groups for E . coli J5 was similar to that observed for serum and whey antibody to ovalbumin . Serum and whey IgG1 and IgG2 concentrations were measured at wk 0, 3, and 6 but differences between groups were not significant . There was no occurrence of mastitis for Group 1 animals in two of the herds . In contrast, Group 1 animals from the third herd had the highest occurrence of mastitis; however, these cases all occurred in first-parity heifers . According to pooled data across all herds, Group 3 animals had the highest occurrence of mastitis . Heritability estimates of serum antibody response to ovalbumin varied between 0.32 to 0.64 depending on week relative to parturition . Heritability estimates of serum antibody response to E . coli J5 also varied between 0.13 to 0.88 depending upon week relative to parturition . These results indicate that high peripartum antibody may be beneficial in some herds. J Mol Endocrinol, 2000 Apr, 24(2), 225 - 32 Analysis of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene for expression in pancreatic islet beta cells using transgenic mice and HIT-T15 cells; Takahashi E et al.; The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells . To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells . The E . coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0 . 5 kb of the 5'-upstream region failed to show reporter expression on histological examination . As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression . When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells . The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells . These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini. Mol Biol Cell, 2000 Apr, 11(4), 1457 - 69 ADAM 23/MDC3, a human disintegrin that promotes cell adhesion via interaction with the alphavbeta3 integrin through an RGD-independent mechanism; Cal S et al.; ADAM 23 (a disintegrin and metalloproteinase domain)/MDC3 (metalloprotease, disintegrin, and cysteine-rich domain) is a member of the disintegrin family of proteins expressed in fetal and adult brain . In this work we show that the disintegrin-like domain of ADAM 23 produced in Escherichia coli and immobilized on culture dishes promotes attachment of different human cells of neural origin, such as neuroblastoma cells (NB100 and SH-S(y)5(y)) or astrocytoma cells (U373 and U87 MG) . Analysis of ADAM 23 binding to integrins revealed a specific interaction with alphavbeta3, mediated by a short amino acid sequence present in its putative disintegrin loop . This sequence lacks any RGD motif, which is a common structural determinant supporting alphavbeta3-mediated interactions of diverse proteins, including other disintegrins . alphavbeta3 also supported adhesion of HeLa cells transfected with a full-length cDNA for ADAM 23, extending the results obtained with the recombinant protein containing the disintegrin domain of ADAM 23 . On the basis of these results, we propose that ADAM 23, through its disintegrin-like domain, may function as an adhesion molecule involved in alphavbeta3-mediated cell interactions occurring in normal and pathological processes, including progression of malignant tumors from neural origin. Mol Biol Cell, 2000 Apr, 11(4), 1345 - 56 A screen for dominant negative mutants of SEC18 reveals a role for the AAA protein consensus sequence in ATP hydrolysis; Steel GJ et al.; An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain . At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs) . Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear . To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken . This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid . Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail . The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro) . Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family . Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p . This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis . Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. J Biol Chem, 2000 Jun 2, 275(22), 16450 - 8 The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain . Characterization of interactions and contribution to chemotaxis; Mukhina S et al.; Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR) . The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC) . The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His(204) to Gln) (r-uPA(H/Q)), urokinase with mutation of His(204) to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r-uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli . We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects . Half-maximal chemotaxis (EC(50)) were apparent at approximately 2 nm with all the uPA variants . The kringle domain induced cell migration with an EC(50) of about 6 nm, whereas the denaturated r-KD and r-uPA(LMW) were without effect . R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain . The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase . Specific binding of r-uPA(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC . uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA(H/Q)-GFD via a single, similar type of binding site . These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r-uPA(LMW) . HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD . Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR. J Biol Chem, 2000 Jul 21, 275(29), 21870 - 6 Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei; Buckner FS et al.; Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs . We have cloned the alpha- and beta-subunits of T . brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites . TB-PFT is expressed in both bloodstream and insect stage parasites . Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli . Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent alpha-helices . Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase . TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase . A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors. J Biol Chem, 2000 Jun 9, 275(23), 17869 - 77 Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability; Lawrence SM et al.; Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized . In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB) . The human gene is ubiquitously expressed and encodes a 40-kDa enzyme . The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E . coli and results in N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection . In vitro the human enzyme uses N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product. J Biol Chem, 2000 Jun 9, 275(23), 17349 - 57 Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase; Abu-Soud HM et al.; We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated . Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0 . 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C) . O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis . The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis . During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate . Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold . Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis . 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role . These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology. Am J Physiol Lung Cell Mol Physiol, 2000 Apr, 278(4), L840 - 7 Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo; Borron P et al.; The role of surfactant-associated protein (SP) A in the mediation of pulmonary responses to bacterial lipopolysaccharide (LPS) was assessed in vivo with SP-A gene-targeted {SP-deficient; SP-A(-/-)} and wild-type {SP-A(+/+)} mice . Concentrations of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein-2, and nitric oxide were determined in recovered bronchoalveolar lavage fluid after intratracheal administration of LPS . SP-A(-/-) mice produced significantly more TNF-alpha and nitric oxide than SP-A(+/+) mice after LPS treatment . Intratracheal administration of human SP-A (1 mg/kg) to SP-A(-/-) mice restored regulation of TNF-alpha, macrophage inflammatory protein-2, and nitric oxide production to that of SP-A(+/+) mice . Other markers of lung injury including bronchoalveolar fluid protein, phospholipid content, and neutrophil numbers were not influenced by SP-A . Data from experiments designed to test possible mechanisms of SP-A-mediated suppression suggest that neither binding of LPS by SP-A nor enhanced LPS clearance are the primary means of inhibition . Our data and others suggest that SP-A acts directly on immune cells to suppress LPS-induced inflammation . These results demonstrate that endogenous or exogenous SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide production in vivo. Biochem J, 2000 Apr 15, 347(Pt 2), 553 - 9 Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose; Witt S et al.; The effects of mutation of key conserved active-site residues (Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and His-563) of glucose oxidase from Penicillium amagasakiense on substrate binding were investigated . Kinetic studies on the oxidation of beta-D-glucose combined with molecular modelling showed the side chain of Arg-516, which forms two hydrogen bonds with the 3-OH group of beta-D-glucose, to be absolutely essential for the efficient binding of beta-D-glucose . The R516K variant, whose side chain forms only one hydrogen bond with the 3-OH group of beta-D-glucose, exhibits an 80-fold higher apparent K(m) (513 mM) but a V(max) only 70% lower (280 units/mg) than the wild type . The complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of beta-D-glucose through the substitution R516Q effected a 120-fold increase in the apparent K(m) for glucose (to 733 mM) and a decrease in the V(max) to 1/30 (33 units/mg) . None of the other substitutions, with the exception of variant F418A, affected the apparent K(m) more than 6-fold . In contrast, the removal of aromatic or bulky residues at positions 73, 418 or 430 resulted in decreases in the maximum rates of glucose oxidation to less than 1/90 . Variants of the potentially catalytically active His-520 and His-563 were completely, or almost completely, inactive . Thus, of the residues forming the active site of glucose oxidase, Arg-516 is the most critical amino acid for the efficient binding of beta-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation. Biochem J, 2000 Apr 15, 347(Pt 2), 527 - 34 Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine; Xu-Welliver M et al.; The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities . All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED(50)) from 100-fold (K165A) to 2400-fold (K165F) . Lys(165) is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA . The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx . 2.5-fold . Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED(50) value for inactivation by BG>200-fold greater than wild-type . Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold . Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG . These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents . These results raise the possibility that the conservation of Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine . They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this therapy. Biochem J, 2000 Apr 15, 347(Pt 2), 519 - 26 Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine-DNA alkyltransferase insensitive to O6-benzylguanine; Xu-Welliver M et al.; The DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, O(6)-benzylguanine (BG) . This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents, and BG is aimed at enhancing cancer treatment in clinical trials . Point mutations in a 24 amino acid sequence likely to form the BG-binding pocket were identified using a screening method designed to identify BG-resistant mutants . It was found that alterations in 21 of these residues were able to render AGT resistant to BG . These included mutations at the highly conserved residues Lys(165), Leu(168) and Leu(169) . The two positions at which changes led to the largest increase in resistance to BG were Gly(156) and Lys(165) . Eleven mutants at Gly(156) were identified, with increases in resistance ranging from 190-fold (G156V) to 4400-fold (G156P) . Two mutants at Lys(165) found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG . Two mutants at the Ser(159) position (S159I and S159V) were >80-fold more resistant than wild-type AGT . Eleven active mutants at Leu(169) were also resistant to BG, but with lower increases (5-86-fold) . Fourteen BG-resistant mutants were found for position Cys(150), with 3-26-fold increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation . Six BG-resistant mutants at Asn(157) were found with increases of 4-13-fold . These results show that many changes can render human AGT resistant to BG without preventing the ability to protect tumour cells from therapeutic alkylating agents. Biochem J, 2000 Apr 15, 347(Pt 2), 383 - 8 Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana; Sanderson SJ et al.; A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity . The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE) . Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages . Maximum enzyme activity accompanied removal of the entire pro-region . This was facilitated by acidification . Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L . mexicana lysates, and had the same N-terminal sequence as the native enzyme . The procedure yielded >3.5 mg of active enzyme per litre of E . coli culture. Dig Dis Sci, 2000 Mar, 45(3), 480 - 6 Effect of Shiga toxin 2 on water and ion transport in human colon in vitro; Fiorito P et al.; Shiga toxin-producing Escherichia coli (STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases . In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined . Net water movement (Jw) and short-circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2 . Stx2 significantly inhibited the absorptive J(w) with no effect on the basal I(sc) after 60 min of exposure . These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells . Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells . These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium. Cancer Res, 2000 Mar 15, 60(6), 1720 - 8 Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1; Recio JA et al.; Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion . As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein . Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts . We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity . However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes . Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway . However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2 . These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH. Pharm Acta Helv, 1999 Dec, 74(1), 1 - 9 Stability of L-asparaginase: an enzyme used in leukemia treatment; Stecher AL et al.; L-asparaginase from Escherichia coli is an important enzyme widely used in leukemia treatment under the trade name Elspar . Up to now, however, the aspects of its stability and storage has not been studied in detail . The aim of this work is to analyze the factors that could interfere in the enzyme's stability . The enzymatic activity was found to be stable in wide pH range (4.5-11.5), showing a slight increase in activity and stability in alkaline pHs, which indicates a more stable conformation of the molecule . The enzyme proved to have a high activity restoration capacity when submitted to temperatures of 65 degrees C, in pH 8.6 buffer and, surprisingly, in physiologic solution . This suggests a positive effect of sodium ions on such restoration capacity . Stability was high in different diluents used as parenteral solutions and in recipients used in medical practice without significant loss of activity for at least 7 days . These results lead us to conclude that the enzyme has a high stability after the lyophilized form has been reconstituted (at least 7 days), since the necessary precautions are taken in terms of sterile manipulation and if it is stored in a suitable parenteral vehicle under low temperature (about 8 degrees C). Nat Biotechnol, 2000 Apr, 18(4), 424 - 8 Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase; van Steensel B et al.; We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes . By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation . Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies . We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies . When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence . Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1 . DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 263 - 74 Monosaccharide transporters in plants: structure, function and physiology; Buttner M et al.; Monosaccharide transport across the plant plasma membrane plays an important role both in lower and higher plants . Algae can switch between phototrophic and heterotrophic growth and utilize organic compounds, such as monosaccharides as additional or sole carbon sources . Higher plants represent complex mosaics of phototrophic and heterotrophic cells and tissues and depend on the activity of numerous transporters for the correct partitioning of assimilated carbon between their different organs . The cloning of monosaccharide transporter genes and cDNAs identified closely related integral membrane proteins with 12 transmembrane helices exhibiting significant homology to monosaccharide transporters from yeast, bacteria and mammals . Structural analyses performed with several members of this transporter superfamily identified protein domains or even specific amino acid residues putatively involved in substrate binding and specificity . Expression of plant monosaccharide transporter cDNAs in yeast cells and frog oocytes allowed the characterization of substrate specificities and kinetic parameters . Immunohistochemical studies, in situ hybridization analyses and studies performed with transgenic plants expressing reporter genes under the control of promoters from specific monosaccharide transporter genes allowed the localization of the transport proteins or revealed the sites of gene expression . Higher plants possess large families of monosaccharide transporter genes and each of the encoded proteins seems to have a specific function often confined to a limited number of cells and regulated both developmentally and by environmental stimuli. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 246 - 62 Sucrose transporters in plants: update on function and structure; Lemoine R; In plants, sucrose is the major transport form for photoassimilated carbon and is both a source of carbon skeletons and energy for plant organs unable to perform photosynthesis (sink organs) . As a molecule translocated over distance, sucrose has to pass through a number of membranes . Membrane transport of sucrose has therefore been considered for a long time as a major determinant of plant productivity . After several decades of physiological and biochemical experiments measuring the activity of sucrose carriers, unequivocal evidence came from the first identification of a cDNA coding a sucrose carrier (SoSUT1, Riesmeier et al . (1992) EMBO J . 11, 4705-4713) . At present 20 different cDNAs encoding sucrose carriers have been identified in different plant species, in both dicots and monocots (one case) . The total number is increasing rapidly and most importantly, it can be guessed from the results obtained for Arabidopsis, that in each species, sucrose transporters represent a gene family . The sequences are highly conserved and those carriers display the typical 12 transmembrane alpha-helices of members of the Major Facilitator superfamily . Yeast expression of those carriers indicate that they are all influx carriers, all cotransport sucrose and proton and that their affinity for sucrose is surprisingly similar (0.2-2 mM) . All their characteristics are in agreement with those demonstrated at the physiological level in plants . These characteristics are discussed in relation to the function in plants and the few data available on the structure of those transporters in relation to their function are presented. Biochim Biophys Acta, 2000 May 1, 1465(1-2), 199 - 218 Anion channels in higher plants: functional characterization, molecular structure and physiological role; Barbier-Brygoo H et al.; Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism . It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions . In contrast, information on the molecular structure of plant anion channels has only recently started to emerge . Only a few genes coding for putative plant anion channels from the large chloride channel (CLC) family have been isolated, and current molecular data on these plant CLCs are presented and discussed . A major challenge remains to identify the genes encoding the various anion channels described so far in plant cells . Future prospects along this line are briefly outlined, as well as recent advances based on the use of knockout mutants in the model plant Arabidopsis thaliana to explore the physiological functions of anion channels in planta. J Biol Chem, 2000 Jun 9, 275(23), 17241 - 8 Functional characterization of yeast mitochondrial release factor 1; Askarian-Amiri ME et al.; The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity . The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes . The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay . The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex . The affinity was strongly dependent on the identity of stop signal . Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded . The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1 . This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions. J Biol Chem, 2000 Jun 2, 275(22), 17106 - 13 The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA . II . Isomerase-independent chaperone activity in vitro; Ramm K et al.; We recently identified FkpA by selecting for the increased yield of antibody single-chain Fv (scFv) fragments in phage display, even of those not containing cis-prolines . We have now investigated the properties of FkpA in vitro . The peptidylprolyl cis-trans-isomerase activity of FkpA was found to be among the highest of any such enzyme with a protein substrate, yet FkpA is not able to enhance the proline-limited refolding rate of the disulfide-free hu4D5-8 scFv fragment, probably due to inaccessibility of Pro-L95 . Nevertheless, the yield of the soluble and functional scFv fragment was dramatically increased in vitro in the presence of FkpA . Similar effects were observed for an scFv fragment devoid of cis-prolines . We are thus forced to conclude that the observed folding-assisting function is independent of the isomerase activity of the protein . The beneficial effect of FkpA was found to be due to two components . First, FkpA interacts with early folding intermediates, thus preventing their aggregation . Additionally, it has the ability to reactivate inactive protein, possibly also by binding to a partially unfolded species that may exist in equilibrium with the aggregated form, which may thus be released on a productive pathway . These in vitro measurements therefore fully reflect the in vivo results from periplasmic overexpression of FkpA. J Biol Chem, 2000 Jun 2, 275(22), 17100 - 5 The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA . I . Increased functional expression of antibody fragments with and without cis-prolines; Bothmann H et al.; The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates . We used a selection system for periplasmic chaperones based on the coexpression of an E . coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Pluckthun, A . (1998) Nature Biotechnol . 16, 376-380) . By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments . This selection strategy was now repeated with a library constructed from the genomic DNA of an skp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA . Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containing cis-prolines . In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage . Together with the in vitro data in the accompanying paper (Ramm, K., and Pluckthun, A . (2000) J . Biol . Chem . 275, 17106-17113), we conclude that the effect of FkpA is independent of its PPIase activity. J Biol Chem, 2000 Jun 2, 275(22), 16518 - 29 The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo; Budd ME et al.; Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease . To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease . We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells . A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease . Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site . Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP . These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP . Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair . Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype. J Biol Chem, 2000 May 19, 275(20), 15512 - 9 Characterization of the unique C terminus of the Escherichia coli tau DnaX protein . Monomeric C-tau binds alpha AND DnaB and can partially replace tau in reconstituted replication forks; Dallmann HG et al.; A contact between the dimeric tau subunit within the DNA polymerase III holoenzyme and the DnaB helicase is required for replication fork propagation at physiologically-relevant rates (Kim, S., Dallmann, H . G., McHenry, C . S., and Marians, K . J . (1996) Cell 84, 643-650) . In this report, we exploit the OmpT protease to generate C-tau, a protein containing only the unique C-terminal sequences of tau, free of the sequences shared with the alternative gamma frameshifting product of dnaX . We have established that C-tau is a monomer by sedimentation equilibrium and sedimentation velocity ultracentrifugation . Monomeric C-tau binds the alpha catalytic subunit of DNA polymerase III with a 1:1 stoichiometry . C-tau also binds DnaB, revealed by a coupled immunoblotting method . C-tau restores the rapid replication rate of inefficient forks reconstituted with only the gamma dnaX gene product . The acceleration of the DnaB helicase can be observed in the absence of primase, when only leading-strand replication occurs . This indicates that C-tau, bound only to the leading-strand polymerase, can trigger the conformational change necessary for DnaB to assume the fast, physiologically relevant form. J Biol Chem, 2000 May 19, 275(20), 15254 - 64 Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor; Hu E et al.; Histone acetylation alters chromatin state by modifying lysines on histone and plays an important role in modulating gene transcription . A dynamic balance of histone acetylation/deacetylation is maintained by histone acetyltransferases and histone deacetylases . Emerging evidence suggests that a family of histone deacetylases may exist to regulate diverse cellular functions, including chromatin structure, gene expression, cell cycle progression, and oncogenesis . We describe here a novel human histone deacetylase, named HDAC8, cloned from human kidney . HDAC8 encodes 377 amino acid residues and shares extensive homology to several known HDACs, in particular a histone deacetylase from Arabidopsis thaliana . Northern blot analyses revealed that HDAC8 expression pattern for HDAC8 is distinct from that for HDAC1 and HDAC3, and expression of HDAC8 mRNA occurs in multiple organs including heart, lung, kidney, and pancreas . HDAC8 mRNA was also observed in several cell lines derived from cancerous tissues . When expressed in HEK293 cells, HDAC8 exhibited deacetylase activity toward acetylated histone, indicating that this protein is a bona fide histone deacetylase . Its histone deacetylase activity was inhibited by trichostatin and other known histone deacetylase inhibitors . Furthermore, active recombinant HDAC8 was expressed and purified from Escherichia coli . When ectopically expressed in cells, HDAC8 was found to be localized to the nucleus . Co-transfection experiments demonstrated that expression of HDAC8 repressed a viral SV40 early promoter activity . These results indicate that HDAC8 is a novel member of the histone deacetylase family, which may play a role in the development of a broad range of tissues and potentially in the etiology of cancer. J Biol Chem, 2000 Jun 23, 275(25), 19218 - 23 The two toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers; White EL et al.; Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing . Western blotting of total T . gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed . Both form enzymatically active homotetramers when overexpressed in Escherichia coli . The specific activity of HGPRT-I is five times that of HGPRT-II . When both isozymes are co-expressed in E . coli, HGPRT-I.HGPRT-II heterotetramers form . The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II . Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits . The implications of this finding are discussed. J Biol Chem, 2000 May 19, 275(20), 15287 - 94 Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides . Localization of regions essential for interaction with the three-subunit core complex; Tso SC et al.; Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized . Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential . IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex . Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex . The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV . These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively. J Biol Chem, 2000 May 19, 275(20), 15074 - 81 Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding; Das B et al.; Vav and Sos1 are Dbl family guanine nucleotide exchange factors, which activate Rho family GTPases in response to phosphatidylinositol 3-kinase products . A pleckstrin homology domain adjacent to the catalytic Dbl homology domain via an unknown mechanism mediates the effects of phosphoinositides on guanine nucleotide exchange activity . Here we tested the possibility that phosphatidylinositol 3-kinase substrates and products control an interaction between the pleckstrin homology domain and the Dbl homology domain, thereby explaining the inhibitory effects of phosphatidylinositol 3-kinase substrates and stimulatory effects of the products . Binding studies using isolated fragments of Vav and Sos indicate phosphatidylinositol 3-kinase substrate promotes the binding of the pleckstrin homology domain to the Dbl homology domain and blocks Rac binding to the DH domain, whereas phosphatidylinositol 3-kinase products disrupt the Dbl homology/pleckstrin homology interactions and permit Rac binding . Additionally, Lck phosphorylation of Vav, a known activating event, reduces the affinities between the Vav Dbl homology and pleckstrin homology domains and permits Rac binding . We also show Vav activation in cells, as monitored by phosphorylation of Vav, Vav association with phosphatidylinositol 3,4,5-trisphosphate, and Vav guanine nucleotide exchange activity, is blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin . These results suggest the molecular mechanisms for activation of Vav and Sos1 require disruption of inhibitory intramolecular interactions involving the pleckstrin homology and Dbl homology domains. J Biol Chem, 2000 Jun 9, 275(23), 17338 - 43 Glycosylation efficiency of Asn-Xaa-Thr sequons depends both on the distance from the C terminus and on the presence of a downstream transmembrane segment; Nilsson I et al.; Statistical studies of N-glycosylated proteins have indicated that the frequency of nonglycosylated Asn-Xaa-(Thr/Ser) sequons increases toward the C terminus (Gavel, Y., and von Heijne, G . (1990) Protein Eng . 3, 433-442) . Using in vitro transcription/translation of a truncated model protein in the presence of dog pancreas microsomes, we find that glycosylation efficiency of Asn-Xaa-Thr sequons indeed is reduced when the sequon is within approximately 60 residues of the C terminus . Surprisingly, the presence of a hydrophobic stop transfer sequence between the Asn-Xaa-Thr sequon and the C terminus results in a very different dependence of glycosylation efficiency on the distance to the C terminus, where the presence of the stop transfer segment inside the ribosome appears to cause a drastic drop in the level of glycosylation . We speculate that this may reflect a change in the structure of the ribosome/translocon complex induced by the stop transfer segment. J Biol Chem, 2000 Jun 2, 275(22), 16414 - 9 The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase; Caldas T et al.; Ribosomal RNAs undergo several nucleotide modifications including methylation . We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA . The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S(150) supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ-deficient strain . In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ . FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle . We identified the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments . In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ. J Biol Chem, 2000 Jun 16, 275(24), 18000 - 10 Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain; Peng H et al.; The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins . KRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1 . Little is known about the biochemical properties of the KRAB domain or the KRAB.KAP-1 complex . Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution . The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity . The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K(d) of 142 nm . The fitted R(max) indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylamide gel electrophoresis analysis of the complex . These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains. J Biol Chem, 2000 Jul 7, 275(27), 20361 - 7 The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins; Thanedar S et al.; Formylation of the initiator tRNA is essential for normal growth of Escherichia coli . The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon . However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation . In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation . The U1G72/U35A36 tRNA is formylated and participates in initiation . More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient . The amino acid attached to the initiator tRNA is also important in conferring protection against PTH . Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis . These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms. J Biol Chem, 2000 May 26, 275(21), 15962 - 8 Characterization and functional expression of cDNAs encoding methionine-sensitive and -insensitive homocysteine S-methyltransferases from Arabidopsis; Ranocha P et al.; Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (AdoMet), and methionine (Met) by a unique reaction and, like other organisms, use SMM as a methyl donor for Met synthesis from homocysteine (Hcy) . These reactions comprise the SMM cycle . Two Arabidopsis cDNAs specifying enzymes that mediate the SMM --> Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by homology and authenticated by complementing an Escherichia coli yagD mutant and by detecting HMT activity in complemented cells . Gel blot analyses indicate that these enzymes, AtHMT-1 and -2, are encoded by single copy genes . The deduced polypeptides are similar in size (36 kDa), share a zinc-binding motif, lack obvious targeting sequences, and are 55% identical to each other . The recombinant enzymes exist as monomers . AtHMT-1 and -2 both utilize l-SMM or (S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM . Both enzymes also use either methyl donor in vivo because both restore the ability to utilize AdoMet or SMM to a yeast HMT mutant . However, AtHMT-1 is strongly inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to the control of flux through the HMT reaction and the SMM cycle . Plant HMT is known to transfer the pro-R methyl group of SMM . This enabled us to use recombinant AtHMT-1 to establish that the other enzyme of the SMM cycle, AdoMet:Met S-methyltransferase, introduces the pro-S methyl group . These opposing stereoselectivities suggest a way to measure in vivo flux through the SMM cycle. J Biol Chem, 2000 Jun 2, 275(22), 17180 - 6 Functional redundancy in the nonspecific RNA binding domain of a class I tRNA synthetase; Wang CC et al.; The sequence of a 228-amino acid nonspecific RNA binding domain appended to the N terminus of a eukaryote tRNA synthetase is shown here to have two lysine-rich clusters (LRCs) that are functionally significant in vivo and in vitro . These two LRCs have unrelated sequences and are separated by a spacer of over 100 amino acids . By using a sensitive test for function in vivo, each LRC is shown to be sufficient in the absence of the other . This sufficiency requires fusion of the spacer to either of the LRCs . Experiments in vitro confirmed that the LRCs are each important for RNA binding . Thus, this nonspecific RNA binding domain has two dissimilar lysine-rich sequence elements that are functionally redundant . Further experiments suggest that this redundancy is not used to dock two molecules of RNA but rather to enhance the overall affinity for a single RNA molecule. J Biol Chem, 2000 Jul 21, 275(29), 22147 - 56 Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum; Carucci DJ et al.; We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum . Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2) . However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P . falciparum proteins and vice versa . In addition, certain key residues in these domains are more characteristic of guanylyl cyclases . Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli . In P . falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes . The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct. J Biol Chem, 2000 Jun 2, 275(22), 16717 - 22 TatD is a cytoplasmic protein with DNase activity . No requirement for TatD family proteins in sec-independent protein export; Wexler M et al.; The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides . Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD . The tatD gene product has two homologues in E . coli coded by the unlinked ycfH and yjjV genes . An E . coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides . Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E . coli Tat pathway substrate . It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system . TatD is shown to be a cytoplasmic protein . TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity . Features of the tatA operon that may control TatD expression are discussed. J Biol Chem, 2000 May 19, 275(20), 15440 - 8 Nucleotide binding activity of SecA homodimer is conformationally regulated by temperature and altered by prlD and azi mutations; Schmidt M et al.; SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane . To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized . We found that at 4 degrees C SecA homodimer binds one ADP molecule with high affinity . This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true . Azi- and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA . Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA . The level of Azi- and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity . These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export. J Biol Chem, 2000 May 26, 275(21), 15809 - 19 Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41; Lee SF et al.; We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex . We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope . These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity . To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coli malE gene, which encodes a monomeric maltose-binding protein . The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration . The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex . The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells . A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety . We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828-856 and 770-795, respectively, play a critical role in cytoplasmic domain self-assembly . Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure . This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity. J Biol Chem, 2000 Jul 14, 275(28), 21017 - 24 Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP) . A conserved acidic residue in the complement-type repeats is important for recognition of RAP; Andersen OM et al.; The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands . The ligand binding sites are located in different regions of clusters consisting of approximately 40 residues, cysteine-rich complement-type repeats (CRs) . The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands . To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10) . By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910 . CR10 differs from other repeats in cluster II by not containing a surface-exposed conserved acidic residue between Cys(IV) and Cys(V) . By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding . We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP. J Biol Chem, 2000 Jun 2, 275(22), 17058 - 63 Mutations that increase the activity of the promoter of the Escherichia coli melibiose operon improve the binding of MelR, a transcription activator triggered by melibiose; Tamai E et al.; MelR is an Escherichia coli transcription factor that activates expression of the melAB operon in response to the presence of melibiose in the environment . MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions -120.5, -100.5, -62.5, and -42.5 upstream of the transcript start point . In a previous study, we described a spontaneous mutant that exhibited increased melAB expression . Sequence analysis showed that this mutant carries five consecutive base changes at positions -49, -50, -51, -52, and -53 upstream of the melAB transcript start . Here we show that these changes improve MelR binding to the target site centered at position -42.5 at the melAB promoter and that this improvement is responsible for increased promoter activity . Thus, the activity of the melAB promoter is fixed by the occupation by MelR of a DNA site that overlaps the -35 hexamer: MelR appears to be a typical class II-type transcription activator. J Biol Chem, 2000 May 19, 275(20), 15049 - 59 The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum delta H minichromosome maintenance protein; Shechter DF et al.; Minichromosome maintenance proteins (MCMs) form a family of conserved molecules that are essential for initiation of DNA replication . All eukaryotes contain six orthologous MCM proteins that function as heteromultimeric complexes . The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea . The archaea Methanobacterium thermoautotrophicum contains a single MCM-related sequence . Here we report on the expression and purification of the recombinant M . thermoautotrophicum MCM protein (MtMCM) in both Escherichia coli and baculovirus-infected cells . We show that purified MtMCM protein assembles in large macromolecular complexes consistent in size with being double hexamers . We demonstrate that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities . The intrinsic helicase activity of MtMCM is abolished when a conserved lysine in the helicase domain I/nucleotide binding site is mutated . MtMCM helicase unwinds DNA duplexes in a 3' --> 5' direction and can unwind up to 500 base pairs in vitro . The kinetics, processivity, and directionality of MtMCM support its role as a replicative helicase in M . thermoautotrophicum . This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified. J Biol Chem, 2000 Jun 9, 275(23), 17571 - 7 Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase; McLachlin DT et al.; The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates . Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined . We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain . The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted . Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only) . Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483 . This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface . A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a . Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex. J Biol Chem, 2000 Jun 30, 275(26), 19747 - 51 Sugar transport through maltoporin of Escherichia coli . Role of polar tracks; Dumas F et al.; The three-dimensional structure of the maltooligosaccharide specific outer membrane channel LamB of Escherichia coli complexed with sugar molecules revealed a hypothetical transport pathway . Sugars are supposed to slide over a stretch of aromatic residues facilitated by continuous making/breaking of hydrogen bonds between the hydroxyl groups of the sugars and charged amino acids, the "polar tracks." The effect of nine single and three multiple mutations in the polar track residues was investigated by current fluctuations, liposome swelling assays, and in vivo uptake of radiolabeled substrates . Additionally, sugar transport through wild-type LamB was investigated by current fluctuation analysis in water and deuterium . This way the effects on k(on) and k(off) could be investigated separately . Analyses of the various mutants revealed a strong effect on the k(on) values . Because steering to the binding site requires only a few interactions, consequently the loss of even one bond will have a strong effect . Deuterium experiments, which changed the characteristic of all hydrogen bonds, showed a strong effect on k(off) rates, because at this stage the sugar has numerous interactions with the channel . Furthermore, all the mutations induces a strong decrease of in vivo uptake of sugars . These results clearly demonstrate the importance of the polar track residues on both on and off rates in sugar transport and reveal a strong cooperative effect of hydrogen bond formation. J Biol Chem, 2000 May 19, 275(20), 15449 - 57 Evidence that the NH2 terminus of vph1p, an integral subunit of the V0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector; Landolt-Marticorena C et al.; The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex . V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation . Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components . The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus . Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p . Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction . Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1) . Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction . Limited trypsin digests cleaves both Nt-Vph1p and Vma13p . The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1)-sensitive ATP hydrolysis . Trypsin cleaved Vph1p at arginine 53 . Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion . These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation. J Biol Chem, 2000 May 19, 275(20), 14795 - 8 RpoS-dependent promoters require guanosine tetraphosphate for induction even in the presence of high levels of sigma(s); Kvint K et al.; RpoS-dependent promoters require ppGpp for induction in the stationary phase . This has been thought to be a simple consequence of final sigma(S) itself requiring ppGpp for its production . By using four model promoters requiring final sigma(S) for normal induction in the stationary phase, we demonstrate that final sigma(S)-dependent promoters require ppGpp even in the presence of high levels of final sigma(S) produced ectopically . Similar to final sigma(70)-dependent promoters under positive control by ppGpp, the requirement of final sigma(S)-dependent promoters for this alarmone is bypassed by specific "stringent" mutations in the beta-subunit of RNA polymerase . The results suggest that stationary phase induction of both final sigma(S)- and final sigma(70)-dependent genes requires the stringent control modulon and that stringency confers dual control on the RpoS regulon by affecting promoter activity and the levels of the required final sigma-factor. Biochemistry, 2000 Apr 11, 39(14), 4191 - 8 Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies; Denninger JW et al.; The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO) . YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion . In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone . We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase . In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold . While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1) . Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC . In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1 . Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385) . The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket. Biochemistry, 2000 Apr 11, 39(14), 4165 - 73 Iron-sulfur center of biotin synthase and lipoate synthase; Ollagnier-De Choudens S et al.; Biotin synthase and lipoate synthase are homodimers that are required for the C-S bond formation at nonactivated carbon in the biosynthesis of biotin and lipoic acid, respectively . Aerobically isolated monomers were previously shown to contain a (2Fe-2S) cluster, however, after incubation with dithionite one (4Fe-4S) cluster per dimer was obtained, suggesting that two (2Fe-2S) clusters had combined at the interface of the subunits to form the (4Fe-4S) cluster . Here we report Mossbauer studies of (57)Fe-reconstituted biotin synthase showing that anaerobically prepared enzyme can accommodate two (4Fe-4S) clusters per dimer . The (4Fe-4S) cluster is quantitatively converted into a (2Fe-2S)(2+) cluster upon exposure to air . Reduction of the air-exposed enzyme with dithionite or photoreduced deazaflavin yields again (4Fe-4S) clusters . The (4Fe-4S) cluster is stable in both the 2+ and 1+ oxidation states . The Mossbauer and EPR parameters were DeltaE(q) = 1.13 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and DeltaE(q) = 0.51 mm/s, delta = 0.85 mm/s, g(par) = 2.035, and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+) . Considering that we find two (4Fe-4S) clusters per dimer, our studies argue against the early proposal that the enzyme contains one (4Fe-4S) cluster bridging the two subunits . Our study of lipoate synthase gave results similar to those obtained for BS: under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit {DeltaE(q) = 1.20 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and g(par) = 2.039 and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+)}, which reacts with oxygen to generate a (2Fe-2S)(2+) center. Biochemistry, 2000 Apr 11, 39(14), 4154 - 64 Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: homologous sites in the transmembrane domain; Nagy JK et al.; This work represents the first stage of thiol-based cross-linking studies to map the oligomeric interface of the homotrimeric membrane protein diacylglycerol kinase (DAGK) . A total of 53 single-cysteine mutants spanning DAGK's three transmembrane segments and the first part of a cytoplasmic domain were purified and subjected to catalytic oxidation in mixed micelles . Four mutants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer disulfide bond formation between homologous sites on adjacent subunits . To establish whether the homologous sites are proximal in the ground-state conformation of DAGK or whether the disulfide bonds formed as a result of motions that brought normally distal sites into transient proximity, additional cross-linking experiments were carried out in three different milieus of varying fluidity {mixed micelles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles, and Escherichia coli membranes} . Cross-linking experiments included disulfide bond formation under three different catalytic conditions {Cu(II)-phenanthroline oxidation, I(2) oxidation, and thionitrobenzoate-based thiol exchange} and reactions with a set of bifunctional thiol-reactive chemical cross-linkers presenting two different reactive chemistries and several spacer lengths . On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a result of protein motions . Results for position 74 were ambiguous . In lipid vesicles and mixed micelles DAGK appears to execute motions that are not present in native membranes, with mobility also being higher for DAGK in mixed micelles than in POPC vesicles. Biochemistry, 2000 Apr 11, 39(14), 4122 - 8 Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin? Levert KL, Lloyd RB, Waldrop GL. Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis . For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position . Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin . The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin . Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue . Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair . To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q) . Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP . The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin . However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction . N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230 . The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base . Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin. Biochemistry, 2000 Apr 11, 39(14), 4105 - 11 Conservative mutations of glutamine-125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities; Hinds TA et al.; The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer . One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures . To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced . These three mutants and wild-type HSV-1 TK were expressed in E . coli and partially purified and their enzymatic properties compared . In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90% . For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant . In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant . The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics . Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding . When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines. Biochemistry, 2000 Apr 11, 39(14), 4090 - 5 On the role of the carboxyl-terminal helix of RXR in the interactions of the receptor with ligand; Budhu AS et al.; The retinoid X receptor (RXR), a ligand-inducible transcription factor that is activated by 9-cis-retinoic acid, is a member of the superfamily of nuclear hormone receptors . The ligand-induced transcriptional activity of nuclear receptors is coordinated by their C-terminal region termed the ligand-binding domain . Structural analyses of several nuclear receptors showed that the most dramatic ligand-induced conformational change in these proteins involves a positional shift in the receptors' C-terminal helix, termed helix 12 . Consequently, in the liganded state, helix 12 is folded over the entrance to the ligand-binding pocket where it serves as a lid, and it has been proposed that this region functions to stabilize ligand binding by at least some nuclear receptors . Here, to examine the possible role of helix 12 in contributing to the association of RXR with its ligand, the equilibrium and kinetic parameters of the interactions of 9-cis-retinoic acid with RXR and with a deletion mutant lacking helix 12 were measured . Deletion of the region did not significantly alter the ligand-binding affinity of RXR at equilibrium . However, both the rate of dissociation and the rate of association of the RXR-9-cis-retinoic acid complex were significantly slower in the absence of helix 12 . Taken together, these observations suggest that helix 12 of RXR facilitates both the entry and the exit of the ligand from the binding pocket without affecting the equilibrium ligand-binding affinity . The results thus point at a previously unsuspected function for this region. Biochemistry, 2000 Apr 11, 39(14), 4075 - 81 Deletion of C-terminal residues of Escherichia coli ribosomal protein L10 causes the loss of binding of one L7/L12 dimer: ribosomes with one L7/L12 dimer are active; Griaznova O et al.; Escherichia coli ribosomal protein L10 binds the two L7/L12 dimers and thereby anchors them to the large ribosomal subunit . C-Terminal deletion variants (Delta10, Delta20, and Delta33 amino acids) of ribosomal protein L10 were constructed in order to define the binding sites for the two L7/L12 dimers and then to make and test ribosomal particles that contain only one of the two dimers . None of the deletions interfered with binding of L10 variants to ribosomal core particles . Deletion of 20 or 33 amino acids led to the inability of the proteins to bind both dimers of protein L7/L12 . The L10 variant with deletion of 10 amino acids bound one L7/L12 dimer in solution and when reconstituted into ribosomes promoted the binding of only one L7/L12 dimer to the ribosome . The ribosomes that contained a single L7/L12 dimer were homogeneous by gel electrophoresis where they had a mobility between wild-type 50S subunits and cores completely lacking L7/L12 . The single-dimer ribosomal particles supported elongation factor G dependent GTP hydrolysis and protein synthesis in vitro with the same activity as that of two-dimer particles . The results suggest that amino acids 145-154 in protein L10 determine the binding site ("internal-site") for one L7/L12 dimer (the one reported here), and residues 155-164 ("C-terminal-site") are involved in the interaction with the second L7/L12 dimer . Homogeneous ribosomal particles containing a single L7/L12 dimer in each of the distinct sites present an ideal system for studying the location, conformation, dynamics, and function of each of the dimers individually. Biochemistry, 2000 Apr 11, 39(14), 4062 - 7 Function of the extra 5'-phosphate carried by histidine tRNA; Fromant M et al.; Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1 . This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase . Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase . In the case of the reaction of E . coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable . Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His) . Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs. Biochemistry, 2000 Apr 11, 39(14), 4046 - 52 Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase; Temple CA et al.; Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center . Quantitation of the guanine and the molybdenum as compared to that found in R . sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor . UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme . EPR spectra were obtained for the Mo(V) state of the enzyme . X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme . The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A . The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A. Biochemistry, 2000 Apr 11, 39(14), 4004 - 11 Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase; Vitart V et al.; Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase . Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain . Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin . Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771) . Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects . Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site) . Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444 . To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody . Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection. Biochemistry, 2000 Apr 11, 39(14), 3988 - 4003 Evidence that beta-tubulin induces a conformation change in the cytosolic chaperonin which stabilizes binding: implications for the mechanism of action; Dobrzynski JK et al.; The class II chaperonin CCT facilitates protein folding by a process that is not well-understood . One striking feature of this chaperonin is its apparent selectivity in vivo, folding only actin, tubulin, and several other proteins . In contrast, the class I chaperonin GroEL is thought to facilitate the folding of many proteins within Escherichia coli . It has been proposed that this apparent selectivity is associated with certain regions of a substrate protein's primary structure . Using limiting amounts of beta-tubulin, beta-tubulin mutants, and beta-tubulin/ftsZ chimeras, we assessed the contribution of select regions of beta-tubulin to CCT binding . In a complementary study, we investigated inter-ring communication in CCT where we exploited polypeptide binding sensitivity to nucleotide to quantitate nucleotide binding . beta-Tubulin bound with a high apparent affinity to CCT in the absence of nucleotide (apparent K(D) approximately 3 nM; its apparent binding free energy, DeltaG, ca . -11.8 kcal/mol) . Despite this, the interactions appear to be weak and distributed throughout much of the sequence, although certain sites ("hot spots") may interact somewhat more strongly with CCT . Globally averaged over the beta-tubulin sequence, these interactions appear to contribute ca . -9 to -11 cal/mol per residue, and to account for no more than 50-60% of the total binding free energy . We propose that a conformation change or deformation induced in CCT by substrate binding provides the missing free energy which stabilizes the binary complex . We suggest that by coupling CCT deformation with polypeptide binding, CCT avoids the need for high "intrinsic" affinities for its substrates . This strategy allows for dynamic interactions between chaperonin and bound substrate, which may facilitate folding on the interior surface of CCT in the absence of nucleotide and/or productive release of bound polypeptide into the central cavity upon subsequent MgATP binding . CCT displayed negative inter-ring cooperativity like GroEL . When ring 1 of CCT bound MgATP or beta-tubulin, the affinity of ring 2 for polypeptide or nucleotide was apparently reduced approximately 100-fold. Arch Pathol Lab Med, 2000 Apr, 124(4), 619 - 24 Pseudomembranous gastritis: a novel complication of Aspergillus infection in a patient with a bone marrow transplant and graft versus host disease; Yong S et al.; A 36-year-old Hispanic man who had undergone allogeneic bone marrow transplantation, complicated by graft versus host disease, was admitted with acute gastrointestinal symptoms, including severe diarrhea and diffuse abdominal pain . He also had a persistent cough with sputum production . Blood cultures yielded Escherichia coli, and sputum cultures grew Apergillus species . The patient was treated with antifungal agents and broad-spectrum antibiotics . Despite aggressive medical therapy, the patient died 10 days after admission . Postmortem examination disclosed severe, bilateral confluent bronchopneumonia, with numerous septated branching hyphae consistent with Aspergillus species fungal organisms that involved the pulmonary parenchyma and tracheobronchial tree . Although the small and large bowels were only mildly congested, the entire gastric mucosa was covered with a 1.5-cm-thick pseudomembrane that contained numerous Aspergillus organisms . Our report represents the first description, to our knowledge, of a diffuse inflammatory pseudomembrane in the stomach, a complication that to date has only been associated with small and large bowel involvement. J Clin Microbiol, 2000 Apr, 38(4), 1684 - 7 Selective isolation of eae-positive strains of shiga toxin-producing Escherichia coli; Fukushima H et al.; Culture on cefixime, tellurite, and sorbitol-MacConkey agar after HCl treatment facilitated the growth of 410 (94%) of 436 eae-positive Shiga toxin-producing Escherichia coli (STEC) strains and 17 (16%) of 107 eae-negative STEC strains . This selectivity was closely related to acid resistance in E . coli and tellurite resistance in eae-positive STEC strains. J Clin Microbiol, 2000 Apr, 38(4), 1592 - 8 Comparison of polyvinyl alcohol fixative with three less hazardous fixatives for detection and identification of intestinal parasites; Jensen B et al.; Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the "gold standard" for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens . However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems . We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears . Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian) . Specimens were processed and stained according to each manufacturer's directions . Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS . Blastocystis hominis and Iodamoeba butschlii were preserved in a readily identifiable state by all methods of fixation . However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion . For example, Entamoeba histolytica (Entamoeba dispar) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS . Likewise, Chilomastix mesnili was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while Entamoeba coli was seen much less frequently with PS than with the other three fixatives . A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives . Sample preparation was most rapid with PS, although the EC method involved the fewest steps . In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of E . histolytica, E . coli, and C . mesnili . Both PF and EC appear to be acceptable, environmentally safe substitutes for PVA. J Clin Microbiol, 2000 Apr, 38(4), 1472 - 5 Comparison of a baculovirus-based VP2 enzyme immunoassay (EIA) to an Escherichia coli-based VP1 EIA for detection of human parvovirus B19 immunoglobulin M and immunoglobulin G in sera of pregnant women; Jordan JA; A split-sample study was conducted to evaluate the clinical performance of an enzyme immunoassay that detects the human parvovirus B19 virus (B19V) immunoglobulin M (IgM) or IgG in the sera of pregnant women . The initial study compared a baculovirus-expressed VP2 enzyme immunoassay (BVP2 EIA) (Biotrin International Inc., Dublin, Ireland) with the currently available and commonly used Escherichia coli-expressed VP1 enzyme immunoassay (EVP1 EIA) (MRL Diagnostics, Cypress, Calif.) . There was a high degree of agreement between the two assays in the detection of IgM antibodies (283 of 307 {92.2%}) or IgG antibodies (279 of 311 {89 . 7%}), with the majority of discrepancies (IgM, 17 of 24 {71%}; IgG, 16 of 31 {50%}) being due to equivocal data obtained with the EVP1 EIA . Specimens with discordant BVP2 EIA and EVP1 EIA results (23 of 24 IgM and 32 of 32 IgG results) were analyzed further by baculovirus-based VP1 immunofluorescence assays (BVP1 IFAs) (Biotrin International) . The BVP2 EIA and BVP1 IFA results for 20 of 23 and 28 of 32 specimens for IgM and IgG, respectively, were concordant . In contrast, the EVP1 EIA and BVP1 IFA data for only 3 of 23 and 4 of 32 specimens for IgM and IgG, respectively, were in agreement, despite the fact that the same capsid antigen was used . Both the BVP2 EIAs and BVP1 IFAs utilize a conformational viral capsid antigen, while the EVP1 EIA uses a denatured viral capsid antigen . In conclusion, the BVP2 EIAs produced far fewer equivocal results for IgM and IgG, correlating more closely to the confirmatory BVP IFAs, than did the EVP1 EIAs and proved to be more accurate for detecting B19V antibodies in the sera of pregnant women. Genetics, 2000 Apr, 154(4), 1597 - 610 Genetic, behavioral and environmental determinants of male longevity in Caenorhabditis elegans; Gems D et al.; Males of the nematode Caenorhabditis elegans are shorter lived than hermaphrodites when maintained in single-sex groups . We observed that groups of young males form clumps and that solitary males live longer, indicating that male-male interactions reduce life span . By contrast, grouped or isolated hermaphrodites exhibited the same longevity . In one wild isolate of C . elegans, AB2, there was evidence of copulation between males . Nine uncoordinated (unc) mutations were used to block clumping behavior . These mutations had little effect on hermaphrodite life span in most cases, yet many increased male longevity even beyond that of solitary wild-type males . In one case, the neuronal function mutant unc-64(e246), hermaphrodite life span was also increased by up to 60% . The longevity of unc-4(e120), unc-13(e51), and unc-32(e189) males exceeded that of hermaphrodites by 70-120% . This difference appears to reflect a difference in sex-specific life span potential revealed in the absence of male behavior that is detrimental to survival . The greater longevity of males appears not to be affected by daf-2, but is influenced by daf-16 . In the absence of male-male interactions, median (but not maximum) male life span was variable . This variability was reduced when dead bacteria were used as food . Maintenance on dead bacteria extended both male and hermaphrodite longevity. EMBO J, 2000 Apr 3, 19(7), 1731 - 42 DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells; Dominguez O et al.; A novel DNA polymerase has been identified in human cells . Human DNA polymerase mu (Pol mu), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT) . Human Pol mu, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn(2+) ions . Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol mu was enhanced by the presence of a template strand . Using activating Mg(2+) ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values . In the presence of Mn(2+) ions, template-enhanced polymerization produced a random insertion of nucleotides . Northern-blotting and in situ analysis showed a preferential expression of Pol mu mRNA in peripheral lymphoid tissues . Moreover, a large proportion of the human expressed sequence tags corresponding to Pol mu, present in the databases, derived from germinal center B cells . Therefore, Pol mu is a good candidate to be the mutator polymerase responsible for somatic hyper- mutation of immunoglobulin genes. EMBO J, 2000 Apr 3, 19(7), 1555 - 66 The Escherichia coli RNA polymerase alpha subunit linker: length requirements for transcription activation at CRP-dependent promoters; Meng W et al.; The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alphaCTD) plays a key role in transcription initiation at many activator-dependent promoters . This domain is connected to the N-terminal domain by an unstructured linker, which is proposed to confer a high degree of mobility on alphaCTD . To investigate the role of this linker in transcription activation we tested the effect of altering the linker length on promoters dependent on the cyclic AMP receptor protein (CRP) . Short deletions within the alpha linker decrease CRP-dependent transcription at a Class I promoter while increasing the activity of a Class II promoter . Linker extension impairs CRP-dependent transcription from both promoters, with short extensions exerting a more marked effect on the Class II promoter . Activation at both classes of promoter was shown to remain dependent upon activating region 1 of CRP . These results show that the response to CRP of RNA polymerase containing linker-modified alpha subunits is class specific . These observations have important implications for the architecture of transcription initiation complexes at CRP-dependent promoters. EMBO J, 2000 Apr 3, 19(7), 1450 - 7 Regulatory cross-talk between adhesin operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein; Xia Y et al.; Pathogenic Escherichia coli often carry determinants for several different adhesins . We show a direct communication between two adhesin gene clusters in uropathogenic E.coli: type 1 fimbriae (fim) and pyelonephritis-associated pili (pap) . A regulator of pap, PapB, is a key factor in this cross-talk . FimB recombinase turns on type 1 fimbrial expression, and PapB inhibited phase transition by FimB in both off-to-on and on-to-off directions . On-to-off switching requiring FimE was increased by PapB . By analysis of FimB- and FimE-LacZ translational fusions it was concluded that the increase in on-to-off transition rates was via an increase in FimE expression . Inhibition of FimB-promoted switching was via a different mechanism: PapB inhibited FimB-promoted in vitro recombination, indicating that FimB activity was blocked at the fim switch . In vitro analyses showed that PapB bound to several DNA regions of the type 1 fimbrial operon, including the fim switch region . These data show that Pap expression turns off type 1 fimbriae expression in the same cell . Such cross-talk between adhesin gene clusters may bring about appropriate expression at the single cell level. Microbiology, 2000 Mar, 146 ( Pt 3), 591 - 8 Mu DNA reintegration upon excision: evidence for a possible involvement of nucleoid folding; Paolozzi L et al.; Mutations induced by the integration of a Mugem2ts prophage can revert at frequencies around 1x10(-6) . In these revertant clones, the prophage excised from its original localization is not lost but reintegrated elsewhere in the host genome . One of the most intriguing aspects of this process is that the prophage reintegration is not randomly distributed: there is a strong correlation between the original site of insertion (the donor site) and the target site of the phage DNA migration (the receptor site) . In this paper, it is shown that in the excision-reintegration process mediated by Mugem2ts, the position of the initial prophage site strongly influences the location of the reintegration site . In addition, for each donor site, the receptor site is a discrete DNA region within which the excised Mu DNA can reintegrate and the two sites implicated in phage DNA migration must be located on the same DNA molecule . These data suggest the involvement of nucleoid folding in the excision-reintegration process. Microbiology, 2000 Mar, 146 ( Pt 3), 581 - 9 Stability by multimer resolution of pJHCMW1 is due to the Tn1331 resolvase and not to the Escherichia coli Xer system; Tolmasky ME et al.; The plasmid pJHCMW1 encodes resistance to several aminoglycosides and beta-lactams and consists of a copy of the transposon Tn1331, a region including the replication functions, and a sequence with homology to ColE1 cer, designated mwr . In this work, the role of this cer-like site in ensuring the stable inheritance of pJHCMW1 by multimer resolution was studied . The Escherichia coli Xer site-specific recombination system acts at sites such as ColE1 cer to resolve plasmid multimers formed by homologous recombination, thereby maintaining plasmids in a monomeric state and helping to ensure stable plasmid inheritance . Despite its high similarity to ColE1 cer, the pJHCMW1 mwr was a poor substrate for Xer recombination in E . coli and did not contribute significantly to plasmid stability . Instead, the Tn1331 co-integrate resolution system was highly active at resolving pJHCMW1 multimers and ensured the stable inheritance of pJHCMW1 . Although Xer recombination at pJHCMW1 mwr was inefficient in E . coli, the recombination that did occur was dependent on ArgR, PepA, XerC and XerD . A supercoiled circular DNA molecule containing two pJHCMW1 mwr sites in direct repeat yielded Holliday-junction-containing product when incubated with ArgR, PepA, XerC and XerD in vitro, confirming that pJHCMW1 mwr is a functional recombination site . However, unlike cer, some Holliday-junction-containing product could be detected for mwr in the absence of ArgR, although addition of this protein resulted in formation of more Holliday junctions . Binding experiments demonstrated that XerD bound to pJHCMW1 mwr core with a high affinity, but that XerC bound to this site very poorly, even in the presence of XerD. Nature, 2000 Mar 23, 404(6776), 355 - 62 Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex; Misura KM et al.; Syntaxin 1a and neuronal Sec1 (nSec1) form an evolutionarily conserved heterodimer that is essential for vesicle trafficking and membrane fusion . The crystal structure of the nSec1-syntaxin 1a complex, determined at 2.6 A resolution, reveals that major conformational rearrangements occur in syntaxin relative to both the core SNARE complex and isolated syntaxin . We identify regions of the two proteins that seem to determine the binding specificity of particular Sec1 proteins for syntaxin isoforms, which is likely to be important for the fidelity of membrane trafficking . The structure also indicates mechanisms that might couple the action of upstream effector proteins to conformational changes in syntaxin 1a and nSec1 that lead to core complex formation and membrane fusion. Int J Food Microbiol, 2000 Mar 10, 54(1-2), 1 - 8 Development and evaluation of a PCR-microplate capture hybridization method for direct detection of verotoxigenic Escherichia coli strains in artificially contaminated food samples; Cocolin L et al.; For the purpose of detecting, directly in food, verotoxigenic Escherichia coli, a microplate hybridization method for the detection of PCR products from the SLT I and SLT II genes, was developed and evaluated . Two pairs of primers and two probes, specific for the SLT I gene and for the SLT II gene, were designed and tested . For the strains containing both genes, two PCR products of different molecular weights were obtained, whereas when only one gene was present only one fragment resulted from PCR . The use of the biotin-labeled probes allowed the immobilization of the PCR products in the microtiter plate wells and by this means their detection was possible using an ELISA-based technique . Forty artificially contaminated and fifty naturally contaminated food samples were analyzed by using the PCR-microplate hybridization technique developed in this study . All the artificially contaminated food samples were positive, independently of the number of cells inoculated before the enrichment step, whereas the naturally contaminated food samples were all negative. Bioinformatics, 1999 Dec, 15(12), 987 - 93 Modeling and predicting transcriptional units of Escherichia coli genes using hidden Markov models; Yada T et al.; MOTIVATION: The hidden Markov model (HMM) is a valuable technique for gene-finding, especially because its flexibility enables the inclusion of various sequence features . Recent programs for bacterial gene-finding include the information of ribosomal binding site (RBS) to improve the recognition accuracy of the start codon, using this feature . We report here our attempt to extend the model into the total transcriptional unit, enabling the prediction of operon structures . RESULTS: First, we improved the prediction accuracy of coding sequences (CDSs) by employing the models of 'typical', 'atypical' and 'negative (false-positive)' classes as well as the models of RBS and its downstream spacer . The sensitivity of exactly predicting the 204 experimentally confirmed CDSs reached 90.2% in an objective test . Based on the prediction result of CDSs, the positions of the promoters and terminators were predicted . Our model could exactly recognize 60% of 390 known transcriptional units . Thus, the accuracy and significance of this prediction problem is far from trivial . We would like to propose this problem as an open theme in bioinformatics because the ongoing or planned post-sequencing projects will produce much data for future improvements. Bioinformatics, 1999 Dec, 15(12), 974 - 9 SEGMENT: identifying compositional domains in DNA sequences; Oliver JL et al.; MOTIVATION: DNA sequences are formed by patches or domains of different nucleotide composition . In a few simple sequences, domains can simply be identified by eye; however, most DNA sequences show a complex compositional heterogeneity (fractal structure), which cannot be properly detected by current methods . Recently, a computationally efficient segmentation method to analyse such nonstationary sequence structures, based on the Jensen-Shannon entropic divergence, has been described . Specific algorithms implementing this method are now needed . RESULTS: Here we describe a heuristic segmentation algorithm for DNA sequences, which was implemented on a Windows program (SEGMENT) . The program divides a DNA sequence into compositionally homogeneous domains by iterating a local optimization procedure at a given statistical significance . Once a sequence is partitioned into domains, a global measure of sequence compositional complexity (SCC), accounting for both the sizes and compositional biases of all the domains in the sequence, is derived . SEGMENT computes SCC as a function of the significance level, which provides a multiscale view of sequence complexity. Biotechnol Bioeng, 2000 May 5, 68(3), 316 - 27 Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS; Noronha SB et al.; Acetate accumulation is a common problem observed in aerobic high cell density Escherichia coli cultures . A previous report has hypothesized that the glyoxylate shunt is active in a low acetate producer, E . coli BL21, and inactive in a high acetate producer, JM109 . To further investigate this hypothesis, we now develop a model for the incorporation of (13)C from uniformly labeled glucose into key TCA cycle intermediates . The (13)C isotopomer distributions of oxaloacetate and acetyl-CoA are first determined using NMR and MS techniques . These distributions are next validated by predicting the NMR spectrum of glutamate . Under steady state isotopic conditions, and with knowledge of the full isotopomer distributions of oxaloacetate and acetyl-CoA, the flux ratios through the TCA cycle and the glycoxylate shunt are obtained with respect to the flux through the PPC anaplerotic shunt . We conclude that in BL21, the glyoxylate shunt is active at 22% of the flux through the TCA cycle, and is inactive in JM109 . Further, in BL21, the flux through the TCA cycle equals the flux through the PPC shunt, while in JM109 the TCA cycle flux is only third of the flux through the PPC shunt . FEBS Lett, 2000 Mar 31, 470(3), 345 - 9 Three of four pseudoknots in tmRNA are interchangeable and are substitutable with single-stranded RNAs; Nameki N et al.; A novel translation, trans-translation, is facilitated by a highly structured RNA molecule, tmRNA . This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4) . Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA . Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions . These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans-translation process . Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional. FEBS Lett, 2000 Mar 31, 470(3), 315 - 8 Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase; Horstkorte R et al.; UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates . UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate . So far, regulation of this essential enzyme by posttranslational modification has not been shown . Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC . We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo . Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity. FEBS Lett, 2000 Mar 31, 470(3), 244 - 8 Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact; Tsunoda SP et al.; F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits . A ring structure of F(o)c subunits is widely believed to be a part of the rotor . Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli . Similar studies have been performed and reported recently {Sambongi et al . (1999) Science 286, 1722-1724} . However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor . We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o) . Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al. Am J Respir Cell Mol Biol, 2000 Apr, 22(4), 451 - 9 A novel karyopherin-beta homolog is developmentally and hormonally regulated in fetal lung; Zhang C et al.; To investigate molecular mechanisms of lung organogenesis, we used representational difference analysis to search for glucocorticoid-inducible genes in developing lung in a fetal rat model . Messenger RNA prepared from fetal and adult rat lung was used to prepare "representative amplicons." Adult-lung complementary DNA (cDNA) amplicons were used as "driver" in successive rounds of subtractive hybridization/amplification to isolate target fetal lung-specific cDNAs . A single clone, which was conserved and had near-perfect homology to eight human/rodent expressed sequence tags, was used as template for 5' and 3' rapid amplification of cDNA ends and SPICE (system for polymerase chain reaction amplification of cDNA ends) reactions to obtain the 3.6-kb cDNA, LGL2 (Genbank, AF 110195) encoding a deduced polypeptide (lgl2) of 963 amino acids . Northern analysis confirmed that LGL2 is differentially expressed in fetal lung (maximal during the pseudoglandular stage, gestational Days 14 to 16), induced by glucocorticoid, and enriched in epithelium relative to the mesenchyme . LGL2 was also detected in human fetal lung at gestational Week 16 as well as in human and rat fetal brain, heart, intestine, and kidney . We mapped LGL2 to chromosome 1p33-34.2 . Comparison with sequences in the genome database identified lgl2 as a member of the karyopherin-beta family of nuclear import proteins, with greatest homology to transportin SR . Maximal expression of LGL2 in the pseudoglandular stage of development is coordinate with that of key transcription factors that regulate prominent signal transduction pathways in fetal lung organogenesis . We propose a role for lgl2 in nuclear import of transcription factors that regulate signal transduction during fetal lung development. Curr Opin Microbiol, 2000 Apr, 3(2), 197 - 202 Protein folding and unfolding by Escherichia coli chaperones and chaperonins; Gottesman ME et al.; The folding of proteins from their initial unstructured state to their mature form has long been known to be promoted by other proteins known as chaperones and chaperonins . Recent biochemical and structural discoveries have provided dramatic insight into how these folding proteins work . This review will discuss these findings and suggest future experimental directions. Curr Opin Microbiol, 2000 Apr, 3(2), 159 - 64 U-turns and regulatory RNAs; Franch T et al.; Conventional antisense RNAs, such as those controlling plasmid replication and maintenance, inhibit the function of their target RNAs rapidly and efficiently . Novel findings show that a common U-turn loop structure mediates fast RNA pairing in the majority of these RNA controlled systems . Usually, an antisense RNA regulates a single, cognate target RNA only . Recent reports, however, show that antisense RNAs can act as promiscuous regulators that control multiple genes in concert to integrate complex physiological responses in Escherichia coli. Curr Opin Microbiol, 2000 Apr, 3(2), 118 - 25 RNA polymerase structure-function: insights into points of transcriptional regulation; Severinov K; The crystal structure of Thermus aquaticus RNA polymerase (RNAP) with 3.3 A resolution has recently been described . The high degree of sequence similarity between T . aquaticus RNAP and the prototypical RNAP from Escherichia coli invites comparison of the new structural data with genetic and biochemical results that defined the interaction sites of E . coli RNAP with transcription regulators. Genetics, 2000 Apr, 154(4), 1427 - 37 Evidence that stationary-phase hypermutation in the Escherichia coli chromosome is promoted by recombination; Bull HJ et al.; Adaptive (or stationary-phase) mutation is a group of phenomena in which mutations appear to occur more often when selected than when not . They may represent cellular responses to the environment in which the genome is altered to allow survival . The best-characterized assay system and mechanism is reversion of a lac allele on an F' sex plasmid in Escherichia coli, in which the stationary-phase mutability requires homologous recombination functions . A key issue has concerned whether the recombination-dependent mutation mechanism is F' specific or is general . Hypermutation of chromosomal genes occurs in association with adaptive Lac(+) mutation . Here we present evidence that the chromosomal hypermutation is promoted by recombination . Hyperrecombinagenic recD cells show elevated chromosomal hypermutation . Further, recG mutation, which promotes accumulation of recombination intermediates proposed to prime replication and mutation, also stimulates chromosomal hypermutation . The coincident mutations at lac (on the F') and chromosomal genes behave as independent events, whereas coincident mutations at lac and other F-linked sites do not . This implies that transient covalent linkage of F' and chromosomal DNA (Hfr formation) does not underlie chromosomal mutation . The data suggest that recombinational stationary-phase mutation occurs in the bacterial chromosome and thus can be a general strategy for programmed genetic change. Gastrointest Endosc, 2000 Apr, 51(4 Pt 1), 391 - 5 Endoscopic transpapillary drainage of pancreatic abscess: technique and results; Venu RP et al.; BACKGROUND: Pancreatic abscess is one of the serious complications of acute pancreatitis . Traditionally, pancreatic abscess has been treated by operative drainage . Based on experience with endoscopic transpapillary drainage of pseudocysts, a similar technique was used in patients with pancreatic abscess . METHOD: Patients were evaluated by endoscopic retrograde cholangiopancreatography . In those with pancreatic abscess communicating with the main pancreatic duct, pancreatic sphincterotomy, saline irrigation of the abscess cavity, and catheter dilation followed by 10F pancreatic stent placement were done . Instillation of gentamicin and nasopancreatic catheter drainage were used in difficult cases . RESULTS: Of 22 patients with pancreatic abscess, 11 underwent endoscopic transpapillary drainage with technical success in 10 patients (90%); 8 patients (74%) had resolution of pancreatic abscess, clinically and radiographically . Intracavitary instillation of gentamicin and nasopancreatic catheter drainage were used in 2 patients . Two patients in whom endoscopic transpapillary drainage failed underwent operative drainage with a favorable outcome, and the one patient in whom endoscopic treatment was technically unsuccessful underwent successful percutaneous drainage . One patient had mild pancreatitis . CONCLUSION: Endoscopic transpapillary drainage is an effective nonoperative therapy for selected cases of pancreatic abscess and is associated with minimal morbidity and no mortality. J Lipid Res, 2000 Apr, 41(4), 629 - 36 Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-coAs and requirement for GTP or ATP and Mg(2+) in addition to its known hydroxylation cofactors; Croes K et al.; Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs . A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E . coli and subsequently purified as an active protein . The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate . The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3-methylhexadecanoic acid . Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated . Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively . Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4-methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain . In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors . Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl-CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway. J Biol Chem, 2000 Apr 7, 275(14), 10702 - 8 Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase; Kozaki A et al.; Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis . This enzyme in plants is localized in plastids and is believed to be composed of biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been purified yet . Accumulated evidence shows that pea plastidic acetyl-CoA carboxylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a redox cascade . To understand the reductive activation of carboxyltransferase at the molecular level here, we obtained the active enzyme composed of decahistidine-tagged (His tag) alpha and beta polypeptides through the expression of the pea plastidic carboxyltransferase gene in Escherichia coli . Gel filtration showed that the molecular size of the recombinant carboxyltransferase is in agreement with that of partially purified carboxyltransferase from pea chloroplasts . The catalytic activity of the recombinant enzyme was similar to that of native carboxyltransferase . These results indicate that the molecular structure and conformation of recombinant carboxyltransferase resemble those of its native counterpart and that native carboxyltransferase is indeed composed of alpha and beta polypeptides . This recombinant enzyme was activated by dithiothreitol, a known reductant of S-S bonds, with a profile similar to that of its native counterpart . The recombinant enzyme was activated by reduced thioredoxin-f, a signal transducer of redox potential in chloroplasts under irradiation . Thus, this enzyme was redox-regulated, like that of the native carboxyltransferase. J Biol Chem, 2000 Apr 7, 275(14), 10582 - 9 Extracellular DsbA-insensitive folding of Escherichia coli heat-stable enterotoxin STa in vitro; Batisson I et al.; To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide . We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity . These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo . In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity . This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted . Periplasmic and lysate fractions of dsbA(+) and dsbA(-) cells did not have any STh activity . In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background . Subsequently, a time course experiment in regard to the secretion of STh from both dsbA(+) and dsbA(-) cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time . Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell. J Biol Chem, 2000 Apr 7, 275(14), 10577 - 81 The Arabidopsis thaliana PIN1At gene encodes a single-domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase; Landrieu I et al.; A homologue of the human site-specific prolyl cis/trans isomerase PIN1 was identified in Arabidopsis thaliana . The PIN1At gene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin . Steady-state PIN1At mRNA is found in all plant tissues tested . We show by two-dimensional NMR spectroscopy that the PIN1At is a prolyl cis/trans isomerase with specificity for phosphoserine-proline bonds . PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions . The N-terminal WW domain of 40 amino acids, typical of all the phosphorylation-dependent eukaryotic parvulins, is absent . However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the alpha1 helix observed in PIN1 that could mediate the protein-protein interactions. J Biol Chem, 2000 Apr 7, 275(14), 10477 - 83 Reciprocal regulation via protein-protein interaction between c-Myc and p21(cip1/waf1/sdi1) in DNA replication and transcription; Kitaura H et al.; The c-myc protooncogene product (c-Myc) is a transcription factor and is rapidly induced in resting cells following various mitogenic stimuli . c-Myc is thus suggested to play an important role in the transition from quiescence to proliferation . Despite numerous studies, including those on the connection between cyclin E/cyclin-dependent kinase 2 and c-Myc, little has been clarified about c-Myc in terms of the cell cycle regulation . Here we show that c-Myc can directly bind to the carboxyl-terminal region of the cyclin-dependent kinase inhibitor p21(cip1/waf1/sdi1) and thus partially relieves the p21 of the inhibitory effect on DNA synthesis directed by the proliferating cell nuclear antigen-dependent DNA polymerase delta . As for transcription, on the other hand, the p21 binding to the Myc box II region of c-Myc blocks c-Myc-Max complex formation on the E-box and thereby suppresses the transcriptional activation from the E-box by c-Myc . These results suggest that c-Myc activates DNA replication via inactivation of p21 and that p21, vice versa, represses the transcriptional activity of c-Myc . The balance of the reciprocal inactivation between c-Myc and p21 may determine the course of cellular processes such as cell proliferation, differentiation, and apoptosis. J Biol Chem, 2000 Apr 7, 275(14), 10331 - 41 Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut; Vinetz JM et al.; The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut . Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions . We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it . PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases . Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides . Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P . gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively . These two chitinase activities also had different pH activity profiles . These data suggest that the P . gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion. J Biol Chem, 2000 Apr 7, 275(14), 10196 - 201 Binding relationships of membrane tethering components . The giantin N terminus and the GM130 N terminus compete for binding to the p115 C terminus; Linstedt AD et al.; By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking . The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane . To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex . We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane . We then generated glutathione S-transferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively . Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GST-giantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin . To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130 . The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115 . These results question a simple tethering model involving a ternary giantin-p115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events. J Biol Chem, 2000 Apr 7, 275(14), 10154 - 9 Signal peptide determinants of SecA binding and stimulation of ATPase activity; Wang L et al.; A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown . In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites . Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated . These were used to establish the requirements for interaction with SecA . Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones . The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region . For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge . The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer . We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo. J Biol Chem, 2000 Apr 7, 275(14), 9963 - 9 Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro; Yusof R et al.; Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids . In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus . The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions . The denatured precursor was refolded to yield a purified active protease complex . Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain . The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position . The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent . These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates. J Biol Chem, 2000 Apr 7, 275(14), 9924 - 9 Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein; Sidorkina OM et al.; The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase . It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG) . The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein . The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E) . The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity . FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA . FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate . FpgP2E was completely inactive in all the assays . The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable . The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C --> T/A in E . coli BH990 (fpg mutY) cells . These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity. Carbohydr Res, 2000 Mar 10, 324(4), 225 - 30 Synthesis and biological activities of lipid A-type pyrancarboxylic acid derivatives; Mochizuki T et al.; The synthesis of lipid A-type pyrancarboxylic acid derivatives, which have a carboxylic acid group in the anomeric position of the reducing part of the disaccharide instead of the phosphate group in lipid A, is described . One of the compounds thus synthesized, which has an acyl substitution pattern similar to that of Escherichia coli lipid A, showed lipopolysaccharide (LPS)-agonistic activity . The other, which contains four lipid chains in the molecule, exhibited strong LPS-antagonistic activity toward human monoblastic U937 cells. Cancer Epidemiol Biomarkers Prev, 1999 Aug, 8(8), 669 - 74 hOGG1 Ser326Cys polymorphism and lung cancer susceptibility; Sugimura H et al.; The human homologue of the yeast OGG1 gene, hOGG1, has been cloned, and its genetic structure has been determined . Several polymorphisms in the hOGG1 gene were detected in the Japanese populations, and among them, the Ser-Cys polymorphism at codon 326 has been shown to have a functional difference in complementation of mutant Escherichia coli that is defective in the repair of 8-hydroxyguanine . Activity in the repair of 8-hydroxyguanine is greater in hOGG1-Ser326 protein than in hOGG1(326) protein . Because many environmental carcinogens produce 8-hydroxyguanine residue and mismatching to this modified base potentially causes oncogenic mutations, the capacity to repair these lesions can be involved in cancer susceptibility in human beings . We, therefore, examined allele distributions of the Ser326Cys polymorphism in a case-control study of male lung cancer in Okinawa . The analyses based on 241 cases and 197 hospital controls disclosed the following findings . (a) Those with the Cys/Cys genotype were at an increased risk of squamous cell carcinoma and nonadenocarcinoma compared to those with the Ser/Cys and those with the Ser/Ser genotypes combined . The odds ratios adjusted for age and smoking history were 3.01 (95% confidence interval, 1.33-6.83) and 2.18 (95% confidence interval, 1.05-4.54), respectively . (b) The odds ratios for other histological subtypes of lung cancer or those in total were not significant . Those for Cys/Cys or Ser/Cys genotype against Ser/Ser did not reach statistical significance in any cell type . (c) The distributions of this polymorphism varied for different populations (Chinese, Japanese, Micronesians, Melanesians, Hungarians, and Australian Caucasians), with much less prevalence of Cys allele in the latter three populations . Although our sample size was limited, these results indicate that the Ser326Cys variant may be related to squamous cell lung cancer susceptibility . The Cys/Cys genotype appears to be more susceptible to squamous cell carcinoma, although the risk is less than that previously reported to be associated with the CYP1A1 gene . Further studies are needed to assess the importance of the interpopulation variation to cancer susceptibility.
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