Escherichia coli

Hum Mol Genet, 2000 Jun 12, 9(10), 1501 - 13
Human adenylosuccinate lyase (ADSL), cloning and characterization of full-length cDNA and its isoform, gene structure and molecular basis for ADSL deficiency in six patients; Kmoch S et al.; Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling . ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits . Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity . We studied the disorder at various levels in a group of six patients with ADSL deficiency . We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping . Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant . Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level . The results showed only the unspliced ADSL to be active . The gene consists of 13 exons spanning 23 kb . The promotor region shows typical features of the housekeeping gene . Eight mutations were identified in a group of six patients . The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype . All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates . However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients . This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.

J Agric Food Chem, 2000 Jun, 48(6), 2614 - 24
Derivation and properties of recombinant Fab antibodies to coplanar polychlorinated biphenyls; Chiu YW et al.; Recombinant Fab antibodies (rFabs) specific for coplanar polychlorinated biphenyls (PCBs) were derived from a hybridoma cell line (Chiu et al . Anal . Chem . 1995, 67, 3829-3839) . Immunoglobulin V(H)-C(H1) and V(L)-C(L) sequences from S2B1 messenger RNA were amplified by PCR and cloned into the M13 phagemid vector pComb3H . Phage displaying rFab were enriched by panning on a PCB hapten conjugate and expressed as soluble rFabs in Escherichia coli XL-1 Blue . Two rFab clones competitively bound PCBs 77 and 126 with half-maximal inhibition (I(50)) of 10-13 ppb in indirect and direct enzyme immunoassays (EIAs), with selectivity nearly identical to that of whole S2B1 IgG and its Fab fragments prepared by papain digestion . These results, and comparison of N-terminal amino acid sequences of MAb S2B1 and the rFab, indicated that rFab S2B1 is a functional copy of the MAb . The rFab S2B1 sequences have 75-89% sequence identity with antibodies that bind nitrophenyl haptens and are being used to construct a three-dimensional computational model of the PCB binding site.

J Agric Food Chem, 2000 Jun, 48(6), 2602 - 7
Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification; Chen GH et al.; A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host . An active soluble form of cystatin was expressed in the cytoplasm of E . coli induced by isopropyl beta-D-thiogalactopyranoside . The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography . The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.

J Agric Food Chem, 2000 Jun, 48(6), 2092 - 6
Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio); Ken CF et al.; A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA . Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da . The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%) . The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species . Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS . A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.

J Agric Food Chem, 2000 Jun, 48(6), 2087 - 91
Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase; Vega-Warner AV et al.; Polyclonal antibodies (PAb) prepared against bovine milk alkaline phosphatase (ALP) were used to develop a competitive indirect (CI) ELISA . Anti-ALP PAb were specific for milk ALP and did not react with ALP from E . coli or bovine and calf intestinal mucosa . Anti-ALP PAb were 20% cross-reactive with bovine placenta ALP . The anti-ALP antibodies also did not recognize bovine serum albumin, acid glycoprotein, ovalbumin, ferritin, and casein, although some cross-reactivity was observed with whey protein isolate . Anti-ALP PAbs reacted with deglycosylated native ALP, but did not recognize ALP denatured at 100 degrees C in 2% SDS or deglycosylated denatured ALP . When buffered solutions of milk ALP were heated at 70 degrees C, ALP activity decreased at a faster rate than ALP content determined by CI-ELISA . The ELISA differentiated between native and heat denatured ALP . Further studies are warranted to determine if an ELISA can be used to verify pasteurization of fluid milk.

Yi Chuan Xue Bao, 2000, 27(2), 176 - 82
{Site-directed mutagenesis of melittin gene and its expression in Escherichia coli}; Wang GL et al.; The cDNA encoding promelittin was obtained from the total RNA of bee poison gland by RT-PCR . Moreover, hydroxylamine clearage site was arranged before the melittin sequences by site-directed mutagenesis . The expression vector containing the mutagenic promelittin sequence with partial sequence of beta-galactosidase was constructed . The result of DNA sequence analysis demonstrated that the obtained cDNA sequence include the desired codon and the reading frame of fusion gene was correct . The induced protein was expressed in Escherichia coli.

Biochemistry (Mosc), 2000 Jun, 65(6), 690 - 5
A role of iron ions in the SOS DNA repair response induced by nitric oxide in Escherichia coli; Stupakova MV et al.; An induction of the SOS DNA repair response by physiological nitric oxide donors (dinitrosyl iron complexes (DNIC) with thiols and S-nitrosothiols (RSNO)) was studied in E . coli cells . DNIC with thiols were the most effective SOS-inducers . Being more toxic, RSNO mediated a similar response at 10-100 microM, but they were inactive at concentrations above 0.5 mM . Pretreatment of the cells with chelating agents, o-phenanthroline and picolinic acid, prevented induction of the SOS response by all NO-donors used and led to a decrease in the DNIC-type EPR signal that appeared after incubation of the cells with DNIC or S-nitrosoglutathione (GSNO) . Analysis of these effects revealed a dual role of iron ions in reactivity and toxicity of the NO-donating agents . On one hand, they could stabilize GSNO in the form of less toxic DNIC, and, on the other hand, they took part in the formation of the SOS-inducing signal by NO-donating agents.

J Biol Chem, 2000 Jul 14, 275(28), 21668 - 77
Functional interaction between the HIV transactivator Tat and the transcriptional coactivator PC4 in T cells; Holloway AF et al.; The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication . Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators . Here we identify the cellular coactivator PC4 as a Tat-interacting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation . We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tat-dependent manner . The domains of PC4 and Tat required for the interaction were mapped . In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4 . The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat . Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro . We propose that PC4 may be involved in linking Tat to the basal transcription machinery.

J Biol Chem, 2000 Sep 15, 275(37), 29000 - 10
Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum; Shenai BR et al.; Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis . Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process . A principal trophozoite cysteine protease was purified by affinity chromatography . Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2 . Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity . A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme . Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment . Under physiological conditions (pH 5.5, 1 mm glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin . Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P . falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.

J Biol Chem, 2000 Oct 6, 275(40), 30826 - 32
Mutational analysis of the N-methyltransferase domain of the multifunctional enzyme enniatin synthetase; Hacker C et al.; N-Methylcyclopeptides like cyclosporins and enniatins are synthesized by multifunctional enzymes representing hybrid systems of peptide synthetases and S-adenosyl-l-methionine (AdoMet)-dependent N-methyltransferases . The latter constitute a new family of N-methyltransferases sharing high homology within procaryotes and eucaryotes . Here we describe the mutational analysis of the N-methyltransferase domain of enniatin synthetase from Fusarium scirpi to gain insight into the assembly of the AdoMet-binding site . The role of four conserved motifs (I, (2085)VLEIGTGSGMIL; II/Y, (2105)SYVGLDPS; IV, (2152)DLVVFNSVVQYFTPPEYL; and V, (2194)ATNGHFLAARA) in cofactor binding as measured by photolabeling was studied . Deletion of the first 21 N-terminal amino acid residues of the N-methyltransferase domain did not affect AdoMet binding . Further shortening close to motif I resulted in loss of binding activity . Truncation of 38 amino acids from the C terminus and also internal deletions containing motif V led to complete loss of AdoMet-binding activity . Point mutations converting the conserved Tyr(223) (corresponding to position 2106 in enniatin synthetase) in motif II/Y (close to motif I) into Val, Ala, and Ser, respectively, strongly diminished AdoMet binding, whereas conversion of this residue to Phe restored AdoMet-binding activity to approximately 70%, indicating that Tyr(223) is important for AdoMet binding and that the aromatic Tyr(223) may be crucial for AdoMet binding in N-methylpeptide synthetases.

Genes Dev, 2000 Jul 1, 14(13), 1589 - 94
Error-prone bypass of certain DNA lesions by the human DNA polymerase kappa; Ohashi E et al.; The Escherichia coli protein DinB is a newly identified error-prone DNA polymerase . Recently, a human homolog of DinB was identified and named DINB1 . We report that the DINB1 gene encodes a DNA polymerase (designated polkappa), which incorporates mismatched bases on a nondamaged template with a high frequency . Moreover, polkappa bypasses an abasic site and N-2-acetylaminofluorene (AAF)-adduct in an error-prone manner but does not bypass a cis-syn or (6-4) thymine-thymine dimer or a cisplatin-adduct . Therefore, our results implicate an important role for polkappa in the mutagenic bypass of certain types of DNA lesions.

Blood, 2000 Jul 15, 96(2), 540 - 5
Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain; van den Brink EN et al.; One of the major binding sites for factor VIII inhibitors is located within the A2 domain . In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII . The V(H) domain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10 . Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg(484)-Ile(508), a previously identified binding site for factor VIII inhibitors in the A2 domain . ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg . The V(H) domain of VK41 was encoded by germline gene segment DP-47 . A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp(712)-Ala(736) in the acidic region adjacent to the A2 domain . Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp(712)-Ala(736) for the corresponding region of heparin cofactor II was strongly reduced . In addition, substitution of Tyr(718719723) for Phe abrogated binding of VK41 to factor VIII . ScFv VK41 did not inhibit factor VIII activity . This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor . (Blood . 2000;96:540-545)

Development, 2000 Aug, 127(15), 3305 - 12
Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo; Ashe HL et al.; The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGF(&bgr;) signaling molecules, Decapentaplegic (Dpp) and Screw (Scw) . Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation (Sog) and a newly identified transcriptional repressor, Brinker (Brk), which are expressed in neurogenic regions that abut the dorsal ectoderm . Here we examine the expression of a number of Dpp target genes in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression . These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos . Brk was found to repress two target genes, tailup and pannier, that exhibit different limits of expression within the dorsal ectoderm . These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression . We also present evidence that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase.

Genes Cells, 2000 May, 5(5), 327 - 41
Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli; Hiraga S et al.; BACKGROUND: We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells . A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions . RESULTS: The present study shows that (i) appearance of SeqA foci depends on continuous DNA replication, suggesting that the SeqA foci represent clusters consisting of SeqA and newly replicated hemimethylated DNA, (ii) in a synchronous round of replication, a single SeqA focus at midcell separates into two foci and these foci abruptly migrate in opposite directions midway through replication from oriC to the terminus, and (iii) oriC is replicated at midcell but replicated oriC copies remain linked with each other at midcell for 40 min after replication at 30 degrees C . Subsequently, the linked oriC copies separate and migrate gradually towards both borders of the nucleoid before cell division . CONCLUSIONS: A single cluster of SeqA-bound hemimethylated DNA segment separates into two clusters and these clusters migrate abruptly in a bipolar fashion during progress of replication and prior to separation of linked sister oriC copies.

FEBS Lett, 2000 Jun 23, 475(3), 192 - 6
PAI-1 stability: the role of histidine residues; Mangs H et al.; The role of the 13 histidine residues in plasminogen activator inhibitor 1 (PAI-1) for the stability of the molecule was studied by replacing these residues by threonine, using site-directed mutagenesis . The generated mutants were expressed in Escherichia coli, purified and characterized . All variants had a normal activity and formed stable complexes with tissue-type plasminogen activator . Most of these PAI-1 variants displayed a similar pH-dependency in stability as wild-type PAI-1, with increased half-lives at lower pH . However, the variant His364Thr had a half-life of about 50 min at 37 degrees C and had almost completely lost its pH-dependency . Therefore, our data suggest that His(364), in the COOH-terminal end of the molecule might be responsible for the pH-dependent stability of PAI-1.

Microbios, 2000, 102(402), 103 - 12
Isolation and characterization of transposon-induced chlorate resistant mutants of the cyanobacterium Anabaena species PCC 7120; Rai AK et al.; Mutants of Anabaena sp . PCC 7120 resistant to chlorate were isolated using transposon mutagenesis . The Anabaena population of 5 x 10(7) cells ml(-1) and log phase Escherichia coli cultures in undisturbed conditions produced maximum exconjugants . Nitrate-promoted growth and cellular constituents observed in the parent were absent in the mutants . Nitrate repressed heterocyst formation and N2-fixation in the parent, but had little or no effect on the mutants.

Biophys Chem, 2000 May 31, 85(1), 59 - 78
The positive role of voids in the plasma membrane in growth and energetics of Escherichia coli; Natesan S et al.; Bacterial respiration, endogenous as well as induced respiration by glucose, lactose and glycine betaine, was found to be sensitive to external solute concentration . Permeability of hydrogen peroxide, a non-electrolyte of molecular size between water and urea, through the bacterial membranes changed directly with the rate of respiration (an activity residing in the bacterial plasma membrane) in E . coli and the enhanced permeability and respiratory activity were highly correlated . Hydrogen peroxide permeability and induction of voids (spaces in the matrix of the bilayer into which hydrophobic fluorescent probes partition, which in turn were used to assess the modulation of these cavities) were shown to be a direct and excellent measure of leak conductance . Fluorescence intensity and anisotropy of the extrinsic fluorescent probes (incorporated by growing bacteria in their presence) decreased with increased respiration in bacteria, consistent with lowered molecular restriction and enhanced hydration in the membrane phase for these probes as seen in dimyristoylphosphatidylcholine bilayers due to phase transition . The physical basis of osmotic phenomena, as a relevant (thermodynamic) volume, could relate to water exchange or compression, depending on the osmotic domain . In the domain of compression in bacteria, i.e . well above the isotonic range, the computed activation volume was consistent with voids in the membrane . This study emphasises a major role of leak conductance in bacterial physiology and growth.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7808 - 13
Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain; Zhu Y et al.; Most histidine kinases are bifunctional enzymes having both kinase and phosphatase activities . The cytoplasmic kinase domain of EnvZ, a transmembrane histidine kinase functioning as an osmosensor in Escherichia coli, consists of two distinct functional subdomains: domain A {EnvZc(223-289)} and domain B {EnvZc(290-450)} . NMR studies demonstrated that domain A consists of a four-helix bundle serving as a dimerization and phosphotransfer domain, and domain B functions as the ATP-binding and catalytic domain . Here we demonstrate that domain A by itself has the phosphatase activity both in vitro and in vivo . This phosphatase activity is Mg(2+) dependent but is not activated by ADP, ATP, or adenosine 5'-{beta, gamma-imido}triphosphate (AMPPNP), each of which may serve as a cofactor for the EnvZ phosphatase activity . Domain B showed a small but distinct effect on the domain A phosphatase activity only in the presence of ADP or AMPPNP . However, when domain B was covalently linked to domain A, dramatic cofactor-dependent enhancement of the phosphatase activity was observed . Extending domain A for another 75 residues at the C terminus or 44 residues at the N terminus did not enhance its phosphatase activity . Substitution mutations at His-243, the autophosphorylation site, demonstrate that the His residue plays an essential role in the phosphatase activity . The so-called X-region mutant L288P that is known to specifically abolish the phosphatase activity in EnvZ had no effect on the domain A phosphatase function . We propose that the EnvZ phosphatase activity is regulated by relative positioning of domains A and B, which is controlled by external signals . We also propose that the His-243 residue participates in both kinase and phosphatase reactions.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7784 - 9
Escherichia coli CspA-family RNA chaperones are transcription antiterminators; Bae W et al.; CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone, which is thought to facilitate translation at low temperature by destabilizing mRNA structures . Here we demonstrate that CspA, as well as homologous RNA chaperones CspE and CspC, are transcription antiterminators . In vitro, the addition of physiological concentrations of recombinant CspA, CspE, or CspC decreased transcription termination at several intrinsic terminators and also decreased transcription pausing . In vivo, overexpression of cloned CspC and CspE at 37 degrees C was sufficient to induce transcription of the metY-rpsO operon genes nusA, infB, rbfA, and pnp located downstream of multiple transcription terminators . Similar induction of downstream metY-rpsO operon genes was observed at cold shock, a condition to which the cell responds by massive overproduction of CspA . The products of nusA, infB, rbfA, and pnp-NusA, IF2, RbfA, and PNP-are known to be induced at cold shock . We propose that the cold-shock induction of nusA, infB, rbfA, and pnp occurs through transcription antitermination, which is mediated by CspA and other cold shock-induced Csp proteins.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7721 - 6
The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity; Deneke J et al.; Escherichia coli phage N15 encodes the slightly acidic, 630-residue protein of 72.2 kDa called protelomerase (TelN) . TelN is a component of the N15 replication system proposed to be involved in the generation of the linear prophage DNA . This linear DNA molecule has covalently closed ends . The reaction converting circular plasmids into linear molecules was catalyzed in vitro . We demonstrate that the product of telN functions as the protelomerase in the absence of other N15-encoded factors . Purified TelN processes circular and linear plasmid DNA containing the proposed target site telRL to produce linear double-stranded DNA with covalently closed ends . The 56-bp telRL target site consists of a central telO palindrome of 22 bp and two 14-bp flanking sequences comprising inverted repeats . telO is separated from these repeats by 3 bp on each side . The telRL sequence is sufficient for TelN-mediated processing . The ends of the DNA molecules generated in vitro have the same configuration as do those observed in vivo . TelN exerts its activity as cleaving-joining enzyme in a concerted action.

J Biol Chem, 2000 Oct 20, 275(42), 33021 - 6
Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase; Reichard P et al.; We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the "activity site" (D57N) of mouse ribonucleotide reductase . Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity . Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site . In all these respects, mouse R1 resembles Escherichia coli R1 . Results with D57N were complicated by the instability of the protein, but two major changes were apparent . First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides . Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1 . Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.

J Biol Chem, 2000 Sep 29, 275(39), 30088 - 91
Covalent heterogeneity of the human enzyme galactose-1-phosphate uridylyltransferase; Henderson JM et al.; Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism . Impairment of this enzyme results in the potentially lethal disorder, galactosemia . Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure . We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast . In both contexts, two predominant GALT species were observed . To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT . The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate . Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern . In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern . Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.

J Biol Chem, 2000 Oct 6, 275(40), 31178 - 82
Characterization of the interaction of calcyclin (S100A6) and calcyclin-binding protein; Nowotny M et al.; Calcyclin (S100A6) is an S100 calcium-binding protein whose expression is up-regulated in proliferating and differentiating cells . A novel 30-kDa protein exhibiting calcium-dependent calcyclin-binding (calcyclin-binding protein, CacyBP) had been identified, purified, and cloned previously (Filipek, A., and Kuznicki, J . (1998) J . Neurochem . 70, 1793-1798) . Here, we have defined the calcyclin binding region using limited proteolysis and a set of deletion mutants of CacyBP . A fragment encompassing residues 178-229 (CacyBP-(178-229)) was capable of full binding to calcyclin . CacyBP-(178-229) was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified . The protein fragment cleaved from the glutathione S-transferase fusion protein was shown by CD to contain 5% alpha-helix, 15% beta -sheet, and 81% random coil . Fluorescence spectroscopy was used to determine calcyclin dissociation constants of 0.96 and 1.2 microm for intact CacyBP and CacyBP-(178-229), respectively, indicating that the fragment can be used for characterization of calcyclin-CacyBP interactions . NMR analysis of CacyBP-(178-229) binding-induced changes in the chemical shifts of (15)N-enriched calcyclin revealed that CacyBP binding occurs at a discrete site on calcyclin with micromolar affinity.

J Mol Biol, 2000 Jul 14, 300(3), 469 - 79
A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi; Arnold DA et al.; In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination . However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type . In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3') . Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi . This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi . Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition . In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme .

J Lipid Res, 2000 Jul, 41(7), 1087 - 95
Apolipoprotein E;-low density lipoprotein receptor interaction . Influences of basic residue and amphipathic alpha-helix organization in the ligand; Zaiou M et al.; Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR) . To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191) . Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions . Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value . This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR . Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

J Arthroplasty, 2000 Jun, 15(4), 430 - 6
Infected total knee arthroplasty treated by arthroscopic irrigation and débridement; Waldman BJ et al.; Sixteen patients with infected total knee arthroplasties (4 postoperative and 12 late hematogenous) were treated by arthroscopic irrigation and debridement . All patients had < or = 7 days of knee symptoms, and there were no radiographic signs of osteitis or prosthetic loosening . Six of the 16 original total knee arthroplasties (38%) did not need prosthesis removal at a mean follow-up of 64 months (range, 36-151 months) . Ten other knees were treated with irrigation, debridement, and hardware removal within 7 weeks of the latest procedure used to try to retain components . Two (13%) of these cases ultimately required an arthrodesis for persistent infection . Although we still believe that this method is preferable to resorting immediately to implant removal for acute infections, arthroscopic debridement was less efficacious for most situations when compared with open treatment . We would use arthroscopic irrigation and debridement only under selected circumstances (medically unstable or anticoagulated patients).

J Endovasc Ther, 2000 Jun, 7(3), 192 - 7
Repair of mycotic paravisceral aneurysm with a fenestrated stent-graft; Kinney EV et al.; PURPOSE: To report the successful endovascular repair of a mycotic paravisceral aneurysm using a fenestrated stent-graft . METHODS AND RESULTS: A 55-year-old white female with a history of rheumatoid arthritis presented with an 8-cm paravisceral aneurysm secondary to pneumonia complicated by empyema . Intravascular ultrasound identified a defect in the aortic wall at the level of the celiac axis . Repair was accomplished with a fenestrated stent-graft that excluded the aneurysm and maintained flow to the celiac axis and superior mesenteric artery . Recovery was uneventful and the patient was discharged in 2 days . Six-month follow-up computed tomographic scanning confirmed aneurysm exclusion and flow to the celiac and superior mesenteric arteries . There was no evidence of graft infection . The patient died from a clinically diagnosed myocardial infarction 10 months after the stent-graft repair . CONCLUSIONS: Fenestrated stent-graft repair may evolve into a useful technique for the treatment of mycotic paravisceral aneurysms.

Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 86 - 90
{Construction and expression of a hepatocellular carcinoma specific rodent and its humanized single-chain Fv fragments in Escherichia coli}; Yuan QA et al.; The aim of this research is to exploit the expression strategies for two genes encoding a rodent and its humanized version single-chain fragments(scFv) specific for hepatocellular carcinoma (HCC) and compare these two scFves in antigen binding activity . Three vectors were used to express these two genes in different routes of fusion pathway, secrete pathway and intracellular pathway . The refolded single-chain antibodies were examined by antigen capture ELISA . Results showed that inclusion bodies were produced in all of the applied vectors despite of variation of IPTG concentration and culture temperature . Cell ELISA and binding competition inhibition indicated that the humanized single-chain Fv retained the similar affinity as its rodent counterpart . These results implied that the solubility of genetic antibodies are determined primarily by its amino acids sequence; The adopted humanization strategy in previous design made little effect on the natural conformation of complementarity-determining regions(CDR) of the parent antibody . The humanized HCC specific scFv can be further evaluated and developed as therapeutics.

Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 82 - 5
{Construction and expression of anticolonic cancer scFv fragment}; Song LX et al.; Anticolonic cancer scFv fragment with a VH-linker-VL structure was constructed and expressed in E . coli . SDS-PAGE analysis showed the fragment cloned in pComb3 was not expressed efficiently in JM83, while cloned in pET-22b(+) highly expressed in BL21(DE3) up to 35.5% of the total bacterial protein obtained, if the culture was incubated under 30 degrees C.

Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 42 - 5
{Cloning and expression of the detoxifying gene in cyanobacteria}; Yan YC et al.; Plasmid pRL-B1 was constructed from detoxifying gene(called B1) of pesticide resistant Culex and from plasmid pRL-439 containing the strong promoter PpsbA . E . coli-cyanobacteria shuttle expression plasmid pDC-B1 was constructed from shuttle vector pDC-8 and from recombinant plasmid pRL-B1, then it was transferred into Synechococcus sp . PCC7942 by triparental conjugative transfer . The existence of B1 was detected by Southern analysis, and the expression of B1 was confirmed by enzyme activity analysis of detoxification of transgenic cyanobacteria . Experimental results indicated that the transgenic cyanobacteria could degrade beta-naphthyl acetate(beta-NA), a specific substrate of esterase . The enzyme activity of transgenic strain was higher than that of the wild type . It may be the first report on transformation of detoxify gene of pesticide resistant culex into Synechococcus strain.

Mutat Res, 2000 Jul 10, 468(2), 173 - 82
Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells; Murata-Kamiya N et al.; We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains . To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189 . We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells . The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal . In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites . Among them, G:C-->C:G and G:C-->T:A transversions were predominant . The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations . These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.

Mutat Res, 2000 Jul 25, 460(2), 117 - 25
The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZalpha gene; Kuipers GK et al.; Base excision repair (BER) is a very important repair mechanism to remove oxidative DNA damage . A major oxidative DNA damage after exposure to ionizing radiation is 7,8-dihydro-8-oxoguanine (8oxoG) . 8oxoG is a strong mutagenic lesion, which may cause G:C to T:A transversions if not repaired correctly . Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG . In the past years, evidence evolved that nucleotide excision repair (NER), a repair system originally thought to repair only bulky DNA lesions, can also repair some oxidative DNA damages . Examples of DNA damages which are recognized by NER are thymine glycol and abasic sites (AP sites) . The main objective of this study is to determine if NER can act as a backup system for the repair of spontaneous and gamma-radiation-induced damages when Fpg is deficient . For that purpose, the effect of a NER-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene was determined, using double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target sequence . Subsequently the DNA was transfected into a fpg(-)uvrA(-) Escherichia coli strain (BH420) and the mutational spectra were compared with the spectra of a fpg(-) E . coli strain (BH410) and a wild type E . coli strain (JM105), which were determined in an earlier study . Furthermore, to examine effects which are caused by UvrA-deficiency, and not by Fpg-deficiency, the spontaneous and gamma-radiation-induced mutation spectra of an E . coli strain in which only UvrA is deficient (BH430) were also determined and compared with a wild type E . coli strain (JM105) . The results of this study indicate that if only UvrA is deficient, there is an increase in spontaneous G:C to T:A transversions as compared to JM105 and a decrease in A:T to G:C transitions . The gamma-radiation-induced mutation spectrum of BH420 (fpg(-)uvrA(-)) shows a significant decrease in G:C to A:T and G:C to T:A mutations, as compared to BH410 where only Fpg is deficient . Based on these results, we conclude that in our experiments NER is not acting as a backup system if Fpg is deficient . Instead, NER seems to make mistakes, leading to the formation of mutations.

Mutat Res, 2000 Jul 25, 460(2), 95 - 104
Cell-cycle regulation, intracellular sorting and induced overexpression of the human NTH1 DNA glycosylase involved in removal of formamidopyrimidine residues from DNA; Luna L et al.; Endonuclease III (Nth) of Escherichia coli is a DNA glycosylase essential for the removal of oxidised pyrimidine base residues from DNA . Several eukaryotic homologues have recently been identified and shown to have biochemical properties similar to those of Nth . However, some of the eukaryotic counterparts also appear to remove imidazole ring-opened purine residues (faPy), a property not shared by the enzymes of bacterial origin . Here, we show that the human enzyme also possesses efficient faPy DNA glycosylase activity as indicated both from studies of the purified protein and induced overexpression of the human NTH1 cDNA in HeLa cells . We constructed green fluorescent protein-tagged hNTH1 fusion proteins to study the cellular localisation of hNTH1 and found strong and exclusive sorting to the nucleus . Studies with synchronised cells showed that the expression of hNTH1 is regulated during the cell cycle with increased transcription during early and mid S-phase.

J Biol Chem, 2000 Oct 6, 275(40), 30817 - 25
Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin; Harbeck B et al.; The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells . It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells . The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions . Hence, phosphorylation may significantly alter the actin binding properties of VASP . This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays . cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling . Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions . In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin . When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers . Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.

J Biol Chem, 2000 Sep 22, 275(38), 29840 - 6
Mechanism of I kappa B alpha binding to NF-kappa B dimers; Phelps CB et al.; X-ray crystal structures of the NF-kappa B.I kappa B alpha complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both I kappa B alpha and NF-kappa B . In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity . I kappa B alpha preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker . Our results indicate that the nuclear localization sequence and the region C-terminal to it of the NF-kappa B p65 subunit is a major contributor to NF-kappa B . I kappa B alpha complex formation . Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and I kappa B alpha . A second set of interactions involving the acidic C-terminal/PEST-like region of I kappa B alpha and the NF-kappa B p65 subunit N-terminal domain also contributes binding energy toward formation of the complex . This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking . Phosphorylation of the C-terminal/PEST-like region by casein kinase II further enhances binding.

J Biol Chem, 2000 Nov 10, 275(45), 35006 - 12
Influence of HMG-1 and adenovirus oncoprotein E1A on early stages of transcriptional preinitiation complex assembly; Lu W et al.; The TATA-binding protein (TBP) in the TFIID complex binds specifically to the TATA-box to initiate the stepwise assembly of the preinitiation complex (PIC) for RNA polymerase II transcription . Transcriptional activators and repressors compete with general transcription factors at each step to influence the course of the assembly . To investigate this process, the TBP.TATA complex was titrated with HMG-1 and the interaction monitored by electrophoretic mobility shift assays . The titration produced a ternary HMG-1.TBP . TATA complex, which exhibits increased mobility relative to the TBP . TATA complex . The addition of increasing levels of TFIIB to this complex results in the formation of the TFIIB.TBP.TATA complex . However, in the reverse titration, with very high mole ratios of HMG-1 present, TFIIB is not dissociated off and a complex is formed that contains all factors . The simultaneous addition of E1A to a mixture of TBP and TATA; or HMG-1, TBP, and TATA; or TFIIB, TBP, and TATA inhibits complex formation . On the other hand, E1A added to the pre-established complexes shows a significantly reduced capability to disrupt the complex . In add-back experiments with all complexes, increased levels of TBP re-established the complexes, indicating that the primary target for E1A in all complexes is TBP.

J Biol Chem, 2000 Sep 22, 275(38), 29618 - 22
The replacement of ATP by the competitive inhibitor emodin induces conformational modifications in the catalytic site of protein kinase CK2; Battistutta R et al.; The structure of a complex between the catalytic subunit of Zea mays CK2 and the nucleotide binding site-directed inhibitor emodin (3-methyl-1,6,8-trihydroxyanthraquinone) was solved at 2.6-A resolution . Emodin enters the nucleotide binding site of the enzyme, filling a hydrophobic pocket between the N-terminal and the C-terminal lobes, in the proximity of the site occupied by the base rings of the natural co-substrates . The interactions between the inhibitor and CK2 alpha are mainly hydrophobic . Although the C-terminal domain of the enzyme is essentially identical to the ATP-bound form, the beta-sheet in the N-terminal domain is altered by the presence of emodin . The structural data presented here highlight the flexibility of the kinase domain structure and provide information for the design of selective ATP competitive inhibitors of protein kinase CK2.

J Biol Chem, 2000 Oct 6, 275(40), 31340 - 6
Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme; Jones PC et al.; The transmembrane sector of the F(0)F(1) rotary ATP synthase is proposed to organize with an oligomeric ring of c subunits, which function as a rotor, interacting with two b subunits at the periphery of the ring, the b subunits functioning as a stator . In this study, cysteines were introduced into the C-terminal region of subunit c and the N-terminal region of subunit b . Cys of N2C subunit b was cross-linked with Cys at positions 74, 75, and 78 of subunit c . In each case, a maximum of 50% of the b subunit could be cross-linked to subunit c, which suggests that either only one of the two b subunits lie adjacent to the c-ring or that both b subunits interact with a single subunit c . The results support a topological arrangement of these subunits, in which the respective N- and C-terminal ends of subunits b and c extend to the periplasmic surface of the membrane and cAsp-61 lies at the center of the membrane . The cross-linking of Cys between bN2C and cV78C was shown to inhibit ATP-driven proton pumping, as would be predicted from a rotary model for ATP synthase function, but unexpectedly, cross-linking did not lead to inhibition of ATPase activity . ATP hydrolysis and proton pumping are therefore uncoupled in the cross-linked enzyme . The c subunit lying adjacent to subunit b was shown to be mobile and to exchange with c subunits that initially occupied non-neighboring positions . The movement or exchange of subunits at the position adjacent to subunit b was blocked by dicyclohexylcarbodiimide . These experiments provide a biochemical verification that the oligomeric c-ring can move with respect to the b-stator and provide further support for a rotary catalytic mechanism in the ATP synthase.

J Biol Chem, 2000 Sep 22, 275(38), 29800 - 7
The Brf and TATA-binding protein subunits of the RNA polymerase III transcription factor IIIB mediate position-specific integration of the gypsy-like element, Ty3; Yieh L et al.; Ty3 integrates into the transcription initiation sites of genes transcribed by RNA polymerase III . It is known that transcription factors (TF) IIIB and IIIC are important for recruiting Ty3 to its sites of integration upstream of tRNA genes, but that RNA polymerase III is not required . In order to investigate the respective roles of TFIIIB and TFIIIC, we have developed an in vitro integration assay in which Ty3 is targeted to the U6 small nuclear RNA gene, SNR6 . Because TFIIIB can bind to the TATA box upstream of the U6 gene through contacts mediated by TATA-binding protein (TBP), TFIIIC is dispensable for in vitro transcription . Thus, this system offers an opportunity to test the role of TFIIIB independent of a requirement of TFIIIC . We demonstrate that the recombinant Brf and TBP subunits of TFIIIB, which interact over the SNR6 TATA box, direct integration at the SNR6 transcription initiation site in the absence of detectable TFIIIC or TFIIIB subunit B" . These findings suggest that the minimal requirements for pol III transcription and Ty3 integration are very similar.

J Biol Chem, 2000 Nov 3, 275(44), 34046 - 53
Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A); Norlin M et al.; A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids . According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction . CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone . In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated . Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol . This enzyme has previously been thought to be specific for cholesterol and cholestanol . A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A . The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A . From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B . These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis . A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.

Clin Diagn Lab Immunol, 2000 Jul, 7(4), 662 - 8
Serodiagnostic potential of culture filtrate antigens of Mycobacterium tuberculosis; Samanich KM et al.; Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K . Samanich et al., J . Infect . Dis . 178:1534-1538, 1998) . We have identified several antigens with strong serodiagnostic potential . In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A . We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts . The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S . Laal et al., Clin . Diag . Lab . Immunol . 4:49-56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies . Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E . coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals . Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies.

Clin Diagn Lab Immunol, 2000 Jul, 7(4), 652 - 7
Comparison of two recombinant major outer membrane proteins of the human granulocytic ehrlichiosis agent for use in an enzyme-linked immunosorbent assay; Tajima T et al.; Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated . Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA . Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls . One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA) . All IFA-negative sera were negative by both ELISAs . Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive . Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive . There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector . There was a significant positive correlation between IFA titers and optical densities of ELISAs . Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA . However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein . Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.

Clin Diagn Lab Immunol, 2000 Jul, 7(4), 607 - 11
Enzyme-linked immunosorbent assays using the recombinant dense granule antigens GRA6 and GRA1 of Toxoplasma gondii for detection of immunoglobulin G antibodies; Lecordier L et al.; The potential of the dense granule antigens GRA1 and GRA6 of Toxoplasma gondii to be used as diagnosis reagents in a recombinant form was evaluated . Both proteins were expressed in Escherichia coli as glutathione-S-transferase (GST) fusions . The GST-GRA1 fusion comprises the entire GRA1 sequence devoid of its N-terminal signal peptide . Separate expression of the two N- and C-terminal hydrophilic regions of GRA6 showed that only the N-terminal hydrophilic part of the protein was recognized by a pool of positive human sera in an immunoblot . One hundred T . gondii-positive and 98 negative human sera were tested in two separate immunoglobulin G (IgG)-direct enzyme-linked immunosorbent assays (ELISAs) using either GST-GRA1 or GST-GRA6-Nt recombinant protein . Whereas the sensitivity of the GST-GRA1 IgG ELISA was low (68%), the GST-GRA6-Nt IgG ELISA reached a sensitivity of 96% . The reactivity to GRA6-Nt was shown to be high even with human sera of low IgG titers . In addition, comparison of the optical density values for each serum revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98% . Therefore, the GST-GRA6-Nt ELISA could be used together with another antigen like GRA1 for the development of a recombinant antigen-based test for serodiagnosis of toxoplasmosis.

Clin Diagn Lab Immunol, 2000 Jul, 7(4), 557 - 62
Cloning of the bovine immunodeficiency virus gag gene and development of a recombinant-protein-based enzyme-linked immunosorbent assay; Zheng L et al.; An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen . The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli . The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein . A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies . Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests . The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively . ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.

J Infect Dis, 2000 Jul, 182(1), 180 - 90 Epub 2000 Jun 30.
Enterotoxin adjuvants have direct effects on T cells and antigen-presenting cells that result in either interleukin-4-dependent or -independent immune responses; Yamamoto M et al.; In an in vitro study, Escherichia coli heat-labile toxin (LT) was shown to directly affect activated CD4(+) T cells and support interleukin (IL)-5 production in IL-4-deficient (IL-4(-/-)) mice, whereas cholera toxin (CT) did not . Both LT and CT enhanced B7-2 expression on B cells and macrophages . These effects were not influenced by CD40-CD40 ligand cosignaling . Addition of LT- or CT-treated antigen-presenting cells to anti-CD3-triggered CD4(+) T cells resulted in the induction of T cell proliferative responses . Further, these responses were inhibited by anti-B7-2 monoclonal antibody . Cocultivation of CD4(+) T cells with LT- or CT-treated antigen-presenting cells and anti-CD3 enhanced Th1- and IL-4-mediated Th2-type cytokine production . The results from in vitro studies were supported by in vivo studies in IL-4(-/-) mice, in which LT induced mucosal IgA responses but CT did not . Thus, although both LT and CT induce mucosal adjuvant responses via IL-4-dependent Th2-type responses, LT also elicits Th1- and IL-4-independent Th2-type responses.

J Protein Chem, 2000 Jan, 19(1), 59 - 66
A comparative study of human muscle and brain creatine kinases expressed in Escherichia coli; Chen LH et al.; We report the expression of the human muscle (CK-MM) and brain (CK-BB) creatine kinases in Escherichia coli . The proteins have been purified to apparent homogeneity and several of their physical and kinetic properties investigated . In the process, we have conclusively verified the correct DNA sequence of the genes encoding the respective isozymes, and determined the correct primary structure and mass of the gene products . Alignment of the primary sequences of these two enzymes shows 81% sequence identity with each other, and no obvious gross structural differences . However, Western blot analyses demonstrated the general lack of antigenic cross-reactivity between these isozymes . Preliminary kinetic analyses show the K(m) and k(cat) values for the creatine and MgATP substrates are similar to values reported for other isozymes from various tissues and organisms . The human muscle and brain CKs do not, however, exhibit the synergism of substrate binding that is observed, for example, in rabbit muscle creatine kinase.

Mol Cell, 2000 Mar, 5(3), 557 - 67
Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16; Chen XS et al.; The papillomavirus major late protein, L1, forms the pentameric assembly unit of the viral shell . Recombinant HPV16 L1 pentamers assemble in vitro into capsid-like structures, and truncation of ten N-terminal residues leads to a homogeneous preparation of 12-pentamer, icosahedral particles . X-ray crystallographic analysis of these particles at 3.5 A resolution shows that L1 closely resembles VP1 from polyomaviruses . Surface loops contain the sites of sequence variation among HPV types and the locations of dominant neutralizing epitopes . The ease with which small virus-like particles may be obtained from L1 expressed in E . coli makes them attractive candidate components of a papillomavirus vaccine . Their crystal structure also provides a starting point for future vaccine design.

Mol Cell, 2000 Apr, 5(4), 639 - 48
Dynamics of substrate denaturation and translocation by the ClpXP degradation machine; Kim YI et al.; ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase . Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag . ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate . Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation . These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.

J Med Microbiol, 2000 Jul, 49(7), 657 - 67
Molecular cloning of a gene (poIA) coding for an unusual DNA polymerase I from Treponema pallidum; Rodes B et al.; The gene coding for the DNA polymerase I from Treponema pallidum, Nichols strain, was cloned and sequenced . Depending on which of the two alternative initiation codons was used, the protein was either 997 or 1015 amino acids long and the predicted protein had a molecular mass of either 112 or 114 kDa . Sequence comparisons with other polA genes showed that all three domains expected in the DNA polymerase I class of enzymes were present in the protein (5'-3' exonuclease, 3'-5' exonuclease and polymerase domains) . Additionally, there were four unique insertions of 20-30 amino acids each, not seen in other DNA polymerase I enzymes . Two of the inserts were near the boundary of the two exonuclease domains and the other two interrupted the 3'-5' exonuclease domain which is involved in proofreading . The predicted amino-acid sequence had an exceptionally high content of cysteine (2.4% compared with <0.05% for most other sequenced DNA polymerase I enzymes) . The polA gene was further cloned into pProEXHTa for expression and purification . The transformants expressed a protein of 115 kDa . Antibodies raised against synthetic peptide fragments of the putative DNA polymerase I recognised the 115-kda band in Western blot analysis . No DNA synthesis activity could be demonstrated on a primed single-stranded template . Although significant quantities of the protein were produced in the host Escherichia coli carrying the plasmid, it was not capable of complementing a polA(-) mutant in the replication of a polA-dependent plasmid.

J Med Microbiol, 2000 Jul, 49(7), 643 - 50
Identification of an immunogenic 18-kDa protein of Helicobacter pylori by alkaline phosphatase gene fusions; Oliaro J et al.; The use of alkaline phosphatase fusion methodology to identify Helicobacter pylori exported proteins enabled the identification of an open reading frame (ORF) encoding a highly immunogenic, previously uncharacterised exported protein . The predicted aminoacid sequence displays a typical N-terminal signal peptide and contains regions of C-terminal hydrophobicity consistent with a membrane-associated protein . Southern blot analysis revealed that the gene encoding the protein was absent in several Helicobacter spp . and a combination of PCR and sequence analysis of the amplified gene showed that it is highly conserved amongst isolates of H . pylori . To obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a beta-galactosidase (beta-gal) fusion in Escherichia coli and the protein was purified by affinity chromatography and proteolytic cleavage of the beta-gal portion . The purified protein, which has an apparent mol . wt of 18 kDa, was recognised by antibody present in 71% of sera from patients infected with H . pylori, but in only 16% of sera from patients with unrelated or no gastrointestinal disease, by Western blot assays . These results indicate that the 18-kDa protein from H . pylori is immunogenic and is expressed in vivo.

Arch Virol, 2000, 145(5), 1009 - 19
Molecular evidence that sugarcane yellow leaf virus (ScYLV) is a member of the Luteoviridae family; Maia IG et al.; A previously uncharacterized virus was reported in southeast Brazil causing a yellowing leaf disease in sugarcane . The virus, termed sugarcane yellow leaf virus (ScYLV), shares features typical of the luteoviruses . To start the molecular characterization of ScYLV, the nucleotide sequence of the coat protein (CP), 17 kDa protein and C-terminus of the RNA-dependent RNA polymerase coding regions was determined from an RT-PCR amplification product . Comparisons showed that the deduced amino acid sequences share a considerable degree of identity and similarity with corresponding sequences of known luteoviruses, thus clearly establishing ScYLV as a member of the family Luteoviridae . The authenticity of the CP open reading frame was confirmed by its expression in Escherichia coli . The recombinant CP positively reacted in immunoblot assays with polyclonal antibodies raised against native ScYLV . Furthermore, phylogenetic analyses also suggest that the 5' and 3' coding blocks of the ScYLV genome possess different taxonomic affinities within the Luteoviridae family, as does also the genome of soybean dwarf virus.

Nat Struct Biol, 2000 Jun, 7(6), 497 - 504
Tertiary core rearrangements in a tight binding transfer RNA aptamer; Bullock TL et al.; Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3' . This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type . The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type . The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases . These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.

Nat Struct Biol, 2000 Jun, 7(6), 479 - 81
Observing conformational and activity changes of tet repressor in vivo; Tiebel B et al.; Effector triggered conformational changes of proteins such as regulators of transcription, receptors, or enzymes are the molecular basis for regulation in biology . Most proteins perform their biological functions intracellularly, in the presence of many potential interaction partners . Studies of conformational changes have mainly been performed in vitro using sophisticated physical and biochemical methods that usually require purified proteins . Here we describe the observation of conformational changes of Tet repressor in the cytoplasm of growing Escherichia coli cells, analyzed by ligand dependent disulfide crosslinking of cysteine residues substituted into mobile regions of the protein . The amount of protein undergoing the structural change is quantitatively linked to the concomitant induction of transcription of a reporter gene.

Nat Struct Biol, 2000 Jun, 7(6), 470 - 4
The structure of the transcriptional antiterminator NusB from Escherichia coli; Altieri AS et al.; We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli . The structure reveals a novel, all alpha-helical protein fold . NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions . Residues that are highly conserved among homologs stabilize the protein core . The solution structure of E . coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E . coli NusB reported earlier.

Nat Struct Biol, 2000 Jun, 7(6), 461 - 5
Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase; Sankaranarayanan R et al.; Accurate translation of the genetic code depends on the ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids . In order to investigate the basis of amino acid recognition and to understand the role played by the zinc ion present in the active site of threonyl-tRNA synthetase, we have determined the crystal structures of complexes of an active truncated form of the enzyme with a threonyl adenylate analog or threonine . The zinc ion is directly involved in threonine recognition, forming a pentacoordinate intermediate with both the amino group and the side chain hydroxyl . Amino acid activation experiments reveal that the enzyme shows no activation of isosteric valine, and activates serine at a rate 1,000-fold less than that of cognate threonine . This study demonstrates that the zinc ion is neither strictly catalytic nor structural and suggests how the zinc ion ensures that only amino acids that possess a hydroxyl group attached to the beta-position are activated.

Braz J Med Biol Res, 2000 Jul, 33(7), 771 - 8
Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-alpha receptor; Yoon S et al.; Abnormal production of interferon alpha (IFN-alpha) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-alpha . IFN-alpha can contribute to the pathogenesis of allograft rejection in bone marrow transplants . Therefore, the development of IFN-alpha inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases . We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-alpha receptor (IFNAR 1-EC) in E . coli . The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays . Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E . coli BL21(DE3)pLysS and by IPTG induction . P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis . Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively . Polyclonal anti-P1 and anti-P2 antibodies were produced in mice . P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-alphaB on Daudi cells, but did not block IFN-alphaB activity at higher concentrations (>6 . 25 nM) . On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-alphaB on Hep 2/c cells challenged with encephalomyocarditis virus.

Eur J Biochem, 2000 Jul, 267(14), 4456 - 64
Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KIS; Maucuer A et al.; We present here a first appraisal of the phosphorylation site specificity of KIS (for 'kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM) . In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested . Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS . Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1 . We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif . Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks.

Eur J Biochem, 2000 Jul, 267(14), 4434 - 44
Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN; Geoghegan SM et al.; The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction . The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized . With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin . A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN . The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm . The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV . A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to < or = 4.3 . The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and < or = 5.2, respectively) . The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form . These results suggest that glutamate 60 plays a major role in control of the redox properties of M . elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone.

Eur J Biochem, 2000 Jul, 267(14), 4381 - 9
Physical characterization of plakophilin 1 reconstituted with and without zinc; Hofmann I et al.; Plakophilin 1 (PKP1) belongs to the arm-repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif . Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions . PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes . In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium . Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc . The alpha helix content of PKP1 was considerably higher when reconstituted with zinc than without . By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1 . The zinc-reconstituted protein formed globular particles of 21.9 +/- 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing . In parallel, the average sedimentation coefficient (s20, w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 x 10-7 cm2.s-1 . The molecular mass of 2.44 x 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer . In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation.

Eur J Biochem, 2000 Jul, 267(14), 4355 - 61
Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8 and Mal f 9; Rasool O et al.; Malassezia furfur, formerly known as Pityrosporum orbiculare or P . ovale, is a yeast that colonizes human skin . Normally, this yeast is nonpathogenic but under the influence of predisposing factors it may induce IgE reactivity in patients with atopic dermatitis . Approximately 40-65% of atopic dermatitis patients have IgE antibodies and/or skin reactivity against M . furfur and a higher T-cell response against this yeast is found in atopic dermatitis patients than in healthy individuals . By making a cDNA library displayed on a phage surface, we previously cloned five different IgE-binding proteins, Mal f 5, Mal f 6, MF 7, MF 8 and MF 9, from this yeast . The cDNAs encoding these allergens were sequenced and expressed in Escherichia coli . The sequences of MF 7, MF 8 and MF 9 were not full length (missing their 5'-ends) giving only partial gene products . To obtain complete cDNA sequences, we performed RACE-PCR to amplify the 5'-ends of each cDNA . These PCR products were sequenced and analyzed . The coding sequences of Mal f 7, Mal f 8 and Mal f 9 encode proteins with ORFs of 141 (16.2 kDa), 179 (19.2 kDa) and 126 (14.0 kDa) amino-acid residues, respectively . None of the putative proteins showed significant sequence homology with other known proteins in the searched database . The proteins encoded by the complete cDNA sequences were expressed in E . coli as recombinant proteins . Immunoblotting and radioallergosorbant test data showed that all of the expressed recombinant proteins have the ability to bind serum IgE from atopic dermatitis patients and furthermore, the M . furfur extract could specifically inhibit this IgE binding.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8938 - 43
The central cytoplasmic loop of the major facilitator superfamily of transport proteins governs efficient membrane insertion; Weinglass AB et al.; Deletion of 5 residues (Delta5) from the central cytoplasmic loop of the lactose permease of Escherichia coli has no significant effect on expression or activity, whereas Delta12 leads to increased rates of permease turnover after membrane insertion and decreased transport activity, and Delta20 abolishes insertion and activity . By expressing Delta12 or Delta20 in two halves, both expression and activity are restored to levels approximating wild type . Replacing deleted residues with random hydrophilic amino acids also leads to full recovery . However, introduction of hydrophobic residues decreases expression and activity in a context-dependent manner . Thus, a minimum length of the central cytoplasmic loop is vital for proper insertion, stability, and efficient transport activity, because of constraints at the cytoplasmic ends of helices VI and VII . Furthermore, the results are consistent with the idea that the middle cytoplasmic loop provides a temporal delay between insertion of the first six helices into the membrane before insertion of the second six helices.

Proc Natl Acad Sci U S A, 2000 Jul 18, 97(15), 8278 - 83
A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops; Tomita K et al.; Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3 . Here, we show that colicin D specifically cleaves tRNAs(Arg) including four isoaccepting molecules both in vivo and in vitro . The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2',3'-cyclic phosphate end, and is inhibited by a specific immunity protein . Consistent with the cleavage of tRNAs(Arg), the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine . Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA(Arg)-cleaving activity both in vivo and in vitro . These findings show that colicin D directly cleaves cytoplasmic tRNAs(Arg), which leads to impairment of protein synthesis and cell death . Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp . Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

Proc Natl Acad Sci U S A, 2000 Jul 18, 97(15), 8251 - 6
Biosynthesis of terpenoids: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from tomato; Rohdich F et al.; The putative catalytic domain (residues 81-401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase of Escherichia coli was expressed in a recombinant E . coli strain . The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2-C-methyl-d-erythritol at a rate of 33 micromol small middle dotmg(-1) small middle dotmin(-1) . The structure of the reaction product, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate, was established by NMR spectroscopy . Divalent metal ions, preferably Mg(2+), are required for activity . Neither the tomato enzyme nor the E . coli ortholog catalyzes the phosphorylation of isopentenyl monophosphate.

Genetics, 2000 Jul, 155(3), 1359 - 67
Through a glass, darkly: reflections of mutation from lacI transgenic mice; Stuart GR et al.; The study of mutational frequency (Mf) and specificity in aging Big Blue lacI transgenic mice provides a unique opportunity to determine mutation rates (MR) in vivo in different tissues . We found that MR are not static, but rather, vary with the age or developmental stage of the tissue . Although Mf increase more rapidly early in life, MR are actually lower in younger animals than in older animals . For example, we estimate that the changes in Mf are 4.9x10(-8) and 1.1 x 10(-8) mutations/base pair/month in the livers of younger mice (<1 . 5 months old) and older mice (> or =1.5 months old), respectively (a 4-fold decrease), and that the MR are 3.9 x 10(-9) and 1.3 x 10(-7) mutations/base pair/cell division, respectively ( approximately 30-fold increase) . These data also permit an estimate of the MR of GC --> AT transitions occurring at 5'-CpG-3' (CpG) dinucleotide sequences . Subsequently, the contribution of these transitions to age-related demethylation of genomic DNA can be evaluated . Finally, to better understand the origin of observed Mf, we consider the contribution of various factors, including DNA damage and repair, by constructing a descriptive mutational model . We then apply this model to estimate the efficiency of repair of deaminated 5-methylcytosine nucleosides occurring at CpG dinucleotide sequences, as well as the influence of the Msh2(-/-) DNA repair defect on overall DNA repair efficiency in Big Blue mice . We conclude that even slight changes in DNA repair efficiency could lead to significant increases in mutation frequencies, potentially contributing significantly to human pathogenesis, including cancer.

EMBO J, 2000 Jul 3, 19(13), 3458 - 64
Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome; Mohr D et al.; GTP hydrolysis by elongation factor G (EF-G) is essential for the translocation step in protein elongation . The low intrinsic GTPase activity of EF-G is strongly stimulated by the ribosome . Here we show that a conserved arginine, R29, of Escherichia coli EF-G is crucial for GTP hydrolysis on the ribosome, but not for GTP binding or ribosome interaction, suggesting that it may be directly involved in catalysis . Another conserved arginine, R59, which is homologous to the catalytic arginine of G(alpha) proteins, is not essential for GTP hydrolysis, but influences ribosome binding and translocation . These results indicate that EF-G is similar to other GTPases in that an arginine residue is required for GTP hydrolysis, although the structural changes leading to GTPase activation are different.

Biochem J, 2000 Jul 15, 349(Pt 2), 611 - 21
Targeting and insertion of C-terminally anchored proteins to the mitochondrial outer membrane is specific and saturable but does not strictly require ATP or molecular chaperones; Lan L et al.; A distinct class of proteins contain a C-terminal membrane anchor and a cytoplasmic functional domain . A subset of these proteins is targeted to the mitochondrial outer membrane . Here, to probe for the involvement of a saturable targeting mechanism for this class of proteins, and to elucidate the roles of chaperone proteins and ATP, we have utilized an in vitro targeting system consisting of in vitro-synthesized proteins and isolated mitochondria . To establish the specificity of targeting we have used a closely related protein pair . VAMP-1A and VAMP-1B are splice variants of the vesicle-associated membrane protein/synaptobrevin-1 (VAMP-1) gene . In intact cells VAMP-1B is targeted to mitochondria whereas VAMP-1A is targeted to membranes of the secretory pathway, yet these isoforms differ by only five amino acids at the extreme C-terminus . Here we demonstrate that, in vitro, VAMP-1B is imported into both intact mitochondria and mitochondrial outer-membrane vesicles with a 15-fold greater efficiency than VAMP-1A . We generated and purified bacterially expressed fusion proteins consisting of the C-terminal two-thirds of VAMP-1A or -1B proteins fused to glutathione S-transferase (GST) . Using these fusion proteins we demonstrate that protein targeting and insertion is saturable and specific for the VAMP-1B membrane anchor . To elucidate the role of cytosolic chaperones on VAMP-1B targeting, we also used the purified, Escherichia coli-derived fusion proteins . (33)P-Labelled GST-VAMP-1B(61-116), but not GST-VAMP-1A(61-118), was efficiently targeted to mitochondria in a chaperone-free system . Thus the information required for targeting is contained within the targeted protein itself and not the chaperone or a chaperone-protein complex, although chaperones may be required to maintain a transport-competent conformation . Moreover, ATP was required for transport only in the presence of cytosolic chaperone proteins . Therefore the ATP requirement of transport appears to reflect the participation of chaperones and not any other ATP-dependent step . These data demonstrate that targeting of C-terminally anchored proteins to mitochondria is sequence specific and mediated by a saturable mechanism . Neither ATP nor chaperone proteins are strictly required for either specific targeting or membrane insertion.

Biochem J, 2000 Jul 15, 349(Pt 2), 605 - 10
Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase; Dowler S et al.; We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain . DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), which bind to the PH domain . In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr(139) . Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr(139), and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2), is not phosphorylated at Tyr(139) following agonist stimulation of cells . Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells . We demonstrated that Tyr(139) of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1 . Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr(139) in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P(3), and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr(139) . These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr(139) by a Src-family tyrosine kinase.

Biochem J, 2000 Jul 15, 349(Pt 2), 501 - 7
Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2; Koo HM et al.; Malonamidase (MA) E2 was previously purified and characterized from Bradyrhizobium japonicum USDA 110 . The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli . The recombinant MAE2 was purified to homogeneity from the transformed E . coli . The biochemical properties of the recombinant enzyme are essentially identical to those from wild-type B . japonicum . A database search showed that the MAE2 protein has a high sequence similarity with the common signature sequences of the amidase family . The only exception is that the aspartic residue in these signature sequences is replaced by a glutamine residue . In order to identify amino acid residues essential for enzyme activity, a series of site-directed mutagenesis studies and steady-state kinetic experiments were performed . Gln(195), Ser(199), Cys(207) and Lys(213) of the common signature sequences were selected for site-directed mutagenesis . Among the mutants, Q195D, Q195E and S199C showed less than 0.02% of the k(cat) value of the wild-type enzyme, and S199A, Q195L and Q195N exhibited no detectable catalytic activities . Mutants (K213L, K213R and K213H) obtained by replacement of the only conserved basic residue, Lys(213), in the signature sequences, also displayed significant reductions (approx . 380-fold) in k(cat) value, whereas C207A kept full activity . These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln(195), Ser(199) and Lys(213) are involved.

Biochem J, 2000 Jul 15, 349(Pt 2), 443 - 53
Tissue-specificity, functional characterization and subcellular localization of a rat ubiquitin-specific processing protease, UBP109, whose mRNA expression is developmentally regulated; Park KC et al.; A cDNA encoding an ubiquitin-specific processing protease, UBP109, in rat skeletal muscle was cloned and its product was characterized . Northern analysis revealed that UBP109 mRNA is highly expressed in testis and spleen, compared with other tissues . Furthermore, in situ hybridization showed that the level of UBP109 mRNA in liver, spinal cord and brain dramatically changed during embryonic development, indicating that the expression of UBP109 mRNA is developmentally regulated . UBP109 was expressed in Escherichia coli and purified to apparent homogeneity using a (125)I-labelled ubiquitin-peptide fusion as a substrate . The purified enzyme cleaved at the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes . UBP109 also released free ubiquitin from poly-His-tagged penta-ubiquitin . Moreover, it released free ubiquitin from poly-ubiquitinated protein conjugates of rabbit reticulocytes . In addition, UBP109 localized to both the cytoplasm and the nucleus and, among three putative nuclear localization sequences, only the one located near the C-terminus is responsible for nuclear localization . These results suggest that UBP109 may play an important role in generation of free ubiquitin from its precursors and its recycling from poly-ubiquitinated protein conjugates, and hence in regulation of ubiquitin-mediated cellular processes, particularly related to embryonic development.

Zhonghua Gan Zang Bing Za Zhi, 2000 Jun, 8(3), 153 - 5
Cloning and sequencing of partial genes of hepatitis C virus genome in patients with acute hepatitis C; Jiang D et al.; OBJECTIVE: To explore the etiological role of HCV in patients with acute hepatitis . METHODS: The prevalence of HCV infection in 89 patients with acute hepatitis was investigated by analysis of HCV RNA and HCV second generation antibody . HCV RNAs extracted from the sera of 5 patients with NANB acute hepatitis, which were positive for HCV RNA, were converted to cDNA by reverse transcription with random primer and genotyping by PCR with type-specific primers . The partial genes of HCV genome were amplified . The PCR products were expressed in E . coli with p-GEM-T vector, and their nucleotide sequences were determined by dideoxynucleotide chain-termination method . RESULTS: The incidence of hepatitis virus infection was 47 . 2% in HAV, 28.1% in HBV and 15.7% in HCV, respectively . The incidence of HAV and HBV coinfection was 14.6% and the rate of non-A, non-B and non-C hepatitis was 9% in all patients . The genotype of HCV-RNA positive patients was 85.8% in HCV-II, 7.1% in HCV-III and 7 . 1% in combining HCV-II/III, respectively . The partial sequence of HCV genome in 5 patients with non-A and non-B acute hepatitis was amplified and the fragment was 424 bp in accordance with original design . The homology of the sequences was 98.1%-99.5% in nucleotide acid and 97.6%-99.2% in amino acid among five isolates . The average homology was 91.9% or 94.3%-95.6% for nucleotide sequences between HCV-I or HCV-II and the 5 isolates, and 92.3%-95.8% for amino acid sequences between the 5 isolates and HCV-I or HCV-II, respectively . CONCLUSION: HCV infection is one of the main hepatitis viruses in patients with acute hepatitis, in which the HCV-II genotype is dominant and should be paid attention to it.

Syst Appl Microbiol, 2000 Apr, 23(1), 115 - 23
Riboprints as a tool for rapid preliminary identification of sphingomonads; Busse HJ et al.; Fourtythree strains of the genus Sphingomonas and close relatives were subjected to riboprint analyses generated after digestion of genomic DNA with the restriction enzyme EcoRI and hybridization with E . coli rrnB operon . The majority of strains were characterized by a complex banding pattern in the riboprints . High degrees of similarities in the riboprints were only observed among strains of the same species such as S . yanoikuyae, S . aromaticivorans, S . subarctica and S . chlorophenolica . Strains of different species including close phylogenetic relatives such as S . asaccharolytica, S . mali and S . pruni were easily distinguished by the differences in the riboprints even after visual evaluation . Thus, our data demonstrate that riboprint analysis is useful for preliminary identification of new sphingomonad isolates at the species level.

Avian Dis, 2000 Apr-Jun, 44(2), 379 - 89
A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis; Lillehoj HS et al.; A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523) . The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein . Both proteins were reactive with the F3 antiserum by western blot analysis . A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E . acervulina sporozoites and Eimeria tenella sporozoites and merozoites . By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E . acervulina, E . tenella, and Eimeria maxima . Spleen lymphocytes from E . acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis . Immunization of chickens with either the E . coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E . acervulina . Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity . These results indicate that the recombinant E . acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

Avian Dis, 2000 Apr-Jun, 44(2), 318 - 24
Chicken embryo lethality assay for determining the virulence of avian Escherichia coli isolates; Wooley RE et al.; Multiple isolates of Escherichia coli from clinical cases of colibacillosis and E . coli from the intestinal tracts of normal broilers at slaughter were assayed by the embryo lethality test to determine their virulence . The assay was repeated five times in order to establish reproducibility and determine the statistical parameters of the test . This study showed that the inoculation of approximately 100 colony-forming units in the allantoic cavity of 12-day-old embryos discriminated between virulent and avirulent E . coli isolates . Gross lesions included cranial and skin hemorrhages in addition to encephalomalacia in embryos inoculated with virulent isolates . Abnormalities were observed by microscopic examination of the heart, brain, and liver in embryos inoculated with virulent isolates . Analysis of data indicated that the length of the test should be 4 days . In the virulent group, day 2 postinoculation had the most significant death patterns . Sample size calculations indicated that 11 embryos are sufficient for the assay . On the basis of death rates, isolates considered to be avirulent had an embryo death rate of <10%, moderately or secondary pathogens had a 10%-29% death rate, and virulent isolates had a death rate of >29% . An important aspect of this assay is the accessibility of good-quality fertile embryonated eggs.

Biosci Biotechnol Biochem, 2000 May, 64(5), 972 - 9
End-product regulation and kinetic mechanism of guanosine-inosine kinase from Escherichia coli; Kawasaki H et al.; Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized . The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer . A pI of 6.0 was obtained by isoelectric focusing . In addition to ATP, it was found that deoxyadenosine 5'-triphosphate, UTP, and CTP could serve as phosphate donors . The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine . Maximum activity was attained at an ATP/Mg2+ concentration ratio of 0.5 . In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP . Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released . Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.

Eur J Appl Physiol, 2000 May, 82(1-2), 142 - 50
Endotoxaemia does not limit heat tolerance in rats: the role of plasma lipoproteins; Caputa M et al.; Severe hyperthermia disrupts the intestinal barrier, allowing bacterial lipopolysaccharides (LPS) to enter the bloodstream . Since the symptoms of heat stroke resemble those of endotoxic shock, there is a common belief that endotoxaemia induces heat stroke . Therefore, we studied the effects of different doses, from moderate to sublethal, of Escherichia coli LPS and an antipyretic (indomethacin) upon the temperature equilibrium of the brain and body of rats exposed to a constant ambient temperature of 38 degrees C . The animals were then heated until they developed heat stroke, which was identified using a critical thermal maximum (CTM) behavioural test . In separate experiments on defence against endotoxaemia, we compared plasma lipid composition in rats exposed to a sublethal dose of LPS, hyperthermia and heat stroke . Neither LPS nor indomethacin, injected into rats while they were in a hyperthermic steady-state condition of 40-41 degrees C, influenced their thermal equilibrium . Unexpectedly, moderate doses of LPS significantly elevated the thermal tolerance of rats, such that the mean (SEM) CTM value of body temperature was raised from 42.7 (0.3) degrees C to 43.1 (0.1) degrees C (P < 0.05) . Indomethacin and huge doses of LPS failed to induce any change in this parameter . The sublethal dose of LPS did not induce mortality in rats subjected to heat stroke . Hyperthermic steady-state conditions and heat stroke alone significantly decreased plasma concentrations of cholesterol, triglyceride and high-density lipoproteins, while the concentrations of low-density lipoproteins increased . A similar pattern of changes was recorded in normothermic rats injected with a sublethal dose of LPS . In conclusion, endotoxaemia in heat-stressed rats induces neither a secondary increase in their core temperature nor a decrease in their ultimate thermal tolerance . Low-density lipoproteins are likely to protect heat-stressed animals against endotoxin-induced death.

Int Arch Allergy Immunol, 2000 Jun, 122(2), 115 - 23
Characterization of api g 1.0201, a new member of the Api g 1 family of celery allergens; Hoffmann-Sommergruber K et al.; BACKGROUND: The association of pollinosis with allergy to plant foods occurs in up to 70% of tree pollen-allergic patients . In recent years, some of the relevant cross-reacting proteins have been characterized at the molecular and immunological level . Api g 1 has been identified as the celery homologue of the major birch pollen allergen, Bet v 1 . Although a number of Bet v 1 isoforms have been characterized from birch pollen, little is known about isoforms of food allergens and their allergenic features . METHODS: Api g 1.0201, an isoform of Api g 1, was isolated from a cDNA library, cloned and sequenced . The cDNA was expressed in Escherichia coli and the purified recombinant protein was tested in immunoblots . RESULTS: Api g 1.0201 displays 72% sequence similarity to the previously identified Api g 1.0101 and consists of 159 amino acid residues . The sequence of Api g 1.0201 has five additional amino acid residues at the carboxy-terminus as compared to Api g 1.0101 . Purified recombinant Api g 1.0201 is recognized by IgE from the sera of celery-allergic patients, as well as by the murine monoclonal anti-Bet v 1 antibody . In general, this isoform displays a weaker IgE-binding capacity than Api g 1.0101, as concluded from immunoblotting experiments . Results from inhibition assays revealed that IgE-binding to Api g 1.0201 is only slightly reduced by preincubation with either purified recombinant Api g 1.0101 or purified recombinant Bet v 1a . Total inhibition was only achieved when using purified natural Bet v 1 . CONCLUSIONS: At present, little is known about the IgE-binding capacity of isoforms of Bet v 1 homologues of food allergens . Identification and characterization of such isoforms may help to contribute to a better understanding of food allergy and the observed cross-reactivity to pollen allergy .

Int Arch Allergy Immunol, 2000 Jun, 122(2), 108 - 14
Production of enzymatically and immunologically active Der f 1 in Escherichia coli; Takahashi K et al.; Der f 1 is a major house dust mite allergen belonging to the cysteine protease family . Because of the great demand for clinical and research use of this allergen, much effort to establish an efficient method of preparing purified Der f 1 has been made . We constructed an isopropyl-beta-D-thiogalactopyranoside-inducible expression plasmid to produce the pro-form of Der f 1 in Escherichia coli . The recombinant product was accumulated as insoluble inclusion bodies in cells . The solubilized inclusion bodies were found to be successfully renatured by two-step gel filtration chromatography . About 70 mg of pro Der f 1 were properly refolded by this method from 1 liter of culture . Acid treatment of the renatured pro Der f 1 resulted in the autocatalytic removal of the pro-sequence . The obtained mature form of Der f 1 bound IgE in patient sera and induced the release of histamine from peripheral blood leukocytes equally to native Der f 1 . Furthermore, mature Der f 1 obtained by this method had identical protease activity with native Der f 1 . We also discussed the contribution of the pro-sequence and the sugar chain of Der f 1 to its antigenic and enzymatic activity . This is the first report to produce an active mature form of recombinant Der f 1 in E . coli .

J Immunol, 2000 Jul 15, 165(2), 931 - 40
Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice; Mokuno Y et al.; We recently reported that the number of gamma delta T cells was increased after infection with Escherichia coli in C3H/HeN mice . We here showed that an i.p . injection with native lipid A derived from E . coli induced an increase of gamma delta T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice . The purified gamma delta T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by V gamma 6-J gamma 1/V delta 1-D delta 2-J delta 2 gene segments and proliferated in response to the native lipid A derived from E . coli in a TCR-independent manner . The lipid A-reactive gamma delta T cells bearing canonical V gamma 6/V delta 1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable . Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the gamma delta T cells to the native lipid A . TLR2-deficient mice showed an impaired increase of the gamma delta T cells following injection of native lipid A . These results suggest that TLR2 is involved in the activation of canonical V gamma 6/V delta 1 T cells by native E . coli lipid A.

J Immunol, 2000 Jul 15, 165(2), 888 - 95
Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells; Cochlovius B et al.; The use of anti-CD3 x antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy . Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs . We designed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the TCR complex . After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells . The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma . The CD3 x CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL . The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb . In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments . Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth . The survival time was further prolonged by including the anti-CD28 mAb . The CD3 x CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.

FEBS Lett, 2000 Jun 30, 476(1-2), 93 - 7
A common binding site for substrates and protons in EmrE, an ion-coupled multidrug transporter; Yerushalmi H et al.; EmrE is an Escherichia coli 12-kDa multidrug transporter, which confers resistance to a variety of toxic cations by removing them from the cell interior in exchange with two protons . EmrE has only one membrane-embedded charged residue, Glu-14, that is conserved in more than 50 homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters . We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate binding site . Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions . We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons . The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes.

FEBS Lett, 2000 Jun 30, 476(1-2), 8 - 11
Sulphur islands in the Escherichia coli genome: markers of the cell's architecture?
Rocha EP, Sekowska A, Danchin A.
Two highly contrasted images depict genomes: at first sight, genes appear to be distributed randomly along the chromosome . In contrast, their organisation into operons (or pathogenicity islands) suggests that, at least locally, related functions are in physical proximity . Analysis of the codon usage bias in orthologous genes in the genome of bacteria which diverged a long time ago suggested that some physical (architectural) selection pressure organised the distribution of genes along the chromosome . The metabolism of highly reactive species such as sulphur-containing molecules must be compartmentalised to escape the deleterious actions of diffusible reagents such as gases or radicals . We analysed the distribution of sulphur metabolism genes in the genome of Escherichia coli and found a number of them to be clustered into statistically significant islands . Another interesting feature of these genes is that the proteins they encode are significantly deprived of cysteine and methionine residues, as compared to the bulk proteins . We speculate that this clustering is associated to the organisation of sulphur metabolism proteins into islands where the sensitive sulphur-containing molecules are protected from reacting with elements in the environment such as dioxygen, nitric oxide or radicals.

Microbiology, 2000 Jul, 146 ( Pt 7), 1617 - 26
Dual regulation of catecholate siderophore biosynthesis in Azotobacter vinelandii by iron and oxidative stress; Tindale AE et al.; Azotobacter vinelandii forms both catecholate and azotobactin siderophores during iron-limited growth . Azotobactin is repressed by about 3 microM iron, but catecholate siderophore synthesis continues up to a maximum of 10 microM iron . This suggests that catecholate siderophore synthesis is regulated by other factors in addition to the ferric uptake repressor (Fur) . In this study the first gene required for catecholate siderophore biosynthesis, which encodes an isochorismate synthase (csbC), was isolated . The region upstream of csbC contained a typical sigma(70) promoter, with an iron-box overlapping the -35 sequence and a Sox-box (Box 1) overlapping the -10 sequence . Another Sox-box was found further upstream of the -35 sequence (Box 2) . Also upstream, an unidentified gene (orfA) was detected which would be transcribed from a divergent promoter, also controlled by an iron-box . The activity of csbC and a csbC::luxAB fusion was negatively regulated by iron availability and upregulated by increased aeration and by superoxide stress . The iron-box in the csbC promoter was 74% identical to the Fur-binding consensus sequence and bound the Fur protein of Escherichia coli with relatively high affinity . Both Box 1 and Box 2 were in good agreement with the consensus sequence for binding the SoxS protein of E . coli and Box 1 was in very good agreement with the Sox-box found in the fpr promoter of A . vinelandii, which is also regulated by superoxide stress . Both Sox-boxes bound a protein found in A . vinelandii cell extracts, with Box 1 exhibiting the higher binding affinity . The Sox protein identified in this assay appeared to be constitutive, rather than inducible by superoxide stress . This indicates that the Sox response in A . vinelandii is different from that in E . coli . These data support the hypothesis that catecholate siderophore biosynthesis is under dual control, repressed by a Fur-iron complex and activated by another DNA-binding protein in response to superoxide stress . The interaction between these regulators is likely to account for the delay in ferric repression of catecholate siderophore production, since these siderophores have an additional role to play in the protection of iron-limited cells against oxidative damage.

Microbiology, 2000 Jul, 146 ( Pt 7), 1605 - 16
Functional analysis of the ClpATPase ClpA of Brucella suis, and persistence of a knockout mutant in BALB/c mice; Ekaza E et al.; The protein ClpA belongs to a diverse group of polypeptides named ClpATPases, which are highly conserved, and which include several molecular chaperones . In this study the gene encoding the 91 kDa protein b-ClpA of the facultative intracellular pathogen Brucella suis, which showed 70% identity to ClpA of Rhodobacter blasticus, was identified and sequenced . Following heterologous expression in Escherichia coli strains SG1126 (DeltaclpA) and SG1127 (Deltalon DeltaclpA), b-ClpA replaced the function of E . coli ClpA, participating in the degradation of abnormal proteins . A b-clpA null mutant of B . suis was constructed, and growth experiments at 37 and 42 degrees C showed reduced growth rates for the null mutant, especially at the elevated temperature . The mutant complemented by b-clpA and overexpressing the gene was even more impaired at 37 and 42 degrees C . In intracellular infection of human THP-1 or murine J774 macrophage-like cells, the clpA null mutant and, to a lesser extent, the strain of B . suis overexpressing b-clpA behaved similarly to the wild-type strain . In a murine model of infection, however, the absence of ClpA significantly increased persistence of B . suis . These results showed that in B . suis the highly conserved protein ClpA by itself was dispensable for intramacrophagic growth, but was involved in temperature-dependent growth regulation, and in bacterial clearance from infected BALB/c mice.

Microbiology, 2000 Jul, 146 ( Pt 7), 1513 - 24
Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG hsp60 proteins, and to the Escherichia coli homologue GroEL; De Bruyn J et al.; Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins . Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour . Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene . In addition, the mycobacterial Hsp60-2 was overexpressed in E . coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose . The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M . bovis BCG and entirely different from that of the mycobacterial antigen 85 . Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E . coli GroEL with organic solvents releases various amounts of non-covalently bound lipids . The presence of lipids on Hsp60-2 was confirmed by labelling M . bovis BCG with radioactive palmitate . The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase . Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E . coli GroEL by TLC showed the same pattern for all the samples . Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins . Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.

J Clin Microbiol, 2000 Jul, 38(7), 2766 - 7
Recurrent infections and chronic colonization by an Escherichia coli clone in the respiratory tract of a patient with severe cystic bronchiectasis; Wang JY et al.; A 39-year-old woman with cystic bronchiectasis had repeated pulmonary infections from 1996 to 1999, and 6 of a total of 28 isolates of Escherichia coli from sputum specimens were studied . Their identical antibiotype and randomly amplified polymorphic DNA patterns indicated a single clone of E . coli, which persistently colonized the respiratory tract, causing recurrent infections.

J Clin Microbiol, 2000 Jul, 38(7), 2696 - 700
Recognition of enteropathogenic Escherichia coli virulence determinants by human colostrum and serum antibodies; Parissi-Crivelli A et al.; Human colostra and sera collected from Mexican mothers and their children at birth and 6 months thereafter were studied for the presence of antibodies against the bundle-forming pilus and several chromosomal virulence gene products (intimin and secreted proteins EspA and EspB) of enteropathogenic Escherichia coli (EPEC) . Among 21 colostrum samples studied, 76, 71.5, 57, and 47% of them contained immunoglobulin A (IgA) antibodies against EspA, intimin, EspB, and BfpA, respectively . Interestingly, there was a difference in IgG response to EPEC antigens between the sera from neonates and sera from the same children 6 months later . While the number of neonates reacting to Esps and intimin diminished when they reached 6 months of age, those reacting with BfpA increased from 9 to 71% . Intimin from an enterohemorrhagic E . coli strain was also recognized by most of the samples reacting with EPEC intimin . These data suggest that Bfp and Esps elicit an antibody response during the early days of life of neonates and support the value of breast-feeding in areas of the world where bacterial diarrheal infections are endemic.

J Clin Microbiol, 2000 Jul, 38(7), 2557 - 62
Differentiation of Borrelia burgdorferi sensu lato on the basis of RNA polymerase gene (rpoB) sequences; Lee SH et al.; We determined the nucleotide sequences (329 bp) of the rpoB DNAs from 22 reference strains of Borrelia . No insertions or deletions were observed . Deduced amino acid sequences of amplified rpoB DNA comprised 109 amino acid residues (N(450) to M(558) {Escherichia coli numbering}) . All amino acid sequences were identical with the exception of those of Borrelia lusitaniae PotiB2 (T(461)-->A) and B . bissettii DN127 (I(498)-->V) . Each species of B . burgdorferi sensu lato was differentiated as a distinct entity in the phylogenetic tree constructed by a neighbor-joining method . B . burgdorferi sensu lato could be distinguished from B . turicatae and B . hermsii, which are associated with relapsing fever . Seventeen Korean isolates could be identified by PCR-linked direct sequencing and restriction analysis of the rpoB DNA . These results suggest that rpoB DNA is useful for identification and characterization of Borrelia . In addition, we developed the rapid species identification method using the species-specific primer sets based on rpoB gene sequences.

J Biol Chem, 2000 Sep 15, 275(37), 28386 - 97
Tandem duplication . A novel type of triplet repeat instability; Pluciennik A et al.; Triplet repeat sequence (TRS) inserts containing (CTG.CAG)(n) (17-175 units in length) were tandemly duplicated when propagated in plasmids in Escherichia coli . The products of this novel type of TRS genetic instability are tracts of as many as 34 multiple units, which contain the entire TRS as well as 129 base pairs of nonrepetitive flanking sequence . The duplication process required the presence of two or more TRS-containing units . Close proximity (170 base pairs) of the TRS to the R6K gamma origin of replication of the pUTminiTn5Cm-derived constructs stimulated the tandem duplication process . These events are proposed to occur due to secondary structure formation, stalling of DNA synthesis, and slippage-mediated misalignment of the complementary strands relative to each other during DNA replication . This mechanism may account for the TRS-associated duplications in protein kinase and metalloprotease genes in neuroblastomas and melanomas, as well as the massive repeat expansions in type II triplet repeat neurological diseases.

Protein Eng, 2000 Jun, 13(6), 445 - 52
Green fluorescent antibodies: novel in vitro tools; Casey JL et al.; We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg) . GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells . Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule . We also demonstrated the utility of EGFP-scFv in immunofluorescence studies . The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies . In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv . We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.

Appl Environ Microbiol, 2000 Jul, 66(7), 3113 - 6
Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli; Henne A et al.; Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones . Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E . coli clones . Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases . The genes responsible for the lipolytic activity were identified and characterized.

Appl Environ Microbiol, 2000 Jul, 66(7), 2829 - 34
Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains; Pagan R et al.; The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003 . These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase . Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide . In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression . Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again . Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures . Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions . Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal . Growth in the presence of IPTG (isopropyl-beta-D-thiogalactopyranoside) increased resistance under high-pressure conditions . The mechanisms of inactivation may thus differ at high and low pressures . These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.

Jpn J Pharmacol, 2000 Feb, 82(2), 144 - 9
Characterization of recombinant human chymase expressed in Escherichia coli; Takai S et al.; We compared recombinant human chymase expressed in Escherichia coli with human chymase purified from vascular tissues . The recombinant chymase, the structure of which was NH2-enterokinase cleavage site-chymase-COOH, was expressed in Escherichia coli and then was solubilized and renatured . The protein did not have a chymase activity, but gained this activity after the cleavage of the N-terminal site by enterokinase . The enzyme was purified by heparin affinity and gel filtration columns . The N-terminal sequence of the protein was identical to the sequence for human chymase . The molecular weights of the recombinant chymase and chymase purified from human vascular tissues were 26 and 30 kDa, respectively, and the 4 kDa difference was thought to be due to the presence or absence of glycan . The optimum pH of the recombinant enzyme activity was between 7.5 and 9.0 . The activity of the recombinant enzyme was inhibited by chymostatin, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, but not by ethylenediaminetetraacetic acid and aprotinin . This enzyme cleaved specifically the Phe8-His9 bond of angiotensin (Ang) I to form Ang II and that of big endothelin (ET)-1 to form ET-1-(1-31) . These findings demonstrated that the enzymatic characteristics of the recombinant enzyme were identical to that of native human chymase.

J Crit Care, 2000 Jun, 15(2), 73 - 83
Role of poly-(ADP-ribose) synthetase in lipopolysaccharide-induced vascular failure and acute lung injury in pigs; Albertini M et al.; PURPOSE: To assess the contribution of poly (adenosine 5'-diphosphate ribose) synthetase (PARS) to the development of bacterial lipopolysaccharide (LPS)-induced acute lung injury and vascular failure in pigs . MATERIALS AND METHODS: Four groups of anesthetized, paralyzed, and mechanically ventilated domestic white pigs . Group 1 served as control, whereas Escherichia coli LPS (20 microg/kg/h) was continuously infused in group 2 . Group 3 received 20 mg/kg injection of 3-aminobenzamide (a selective inhibitor of PARS activity) 15 minutes before LPS infusion . Only 3-aminobenzamide and not LPS was injected in group 4 . All animals were examined for 180 minutes . Systemic and pulmonary hemodynamics and lung mechanics were measured during the experimental period . Lung wet/dry ratio, bronchoalveolar lavage (BAL) protein levels and cell counts and lung nitrotyrosine (footprint of peroxynitrite) immunostaining were also measured in a few animals . RESULTS: LPS infusion evoked a progressive decline in systemic arterial pressure, a small increase in cardiac output, and biphasic elevation of pulmonary arterial pressure . Lung compliance declined progressively, whereas lung and total respiratory resistance rose significantly after LPS infusion . Prominent nitrotyrosine immunostaining was detected around small airways and pulmonary endothelium of LPS-infused animals . No significant changes in lung wet/dry ratio and BAL protein levels and cell counts were produced by LPS infusion . Pretreatment with 3-aminobenzamide did not alter the systemic and pulmonary hemodynamic responses to LPS infusion but eliminated the rise in pulmonary and total respiratory resistance . CONCLUSIONS: We concluded that PARS activation plays an important role in the changes of lung mechanics associated with LPS-induced acute lung injury but had no role in vascular failure.

J Photochem Photobiol B, 2000 Mar, 55(1), 43 - 8
Inducible stable DNA replication (iSDR) and the uvr-dependent tolerance of pyrimidine dimers in UV-irradiated Escherichia coli are two uncoupled processes; Slezarikova V et al.; Inducible stable DNA replication (iSDR) is dependent on recombination and is supposed to play a role in DNA repair of Escherichia coli . Our previous data suggested that iSDR may be involved in the tolerance of UV lesions, which remain unexcised in excision-proficient E . coli exposed to some UV pretreatments . Now, the tolerance of unexcised lesions has been followed in E . coli recB21 and in E . coli priA1 sup mutants, incapable of iSDR . The obtained data do not confirm the previous hypothesis about the involvement of iSDR in the putative uvr-dependent lesion tolerance . They rather suggest that iSDR and the uvr-dependent lesion tolerance are two uncoupled processes.

Nat Struct Biol, 2000 Jul, 7(7), 586 - 93
Multistep mechanism of substrate binding determines chaperone activity of Hsp70; Mayer MP et al.; The 70 kDa heat shock proteins (the Hsp70 family) assist refolding of their substrates through ATP-controlled binding . We have analyzed mutants of DnaK, an Hsp70 homolog, altered in key residues of its substrate binding domain . Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered closing device involving independent action of an alpha-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a beta-structured subdomain . Correlative evidence from mutational analysis suggests that the ADP and ATP states of DnaK differ in the frequency of the conformational changes in the alpha-helical lid and beta-domain that cause opening of the substrate binding cavity . The affinity for substrates, as defined by this mechanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis mediated locking-in of substrates and chaperone activity of DnaK.

Nat Struct Biol, 2000 Jul, 7(7), 580 - 5
Systematic circular permutation of an entire protein reveals essential folding elements; Iwakura M et al.; The importance of chain connectivity in determining protein function and stability can be examined by breaking the peptide backbone using a technique such as circular permutation . Cleavage at certain positions results in a complete loss of the ability of the protein to fold . When such cleavage sites occur sequentially in the primary structure, we call the region a 'folding element', a new concept that could assist in our understanding of the protein folding problem . The folding elements of dihydrofolate reductase have been assigned by conducting a systematic circular permutation analysis in which the peptide backbone was sequentially broken between every pair of residues in the protein . The positions of folding elements do not appear to correspond to secondary structure motifs, substrate or coenzyme binding sites, or accessible surface area . However, almost all of the amino acid residues known to be involved in early folding events are located within the folding elements.

Nat Struct Biol, 2000 Jul, 7(7), 555 - 9
Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme; Li J et al.; Here we report the solution and refinement at 1.9 A resolution of the crystal structure of the Escherichia coli medium chain length acyl-CoA thioesterase II . This enzyme is a close homolog of the human protein that interacts with the product of the HIV-1 Nef gene, sharing 45% amino acid sequence identity with it . The structure of the E . coli thioesterase II reveals a new tertiary fold, a 'double hot dog', showing an internal repeat with a basic unit that is structurally similar to the recently described beta-hydroxydecanoyl thiol ester dehydrase . The catalytic site, inferred from the crystal structure and verified by site directed mutagenesis, involves novel chemistry and includes Asp 204, Gln 278 and Thr 228, which synergistically activate a nucleophilic water molecule.

J Biochem (Tokyo), 2000 Jul, 128(1), 129 - 37
Two SecG molecules present in a single protein translocation machinery are functional even after crosslinking; Nagamori S et al.; SecG, a membrane component of the protein translocation apparatus of Escherichia coli, undergoes membrane topology inversion, which is coupled to the membrane insertion and deinsertion cycle of SecA . Eighteen SecG derivatives possessing a single cysteine residue at various positions were constructed and expressed in a secG null mutant . All the SecG-Cys derivatives retained the SecG function, and stimulated protein translocation both in vivo and in vitro . Inverted membrane vesicles containing a SecG-Cys derivative were labeled with a membrane-permeable or -impermeable sulfhydryl reagent before or after solubilization with a detergent . The accessibility of these reagents to the cysteine residue of each derivative determined the topological arrangement of SecG in the membrane . Derivatives having the cysteine residue in the periplasmic region each existed as a homodimer crosslinked through disulfide bonds, indicating that two SecG molecules closely co-exist in a single translocation machinery . The crosslinking did not abolish the SecG function and the crosslinked SecG dimer underwent topology inversion upon protein translocation.

J Biochem (Tokyo), 2000 Jul, 128(1), 29 - 38
Three-dimensional structure of 4-amino-4-deoxychorismate lyase from Escherichia coli; Nakai T et al.; 4-Amino-4-deoxychorismate lyase (ADCL) is a member of the fold-type IV of PLP dependent enzymes that converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate . The crystal structure of ADCL from Escherichia coli has been solved using MIR phases in combination with density modification . The structure has been refined to an R-factor of 20.6% at 2.2 A resolution . The enzyme is a homo dimer with a crystallographic twofold axis, and the polypeptide chain is folded into small and large domains with an interdomain loop . The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein . Although the main chain folding of the active site is homologous to those of D-amino acid and L-branched-chain amino acid aminotransferases, no residues in the active site are conserved among them except for Arg59, Lys159, and Glu193, which directly interact with the coenzyme and play critical roles in the catalytic functions . ADC was modeled into the active site of the unliganded enzyme on the basis of the X-ray structures of the unliganded and liganded forms in the D-amino acid and L-branched-chain amino acid aminotransferases . According to this model, the carboxylates of ADC are recognized by Asn256, Arg107, and Lys97, and the cyclohexadiene moiety makes van der Waals contact with the side chain of Leu258 . ADC forms a Schiff base with PLP to release the catalytic residue Lys159, which forms a hydrogen bond with Thr38 . The neutral amino group of Lys159 eliminates the a-proton of ADC to give a quinonoid intermediate to release a pyruvate in accord with the proton transfer from Thr38 to the olefin moiety of ADC.

J Biochem (Tokyo), 2000 Jul, 128(1), 21 - 7
Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase; Gekko K et al.; To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (v(o)) and adiabatic compressibility (beta(s)(o)) were determined for a series of mutants with amino acid replacements at Gly67 (7 mutants), Gly121 (6 mutants), and Ala145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15 degrees C . These mutations induced large changes in v(o) (0.710-0.733 cm(3) . g(-1)) and beta(s)(o) (-1.8 x 10(-6)-5.5 x 10(-6) bar(-1)) from the corresponding values for the wild-type enzyme (v(o)=0.723 cm(3) . g(-1), beta(s)(o) = 1.7 x 10(-6) bar(-1)), probably due to modifications of internal cavities . The beta(s)(o) value increased with increasing v(o), but showed a decreasing tendency with the volume of the amino acid introduced . There was no significant correlation between beta(s)(o) and the overall stability of the mutants determined from urea denaturation experiments . However, a mutant with a large beta(s)(o) value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (k(cat)) and in part due to increased affinity for the substrate (K(m)), despite the fact that the mutation sites are far from the catalytic site . These results demonstrate that the flexibility of the DHFR molecule is dramatically influenced by a single amino acid substitution in one of these loops and that the flexible loops of this protein play important roles in determining the enzyme function.

J Biochem (Tokyo), 2000 Jul, 128(1), 11 - 20
Differential scanning calorimetry of light meromyosin fragments having various lengths of carp fast skeletal muscle isoforms; Kakinuma M et al.; Various recombinant light meromyosin (LMM) fragments were prepared from cDNAs encoding the 10 degrees C and 30 degrees C types of myosin heavy chain isoforms predominantly expressed in fast skeletal muscles of the 10 degrees C- and 30 degrees C-acclimated carp, respectively . These included three kinds of quarter fragments, 1/4-, 2/4-, and 4/4-quarter, composed of residues 1-130, 131-270, and 401-563 from the N-terminus, respectively, as well as three halves, N-, M-, and C-half fragments, containing residues 1-301, 131-400, and 302-563, respectively, and 69K fragments of residues 1-525 . Unfortunately, in spite of extensive efforts, the 3/4-quarter fragment was not expressed for both 10 degrees C and 30 degrees C types in our expression system using Escherichia coli . All the LMM fragments except for the 10- and 30-2/4 quarters for the 10 degrees C and 30 degrees C types, respectively, exhibited a typical pattern of a-helix in CD spectrometry . When these were subjected to differential scanning calorimetry (DSC), 30 degrees C-type LMM fragments were all found to be more thermostable than the 10 degrees C-type counterparts . To identify amino acid substitutions responsible for different thermostabilities between the 10 degrees C- and 30 degrees C-type LMMs, six mutant proteins were prepared, mainly focusing on substitutions in the C-terminal half of LMM, and subjected to DSC and CD analyses . For three mutants in which two residues of the 10 degrees C type were replaced by those of the 30 degrees C type, 10-S355T/T361A, 10-M415L/L417V, and 10-S535A/H536Q, the endothermic peaks in DSC increased by 1.4-2.0 degrees C from that of the original 10 degrees C type . The T(m) values for two single-residue substitutions, 10-H449R and 10-T491I, shifted 0.8 and 1.3 degrees C higher than that for the 10 degrees C-type LMM, respectively, whereas the last mutant, 10-G61V, showed no change in thermostability . The finding that the difference in T(m) values for major endothermic peaks from the 10-69K and 30-69K fragments was 4.6 degrees C, which roughly corresponds to that between the original 10 degrees C and 30 degrees C types, suggested that the eight substitutions located in the C-terminal region of the 69K fragments (residues 302-525) are major candidates for the residues responsible for the difference in thermostability between the 10 degrees C- and 30 degrees C-type LMMs.

Insect Biochem Mol Biol, 2000 Aug-Sep, 30(8-9), 839 - 45
Intracellular localization and tissue specificity of the Methoprene-tolerant (Met) gene product in Drosophila melanogaster; Pursley S et al.; The Methoprene-tolerant (Met) gene product in Drosophila melanogaster facilitates the action of juvenile hormone (JH) and JH analog insecticides . Previous work resulted in the cloning and identification of the gene as a member of the bHLH-PAS family of transcriptional regulators . A Met(+) cDNA was expressed in Escherichia coli, and polyclonal antibody was prepared against the purified protein . A single band on a Western blot at the expected size of 79kD was detected in extracts from Met(+) larvae but not from Met(27) null mutant larvae, demonstrating the antibody specificity . Antibody detected MET in all stages of D . melanogaster development and showed tissue specificity of its expression . MET is present in all cells of early embryos but dissipates during gastrulation . In larvae it is present in larval fat body, certain imaginal cells, and immature salivary glands . In pupae it persists in fat body cells and imaginal cells, including abdominal histoblast cells . In adult females MET is present in ovarian follicle cells and spermathecae; in adult males it is present in male accessory gland and ejaculatory duct cells . In all of these tissues MET is found exclusively in the nucleus . Some of these tissues are known JH target tissues but others are not, suggesting either the presence of novel JH target tissues or another function for MET.

Gene, 2000 Jun 27, 251(2), 131 - 9
Loss of a member of the aquaporin gene family, aqpA affects spore dormancy in Dictyostelium; Mitra BN et al.; We isolated and characterized a gene from Dictyostelium discoideum, which encodes a protein of 279 amino acids (30.6kDa) containing six transmembrane domains with two highly conserved motifs of asparagine-proline-alanine (NPA) found in the aquaporin family of water-channel proteins, although the second motif of the protein has been modified into NPV (asparagine-proline-valine) . The deduced amino acid sequence of the gene, which we have named aqpA, is 39% identical to D . discoideum WacA, 26% identical to human Aqp5, 26% identical to Oryza sativa PIP2a, 25% identical to yeast Aqy1 and 24% identical to E.coli AqpZ . Southern analyses indicated that aqpA is present as a single copy in the genome . Northern blot analysis showed that the developmentally regulated 1kb mRNA transcript first appears at the tight mound stage (12h), and is abundant in fingers (16h) and late culminants (20h) . In-situ hybridization of slugs revealed that aqpA mRNA accumulated in cells of the prespore region but not in those of the prestalk region . Disruption of aqpA by homologous recombination did not significantly affect growth or developmental morphogenesis . Although mutant spores were viable, when assayed soon after encapsulation, they became permeable to propidium iodide and lost viability after a week on the top of a fruiting body . Thus, AqpA is essential to maintain spore dormancy perhaps through the regulation of water flow.

J Biol Chem, 2000 Sep 15, 275(37), 28701 - 7
The contribution of individual interchain interactions to the stabilization of the T and R states of Escherichia coli aspartate transcarbamoylase; Sakash JB et al.; Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions . The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state . In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state . Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity . The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions . Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties . From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme . As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state . In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state . The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme . The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.

J Biol Chem, 2000 Sep 8, 275(36), 27865 - 73
Escherichia coli replicative helicase PriA protein-single-stranded DNA complex . Stoichiometries, free energy of binding, and cooperativities; Jezewska MJ et al.; Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique . The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers . The total site-size of the PriA.ssDNA complex, i.e . the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer . However, the protein can efficiently form a complex with a minimum of 8 nucleotides . Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size . The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site . The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries . The intrinsic affinity depends little upon the length of the ssDNA . Moreover, the binding is accompanied by weak cooperative interactions.

J Biol Chem, 2000 Sep 22, 275(38), 29737 - 42
Expression analysis of phenylketonuria mutations . Effect on folding and stability of the phenylalanine hydroxylase protein; Gamez A et al.; Phenylketonuria is an autosomal recessive human genetic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene . In the present work we have used different expression systems to reveal folding defects of the PAH protein caused by phenylketonuria mutations L348V, S349L, and V388M . The amount of mutant proteins and/or the residual activity can be rescued by chaperonin co-overexpression in Escherichia coli or growth at low temperature in COS cells . Thermal stability profiles and degradation time courses of PAH expressed in E . coli show that the mutant proteins are less stable than the wild-type enzyme, also confirmed by pulse-chase experiments using a coupled in vitro transcription-translation system . Size exclusion chromatography shows altered oligomerization, partially corrected with chaperonins coexpression, except for the S349L mutant protein, which is recovered as inactive aggregates . PAH subunit interaction is affected in the S349L protein, as demonstrated in a mammalian two-hybrid assay . In conclusion, serine 349, located in the three-dimensional structure lining the active site and involved in the structural maintenance of the iron binding site, is essential for the structural stability and assembly and also for the catalytic properties of the PAH enzyme, whereas the L348V and V388M mutations affect the folding properties and stability of the protein . The experimental modulation of mutant residual activity provides a potential explanation for the existing inconsistencies in the genotype-phenotype correlations.

Symp Ser Soc Appl Microbiol, 2000, (29), 1S - 9S
The public health significance of VTEC O157; Parry SM et al.; Surveillance of human VTEC O157 has been reported in several countries, based on laboratory evidence . The incidence is generally less than 10 per 100000 (the highest incidence is in children), with regional variations and a marked seasonality . Laboratory selection criteria and reporting have contributed to, but cannot entirely explain, variations within and between countries . Surveillance data and outbreaks in definable cohorts indicate that the spectrum of illness ranges from diarrhoea through acute bloody diarrhoea, with about 5% of cases developing haemolytic uraemic syndrome; less than 50% of patients report frank blood in their stools . Studies of sporadic cases have associated illness with beef products (particularly if undercooked and eaten outside the home), cooked sliced meat meals and contact with a household member with diarrhoea . Outbreaks have been attributed to contaminated foods (including beefburgers) and water, animal contact and person-to-person spread . Secondary transmission by the primary case in a household is of particular concern, and household transmission has been estimated at 4%, with patients excreting for around 10 days following onset . Recommendations for control have highlighted measures on farms, in slaughterhouses, retail and catering food premises, and in the home.

J Biol Chem, 2000 Sep 15, 275(37), 28984 - 8
Comparison of the two murine terminal {corrected} deoxynucleotidyltransferase terminal isoforms . A 20-amino acid insertion in the highly conserved carboxyl-terminal region modifies the thermosensitivity but not the catalytic activity; Boule JB et al.; Terminal deoxynucleotidyltransferase (TdT) catalyzes the addition of nucleotides to 3'-hydroxyl ends of DNA strands in a template-independent manner and has been shown to add N-regions to gene segment junctions during V(D)J recombination . TdT is highly conserved in all vertebrate species, with a second isoform, characterized by a 20-amino acid insertion near the COOH-terminal end, described only in the mouse . The two murine isoforms differ in their subcellular localization, and the long isoform (TdTL) has previously been found to be unable to add N-regions . Using purified protein produced in a high level expression system in Escherichia coli, we were able to carry out detailed catalytic comparisons of these two TdT isoforms . We discovered that TdTL exhibits terminal transferase activity with kinetic parameters similar to those of the conserved TdT isoform (TdTS) . We observed, however, that TdTL is inactivated at physiologic temperature but stable at lower temperatures . This thermal sensitivity of TdTL polymerase activity is not correlated with a significant change in the circular dichroism spectrum of the protein . Thus, the 20-amino acid insertion in TdTL does not affect the catalytic activity but modifies the thermosensitivity.

Poult Sci, 2000 Jun, 79(6), 871 - 8
The individual and combined effects of fumonisin B1 and moniliformin on performance and selected immune parameters in turkey poults; Li YC et al.; Effects of feeding diets containing fumonisin B1 (FB1) and moniliformin (M), singly or in combination, on performance and immune response were evaluated in poults . Day-old poults were randomly assigned to one of four dietary treatments with four replicates of four poults each . Dietary treatments were 1) control; 2) 200 mg FB1, 0 mg M/kg diet; 3) 0 mg FB1, 100 mg M/kg diet; and 4) 200 mg FB1, 100 mg M/kg diet . In Experiment 1, poults were injected with 0.25 mL Newcastle disease virus (NDV) vaccine on Weeks 2 and 3 of the experiment, and anti-NDV antibody titers were measured 7 d after each injection . Compared with controls, poults fed FB1 had significantly lower (P < 0.05) secondary antibody response . Poults fed M and the combination of FB1 and M had significantly lower (P < 0.05) primary and secondary antibody response . Lower relative thymus weights were observed in poults fed diets containing FB1 or M . Decreased relative bursa and spleen weights were observed in poults fed M . In Experiment 2, poults were placed on dietary treatments for 3 wk . On Day 21, 2 x 10(6) peripheral lymphocytes were incubated with mitogens . Poults fed diets containing FB1 had a significantly lower (P < 0.05) proliferative response to mitogens in comparison to controls . In Experiment 3, poults were placed on the diets for 3 wk and were injected with 4.4 x 10(7) E . coli/kg body weight on Day 21 . Significantly higher (P < 0.05) numbers of E . coli colonies were observed in the blood and tissue homogenates of poults fed M . In all three experiments, feed intake and body weight gains were significantly lower (P < 0.05) in turkeys fed diets containing M . Data from the present study suggest that FB1 and M are immunosuppressive in poults and that M not only suppresses immune response but also performance . However, neither synergistic nor additive effects between FB1 and M were observed for any of the parameters measured.

Poult Sci, 2000 Jun, 79(6), 810 - 6
Immunocompetence and viability under commercial conditions of broiler groups differing in growth rate and in antibody response to Escherichia coil vaccine; Yunis R et al.; Mortality and morbidity due to infectious diseases are an increasing source of losses to the broiler industry . Breeding chickens for improved disease resistance may reduce these losses . A study was designed to evaluate the contribution of selection for immune response to viability of broilers under farm conditions . The experimental populations consisted of six groups: two lines divergently selected for high (HH) or low (LL) antibody (Ab) response to Escherichia coli vaccination; commercial broilers (CC); and the HH x CC, LL x CC, and HH x LL crosses . Chicks were tested under standard vaccination program and management on commercial farms in two years (1997 and 1998) . Mortality was recorded in the whole groups, each consisting of several hundred or thousand of chicks, whereas BW and Ab to natural exposure to E . coli and to vaccination with Newcastle disease virus (NDV) were determined in samples of 50 to 120 chicks/group per yr . Groups were clustered into three levels of BW: CC representing contemporary fast-growing broilers; HH, LL, and HL representing broilers 10 yr earlier; and HC and LC with intermediate BW . The HH and LL groups exhibited the highest and lowest E . coli Ab titers, respectively . Mean Ab of the CC group equaled the average of the selected lines, and all crosses exhibited mid-parent Ab titers, indicating additive genetic control . Group means for Ab to NDV were highly correlated with those of E . coli, suggesting a common genetic control for the immune response to these two antigens . In both years, the highest mortality was found in the fast-growing group (CC), and the lowest mortality was in the slow-growing HH, LL, and HL groups . In the crosses, despite their similar mean BW, mortality was one-third higher among LC vs . HC birds . These results suggest that Ab response and potential growth rate interact in their effect on mortality due to infectious diseases.

Poult Sci, 2000 Jun, 79(6), 799 - 803
Immune competence of chicks from two lines divergently selected for antibody response to sheep red blood cells as affected by supplemental vitamin E; Yang N et al.; Effects of dietary vitamin E on responses to SRBC antigens and Escherichia coli infection were studied in chicks from White Leghorn lines selected for 24 generations for high (HAS) and low (LAS) antibody responses to SRBC . Chicks were fed corn-soybean diets consisting of either high (300 IU per kg feed) or low (10 IU per kg feed) concentrations of vitamin E from the day of hatch through the end of experiment . The LAS chicks were heavier than the HAS chicks at 14 d of age and thereafter; there was no difference in BW between vitamin E concentrations . At 37 d of age, chicks were inoculated via the brachial vein with 0.1 mL of 0.25% SRBC suspension . Antibody titers at 6 and 10 d after inoculation were higher in HAS than in LAS chicks . At 6 and 10 d after inoculation with SRBC, antibody responses were lower in LAS chicks fed the diet containing the higher vitamin E concentration than in those fed the diet containing the lower concentration of vitamin E . At 64 d of age, chicks were injected in the posterior thoracic air sac with 0.1 mL of 10(-2) or 10(-4) dilution of Escherichia coli and scored for pericardial and air sac lesions . The HAS chicks were more susceptible to E . coli infection than LAS chicks as measured by lesion scores and BW changes . Although dietary vitamin E had no effect on lesion scores in either line, BW loss at 24 h after E . coli inoculation was significantly reduced in HAS chicks fed the higher concentration of vitamin E . The dosage of E . coli had no effect on lesion scores and BW changes . These results suggest that genetic selection might have changed immune competence in relation to responses to dietary vitamin E, and the optimum dietary concentration of vitamin E depends on genotype, among other factors.

J Pharm Pharmacol, 2000 Jun, 52(6), 599 - 602
Anthraquinone-oligodeoxynucleotide conjugates as inhibitors of gene transcription; Gibson V et al.; Several anthraquinone-oligodeoxynucleotide conjugates have been synthesized and their inhibition of transcription of the bla gene in E . coli has been determined . All conjugates at 10 microM inhibited transcription and three conjugates inhibited mRNA production by 40 to 50% for periods of up to 60 min.

Biochem Pharmacol, 2000 Aug 15, 60(4), 571 - 9
Reductive activation of mitomycin C by neuronal nitric oxide synthase; Jiang HB et al.; Mitomycin C (MC) requires bioreduction prior to the generation of alkylating moieties . NADPH-cytochrome P450 reductase is predominant in metabolic activation of MC in hypoxic cancer cells . In this study, neuronal nitric oxide synthase (nNOS), whose reductase domain is structurally similar to that of NADPH-cytochrome P450 reductase, was assessed for its ability to activate MC . nNOS under anaerobic conditions catalyzed the reduction of MC, which was measured as the decrease in absorbance at 375 nm . Neither the heme blocker potassium cyanide (1 mM) nor the nNOS competitive inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM) affected the bioreduction of MC, whereas 0.1 mM diphenyleneiodonium chloride, which binds to the reductase domain of nNOS, inhibited MC reduction completely . The reduction of MC by nNOS was influenced by Ca(2+)/calmodulin . In the absence of Ca(2+)/calmodulin, the rate of MC reduction decreased by 28% at pH 6.6 . The formation of an alkylated complex of 4-(p-nitrobenzyl)pyridine occurred in a manner analogous to that observed in MC metabolic experiments . The rate of MC reduction and the formation of the alkylated complex of 4-(p-nitrobenzyl)pyridine at pH 6.6 were increased by 43 and 54%, respectively, as compared with that at pH 7.6 . nNOS-activated MC resulted in the consumption of oxygen in air . The rate of oxygen consumption decreased by 50% in the presence of 2000 U/mL of catalase . MC inhibited nNOS activity in a noncompetitive manner . These findings demonstrate that nNOS is capable of catalyzing the bioreduction of MC.

Biochem Pharmacol, 2000 Aug 15, 60(4), 539 - 44
Selective inhibition of nitric oxide synthase type I by clonidine, an anti-hypertensive drug; Venturini G et al.; Clonidine, clinically used in the treatment of hypertension, is a central alpha(2)-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation . Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated . This was verified by determination of NOS activity in vitro and by analysis of inducible Ca(2+)-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model . Clonidine inactivated neuronal Ca(2+)-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca(2+)-dependent NOS (NOS-III) activity . However, the value of K(i) for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH(4)) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds . In particular, the value of K(i) for clonidine binding to NOS-I increased (from {7 . 9 +/- 0.4} x 10(-5) M to {8.0 +/- 0.4} x 10(-3) M) as BH(4) concentration was increased (between 3.0 x 10(-7) M and 1.0 x 10(-3) M), at pH 7.5 and 37.0 degrees . In addition, clonidine (1.0 x 10(-4) M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) . Finally, clonidine (1.0 x 10(-4) M to 1.0 x 10(-3) M) dose dependently increased the levels of LPS/IFN-gamma-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells . As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

J Biol Chem, 2000 Sep 15, 275(37), 28413 - 20
Molecular mechanism and energetics of clamp assembly in Escherichia coli . The role of ATP hydrolysis when gamma complex loads beta on DNA; Bertram JG et al.; Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex . The gamma complex requires ATP to load beta onto DNA . A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution . The two temporally correlated stopped-flow signals revealed that a preassembled beta . gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step . Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)) . Following hydrolysis, gamma complex dissociates from the DNA ( approximately 22 s(-1)) . Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only approximately 3 s(-1) . Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis . The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type . The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.

J Biol Chem, 2000 Sep 15, 275(37), 28695 - 700
A reaction-induced fourier transform-infrared spectroscopic study of the lactose permease . A transmembrane potential perturbs carboxylic acid residues; Patzlaff JS et al.; In chemiosmotic coupling, a transmembrane ion gradient is used as the source of energy to drive reactions . This process occurs in all cells, but the microscopic mechanism is not understood . Here, Escherichia coli lactose permease was used in a novel spectroscopic method to investigate the mechanism of chemiosmotic coupling in secondary active transporters . To provide a light-triggered electrochemical gradient, bacteriorhodopsin was co-reconstituted with the permease, and reaction-induced Fourier transform-infrared spectra were obtained from the co-reconstituted samples . The bacteriorhodopsin contributions were subtracted from these data to give spectra reflecting permease conformational changes that are induced by an electrochemical gradient . Positive bands in the 1765-1730 cm(-1) region are attributable to carboxylic acid residues in the permease and are consistent with changes of pK(a), protonation state, or environment . This is the first direct information concerning gradient-induced structural changes in the permease at the single amino acid level . Ultimately, these structural changes facilitate galactoside binding and may be involved in the storage of free energy.

J Biol Chem, 2000 Sep 15, 275(37), 28466 - 82
The plastid ribosomal proteins . Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast); Yamaguchi K et al.; We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using two-dimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry) . Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in other types of ribosomes . Orthologues of E . coli L25 and L30 are absent in spinach plastid ribosome . 25 of the plastid 50 S RPs are encoded in the nuclear genome and synthesized on cytosolic ribosomes, whereas eight of the plastid RPs are encoded in the plastid organelle genome and synthesized on plastid ribosomes . Sites for transit peptide cleavages in the cytosolic RP precursors and formyl Met processing in the plastid-synthesized RPs were established . Post-translational modifications were observed in several mature plastid RPs, including multiple forms of L10, L18, L31, and PSRP-5 and N-terminal/internal modifications in L2, L11 and L16 . Comparison of the RPs in gradient-purified 70 S ribosome with those in the 30 and 50 S subunits revealed an additional protein, in approximately stoichiometric amount, specific to the 70 S ribosome . It was identified to be plastid ribosome recycling factor . Combining with our recent study of the proteins in plastid 30 S subunit (Yamaguchi, K., von Knoblauch, K., and Subramanian, A . R . (2000) J . Biol . Chem . 275, 28455-28465), we show that spinach plastid ribosome comprises 59 proteins (33 in 50 S subunit and 25 in 30 S subunit and ribosome recycling factor in 70 S), of which 53 are E . coli orthologues and 6 are plastid-specific proteins (PSRP-1 to PSRP-6) . We propose the hypothesis that PSRPs were evolved to perform functions unique to plastid translation and its regulation, including protein targeting/translocation to thylakoid membrane via plastid 50 S subunit.

J Biol Chem, 2000 Sep 15, 275(37), 28455 - 65
The plastid ribosomal proteins . Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast); Yamaguchi K et al.; Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry) . 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases . 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome . Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described . Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E . coli) . The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa) . PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria . We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

Carcinogenesis, 2000 Jul, 21(7), 1397 - 402
Enhanced in vivo repair of O(4)-methylthymine by a mutant human DNA alkyltransferase; Encell LP et al.; The repair of O(6)-methylguanine (m(6)G) by human O(6)-alkylguanine-DNA alkyltransferase (hAGT) is approximately 5000-fold greater than that for O(4)-methylthymine (m(4)T) . To evaluate each adduct's contribution to mutagenesis, we previously created a mutant hAGT with increased specificity for m(4)T in vitro . The mutant and wild-type (WT) hAGT have now been expressed in bacterial strains that allow for the specific detection of A:T-->G:C and G:C-->A:T mutations induced by m(4)T and m(6)G, respectively . After exposure to the mutagenic methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, A:T-->G:C substitutions were reduced >4-fold in cells expressing the mutant hAGT compared with 1 . 1-fold for WT hAGT . G:C-->A:T substitutions were decreased >2.5-fold in cells expressing the mutant hAGT, whereas WT hAGT totally prevented G:C-->A:T mutations . These results demonstrate that the altered substrate specificity of hAGT observed in vitro also occurs in vivo, and that it is responsible for the observed differences in mutations.

Ann Rheum Dis, 2000 Jul, 59(7), 533 - 8
Decreased T cell precursor frequencies to Epstein-Barr virus glycoprotein Gp110 in peripheral blood correlate with disease activity and severity in patients with rheumatoid arthritis; Toussirot E et al.; OBJECTIVES: Rheumatoid arthritis (RA) is a chronic joint disease associated with certain HLA-DR alleles expressing the QK/RRAA motif or shared epitope . The Epstein-Barr virus (EBV) has been suspected to be a causative factor for RA . The EBV gp110, a glycoprotein of the replicative cycle that contains a copy of the shared epitope, constitutes an important target in the immune control of EBV replication . This study evaluated the specific T cell response to EBV gp110 in patients with RA expressing or not the shared epitope and examined whether this immune cellular response might be related to disease activity and severity . METHODS: 25 patients with RA were studied and compared with 25 healthy controls . Disease activity was assessed by biochemical markers of inflammation (erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) levels) . Disease severity was defined by extra-articular disease (vasculitis, subcutaneous nodules, or other organ disease) . The frequencies of peripheral blood T cells specific for EBV gp110 and a control protein (total protein extract from Escherichia coli) were determined by direct limiting dilution analysis without preliminary bulk culture . RESULTS: The gp110 precursor frequencies ranged from 0 to 20 x 10(-6) in patients with RA and controls . The mean gp110 T cell precursor frequency was lower in patients with RA (SD 3.2 (4.4) x 10(-6)) than in healthy controls (4.1 (3.8) x 10(-6)) (p = 0.02) . No difference was found for the control protein (p = 0.09) . Both shared epitope positive and negative patients with RA responded to gp110, without significant difference . A negative correlation between both ESR and CRP levels and the gp110 T cell response was found (r = -0.71, p<0.0001 and r = -0.42, p = 0.038, respectively) . Finally, patients with extra-articular disease displayed the lowest immune cellular response to EBV gp110 . CONCLUSION: These results suggest that patients with RA have a decreased T cell response to EBV gp110 . Since gp110 is an important protein in the control of EBV replication, this might lead to a poor control of EBV infection, chronic exposure to other EBV antigens, and thus to a chronic inflammatory response in patients with RA.

Curr Biol, 2000 Jun 15, 10(12), R444 - 6
DNA helicases: one small step for PcrA, one giant leap for RecBC?
Wigley DB.
One might imagine that the mechanism of helicases would relate to the number of base pairs that are unwound for each ATP that is hydrolysed . Recent studies, however, suggest the situation can be more complicated than this.

Virology, 2000 Jul 5, 272(2), 302 - 14
The RGD sequence in the cytomegalovirus DNA polymerase accessory protein can mediate cell adhesion; Loh LC et al.; The murine cytomegalovirus (MCMV) polymerase processivity factor ppM44 (also referred to as pp50) is an abundant phosphoprotein found in MCMV-infected cells . Sequence analysis of the MCMV M44 open reading frame revealed an "RGD" motif that is also present in the human cytomegalovirus (HCMV) UL44 open reading frame . In this report, histidine-tagged M44 protein produced in Escherichia coli or the vaccinia/T7 expression system was purified to near homogeneity by metal chelation affinity chromatography using His*Bind resins . We demonstrated that recombinant M44 protein could mediate cell adhesion via its conserved "RGD" motif, because a single amino acid change (RGD to RGE) abolished cell attachment . In addition, cell adhesion was abolished in the presence of EDTA . We next showed that recombinant HCMV UL44, but not human herpesvirus type 6 p41, which lacks the RGD motif, could mediate cell adhesion in a similar manner . We also provided evidence that ppM44 was present in the culture medium during virus infection . Thus these results suggested that in addition to its primary role as the polymerase processivity factor, MCMV ppM44 may serve as a substrate for integrin-binding via its conserved RGD motif, with the potential for a novel role in the MCMV replication cycle .

Virology, 2000 Jun 20, 272(1), 72 - 84
Studies on the attenuation phenotype of polio vaccines: poliovirus RNA polymerase derived from Sabin type 1 sequence is temperature sensitive in the uridylylation of VPg; Paul AV et al.; Determinants of temperature sensitivity and/or attenuation in Sabin type 1 poliovirus reside in the 5' NTR and coding sequences of the capsid proteins and viral RNA polymerase, 3D(pol) . Previous studies have implicated at least two mutations in 3D(pol) of Sabin 1 vaccine strain {PV1(S)}, including a Y73H change, as contributing to these phenotypes . We have used an in vitro assay to test the first step in RNA synthesis, the uridylylation of the terminal protein VPg with 3D(pol) isolated from PV1(S) . Wt and two mutant 3D(pol) proteins (Y73H, D53N/Y73H) were expressed in Escherichia coli and were purified, and their activities were measured in the synthesis of VPgpU(pU) and of VPg-linked poly(U) at 30 and 39.5 degrees C . Our results show that at 39.5 degrees C the Y73H mutation leads to a defect in the synthesis of VPgpUp(U) and of VPg-poly(U) but not in the elongation of a (dT)(15) primer . The double mutant protein had the same activities as Y73H 3D(pol) . Using the yeast two-hybrid assay, we detected a reduced interaction between 3D(pol) molecules carrying either the single or double mutations . Tyrosine-73 maps to the finger domain in the three-dimensional structure of 3D(pol) . A model will be presented in which a change of Y73 to H73 may interfere with an interaction between two polymerase molecules that, in turn, may interfere with VPg uridylylation . Alternative explanations, however, cannot be excluded at the present time .

Biochem Biophys Res Commun, 2000 Jul 5, 273(2), 705 - 11
Expression and binding properties of a soluble chimeric protein containing the N-terminal domain of the Duffy antigen; Wasniowska K et al.; The blood group Duffy antigen of human erythrocytes, which exists in two allelic forms, Fy(a) and Fy(b), is a promiscuous chemokine receptor . In this report we describe the expression and purification of a chimeric protein composed of the amino-terminal extracellular domain of the Duffy antigen (aa 3-60), C-terminal intracellular fragment of glycophorin A (GPA, aa 104-131), and the hexahistydyl tag . We obtained two forms of the recombinant protein containing the Fy(a) or Fy(b) epitope, denoted Fy(a)/GPA and Fy(b)/GPA, respectively . These constructs were expressed in Escherichia coli as periplasmic proteins and were purified by affinity chromatography on the Ni-NTA-agarose . Both proteins bound the monoclonal antibodies recognizing the common Fy6 epitope of the Duffy antigen and an epitope of the C-terminal fragment of GPA, and only the Fy(a)/GPA bound anti-Fy(a) antibody . However, binding of IL-8 to the recombinant proteins was not detected, which indicated that an N-terminal domain of the Duffy antigen is not sufficient for an effective chemokine binding . The lack of the chemokine binding was not likely to be due to the lack of glycosylation of the Fy/GPA, since IL-8 was effectively bound to de-N-glycosylated erythrocytes .

Biochem Biophys Res Commun, 2000 Jul 5, 273(2), 642 - 8
Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity; Nakata D et al.; A cDNA of the mouse homologue of Escherichia coli N-acetylneuraminic acid (Neu5Ac) synthase (neuB gene product) was cloned by the PCR-based method . The mouse homologue consists of 359 amino acids, and the cDNA sequence displays 33% identity to that of the E . coli Neu5Ac synthase . The recombinant mouse homologue which is transiently expressed in HeLa cells does not exhibit the Neu5Ac synthase activity, which catalyzes condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) to synthesize Neu5Ac, but the Neu5Ac 9-phosphate (Neu5Ac-9-P) synthase activity, which catalyzes condensation of PEP and ManNAc 6-phosphate (ManNAc-6-P) to synthesize Neu5Ac-9-P . Thus, the mouse homologue of E . coli Neu5Ac synthase is the Neu5Ac-9-P synthase . The Neu5Ac-9-P synthase is a cytosolic enzyme and ubiquitously distributed in mouse various tissues . Notably, the Neu5Ac-9-P synthase can not catalyze the synthesis of deaminoneuraminic acid (KDN) or KDN-9-P from PEP and Man or ManNAc-6-P, thus suggesting that the enzyme is not involved in the synthesis of KDN . This is consistent with the previous observation that only a very low activity to synthesize KDN is found in mouse B16 cells {Angata, T., et al . (1999) Biochem . Biophys . Res . Commun . 261, 326-331} .

Protein Expr Purif, 2000 Jul, 19(2), 312 - 8
Controlled intracellular processing of fusion proteins by TEV protease; Kapust RB et al.; Here we describe a method for controlled intracellular processing (CIP) of fusion proteins by tobacco etch virus (TEV) protease . A fusion protein containing a TEV protease recognition site is expressed in Escherichia coli cells that also contain a TEV protease expression vector . The fusion protein vector is an IPTG-inducible ColE1-type plasmid, such as a T7 or tac promoter vector . In contrast, the TEV protease is produced by a compatible p15A-type vector that is induced by tetracyclines . Not only is the TEV protease regulated independently of the fusion protein, but its expression is highly repressed in the absence of inducer . Certain fusion partners have been shown to enhance the yield and solubility of their passenger proteins . When CIP is used as a purification step, it is possible to take advantage of these characteristics while both eliminating the need for large amounts of pure protease at a later stage and possibly simplifying the purification process . Additionally, we have observed that in some cases the timing of intracellular proteolysis can affect the solubility of the cleaved passenger protein, allowing it to be directed to either the soluble or the insoluble fraction of the crude cell lysate . This method also makes it possible to quickly gauge the efficiency of proteolysis in vivo, before protein purification has begun and in vitro processing is attempted .

Protein Expr Purif, 2000 Jul, 19(2), 304 - 11
Expression of an anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin in a mutant CHO cell line; Liu YY et al.; ADP-ribosylating immunotoxins are generally expressed in Escherichia coli and then refolded in vitro . Because the efficiency of the in vitro refolding process decreases with the number of protein domains and internal disulfide bonds, these immunotoxins have been generally limited to single-chain monovalent structures . We now show that using the hamster cell line CHO K1 RE1.22c (J . M . Moehring and T . J . Moehring, 1979, Somat . Cell Genet . 5, 453-468) that has been mutated to ADP-ribosylation insensitivity, a level of 4 microg/ml of a truncated anti-T cell immunotoxin, DT390-scFvUCHT1, can be secreted into the medium . This immunotoxin is glycosylated at the two potential N-linked glycosylation sites in the toxin moiety: positions 16-18 in the A chain and residues 235-237 in the B chain . The glycosylated immunotoxin is relatively nontoxic (IC(50) 4.8 x 10(-10) M) . Removal of the N-linked oligosaccharides by N-glycosidase F treatment or mutations at the two N-linked glycosylation sites results in a highly active immunotoxin with an IC(50) of 4 x 10(-12) M toward CD3(+) Jurkat cells . This is a 12-fold increase in toxicity over the same immunotoxin harvested from E . coli periplasm without refolding . A single Asn(235) Ala mutation that removed the B chain glycosylation was nearly as toxic as the double mutant . This suggests that B chain glycosylation is the major cause for the loss of toxicity .

Protein Expr Purif, 2000 Jul, 19(2), 284 - 8
Heterodimeric complex of RAR and RXR nuclear receptor ligand-binding domains: purification, crystallization, and preliminary X-ray diffraction analysis; Bourguet W et al.; Both the human retinoic acid receptor alpha (hRARalpha) and a constitutively active mutant (F318A) of the mouse retinoid X receptor alpha (mRXR alpha F318A) ligand-binding domains were separately overexpressed in Escherichia coli, copurified as a heterodimer in a two-step procedure, and cocrystallized with an RAR alpha-specific antagonist by using polyethylene glycol 10,000 as precipitant . The crystals grew in the hexagonal space group P6(1)22 displaying the unit cell parameters a = b = 116.6 A and c = 207.8 A . They diffracted X-ray to a limit of 2.2-A resolution . The asymmetric unit comprises one heterodimer and the crystal contains 60% solvent . The structure was determined by molecular replacement and is currently being refined .

Protein Expr Purif, 2000 Jul, 19(2), 271 - 5
Expression and purification of recombinant neurotensin in Escherichia coli; Williamson PT et al.; An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies . A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein . The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein . Purification of recombinant neurotensin was performed by reverse-phase HPLC . This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (<15 amino acids) with isotopes for NMR or other biophysical techniques .

Protein Expr Purif, 2000 Jul, 19(2), 259 - 64
Expression and characterization of recombinant Rhodocyclus tenuis high potential iron-sulfur protein; Caspersen MB et al.; The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods . UV-visible spectra of the reduced and oxidized recombinant protein were similar to those of the native protein . EPR of the oxidized protein shows g values of 2 . 11, 2.03, and 2.03 . ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a {Fe(4)S(4)} cluster in the holoprotein . The observed mass of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein . The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively . All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating correct folding and incorporation of the iron-sulfur cluster .

Protein Expr Purif, 2000 Jul, 19(2), 246 - 52
An analysis of two refolding routes for a C-terminally truncated human collagenase-3 expressed in Escherichia coli; Hardern IM et al.; We describe here the expression of a C-terminally truncated form of human procollagenase-3 in Escherichia coli . The protein was found almost exclusively in inclusion bodies that were solubilized and refolded by two separate methods and then purified on Ni-NTA agarose . The purified proenzyme could be activated with either trypsin or APMA and active enzyme could be purified on a peptidic hydroxamate affinity column . Competitive elution from the affinity matrix yielded a highly purified preparation .

Protein Expr Purif, 2000 Jul, 19(2), 227 - 34
Expression, purification, and functional analysis of the human serine protease HtrA2; Savopoulos JW et al.; HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product . Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions . In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each . We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E . coli expression systems which were initially unsuccessful . Using E . coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels . By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated .

Protein Expr Purif, 2000 Jul, 19(2), 219 - 26
Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies; Lopez-Vara MC et al.; Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major methyl donor for transmethylation reactions . Attempts to perform structural studies using rat liver MAT have met with problems because the protein purified from cellular extracts is heterogeneous . Overexpression of the enzyme in Escherichia coli rendered most of the protein as inclusion bodies . These aggregates were purified by specific washes using urea and Triton X-100 and used for refolding . Maximal activity was obtained when chaotropic solubilization included the structural cation Mg(2+), the protein concentration was kept below 0.1 mg/ml, and denaturant removal was carried out in a two-step process, namely, a fast dilution followed by dialysis in the presence of 10 mM DTT or GSH/GSSG redox buffers . Refolding by this procedure generated the oligomeric forms, MAT I and III, which were basically indistinguishable from the purified rat liver forms in secondary structure and catalytic properties .

Plasmid, 2000 Jul, 44(1), 94 - 9
Highly conserved DNA sequence present in small plasmids from Selenomonas ruminantium; Fliegerova K et al.; Plasmid pJW1 from Selenomonas ruminantium subsp . lactilytica strain JW13 has been cloned in Escherichia coli vector pBluescriptSK(-) and completely sequenced . The plasmid is only 1410 bp with an overall GC content of 42.2% . Computer analysis of sequence data revealed a single open reading frame (ORF1, 146 amino acids, MW 16,525.5 Da) encoding a putative replication protein which is similar to the Rep protein of Ruminobacter amylophilus plasmid pRAO1 . ORF1 is followed by a long AT-rich (75%) region and a region abundant in direct and inverted repeats . Comparison of DNA sequences revealed the presence of a short (<250 bp) DNA segment which is highly conserved between several small S . ruminantium plasmids including pJDB21 .

Plasmid, 2000 Jul, 44(1), 89 - 93
Tungsten particle-induced nicking of supercoiled plasmid DNA; Mazus B et al.; Small particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used for biotechnological purposes . In such applications, tungsten was observed to affect the integrity of plasmid DNA . Here we present evidence that interaction between tungsten particles and intact circular plasmids pU19, pUC119, and ColE1 may result in generation of a limited number of single-strand DNA breaks . As a consequence, supercoiled DNA is converted into its open circular form and no fragmentation products can be detected . The rate of the tungsten-mediated reaction depends on pH but is not influenced by ascorbate, Tris, or EDTA . No DNA nicking can be observed when the tungsten particles are replaced by substances that can be leached out from these particles with water or incubation buffers . Likewise, commercial sodium tungstate, tungsten (VI) oxide, and tungsten (VI) chloride and products of its decomposition remain DNA undamaged . Native plasmid DNA molecules, upon adsorption on the surface of tungsten microparticles, may undergo some nicking without a need for participation of external catalysts .

Plasmid, 2000 Jul, 44(1), 12 - 23
Nucleotide sequence of a 7-kb fragment of pACM1 encoding an IncM DNA primase and other putative proteins associated with conjugation; Preston KE et al.; A 7-kb fragment of pACM1 (fragment 90 inverted question mark91) containing one or more kor (kill-override) loci was sequenced, and 28 open reading frames (ORFs; >/=50 codons) were identified . The nucleotide sequence has no significant homologs in the GenBank database except for a 1.3-kb region 98.6% identical to the iml (insensitivity to phage PhiM-mediated lysis) determinant fragment of IncM plasmid R446 . Deduced amino acid sequences for several ORFs are homologous to those of known proteins, including the Sog DNA primases of IncI1 plasmids R64 and ColIb-P9 and the TraL, TraM, and TraN products of ColIb-P9 . Two protein products of the putative primase ORF (ORF 1, 1100 amino acids) were detected by SDS-PAGE . The 158- and 107-kDa proteins were designated PriL and PriS, respectively . PriS is apparently produced by an in-frame reinitiation of the ORF 1 transcript at a second start codon located between a Sau96I site and a PstI site . The motif EGYATA, conserved among primases and associated with primase function, occurs in the first one-third of the deduced amino acid sequence of PriL and is not included in PriS . Partial suppression of the temperature-sensitive dnaG3 mutation in BW86 was demonstrated by recombinants that overexpressed both PriL and PriS, but not by constructs overexpressing only PriS . Therefore, primase function can be assigned to PriL . Fragment 90/91 represents a portion of the IncM tra region, which has not previously been examined in detail .

J Mol Biol, 2000 Jul 7, 300(2), 353 - 62
A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases; Podobnik M et al.; Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases . We present the crystal structure of the catalytic unit of a primase enzyme, that of a approximately 320 residue fragment of Escherichia coli primase, determined at 2.9 A resolution . Central to the catalytic unit is a TOPRIM domain that is strikingly similar in its structure to that of corresponding domains in DNA topoisomerases, but is unrelated to the catalytic centers of other DNA or RNA polymerases . The catalytic domain of primase is crescent-shaped, and the concave face of the crescent is predicted to accommodate about 10 base-pairs of RNA-DNA duplex in a loose interaction, thereby limiting processivity .

J Mol Biol, 2000 Jun 23, 299(5), 1373 - 86
The thermodynamic stability of the proteins of the ccd plasmid addiction system; Dao-Thi MH et al.; The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods . The thermodynamic stability was measured at different temperatures combining denaturant and thermally induced unfolding . It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a significantly lower thermodynamic stability . Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins . This concentration dependence may be of physiological significance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation.The influence of DNA on the thermal stability of the two proteins was probed . It was found that cognate DNA increases the melting temperature of CcdA . In the presence of non-specific DNA the thermal stability was not changed . The melting temperature of CcdB was not influenced by the applied double-stranded oligonucleotides, neither cognate nor unspecific .

J Mol Biol, 2000 Jun 23, 299(5), 1363 - 71
Determination of intramolecular distance distribution during protein folding on the millisecond timescale; Ratner V et al.; A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed . The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined . The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument) . At that time the width of that distribution was rather large (16+/-2 A) . The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10(-7) cm(2)/second . In a second slower phase of the refolding transition, the width was reduced to its native value (6+/-4 A) .

J Mol Biol, 2000 Jun 23, 299(5), 1325 - 39
Phosphorylated and dephosphorylated structures of pig heart, GTP-specific succinyl-CoA synthetase; Fraser ME et al.; Succinyl-CoA synthetase (SCS) catalyzes the reversible phosphorylation/dephosphorylation reaction: inverted question mark inverted question mark inverted question markrm succinyl inverted question markhbox inverted question mark- inverted question markCoA+NDP+P_i inverted question markleftrightarrow succinate+CoA+NTP inverted question mark inverted question markwhere N denotes adenosine or guanosine . In the course of the reaction, an essential histidine residue is transiently phosphorylated . We have crystallized and solved the structure of the GTP-specific isoform of SCS from pig heart (EC 6.2.1.4) in both the dephosphorylated and phosphorylated forms . The structures were refined to 2.1 A resolution . In the dephosphorylated structure, the enzyme is stabilized via coordination of a phosphate ion by the active-site histidine residue and the two "power" helices, one contributed by each subunit of the alphabeta-dimer . Small changes in the conformations of residues at the amino terminus of the power helix contributed by the alpha-subunit allow the enzyme to accommodate either the covalently bound phosphoryl group or the free phosphate ion . Structural comparisons are made between the active sites in these two forms of the enzyme, both of which can occur along the catalytic path . Comparisons are also made with the structure of Escherichia coli SCS . The domain that has been shown to bind ADP in E . coli SCS is more open in the pig heart, GTP-specific SCS structure .

J Mol Biol, 2000 Jun 23, 299(5), 1303 - 11
A revised mechanism for the alkaline phosphatase reaction involving three metal ions; Stec B et al.; Here, X-ray crystallography has been used to investigate the proposed double in-line displacement mechanism of Escherichia coli alkaline phosphatase in which two of the three active-site metal ions have a direct role in catalysis . Two new X-ray crystal structures of the wild-type enzyme in the absence and presence of inorganic phosphate have been refined at 1.75 A to final working R-factors of 15.4% and 16.4%, respectively . In the refinement of both structures, residues in the active sites were treated anisotropically . The ellipsoids resulting from the partial anisotropic refinement show a clear route for the binding and release of substrate/product . In addition, a direct comparison of the refined structures with and without phosphate reveal a strong correlation between the occupancy of the third metal-binding site and the conformation of the Ser102 nucleophile . These findings clarify two important and unresolved aspects of the previously proposed catalytic mechanism, how Ser102 is activated for nucleophilic attack and why a magnesium ion in the third metal site is required for catalysis . Analysis of these results suggest that three metal-ion assisted catalysis is a more accurate description of the mechanism of the alkaline phosphatase reaction . A revised mechanism for the catalytic reaction of alkaline phosphatase is proposed on the basis of the two new X-ray crystal structures reported .

J Mol Biol, 2000 Jun 23, 299(5), 1279 - 87
Three-dimensional reconstruction of transcription termination factor rho: orientation of the N-terminal domain and visualization of an RNA-binding site; Yu X et al.; The Escherichia coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid . Unlike hexameric DNA helicases, where a single strand of DNA passes through the central channel, it has been proposed that the RNA wraps around the outside of the ring . We have generated a three-dimensional reconstruction of rho, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring . An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring . Although rho shares a common structural core with the F1-ATPase and other hexameric helicases, there has been a divergence in function due to rho's N-terminal domain, which has no homology to other helicases .

J Mol Biol, 2000 Jun 23, 299(5), 1271 - 8
The aquaporin sidedness revisited; Scheuring S et al.; Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues . All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore . Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass . This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness . Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals . They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy . The shadowing of some surfaces was prevented by protruding aggregates . Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal . Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps . AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1) . Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1 . We conclude that the sidedness we originally proposed for aquaporin density maps was inverted .

J Mol Biol, 2000 Jun 23, 299(5), 1257 - 70
A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation; Tran VK et al.; In bacteria and lower eukaryotes, adaptation to changes in the environment is often mediated by two-component regulatory systems . Such systems provide the basis for chemotaxis, nitrogen and phosphate regulation and adaptation to osmotic stress, for example . In Escherichia coli, the sensor kinase EnvZ detects a change in the osmotic environment and phosphorylates the response regulator OmpR . Phospho-OmpR binds to the regulatory regions of the porin genes ompF and ompC, and alters their expression . Recent evidence suggests that OmpR functions as a global regulator, regulating additional genes besides the porin genes . In this study, we have characterized a previously isolated OmpR2 mutant (V203M) that constitutively activates ompF and fails to express ompC . Because the substitution was located in the C-terminal DNA-binding domain, it had been assumed that the substitution would not affect phosphorylation of the N-terminal domain of OmpR . Our results indicate that this substitution completely eliminates phosphorylation by a small phosphate donor, acetyl phosphate, but not phosphorylation by the kinase EnvZ . The mutant OmpR has altered dephosphorylation kinetics and altered binding affinities to both ompF and ompC sites compared to the wild-type . Thus, a single amino acid substitution in the C-terminal DNA-binding domain has dramatic effects on the N-terminal phosphorylation domain . Most strikingly, we have identified a single base change in the OmpR binding site of ompC that restores high-affinity binding activity by the mutant . We interpret our results in the context of a model for porin gene expression .

J Mol Biol, 2000 Jun 23, 299(5), 1245 - 55
Probing the Escherichia coli transcriptional activator MarA using alanine-scanning mutagenesis: residues important for DNA binding and activation; Gillette WK et al.; The MarA transcriptional activator binds to a 20 bp asymmetric degenerate sequence (marbox) located at different positions and orientations within the promoters of the genes of the Escherichia coli mar regulon . Solution of the MarA-marbox X-ray crystallographic structure suggested the presence of base-specific and non-specific interactions between the marbox and two helix-turn-helix (HTH) motifs on the monomeric MarA . Here, we use alanine-scanning mutagenesis and DNA retardation analysis to: (i) evaluate the contacts between MarA and the marboxes of five differently configured mar regulon promoters; (ii) assess the role of conserved hydrophobic amino acid residues for MarA activity; and (iii) identify residues required for RNA polymerase activation . These analyses revealed that the phosphate-backbone contacts and hydrogen bonds with the bases of the marbox are more significant for DNA binding than are the van der Waals interactions . While both N and C-terminal HTH motifs make essential contributions to binding site affinity, MarA is more sensitive to alterations in the N-terminal HTH . In a similar way, the activity of MarA is more sensitive to alterations in the hydrophobic core of this HTH . Solvent-exposed amino acid residues located at many positions on the MarA surface are important for activity . Some of these residues affect activity on all promoters and thus, are implicated in maintaining MarA structure whereas several solvent-exposed amino acids not involved in DNA binding were important for MarA activity on specific promoters . The pattern of activation defects defined a class II promoter-specific activating region . However, a localized class I activating region was not apparent . These results suggest that MarA activates transcription by at least two distinct mechanisms . Furthermore, the important role of phosphate contacts in marbox affinity suggests that indirect readout contributes to binding site recognition by MarA .

J Mol Biol, 2000 Jun 23, 299(5), 1165 - 77
C-terminal domains of Escherichia coli topoisomerase I belong to the zinc-ribbon superfamily; Grishin NV; Detection of remote evolutionary connections is increasingly difficult with sequence and structural divergence . A combination of sequence and structural analysis, in which statistically supported sequence similarity had a crucial impact, revealed that Escherichia coli topoisomerase I C-terminal fragment is evolutionarily related to the three tetracysteine zinc-binding domains of the enzyme . Spatial structure analysis of this C-terminal fragment indicates that it consists of two structurally similar domains and suggests homology between them . Sequence similarity between the zinc-binding domains of type Ia topoisomerases and transcription regulators of known spatial structure helps to conclude that E . coli topo I contains five copies of a zinc ribbon domain at the C terminus . Two of these domains, corresponding to the C-terminal fragment, lost their cysteine residues and are probably not able to bind zinc . Present analyses lead to the classification of the C-terminal fragment of E . coli topoisomerase I as a member of zinc ribbon superfamily, despite the absence of zinc-binding sites .

J Mol Biol, 2000 Jun 23, 299(5), 1157 - 64
Aspartyl tRNA-synthetase from Escherichia coli: flexibility and adaptability to the substrates; Rees B et al.; The crystal structure of aspartyl-tRNA synthetase from Escherichia coli has been determined to a resolution of 2.7 A . The structure is compared to the same enzyme co-crystallized with tRNA(Asp) and containing aspartyl adenylate or ATP . The asymmetric unit contains three monomers of the enzyme . While most parts of the protein show no significant differences in the three monomers, a few regions cannot be superimposed . Those regions are characterized by a high B-factor, and consist mostly of loops that make contacts with the tRNA in the complexes . The flexibility of the protein is seen at a global level, by the observation of a 10 to 15 degrees rotation of the N-terminal and insertion domains upon tRNA binding, and at the level of the individual amino acid residues, by main-chain and side-chain rearrangements . In contrast to these induced-fit conformational changes, a few residues essential for the tRNA anticodon or aspartyl-adenylate recognition exist in a predefined conformation, ensured by specific interactions within the protein .

Am J Reprod Immunol, 2000 May, 43(5), 305 - 11
Acute intrauterine infection results in an imbalance between pro- and anti-inflammatory cytokines in the pregnant rabbit; Leslie KK et al.; PROBLEM: Intrauterine infection results in an increase in cytokines . This study compared the time courses for the pro- and anti-inflammatory cytokine responses in 33 pregnant rabbits at 70% gestation . Pro-inflammatory markers were activated nuclear factor-kappa B (NF-kappaB) in placenta and tumor necrosis factor-alpha (TNF-alpha) in amniotic fluid . These were compared to the anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra), in placenta and uterus . METHOD OF STUDY: Does were endoscopically inoculated with Escherichia coli through their cervices and sacrificed at six intervals between 0 and 30 hr post-inoculation . RESULTS: Activated NF-kappaB, determined by electromobility gel shift assay, increased significantly 16 hr after bacterial inoculation (P < or = 0.05) . This was directly mirrored by TNF-alpha concentrations, determined by bioassay, in the amniotic fluid . However, IL-1ra levels, determined by enzyme-linked immunosorbent assay, did not increase in response to infection . CONCLUSIONS: Intrauterine infection results in an imbalance between pro- and anti-inflammatory cytokines that may potentiate infection-induced preterm delivery.

EMBO J, 1983, 2(8), 1409 - 15
Electron microscopy and single molecule averaging of subunit-deficient F1-ATPases from Escherichia coli and spinach chloroplasts; Akey CW et al.; The morphology of F1-ATPases lacking one or more small subunits has been investigated by minimal-beam electron microscopy of close-packed monolayers of molecules . Computer-based rotational analyses of single molecules were performed on reconstituted 3-subunit F1-ATPase (-delta epsilon) from Escherichia coli and both 3-subunit (-delta epsilon) and 4-subunit (-delta) F1-ATPase from chloroplasts . Optical diffraction measurements of close-packed arrays revealed maximal dimensions of 122 +/- 4 A and 129 +/- 9 A for 3-subunit ECF1 and 4-subunit CF1, respectively . Molecules which displayed either hollow or solid hexagonal morphologies were observed in all preparations . Averaged reconstructions were obtained from molecules with hollow morphologies in 3-subunit preparations and demonstrated strong hexagonal symmetry in projection with a central, stain-filled cavity . The average reconstruction obtained from molecules with the solid morphology in 4-subunit CF1 preparations, was also strongly hexagonal with six peripheral units ringed about a central subunit . Differences between hollow and solid morphologies cannot be attributed solely to the presence or absence of the delta and epsilon subunits; therefore, the two image types may represent staining variants of a common structure . Overall, the reconstructions are consistent with an alpha 3 beta 3 gamma stoichiometry for the coupling factors from both E . coli and chloroplasts.

Mikrobiol Z, 2000 Mar-Apr, 62(2), 27 - 37
{LPS mutants of Sinorhizobium meliloti and their nodulation competitiveness}; Zatiovskaia TV et al.; Four Tn5-transposon LPS mutants of Sinorhizobium meliloti (Tb9, Tb29, Ts22 and Ts32) have been studied . Each of four mutants has been established to contain a single insertion of Tn5-transposon in its genome . All mutations are located on a chromosome . Nodulation competitiveness (NC) of mutants towards the parent strain of S . meliloti CXM1-188 was investigated by resistant method using coinoculation of mutant and parent strain in the ratio 1:1 . It was shown that NC was only 19-31% and 8-10%, for two strains Tb29 and Ts22, respectively which had lost the capability to synthesize higher molecular weight form of lipopolysaccharide (LPS1) . Nodulation competitiveness of two other strains (Tb9 and Ts32) which retained the capability to synthesize LPS1 although in modified form varied from 49 to 62% and did not differ from NC of strain CXM1-188 . The investigation of nodule formation rate has shown that four LPS-mutants did not differ from the parent strain by the number of root nodules . However the appearance of nodules induced by the mutant Tb29 was registered 7 days later than the nodules formed by other LPS-mutants and CXM1-188 strain . Obtained data concerning a single Tn5-insertion in genome of each of four S . meliloti LPS-mutants testify to the fact that both the disturbance of lipopolysaccharide synthesis and change of nodulation competitiveness in mutants Tb29 and Ts22 are results of a single mutation.

Planta, 2000 May, 210(6), 1006 - 13
Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis; Hirotani M et al.; A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.) . The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones . The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented . The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53,094 Da . The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy . The enzyme activities of recombinant UBGT expressed in E . coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4'-dihydroxyflavone and kaempferol, and phenolic compounds . The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments.

Planta, 2000 May, 210(6), 979 - 84
12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis; Schaller F et al.; In addition to OPR1 and OPR2, two isoenzymes of 12-oxophytodienoate reductase, a third isoform (OPR3) has recently been identified in Arabidopsis thaliana (L.) Heynh . The expression of the OPR3 gene is induced not only by a variety of stimuli, such as touch, wind, wounding, UV-light and application of detergent, but also by brassinosteroids . The three enzymes were expressed in a functional form in Escherichia coli, and OPR2 was additionally expressed in insect cell cultures and overexpressed in A . thaliana . Substrate conversion was analyzed using a stereospecific assay . The results show that OPR3 effectively converts the natural (9S,13S)-12-oxophytodienoic acid {Km = 35 microM, Vmax 53.7 nkat (mg protein)-1} to the corresponding 3-2(2'(Z)-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) stereoisomer while OPR1 and OPR2 convert (9S,13S)-12-oxophytodienoic acid with greatly reduced efficiency compared to OPR3 . Thus, OPR3 is the isoenzyme relevant for jasmonate biosynthesis.

Planta, 2000 May, 210(6), 938 - 46
Blue-light regulation of phytoene dehydrogenase (carB) gene expression in Mucor circinelloides; Velayos A et al.; The carB gene, encoding the phytoene dehydrogenase of Mucor circinelloides, was isolated by heterologous hybridisation with a probe derived from the corresponding gene of Phycomyces blakesleeanus . The cDNA and genomic copies complemented phytoene dehydrogenase defects in Escherichia coli and in carB mutants of M . circinelloides, respectively . Fluence-response curves for transcript accumulation were constructed after different blue-light pulses . The level of carB mRNA accumulation reached values up to 150-fold higher than basal levels in darkness . Several elements in the promoter of this gene resemble a consensus sequence identified in Neurospora crassa (APE) which is essential for blue-light regulation . Comparison of the available phytoene dehydrogenase sequences from plants, fungi, algae and bacteria suggests that the two known types of phytoene dehydrogenase are more closely related to each other than previously thought.

Can J Microbiol, 2000 May, 46(5), 485 - 9
Deletions of mob and tra pJP4 transfer functions after mating of Ralstonia eutropha JMP134 (pJP4) with Escherichia coli harboring F'::Tn10; Clement P et al.; One-tenth of Escherichia coli transconjugants resulting from the transfer of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 to E . coli XL1Blue, contained pJP4 derivatives with deletions (approximately 15-30 kb) . The occurrence of these deletions is probably associated with the presence of Tn10 in the recipient . DNA endonuclease restriction analysis of the pJP4 deletion derivatives showed the absence of SphI and EcoRI fragments previously reported to hybridize with IncP Tra DNA probes . Moreover, these pJP4 deletion derivatives are not able to self-transfer, nor are they able to be mobilized . Accordingly, these pJP4 deletion derivatives lack transfer functions.

Biofizika, 2000 May-Jun, 45(3), 432 - 8
{Analysis of peculiarities in nucleotides disposition in the origin of chromosome replication-oriC from Escherichia coli}; Esipova NG et al.; Along with symmetrical features (palindromes, direct and inverted repeats), periodicities in the disposition of nucleotides in the origin of chromosome replication oriC from E . coli were studied by means of Fourier analysis . Peaks corresponding to the periods T = 2, 17, 95-100 nucleotides are the highest in the Fourier spectrum of oriC . Peaks corresponding to the periods T = 3, 11, 19, 13, 24, 27, 28, 41, 79-81 nucleotides are also prominent, but not so high . Thus, the main periodicities of the oriC spectrum of are not multiple of the B-DNA sugar-phosphate backbone period, which destabilize DNA at oriC and contributes to the spontaneous unwinding of DNA . The differences between the Fourier spectrum of oriC and those of regions adjacent to oriC are demonstrated.

J Biol Chem, 2000 Sep 22, 275(38), 29225 - 32
The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin; Roman LJ et al.; The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR) . However, all nitric-oxide synthase (NOS) isoforms are 20-40 residues longer in the C terminus, forming a "tail" that is absent in CPR . To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively . Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms . With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates . Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants . Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms . Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin . By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus . Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so . We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.

J Biol Chem, 2000 Sep 22, 275(38), 29610 - 7
Identification of the (183)RWTNNFREY(191) region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity; Chung L et al.; Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments . To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end . MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens . After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C . The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity . Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold . Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold . Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity . However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity . It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required . These elements probably act in a concerted manner to cleave the collagen triple helix.

J Biol Chem, 2000 Sep 22, 275(38), 29672 - 84
Global gene expression profiling in Escherichia coli K12 . The effects of integration host factor; Arfin SM et al.; We have used nylon membranes spotted in duplicate with full-length polymerase chain reaction-generated products of each of the 4,290 predicted Escherichia coli K12 open reading frames (ORFs) to measure the gene expression profiles in otherwise isogenic integration host factor IHF(+) and IHF(-) strains . Our results demonstrate that random hexamer rather than 3' ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria . This is explained by the fact that the currently available set of 4,290 unique 3' ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing degradation rates (steady-state levels) are observed for the 25-base pair region of each message complementary to each ORF-specific primer . To evaluate the DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design . These statistical methods allowed us to identify and appropriately correct for experimental variables that affect the reproducibility and accuracy of DNA microarray measurements and allowed us to determine the statistical significance of gene expression differences between our IHF(+) and IHF(-) strains . Our results demonstrate that small differences in gene expression levels can be accurately measured and that the significance of differential gene expression measurements cannot be assessed simply by the magnitude of the fold difference . Our statistical criteria, supported by excellent agreement between previously determined effects of IHF on gene expression and the results reported here, have allowed us to identify new genes regulated by IHF with a high degree of confidence.

J Biol Chem, 2000 Sep 15, 275(37), 29100 - 6
The RAD51 family member, RAD51L3, is a DNA-stimulated ATPase that forms a complex with XRCC2; Braybrooke JP et al.; The Rad51 protein in eukaryotic cells is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination . Several proteins showing sequence similarity to Rad51 have previously been identified in both yeast and human cells . In Saccharomyces cerevisiae, two of these proteins, Rad55p and Rad57p, form a heterodimer that can stimulate Rad51-mediated DNA strand exchange . Here, we report the purification of one of the representatives of the RAD51 family in human cells . We demonstrate that the purified RAD51L3 protein possesses single-stranded DNA binding activity and DNA-stimulated ATPase activity, consistent with the presence of "Walker box" motifs in the deduced RAD51L3 sequence . We have identified a protein complex in human cells containing RAD51L3 and a second RAD51 family member, XRCC2 . By using purified proteins, we demonstrate that the interaction between RAD51L3 and XRCC2 is direct . Given the requirements for XRCC2 in genetic recombination and protection against DNA-damaging agents, we suggest that the complex of RAD51L3 and XRCC2 is likely to be important for these functions in human cells.

J Biol Chem, 2000 Oct 6, 275(40), 31239 - 44
Structural basis of hematopoietic prostaglandin D synthase activity elucidated by site-directed mutagenesis; Pinzar E et al.; Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) and conjugation of glutathione to 1-chloro-2, 4-dinitrobenzene . We introduced site-directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure . Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group . Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme . Y8F, R14K/E, and W104I were completely inactive . C156L/Y selectively lost only PGDS activity . K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH(2) in the PGDS reaction . No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased . Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0 . These results indicate that Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione.

J Biol Chem, 2000 Aug 18, 275(33), 25069 - 72
Stabilization of circular rpsT mRNA demonstrates the 5'-end dependence of RNase E action in vivo; Mackie GA; RNase E is the major intracellular endonuclease in Escherichia coli . Its ability to cleave susceptible substrates in vitro depends on both the cleavage site itself and the availability of an unstructured 5' terminus . To test whether RNase E activity is 5'-end-dependent in vivo in the presence of all the components of the RNA degradative machinery, a known substrate, the rpsT mRNA, has been embedded in a permuted group I intron to permit its efficient, precise circularization in E . coli . Circular rpsT mRNAs are 4-6-fold more stable in vivo than their linear counterparts . Even partial inactivation of RNase E activity further enhances this stability 6-fold . However, the stabilization of circular rpsT mRNAs depends strongly on their efficient translation . These results show unambiguously the importance of an accessible 5'-end in controlling mRNA stability in vivo and support a two-step ("looping") model for RNase E action in which the first step is end recognition and the second is actual cleavage.

Arch Biochem Biophys, 2000 Jun 1, 378(1), 51 - 6
Cloning, chromosomal sublocalization of the human soluble aminopeptidase P gene (XPNPEP1) to 10q25.3 and conservation of the putative proton shuttle and metal ligand binding sites with XPNPEP2; Sprinkle TJ et al.; Human soluble ("cytosolic") aminopeptidase P (hsAmP) is an aminoacylprolyl hydrolase (EC 3.4.11.9) present in all tissues yet examined . hsAmP is related in terms of catalytic specificity to an ectoenzyme, membrane aminopeptidase P (hmAmP), which is largely limited in distribution to endothelia and brush border epithelia . Although both enzymes can degrade oligopeptides having N-terminal Xaa-Pro- moieties, hsAmP and hmAmP are of relatively low sequence homology . Recently, it has been shown that the two enzymes are not products of splice variants of the same gene . How hsAmP relates to hmAmP has clinical significance in that both can inactivate bradykinin, and AmP deficiency states have been described . The hmAmP gene (XPNPEP2) is disposed at chromosome Xq25, a disposition with clear meaning in terms of inheritance of hmAmP deficiencies . To further explore similarities and differences between hsAmP and hmAmP, the present study was begun to determine the chromosomal disposition of the hsAmP gene . Here we show that the gene is sublocalized on chromosome 10q25.3 . We also show that hsAmP and hmAmP contain homologous blocks of sequence common to members of the "pita bread-fold" protein family, of which Escherichia coli methionine aminopeptidase is the prototype . The prototype is known to contain a proton shuttle and five divalent metal ligands, counterparts of which we identify in the homologous blocks of sequence in both hsAmP and hmAmP and compare to E . coli aminopeptidase.

Arch Biochem Biophys, 2000 Jun 1, 378(1), 16 - 24
Tropomodulin-binding site mapped to residues 7-14 at the N-terminal heptad repeats of tropomyosin isoform 5; Vera C et al.; Tropomodulin is a globular protein that caps the pointed end of actin filaments by complexing with the N-terminus of a tropomyosin (TM) molecule . TM consists of coiled coils except for the N-terminus, which may be globular . Here we report that human TM isoform 5 (hTM5) lacking the N-terminal 18 residues lost its binding activity toward tropomodulin . We further characterized the tropomodulin-binding site by creating a series of deletion and missense mutations within this region, followed by a solid-phase binding assay . I7, V10, and I14, hydrophobic residues located at the a and d positions of N-terminal heptad repeats involving intertwine, are essential for tropomodulin binding . R12, a positively charged residue at the f position, is also involved in recognition . In contrast, A2R and G3Y mutations, each creating a bulky N-terminus, did not alter the binding . In addition, rat TM5b, which differs from hTM5 in residues 4-6, exhibits a similar binding affinity . The tropomodulin-binding site, therefore, is mapped to residues 7-14 at the beginning of the long heptad repeats . Column chromatography revealed that hTM5 mutants remained capable of dimerization . Results also suggest tropomodulin has a groove-type, rather than a cavity-type, binding site for hTM5 . We also mapped the epitope of monoclonal antibody LC1 to residues 4-10 of hTM5 and showed the competition between mAb LC1 and tropomodulin in hTM5 binding . Since the N-terminal residues need to overlap with the C-terminus of TM in their head-to-tail association, this investigation elucidates the mechanisms by which the tropomodulin-hTM5 complex is formed and functions in regulating the actin filaments.

Eur J Epidemiol, 2000 Mar, 16(3), 287 - 93
Prevalence of enteroparasites in a residence for children in the Córdoba Province, Argentina; Guignard S et al.; A study of the prevalence of enteroparasites in a population belonging to a substitute home that gives shelter to orphaned and homeless children was done using conventional methods of analysis . This home is located in Cordoba Province, Argentina, and has the following characteristics: It has nine houses located inside the main plot of ground, that shelter 139 individuals, and 25 houses outside this plot distributed randomly in Unquillo city and that shelter 257 individuals . The overall parasitic infection, pathogen and commensal organisms included, yielded 84.8% and the prevalence of the most important parasites was: Enterobius vermicularis (43.4%), Giardia lamblia (23.0%), Ascaris lumbricoides (13.1%), Entamoeba coli (45.5%), Blastocystis hominis (44.4%) and Endolimax nana (34.6%) . We also analyzed the population dividing it according to the residence place (inside or outside the plot), age and sex of the individuals . In reference to the location of the patients, A . lumbricoides and E . coli showed significant prevalence in the individuals living inside the plot (p < 0.001 and p < 0.005, respectively) and of B . hominis in those living outside the main plot (p < 0.005) . Results indicated a greater parasitism level in the outside residents (61.5%, p < 0.001) . When the individuals were studied according to sex, no significant difference was observed, except for E . vermicularis that showed greater prevalence in the male sex (p < 0.04) . When the individuals were grouped according to age ranges, a greater prevalence in individuals from 5 to 14 years was noticed (p < 0.01) . In this study is also included an analysis of the multiparasitism level that comprises the whole population.

Mol Genet Metab, 2000 Apr, 69(4), 269 - 76
Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay; Senoo M et al.; Fetal somatic cell gene therapy could become an attractive solution for some congenital genetic diseases or the disorders which manifest themselves during the fetal period . We performed adenovirus-mediated gene transfer to mice and guinea pig fetuses in utero and evaluated the efficiency of gene transfer by histochemical analysis and a quantitative TaqMan-polymerase chain reaction (TaqMan-PCR) assay . We first injected a replication-deficient recombinant adenovirus containing the Escherichia coli LacZ gene driven by a CAG promoter (AxCALacZ) into pregnant mice through the amniotic space, placenta, or intraperitoneal space of the fetus . Histochemical analysis showed limited transgene expression in fetal tissues . We then administered AxCALacZ to guinea pig fetuses in the late stage of pregnancy through the umbilical vein . The highest beta-galactosidase expression was observed in liver followed by moderate expression in heart, spleen, and adrenal gland . The transgene expression was also present in kidney, intestine, and placenta to a lesser degree . No positively stained cells were observed in lung, muscle, or pancreas except in the vascular endothelium of these organs . Quantitative measurement of recombinant adenoviral DNA by the TaqMan-PCR assay showed that the vast majority of the injected viruses was present in liver . The current study indicated that adenovirus-mediated gene transfer into guinea pig fetus through the umbilical vein is feasible and results in efficient transgene expression in fetal tissues . The experimental procedures using pregnant guinea pigs might serve as a good experimental model for in utero gene transfer . Since our TaqMan-PCR assay detects the LacZ gene, one of the most widely used reporter genes, it may be generally applicable to adenovirus quantification in various gene transfer experiments.

Anal Biochem, 2000 Jun 1, 281(2), 202 - 8
Binding of avian ovomucoid to shiga-like toxin type 1 and its utilization for receptor analog affinity chromatography; Miyake M et al.; Development of a simple and efficient purification procedure for Shiga-like toxin I (Stx1) was attempted . Since it has been suggested that pigeon egg white ovomucoid carries a P1 antigenic determinant, we examined its ability to bind Stx1 . The ovomucoid glycoprotein fraction (GPro) was prepared from pigeon egg white by acetone precipitation, and a portion of the GPro was treated with pronase to obtain the glycopeptide fraction (GPep) . When both GPro and GPep were coupled to CNBr-activated Sepharose 4B and subjected to affinity chromatography, Stx1 specifically bound to both columns . The Stx1 eluted with a buffer containing 4.5 M MgCl2 was shown to be highly purified to homogeneity by polyacrylamide gel electrophoresis under denatured condition; only two protein bands with molecular weights of 32,000 and 8000, which correspond to the A and the B subunits of Stx1, respectively, were recognized . The purified toxin showed cytotoxicity on Vero cells with a specific activity of approximately 6 x 10(8) CD50/mg protein; almost 100% of the activity was recovered from Escherichia coli cell lysate . We propose that the utilization of avian ovmucoid for the affinity chromatography provides a potentially simple, convenient, and widely available method to purify Shiga-like toxins.

Spine, 2000 Jul 1, 25(13), 1729 - 32
Postoperative synergistic gangrene after spinal fusion; Kauffman CP et al.; STUDY DESIGN: A case of synergistic necrotizing gangrenous fasciitis after spinal surgery is reported . OBJECTIVES: To describe this unusual complication, explain the rationale of treatment, and increase awareness of this serious postoperative complication . SUMMARY OF BACKGROUND DATA: Although several cases of postoperative synergistic necrotizing fasciitis have been reported, there are no previously reported cases of this condition after spinal surgery . METHODS: A rapidly progressive necrotizing spinal wound infection after fusion for degenerative disc disease was treated in a 39-year-old man . RESULTS: The infection was successfully treated with serial debridements, appropriate antibiotics, and hyperbaric wound oxygenation . CONCLUSIONS: The authors suggest adherence to the fundamental principles of treatment including radical surgical debridement and appropriate antibiosis for necrotizing gangrene after spinal surgery . In evaluation of aggressive spinal wound infections, diagnosis of synergistic necrotizing fasciitis should be kept in mind . Although hyperbaric wound oxygenation was implemented as an adjunct and appeared to aid in controlling the infection, its effect on outcome is not clear.

Domest Anim Endocrinol, 2000 May, 18(4), 363 - 78
Canine thyrotropin beta-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells; Yanga X et al.; The gene encoding the mature beta subunit of canine thyroid stimulating hormone (cTSH beta) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSH beta purified from E . coli were generated . The gene fragment that encodes mature TSH beta was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species . The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSH beta gene and an intron of 450 bp . The two exons, which encode the mature cTSH beta subunit, was joined together by an overlap PCR and was expressed in E . coli as 6xHis-tagged protein . The purified recombinant cTSH beta with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot . Monoclonal antibodies were raised against the purified cTSH beta and subsequently characterized . For transient expression in CHO cells that are permanently transfected with the bovine common alpha gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5' end of the cTSH beta gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells . The medium from these transfected cells, presumably containing the bovine alpha and canine TSH beta in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.

FEBS Lett, 2000 Jun 23, 475(3), 170 - 4
Identification of an optimal Ncx binding sequence required for transcriptional activation; Shimizu H et al.; The Ncx gene encodes a homeobox containing transcription factor that belongs to the Hox11 gene family . We determined specific Ncx protein binding consensus DNA sequences . Optimal Ncx binding sequences were 5'-CGGTAATTGG-3' (TAAT core) and 5'-CGGTAAGTGG-3' (TAAG core), which coincided with the Hox11 binding sequence . Both Ncx and Hox11 could bind to the TAAT and the TAAG core oligonucleotide in vitro . However, they could efficiently transactivate the reporter plasmid linked to the TAAT core sequence but not to the TAAG core sequence . Thus, Ncx and Hox11 act as transcriptional activators via their target sequence, 5'-CGGTAATTGG-3'.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7790 - 5
Interaction of the iron-sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli; Hoff KG et al.; The iscU gene in bacteria is located in a gene cluster encoding proteins implicated in iron-sulfur cluster assembly and an hsc70-type (heat shock cognate) molecular chaperone system, iscSUA-hscBA . To investigate possible interactions between these systems, we have overproduced and purified the IscU protein from Escherichia coli and have studied its interactions with the hscA and hscB gene products Hsc66 and Hsc20 . IscU and its iron-sulfur complex (IscU-Fe/S) stimulated the basal steady-state ATPase activity of Hsc66 weakly in the absence of Hsc20 but, in the presence of Hsc20, increased the ATPase activity up to 480-fold . Hsc20 also decreased the apparent K(m) for IscU stimulation of Hsc66 ATPase activity, and surface plasmon resonance studies revealed that Hsc20 enhances binding of IscU to Hsc66 . Surface plasmon resonance and isothermal titration calorimetry further showed that IscU and Hsc20 form a complex, and Hsc20 may thereby aid in the targeting of IscU to Hsc66 . These results establish a direct and specific role for the Hsc66/Hsc20 chaperone system in functioning with isc gene components for the assembly of iron-sulfur cluster proteins.

J Pharmacol Exp Ther, 2000 Jun, 293(3), 996 - 1001
Preclinical and clinical evidence for disappearance of long-circulating characteristics of polyethylene glycol liposomes at low lipid dose; Laverman P et al.; This study describes the effect of the lipid dose of (99m)Tc-polyethylene glycol (PEG) liposomes in the low-dose range (0 . 02-1.0 micromol/kg) on the pharmacokinetics and biodistribution in rats, rabbits, and humans . The biodistribution and pharmacokinetics of (99m)Tc-PEG liposomes at various dose levels were studied in rats and rabbits with a focal Escherichia coli infection . Scintigraphic images were recorded on a gamma camera . In addition, the role of macrophages in the biodistribution of a low-dose PEG liposome injection was studied . Finally, the pharmacokinetics of (99m)Tc-PEG liposomes at two lipid dose levels was studied in four patients . At a dose level of 0.03 micromol/kg, the blood level in rats at 4 h postinjection was significantly lower than at the highest dose level (1.1 micromol/kg) . The same effect was observed in rabbits where enhanced clearance was observed at a dose level of 0.02 micromol/kg . The circulatory half-life decreased from 10.4 to 3.5 h (at 1.0 and 0 . 02 micromol/kg, respectively) . At the lowest dose level, liposomes were mainly taken up by the liver and to a lesser extent by the spleen . Injection of a low dose of PEG liposomes in macrophage-depleted rabbits resulted in normal pharmacokinetics, suggesting involvement of macrophages in the effectuation of the rapid elimination of the liposomes from the circulation . Most importantly, the rapid clearance of low-dose PEG liposomes was also observed in humans when relatively low lipid doses were administered . This study showed that at very low lipid doses the biodistribution of PEG liposomes is dramatically altered.

Biochemistry, 2000 Jul 4, 39(26), 7764 - 71
Identification of catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by gamma-monofluorophosphono derivative of glutamic acid: N-terminal thr-391 in small subunit is the nucleophile; Inoue M et al.; gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione metabolism and catalyzes the hydrolysis and transpeptidation of gamma-glutamyl compounds such as glutathione and its derivatives . The reaction is thought to proceed via a gamma-glutamyl-enzyme intermediate where a hitherto unknown catalytic nucleophile is gamma-glutamylated . Neither affinity labeling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile . We describe here the identification of the catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride derivative of glutamic acid . Compound 1 rapidly inactivated the enzyme in a time-dependent manner (k(on) = 4.83 x 10(4) M(-1) s(-1)) . The inactivation rate was decreased by increasing the concentration of the substrate . The inactivated enzyme did not regain its activity after prolonged dialysis, suggesting that 1 served as an active-site-directed affinity label by phosphonylating the putative catalytic nucleophile . Ion-spray mass spectrometric analyses revealed that one molecule of 1 phosphonylated one molecule of the small subunit . LC/MS experiments of the proteolytic digests of the phosphonylated small subunit identified the N-terminal peptide Thr391-Lys399 as the phosphonylation site . Subsequent MS/MS experiments of this peptide revealed that the phosphonylated residue was Thr-391, the N-terminal residue of the small subunit . We conclude that the N-terminal Thr-391 is the catalytic nucleophile of E . coli gamma-glutamyltranspeptidase . This result strongly suggests that gamma-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family.

Nucleic Acids Res, 2000 Jul 1, 28(13), 2535 - 40
Recognition of GT mismatches by Vsr mismatch endonuclease; Fox KR et al.; The Vsr mismatch endonuclease recognises the sequence CTWGG (W = A or T) in which the underlined thymine is paired with guanine and nicks the DNA backbone on the 5'-side of the mispaired thymine . By using base analogues of G and T we have explored the functional groups on the mismatch pair which are recognised by the enzyme . Removal of the thymine 5-methyl group causes a 60% reduction in activity, while removing the 2-amino group of guanine reduces cleavage by 90% . Placing 2-amino-purine or nebularine opposite T generates mis-matches which are cut at a much lower rate (0.1%) . When either base is removed, generating a pseudoabasic site (1', 2'-dideoxyribose), the enzyme still produces site-specific cleavage, but at only 1% of the original rate . Although TT and CT mismatches at this position are cleaved at a low rate (approximately 1%), mismatches with other bases (such as GA and AC) and Watson-Crick base pairs are not cleaved by the enzyme . There is also no cleavage when the mismatched T is replaced with difluorotoluene.

Nucleic Acids Res, 2000 Jul 1, 28(13), 2527 - 34
Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix(Ala) variants; Nagan MC et al.; Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA(Ala) has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the 'discriminator' base at position 73 . Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix . We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M) . Despite lacking N7, this analog is well tolerated by AlaRS . Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis . Additional MD simulations of wild-type microhelix(Ala) and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity . For the two DeltaN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.

Nucleic Acids Res . 2000 Jun 15;28(12):E65.
Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli; Imam AM et al.; We report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host . High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations . No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.COLI: RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.

Nucleic Acids Res . 2000 Jun 15;28(12):E64.
New chromatographic and biochemical strategies for quick preparative isolation of tRNA; Cayama E et al.; A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity . The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation . Optimal combinations are presented for three different cases . (i) tRNA(Phe) from Escherichia coli . This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC . (ii) tRNA(Ile) from E . coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC . The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity . (iii) tRNA(i)(Met) from Saccharomyces cerevisiae . The aminoacylated form of this tRNA is unstable . To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation . These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.

Nucleic Acids Res . 2000 Jun 15;28(12):E57.
Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method; Levy MS et al.; A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described . The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA . Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation . For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared . The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative . During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations . Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form . In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.

Nucleic Acids Res, 2000 Jun 15, 28(12), 2324 - 32
Characterisation of the catalytically active form of RecG helicase; McGlynn P et al.; Replication of DNA is fraught with difficulty and chromosomes contain many lesions which may block movement of the replicative machinery . However, several mechanisms to overcome such problems are beginning to emerge from studies with Escherichia coli . An important enzyme in one or more of these mechanisms is the RecG helicase, which may target stalled replication forks to generate a four-stranded (Holliday) junction, thus facilitating repair and/or bypass of the original lesion . To begin to understand how RecG might catalyse regression of fork structures, we have analysed what the catalytically active form of the enzyme may be . We have found that RecG exists as a monomer in solution as measured by gel filtration but when bound to junction DNA the enzyme forms two distinct protein-DNA complexes that contain one and two protein molecules . However, mutant inhibition studies failed to provide any evidence that RecG acts as a multimer in vitro . Additionally, there was no evidence for cooperativity in the junction DNA-stimulated hydrolysis of ATP . These data suggest that RecG functions as a monomer to unwind junction DNA, which supports an 'inchworm' rather than an 'active rolling' mechanism of DNA unwinding . The observed in vivo inhibition of wild-type RecG by mutant forms of the enzyme was attributed to occlusion of the DNA target and correlates with the very low abundance of replication forks within an E.COLI: cell, even during rapid growth.

Nucleic Acids Res . 2000 Jun 1;28(11):E51.
Early melting of supercoiled DNA topoisomers observed by TGGE; Viglasky V et al.; We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (sigma) . We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs . The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity . Supercoiled topo-isomers represent a system of molecules that is sensitive to changes in temperature . We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values . Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress . The estimated melting temperature for plasmids with sigma < -0.05 is very significant because these temperatures for early melting are below physiological temperatures.

Nucleic Acids Res . 2000 Jun 1;28(11):E50.
Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates; Fromenty B et al.; Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology . However, long PCR amplification fails whenever the DNA template is imperfectly preserved . We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates . Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99 degrees C), depurination at low pH and near-UV radiation . Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method . Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome . Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or TAQ: plus PWO: DNA polymerases . However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples . In conclusion, supplementation of long PCR mixes with E.COLI: exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.

Nucleic Acids Res, 2000 Jun 1, 28(11), 2207 - 13
Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding; Rothwell DG et al.; HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells . Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme . Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity approximately 500-fold . Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1 . These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1-D210E protein has a 3000-fold reduced K(cat )for AP site cleavage, but an unchanged K(m) . Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1-substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein . We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.

J Bacteriol, 2000 Jul, 182(14), 4124 - 7
All major regions of FtsK are required for resolution of chromosome dimers; Boyle DS et al.; Resolution of chromosome dimers, by site-specific recombination between dif sites, is carried out in Escherichia coli by XerCD recombinase in association with the FtsK protein . We show here that a variety of altered FtsK polypeptides, consisting of the N-terminal (cell division) domain alone or with deletions in the proline-glutamine-rich part of the protein, or polypeptides consisting of the C-terminal domain alone are all unable to carry out dif recombination . Alteration of the putative nucleotide-binding site also abolishes the ability of FtsK to carry out recombination between dif sites.

J Bacteriol, 2000 Jul, 182(14), 4117 - 20
SprE levels are growth phase regulated in a sigma(S)-dependent manner at the level of translation; Gibson KE et al.; SprE regulates sigma(S) levels in response to nutrient availability by promoting ClpXP-mediated degradation . Paradoxically, we observe that SprE is similarly regulated, accumulating preferentially upon starvation . This regulation of SprE levels is sigma(S) dependent, altering SprE synthesis at the level of translation . Thus, we demonstrate that SprE and sigma(S) function within a regulatory feedback loop.

J Bacteriol, 2000 Jul, 182(14), 4108 - 12
SecB dependence of an exported protein is a continuum influenced by the characteristics of the signal peptide or early mature region; Kim J et al.; We have used Escherichia coli alkaline phosphatase to show the interplay among the characteristics of two amino-terminal domains in the preprotein (the signal peptide and the early mature region), the efficiency with which this protein is transported, and its requirement for SecB to accomplish the transport process . The results suggest that although alkaline phosphatase does not normally require SecB for transport, it is inherently able to utilize SecB, and it does so when its ability to interface with the transport machinery is compromised.

J Bacteriol, 2000 Jul, 182(14), 4068 - 76
Green fluorescent protein functions as a reporter for protein localization in Escherichia coli; Feilmeier BJ et al.; The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein {MBP}) and a variant of gfp optimized for fluorescence in bacteria (GFPuv) . These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP . Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence . In contrast, when the MBP signal sequence was deleted, fluorescence was observed . Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space . Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation . Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway . The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations . While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells . Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm . These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.

J Bacteriol, 2000 Jul, 182(14), 4028 - 34
Slow polymerization of Mycobacterium tuberculosis FtsZ; White EL et al.; The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified . The recombinant protein has GTPase activity typical of tubulin and other FtsZs . FtsZ polymerization was studied using 90 degrees light scattering . The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E . coli FtsZ . Depolymerization also occurs slowly, taking 1 h or longer under most conditions . Polymerization requires both Mg(2+) and GTP . The minimum concentration of FtsZ needed for polymerization is 3 microM . Electron microscopy shows that polymerized M . tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments . Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido{4,3-b}{1,4}diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M . tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

J Bacteriol, 2000 Jul, 182(14), 4022 - 7
Mobilization of chimeric oriT plasmids by F and R100-1: role of relaxosome formation in defining plasmid specificity; Fekete RA et al.; Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro . We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo . Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene . The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids . The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays . In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence . Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT . High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization . Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

J Bacteriol, 2000 Jul, 182(14), 4012 - 21
Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype; Hamrick TS et al.; Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized . FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner . Point mutations were located by DNA sequencing and deletion mapping . All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein . All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein . Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants . The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent . Consequently, this mutant was chosen for further study . Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change . Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures . Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C . The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.

J Bacteriol, 2000 Jul, 182(14), 3981 - 8
Rho-dependent transcription termination in the tna operon of Escherichia coli: roles of the boxA sequence and the rut site; Konan KV et al.; Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination . Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon . Induction requires translation of a 24-residue leader peptide-coding region, tnaC, containing a single, crucial Trp codon . Studies with a lacZ reporter construct lacking the tnaC-tnaA spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in cis on the ribosome translating tnaC to inhibit its release . The stalled ribosome is thought to block Rho's access to the transcript . In this paper we examine the roles of the boxA sequence and the rut site in Rho-dependent termination . Deleting six nucleotides (CGC CCT) of boxA or introducing specific point mutations in boxA results in high-level constitutive expression . Some constitutive changes introduced in boxA do not change the TnaC peptide sequence . We confirm that deletion of the rut site results in constitutive expression . We also demonstrate that, in each constitutive construct, replacement of the tnaC start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the tnaC coding sequence . Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action . Combining a boxA point mutation with rut site deletion results in constitutive expression comparable to that of a maximally induced operon . These results support the hypothesis that in the presence of tryptophan the ribosome translating tnaC blocks Rho's access to the boxA and rut sites, thereby preventing transcription termination.

J Bacteriol, 2000 Jul, 182(14), 3972 - 80
Effect of CIS on activity in trans of the replication initiator protein of an IncB plasmid; Praszkier J et al.; RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in cis . The cis activity of RepA is thought to be mediated by CIS, a 166-bp region of DNA separating the coding region of repA from the origin of replication (ori) of pMU720 . To investigate the trans activity of RepA, the repA gene, without its cognate ori, was cloned on a multicopy plasmid, pSU39 . The ori on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) . Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned ori by RepA produced in trans from the pSU39 derivatives . The effect of CIS, when present either on the RepA-producing or the ori plasmid or both, on the efficiency of replication of the ori plasmid in vivo, was determined . The presence of CIS, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of the ori plasmid . This inhibitory activity of CIS was sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA . By contrast, CIS had no effect when present on the ori plasmid . Initiation of replication from the ori in trans was independent of transcription into CIS.

J Bacteriol, 2000 Jul, 182(14), 3965 - 71
Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ; Hu Z et al.; In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by the min system . MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE . In this study we found that MinC consists of two functional domains connected by a short linker . When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro . The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts min function, resulting in a minicell phenotype . We also find that MinC is an oligomer, probably a dimer . Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization . These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD . The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

J Bacteriol, 2000 Jul, 182(14), 3942 - 7
Identification of Escherichia coli dnaE (polC) mutants with altered sensitivity to 2',3'-dideoxyadenosine; Hiratsuka K et al.; Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S . A . Rashbaum and N . R . Cozzarelli, Nature 264:679-680, 1976) . We have used this observation as the basis of a genetic strategy to identify mutations in the dnaE (polC) gene of Escherichia coli that alter sensitivity to 2',3'-dideoxyadenosine (ddA) . Two dnaE (polC) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized . The mutant phenotypes are due to single amino acid substitutions in the alpha subunit, the protein product of the dnaE (polC) gene . Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution . The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype) . All of the amino acid substitutions are in conserved regions near essential aspartate residues . These results prove the effectiveness of the genetic strategy in identifying informative dnaE (polC) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the alpha subunit of the DNA polymerase III holoenzyme.

Bioinformatics, 2000 Mar, 16(3), 212 - 21
Net nearest neighbor analysis (NNNA) summarizes non-compensated dinucleotides within gene sequences; Lang DM; MOTIVATION: Net Nearest Neighbor Analysis (NNNA) measures a previously unexamined aspect of dinucleotide frequency-the non-compensated, non-repetitive dinucleotides in a sequence . Non-compensated dinucleotides are those in excess of their corresponding reverse dinucleotides . RESULTS: NNNA regards dinucleotides as vector quantities, making it possible to summarize any sequence as a group of circuits and tags . The results of NNNA are found to be consistent with traditional analytic methods, yet reveal additional characteristics of the sequences . The NNNA circuits and tags uniquely identify each tRNA in Escherichia coli K-12 and certain structural components of each tRNA, extract function-specific characteristics for each of the sequences involved in the formation of insulin from preinsulin, and exhibit species-specific phylogenetic characterization (demonstrated with MONILINIA:) . AVAILABILITY: Nearest neighbor analysis software has been available for many years and is a component of most gene analysis software packages, including the Staden Package which is available at no charge to academic users .

Hybridoma, 2000 Apr, 19(2), 185 - 9
Mouse monoclonal antibodies to hepatitis B virus preS1 produced after immunization with recombinant preS1 peptide; Ryu CJ et al.; We have efficiently generated mouse monoclonal antibodies (MAbs), which bind specifically to amino acids 21-47 of the preS1 domain of hepatitis B virus (HBV) by immunizing mice with the preS1 peptide (amino acids, aa 1-56) conjugated to keyhole limpet hemocyanin . Hybridomas were screened by an indirect enzyme-linked immunosorbent assay (ELISA) using the purified preS1 peptide as a coated antigen . Eighteen positive hybridomas were selected and subjected to isotyping . Of these, 5 clones secreted immunoglobulin G (IgG) and 13 clones secreted IgM . Four (KR1, KR2, KR3, and KR4) of the 5 IgG MAbs bound to preS1 peptide (aa 21-47) . Epitope mapping using bacterially expressed GST fusion proteins revealed that three clones (KR2, KR3, KR4) (IgG1, K) recognize aa 21-35, while KR1 (IgG2a, K) recognizes aa 35-47 of the preS1 . These MAbs immunoprecipitated HBV particles, demonstrating that they bind to native HBV particles.

Vet Res Commun, 2000 Jul, 24(5), 327 - 38
Studies on the mechanism of diarrhoea induced by Escherichia coli heat-stable enterotoxin (STa) in newborn calves; Al-Majali AM et al.; Enterotoxigenic Escherichia coli (ETEC) produces a heat-stable enterotoxin (STa) that binds to and activates a putative intestinal receptor, guanylate cyclase, causing an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP) . Using flow cytometry and 125I-STa binding assays, we studied the distribution of STa-receptors on enterocytes isolated from different segments of the newborn calf's intestinal tract . We also investigated the effect of STa on the intracellular levels of cGMP and ion transport to the intestinal lumen . More STa-receptors were found on enterocytes prepared from the ileum than on enterocytes obtained from the other segments of the intestinal tract . Guanylate cyclase activity was higher in the ileum of STa-challenged calves than in the ileum of control calves . No changes were observed in the guanylate cyclase activity of the other intestinal segments of the STa-challenged and control calves . Na+ levels, as measured by atomic absorption spectroscopy, were significantly increased in the luminal contents of the ileum of STa-challenged calves, whereas serum Cl- levels were significantly lower in the STa-challenged calves than in control calves . This study supports previous observations on the role of guanylate cyclase in the initiation of STa-induced secretory diarrhoea and suggests that Na+/Cl- coupling may be the major mechanism for the loss of ions in the diarrhoeal response that is mostly induced in the ileum of newborn calves.

Arch Dermatol Res, 2000 May, 292(5), 217 - 24
Development of a system for detection of circulating antibodies against hemidesmosomal proteins in patients with bullous pemphigoid; Husz S et al.; Specific antibodies directed against special hemidesmosomal proteins are involved in the pathogenesis of bullous pemphigoid (BP), and detection of these antibodies is crucial for a correct diagnosis . As the BP autoantigen primary structures are known, the question was addressed as to whether it is possible to demonstrate circulating antibodies against BP autoantigens (BPAG1 and BPAG2) by means of an ELISA system, using antigenic epitopes . With the help of the programs Peptidestructure and Plotstructure, antigenic epitopes of BP antigens were predicted, chemically synthesized and screened using serum from 43 proven BP patients . The coding sequences of the best antigenic epitopes were then chemically synthesized and inserted as monomer and homo- or hetero-oligomer forms into fusion-expression plasmids (PGEX-4T, Pharmacia) in-frame to the C-terminus of glutathione-S-transferase . Fusion products were expressed and purified from Escherichia coli cells by affinity chromatography . The recombinant proteins were used for the detection of antibodies in the serum of 43 BP patients and of 60 controls (including 30 healthy persons, 22 patients with pemphigus vulgaris and 8 patients with other bullous dermatoses) . Use of the homo- and hetero-oligomers of the recombinant fusion peptides increased the sensitivity of the disease-specific antibody detection . When a mixture of the best recombinant fusion proteins was used, the sensitivity of the ELISA assays in the case of the BP patients' serum was 0.90 . This system could form the basis of a rapid and simple system for the diagnosis of BP.

FEMS Microbiol Lett, 2000 Jul 1, 188(1), 97 - 101
Interaction of omega-conotoxin and the membrane calcium transport system of Escherichia coli; Tisa LS; omega-Conotoxin, a calcium channel blocker, inhibits chemotaxis by Escherichia coli . To test whether omega-conotoxin acts at the cytoplasmic membrane, the kinetics of 125I-omega-conotoxin binding was investigated . 125I-omega-Conotoxin bound to Tris-EDTA-permeabilized cells or right-side-out membrane vesicles with saturation kinetics . Binding of 125I-omega-conotoxin to membrane vesicles was inhibited by Ca(2+) ions, but not by Mg(2+) ions . The calA mutant, defective in calcium transport, was more resistant to omega-conotoxin inhibition of chemotaxis than the parental wild-type . 125I-omega-Conotoxin binding to membrane vesicles indicated that both the wild-type and the calA mutant had similar K(D)s for omega-conotoxin binding . However, the saturation level was higher with the calA mutant, indicating that there are more binding sites in the calA mutant . Thus, calA does not directly affect the affinity of the omega-conotoxin binding site . Chemical cross-linking experiments identified two proteins as potential omega-conotoxin receptors.

FEMS Microbiol Lett, 2000 Jul 1, 188(1), 55 - 61
Cloning, sequencing, and expression in Escherichia coli of a cytochrome P450 gene from Cunninghamella elegans; Wang RF et al.; A polyclonal antibody against microsomes of a fungus, Cunninghamella elegans, was used to screen a C . elegans cDNA library . A cDNA clone, containing an open reading frame (ORF) encoding a protein of 389 amino acids (aa), was obtained . GenBank comparison (BLAST) showed that the protein was closely related to P450 because a heme-binding region, which is highly conserved in all P450 sequences, was found in the ORF protein . Using an oligo probe designed from this C . elegans heme-binding region to rescreen the cDNA library, we obtained three new clones . Sequence comparison showed that the three clones, with different length cDNA inserts, were from the same mRNA of the C . elegans P450 gene . One clone had the full C . elegans P450 gene, encoding 473 aa with a molecular mass of 54958.60, whereas the 389 was a part of the 473 aa without the N-terminal . The entire C . elegans P450 gene was successfully subcloned and overexpressed in a plasmid-Escherichia coli system (pQE30) . Immunostaining with three antibodies (CYP1A1, CYP2E1, and CYP3A1) against mammalian P450 enzymes and benzidine staining for hemoproteins showed positive results for the recombinant protein expressed in E . coli . A phylogenetic tree was constructed by comparison of other fungal P450s to the C . elegans sequence . The C . elegans P450 clustered close to the cyp51 family and was named cyp509A1 by the International Committee on the Nomenclature for Cytochrome P450 Enzymes.

Virus Res, 2000 Apr, 67(2), 119 - 25
Selection of a specific peptide from a nona-peptide library for in vitro inhibition of grass carp hemorrhage virus replication; Wang B et al.; Grass carp hemorrhage virus (GCHV), a member of Reoviridae, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon idellus) in China . Icosahedral virions of GCHV were used as to assay the effect of specific peptides for the inhibition of GCHV infectivity . A random nona-peptide library displayed on phage fUSE5 was constructed, and the expressed peptides were fused onto the amino terminus of the minor coat protein III . By biopanning, the fused peptides were bound to the biotinylated GCHV . Phages containing specific peptides bound to GCHV were eluted and amplified in Escherichia coli K91 . Three rounds of affinity selection enriched the pool of inhibiting peptides . Sixteen clones which inhibited the replication of GCHV in a grass carp kidney cell line were selected . The TCID(50) of GCHV was decreased over 10000x . Six clones having the strongest inhibitory effect shared the same DNA sequence, with a deduced amino acid sequence of NH(2)-Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala-COOH . A synthesized nona-peptide of identical sequence exhibited similar inhibitory activity towards GCHV replication in vitro.

Antiviral Res, 2000 Jun, 46(3), 181 - 93
A novel high throughput screening assay for HCV NS3 helicase activity; Hicham Alaoui-Ismaili M et al.; A novel assay for measurement of Hepatitis C virus (HCV) NS3 helicase activity was developed using Flashplate technology . This assay involves the use of a DNA duplex substrate and recombinant HCV NS3 produced in Escherichia coli . The DNA duplex consisted of a pair of oligonucleotides, one biotinylated, the other radiolabeled at their respective 5' termini . This DNA duplex was immobilized, via the biotin molecule, on the surface of a neutravidin-coated SMP103 Flashplate (NEN Life Science Products) . Helicase activity results in the release of the radiolabeled oligonucleotide, which translates in signal reduction with respect to control wells . Biochemical characterization of the HCV NS3 helicase activity was performed using this assay . We demonstrated that the NS3-mediated unwinding is proportional to both the amount of DNA substrate in the well, and to the NS3 concentration in the reaction . Most of the NS3-mediated unwinding was achieved in the initial 60 min of incubation . As expected the reactions were ATP-dependent and found to be affected by the concentration of MgCl(2), MnCl(2), KCl, EDTA, and by pH . We found this assay to be highly reproducible since only slight variation was observed when a total of 68 helicase reactions were performed on one plate . Therefore, this Flashplate helicase assay is fast, convenient and reproducible . These criteria make it suitable for high throughput screening of potential NS3 helicase inhibitors.

J Biol Chem, 2000 Sep 8, 275(36), 27559 - 65
Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei . Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase; Duffieux F et al.; Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes . So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes . Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e . glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases . These genes were cloned and sequenced . Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic) . However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes . The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.

Biophys J, 2000 Jul, 79(1), 479 - 84
Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy; Berge T et al.; Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences . The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation . DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes . Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme . Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA . Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site . Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization . This event is followed by ATP-dependent translocation and cleavage of the DNA.

Eur J Biochem, 2000 Jul, 267(13), 4264 - 71
Overproduction of Thermus sp . YS 8-13 manganese catalase in Escherichia coli production of soluble apoenzyme and in vitro formation of active holoenzyme; Mizobata T et al.; Overproduction of Thermus sp . YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site . E . coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies . Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion . When the E . coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction . However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures . Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.

Mol Cell Biol, 2000 Jul, 20(14), 5196 - 207
Identification of functionally important domains in the N-terminal region of telomerase reverse transcriptase; Xia J et al.; Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats . The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase-like motifs that presumably mediate catalysis . These motifs are located in the C-terminal region of the polypeptide . Hidden Markov model-based sequence analysis revealed in the N-terminal region of all TERTs the presence of four conserved motifs, named GQ, CP, QFP, and T . Point mutation analysis of conserved residues confirmed the functional importance of the GQ motif . In addition, the distinct phenotypes of the GQ mutants suggest that this motif may play at least two distinct functions in telomere maintenance . Deletion analysis indicates that even the most N-terminal nonconserved region of yeast TERT (N region) is required for telomerase function . This N region exhibits a nonspecific nucleic acid binding activity that probably reflects an important physiologic function . Expression studies of various portions of the yeast TERT in Escherichia coli suggest that the N region and the GQ motif together may constitute a stable domain . We propose that all TERTs may have a bipartite organization, with an N-GQ domain connected to the other motifs through a flexible linker.

Cancer Res, 2000 Jun 15, 60(12), 3147 - 51
Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay; Han SY et al.; The tumor suppressor gene PTEN is frequently mutated in diverse human cancers and in autosomal dominant cancer predisposition disorders . Recent studies have shown that the lipid phosphatase activity of PTEN is critical for its tumor suppressor function and that PTEN negatively regulates the phosphatidylinositol 3'-kinase-protein kinase B pathway . Although more than half of PTEN mutations result in protein truncation, a significant fraction of PTEN mutations are missense mutations . To examine whether tumor-derived and germ-line-derived missense mutations inactivate PTEN lipid phosphatase function, we constructed 42 distinct types of PTEN missense mutations and expressed them in Escherichia coli . The purified (His)6-tagged PTEN proteins were tested for their ability to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-triphosphate . In addition, we examined the effect of mutant PTENs on the ability of PTEN to bind to the phospholipid membrane . The results revealed that the majority of PTEN missense mutations {38 of 42 (90%)} eliminated or reduced phosphatase activity and that all of the mutations examined had no effect on the membrane binding activity of PTEN . Our study indicated that phosphoinositide phosphatase activity is important for the tumor suppressor function of PTEN and that there may be other mechanisms of PTEN inactivation that are not monitored by in vitro phosphatase assay and in vitro membrane binding assay.

J Antibiot (Tokyo), 2000 Apr, 53(4), 373 - 84
ABC transporter genes, kasKLM, responsible for self-resistance of a kasugamycin producer strain; Ikeno S et al.; We previously reported that a 7.6-kb DNA fragment from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM acetyltransferase gene (kac338) and some other genes possibly involved in KSM biosynthesis . As an extension of that study, a 10-kb SacI-KpnI DNA fragment, located approximately 5-15-kb upstream of kac33, was cloned and a 4.2-kb SacI-EcoRI fragment therefrom was sequenced, revealing one incomplete (designated ORF J) and three complete open reading frames (designated kasK, kasL and kasM) . The coding frames of kasK, L and M overlap one another with terminator/initiator ATGA sequence . RT-PCR analysis of a DNA region including kasKLM indicated the presence of one transcript that is long enough to span the three genes . The kasK gene potentially encodes an ATP-binding protein of the ATP-binding cassette (ABC) transporter superfamily . Homology search for the deduced KasK protein shows similarity to other ABC transporters involved in self-resistance of a mithramycin and possibly doxorubicin producer strain . The kasL and kasM genes encode different integral membrane proteins, both having six putative transmembrane helices . An expression plasmid for kasKLM (pTV-KLM) was constructed and these genes were expressed in E . coli JM 109, which had been sensitive to KSM . The transformant acquired resistance to KSM, suggesting that KasK, L and M proteins as a set in S . kasugaensis M338-M1 pump out KSM to protect the producer from its toxic metabolite.

FEMS Immunol Med Microbiol, 2000 Jul, 28(3), 219 - 24
Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis; Maeda H et al.; Porphyromonas gingivalis, a putative pathogen in human periodontal disease, possesses a 60-kDa heat shock protein (hsp60, GroEL) . The GroEL homologs are known to be key molecules in auto-immune reactions because of the sequence similarity with human hsp60 . In this study, B-cell epitopes on P . gingivalis GroEL (PgGroEL) were analyzed by both Western immunoblotting with truncated PgGroEL and by the multi-pin synthetic peptide approach . To examine auto-antibody production in periodontitis patients, Western immunoblotting with human gingival fibroblasts was performed . Deletion mutants were constructed from the cloned PgGroEL gene (P . gingivalis groEL), and four C-terminal truncated PgGroEL and one N-terminal truncated PgGroEL were prepared from the deletants . Sera from periodontitis patients reacted with all truncated PgGroEL used in this study . The results suggest that the B-cell epitopes were overlaid throughout PgGroEL . To determine the detailed locations of the B-cell epitope, 84 decapeptides covering the entire PgGroEL were synthesized and the serum IgG response to the peptides was examined . Epitope mapping using the synthetic peptides confirmed that the B-cell epitopes were overlaid throughout the length of PgGroEL and revealed that highly conserved peptides between PgGroEL and human hsp60 were recognized by the serum antibodies . Immuno-reactivity against human gingival fibroblasts was examined with sera from 30 periodontitis patients and 10 periodontally healthy subjects . IgG antibody against the 65-kDa antigen in human gingival fibroblasts (same molecular mass as human hsp60) was detected in two patients . Although IgG production against human hsp60 may be rare case in periodontitis patients, the results of epitope mapping demonstrated the potential of PgGroEL to cause the cross-reactions with human hsp60.

FEMS Immunol Med Microbiol, 2000 Jul, 28(3), 197 - 203
Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells; Kawai Y et al.; The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported . OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells . In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS . OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum . However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum . OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells . These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.

Vet Microbiol, 2000 Jul 3, 75(1), 99 - 106
Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies; Subramaniam S et al.; The 36kDa L-lactate dehydrogenase (LDH) and a 29kDa partial fragment of an ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potential as diagnostic antigens . Recombinant LDH was genetically engineered to contain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni(2+)-chelate affinity chromatography . A partial 262 amino acid segment representing the C-terminal end of the PR2 protein was cloned as a glutathione S-transferase (GST) fusion protein, expressed in E . coli and purified by urea extraction . Purified recombinant LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assays . Our immunoblots showed that both proteins detected anti-M . hyopneumoniae antibodies in field and experimentally infected pig sera but not in any of the SPF control sera . The two proteins were specific for M . hyopneumoniae as they did not react with sera of pigs infected with the closely related Mycoplasma flocculare and Mycoplasma hyorhinis which are frequently isolated in pigs but are not of particular concern.

Vet Microbiol, 2000 Jul 3, 75(1), 59 - 71
Persistence of cellulitis-associated Escherichia coli DNA fingerprints in successive broiler chicken flocks; Singer RS et al.; Avian cellulitis in broiler chickens is primarily caused by Escherichia coli . Previous research found that the E . coli isolates of cellulitis origin were unique to each ranch, suggesting that these E . coli were endemic within the ranch environment . To test the hypothesis that the E . coli associated with cellulitis are endemic in the litter of the broiler house, we designed a study to determine whether E . coli DNA fingerprints associated with cellulitis persist over successive flocks that are grown in the same house . In addition, we assessed the impact of different cleaning and disinfection strategies on this persistence . Two broiler houses were followed on each of five farms over 3-4 flocks . A total of 353 E . coli isolates from cellulitis lesions were analyzed in this study, and 314 of these isolates (89%) were DNA fingerprinted by PFGE . In each ranch, there were several DNA fingerprint patterns that were present over successive flocks, regardless of the cleaning and disinfection strategy utilized . Isolates persisted as long as 191 days, implying that these E . coli are capable of persisting in the broiler house environment for long periods of time . In addition, these E . coli isolates were associated with cellulitis lesions in successive flocks . Thus, the isolates of E . coli that are associated with cellulitis in broiler chickens appear to be endemic in the litter environment of the broiler house.

Vet Microbiol, 2000 Jul 3, 75(1), 11 - 6
Localization of linear B-cell epitopes on infectious bronchitis virus nucleocapsid protein; Seah JN et al.; The nucleocapsid (N) protein of many viruses is highly conserved, immunogenic, and abundantly expressed during infection . These features make it a suitable candidate for diagnostic applications . The nucleocapsid protein of infectious bronchitis virus (IBV) was dissected into 12 fragments and expressed in Escherichia coli . Sera against Australia T, China Ch5, Singapore P4, USA M41 and China T3 isolates were used to study the conservation and localization of the antigenic region on the IBV nucleocapsid protein . Our results show linear immunodominant epitopes, which were found in three fragments covering amino acid residues 175-241, 310-370 and 360-409.

J Virol, 2000 Jul, 74(14), 6494 - 500
Effects of amino acid substitutions at position 115 on the fidelity of human immunodeficiency virus type 1 reverse transcriptase; Boyer PL et al.; We compared the fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and two RT mutants, Y115F and Y115V . Although neither mutation had a large effect on the overall fidelity of the enzyme, both mutations altered the spectrum of mutations and the precise nature of the mutational hot spots . The effects of Y115V were greater than those of Y115F . When we compared the behavior of the wild-type enzyme with published data, we found that, in contrast to what has been published, misalignment/slippage could account for only a small fraction of the mutations we observed . We also found that a preponderance of the mutations (both transitions and transversions) resulted in the insertion of an A . Because we were measuring DNA-dependent DNA synthesis (plus-strand synthesis), this bias could contribute to the A-rich nature of the HIV-1 genome.

Exp Parasitol, 2000 May, 95(1), 63 - 70
Fasciola hepatica: heterologous expression and functional characterization of a thioredoxin peroxidase; Salazar-Calderon M et al.; A Fasciola hepatica cDNA clone of 779 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens . The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 582 bp which encoded a 194-amino-acid-residue polypeptide (M(r) 21,723 Da) showing a high degree of homology to thioredoxin peroxidases . This putative antioxidant protein gene was expressed in Escherichia coli as a GST fusion protein . The recombinant fusion protein showed in vitro antioxidant properties and protected rabbit muscle enolase and E . coli glutamine synthetase from inactivation by nonenzymatic Fe(3+)/O(2)/DTT or Fe(3+)/O(2)/ascorbate metal-catalyzed oxidation systems.

Exp Parasitol, 2000 May, 95(1), 54 - 62
Trichomonas vaginalis: characterization, expression, and phylogenetic analysis of a carbamate kinase gene sequence; Minotto L et al.; The gene encoding carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) from Trichomonas vaginalis has been sequenced and its expression in this protozoon has been verified using reverse-transcription polymerase chain reaction . The codon usage and percentage nucleotide composition in the coding and noncoding regions are consistent with other genes isolated from this parasite . Phylogenetic analysis of this gene has suggested possible speciation events that are congruent with other studies, with suggestions of lateral gene transfer . The gene was expressed in Escherichia coli using the pQE-30 expression system, and the recombinant protein was purified using affinity chromatography . The expression of the recombinant protein was identified via Western blotting and matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides . Preliminary kinetic assays have revealed that the recombinant enzyme has a K(m) similar to that of the native enzyme and size-exclusion chromatography has shown that the enzyme is active as the homodimer.

J Mol Biol, 2000 Jun 30, 300(1), 197 - 212
Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein; Mueller GA et al.; The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods . The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa . Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues . Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints . Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints . A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively . Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations . As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above . The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS . Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology . When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A .

J Mol Biol, 2000 Jun 30, 300(1), 187 - 96
ATPase cycle of an archaeal chaperonin; Gutsche I et al.; Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT . Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome . Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites . Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*P(i) species prior to the release of the gamma-phosphate group . After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes . The effect of Mg(2+) and K(+) nucleotide cycling is documented . We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins .

J Mol Biol, 2000 Jun 30, 300(1), 113 - 26
Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides; Morosyuk SV et al.; Nucleotides 680 to 710 of Escherichia coli 16 S rRNA form a distinct structural domain required for ribosome function . The goal of this study was to determine the functional significance of pairing interactions in the 690 region . Two different secondary structures were proposed for this hairpin, based on phylogenetic and chemical modification studies . To study the effect of pairing interactions in the 690 hairpin on ribosome function and to determine which of the proposed secondary structures is biologically significant, we performed an instant-evolution experiment in which the nine nucleotides that form the proposed base-pairs and dangling ends of the 690 stem were randomly mutated, and functional mutant combinations were selected . A total of 96 unique functional mutants were isolated, assayed in vivo, and sequenced . Analysis of these data revealed extensive base-pairing and stacking interactions among the mutated nucleotides . Formation of either a Watson-Crick base-pair or G.U pair between positions 688 and 699 is absolutely required for ribosome function . We also performed NMR studies of a 31-nucleotide RNA which indicate the formation of a functionally important base-pair between nucleotides 688 and 699 . Formation of a second base-pair between positions 689 and 698, however, is not essential for ribosome function, but the level of ribosome function correlates with the predicted thermodynamic stability of the nucleotide pairs in these positions . The universally conserved positions G690 and U697 are generally portrayed as forming a G.U mismatch . Our data show co-variation between these positions, but do not support the hypothesis that the G690:U697 pair forms a wobble structure . NMR studies of model 14-nt and 31-nt RNAs support these findings and show that G690 and U697 are involved in unusual stacking interactions but do not form a wobble pair . Preliminary NMR structural analysis reveals that the loop portion of the 690 hairpin folds into a highly structured and novel conformation .

J Mol Biol, 2000 Jun 30, 300(1), 103 - 12
Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus; Ruigrok RW et al.; The matrix protein VP40 of Ebola virus is believed to play a central role in viral assembly as it targets the plasma membrane of infected cells and subsequently forms a tightly packed layer on the inner side of the viral envelope . Expression of VP40 in Escherichia coli and subsequent proteolysis yielded two structural variants differing by a C-terminal truncation 114 amino acid residues long . As indicated by chemical cross-linking studies and electron microscopy, the larger polypeptide was present in a monomeric form, whereas the truncated one formed hexamers . When analyzed for their in vitro binding properties, both constructs showed that only monomeric VP40 efficiently associated with membranes containing negatively charged lipids . Membrane association of truncated, hexameric VP40 was inefficient, indicating a membrane-recognition role for the C-terminal part . Based on these observations we propose that assembly of Ebola virus involves the formation of VP40 hexamers that is mediated by the N-terminal part of the polypeptide .

J Mol Biol, 2000 Jun 30, 300(1), 75 - 82
Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins; Santamaria D et al.; In Escherichia coli plasmids carrying two inversely oriented ColE1 origins, DNA replication initiates at only one of the two potential origins . The other silent origin acts as a replication fork barrier . Whether this barrier is permanent or simply a pausing site remains unknown . Here, we used a repeated primer extension assay to map in vivo, at the nucleotide level, the 5' end of the nascent strand where initiation and blockage of replication forks occurs . Initiation occurred primarily at the previously defined origin, however, an alternative initiation site was detected 17 bp upstream . At the barrier, the lagging strand also terminated at the main initiation site . Therefore, the 5' end of the nascent strand at the barrier was identical to that generated during initiation . This observation strongly suggests that blockage of the replication fork at the silent origin is not just a pausing site but permanent, and leads to a premature termination event .

Arch Biochem Biophys, 2000 Jul 1, 379(1), 137 - 46
Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction; Williams DC et al.; The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol . Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids . Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired . Since the transit peptide-mature enzyme cleavage site could not be determined directly, a series of N-terminally truncated enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated . Deletion of up to 79 residues yielded functional protein; however, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus . The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme . In addition to the major product, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%) . Isotopically sensitive branching experiments utilizing (4R)-{4-(2)H(1)}geranylgeranyl diphosphate confirmed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation . These results, along with the failure of the enzyme to utilize 2, 7-cyclogeranylgeranyl diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure) . Two potential mechanism-based inhibitors were tested with recombinant taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition .

Biol Pharm Bull, 2000 Jun, 23(6), 700 - 3
Study of the beta1 adrenergic receptor expression in human tissues: immunological approach; Hellgren I et al.; Questions exist regarding tissue distribution of the beta1 adrenergic receptor (beta1-AR) . The aim of this study was to investigate relative distribution patterns of the beta1-AR at the protein level in a variety of human tissues by Western blot analysis . The specificity of anti-peptide antibodies was confirmed both by Western blot with recombinant beta1-AR expressed as a membrane protein in E . coli and by immunoprecipitation of membranes from Sf9 cells infected with baculovirus to express the human recombinant beta1-AR . beta1-AR was found in all tissues examined . The relative amount of protein varied significantly between the tissues, from highest in lung and testis to very low in liver . beta1-ARs were rather abundant in heart, kidney, placenta, spleen and thyroid . These results reveal unique distribution of beta1-AR protein that suggests its tissue specific role . Moreover, our data demonstrate a high sensitivity of immunological detection that allows direct comparison of beta1-AR subtype expression and could be used for receptor study in biopsies available in limited amounts, such as human heart biopsy.

Biol Pharm Bull, 2000 Jun, 23(6), 695 - 9
Inhibitory effects of green tea and grape juice on the phenol sulfotransferase activity of mouse intestines and human colon carcinoma cell line, Caco-2; Tamura H et al.; Tea and fruit juices are beverages consumed daily all over the world . The present study reports the inhibitory effects of these beverages on the activity of mammalian intestinal phenol sulfotransferases (P-STs) . Green tea strongly inhibited the E . coli-expressed mouse intestinal P-ST activity in vitro . (-)-Epigallocatechin gallate (EGCG) was found to be the most potent inhibitor among the catechins tested (IC50=0.93 microM) . (-)EGCG also inhibited the P-ST activity of the human colon carcinoma cell line, Caco-2 . Kinetic analysis showed that the inhibition was competitive . Among fruit juices examined (apple, grape, grapefruit and orange), grape juice exhibited the most potent inhibitory action on the P-ST activity of mouse intestines and human colon carcinoma cells . The inhibitory activity of grape juice was located mainly in the skin and seeds . Flavonols, such as quercetin and kaempferol, inhibited the P-ST activity at low concentrations . These observations suggest the possible inhibition of P-ST activity in human intestines by green tea or grape juice.

Ann N Y Acad Sci, 2000, 899, 69 - 87
Genetic responses to free radicals . Homeostasis and gene control; Gonzalez-Flecha B et al.; Gene regulation mechanisms have evolved allowing cells to finetune the level of "endogenous" oxidative stress and to cope with increased free radicals from external sources . Levels of H2O2 are tightly controlled in E . coli by OxyR, which is activated by H2O2 to increase scavenging activities and limit H2O2 generation by the respiratory chain . Sub-micromolar levels of H2O2 are maintained in mammalian tissues, though the regulatory systems that govern this control are unknown . Excess superoxide triggers the soxRS system in E . coli, which is controlled by the oxidant-sensitive iron-sulfur centers of the SoxR protein . Nitric oxide activates SoxR by a different modification of the iron-sulfur centers . The soxRS regulon mobilizes diverse functions to scavenge free radicals and repair oxidative damage in macromolecules, and other mechanisms that exclude many environmental agents from the cell . Mammalian cells also sense and respond to sub-toxic levels of nitric oxide, activating expression of heme oxygenase 1 through stabilization of its mRNA . These inductions give rise to adaptive resistance to nitric oxide in neuronal and other cell types.

Chin J Dent Res, 1999 May, 2(2), 14 - 8
cDNA cloning and sequencing of MH2 domain of Smad2 from human dental pulp cells; Chen J et al.; OBJECTIVE: The purpose of this study was to clone and sequence the cDNA of MH2 domain of Smad2 from human dental pulp cells . METHODS: In this study, total RNA was isolated from primary cultured human dental pulp cells and reverse-transcribed into cDNA . The desired DNA product was obtained by nested PCR with 4 smad2 MH2 domain-specific primers . The segment was inserted into pBluescript II SK vector and the resulting plasmid was transformed into E . coli JM109 . The double-strand cDNA of positive clone was sequenced by PE317-A automatic sequencing . RESULTS: cDNA of MH2 domain of Smad2 was obtained from human dental pulp cells . The sequence was consistent with that cloned from a human kidney cDNA library . No mutation was found . CONCLUSION: This study provides the first evidence of expression of smad2 in human dental pulp cells, and indicates that TGF-beta signaling may be mediated by Smad2 in human dental pulp cells . The cDNA cloned in pBluescript/S2MH2 could be used for further functional studies of Smad2 and MH2 domain in dental pulp cells.

Biochimie, 2000 Mar, 82(3), 211 - 9
Nucleotide sequence analysis, overexpression in Escherichia coli and kinetic characterization of Anacystis nidulans catalase-peroxidase; Engleder M et al.; Bifunctional catalase-peroxidases are the least understood type of peroxidases . A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301) is reported . Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the examination of both the spectral characteristics and the reactivity of its redox intermediates by using the multi-mixing stopped-flow technique . The homodimeric acidic protein (pI = 4.6) contained high catalase activity (apparent K(m) = 4.8 mM and apparent k(cat) = 8850 s(-1)) . Cyanide is shown to be an effective inhibitor of the catalase reaction . The second-order rate constant for cyanide binding to the ferric protein is (6.9 +/- 0.2) x 10(5) M(-1 )s(-1) at pH 7.0 and 15 degrees C and the dissociation constant of the cyanide complex is 17 microM . Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation . The apparent second-order rate constant for formation of compound I from the ferric enzyme and peroxoacetic acid is (1.3 +/- 0.3) x 10(4 )M(-1 )s(-1) at pH 7.0 and 15 degrees C . The spectrum of compound I is characterized by about 40% hypochromicity, a Soret region at 406 nm, and isosbestic points between the native enzyme and compound I at 355 and 428 nm . Rate constants for reduction of KatG compound I by o-dianisidine, pyrogallol, aniline and isoniazid are shown to be (7.3 +/- 0.4) x 10(6) M(-1 )s(-1), (5.4 +/- 0.3) x 10(5) M(-1 )s(-1), (1.6 +/- 0.3) x 10(5) M(-1 )s(-1) and (4.3 +/- 0.2) x 10(4) M(-1 )s(-1), respectively . The redox intermediate formed upon reduction of compound I did not exhibit the classical red-shifted peroxidase compound II spectrum which characterizes the presence of a ferryl oxygen species . Its spectral features indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.

Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 237 - 46
Reactive-site specificity of human kallistatin toward tissue kallikrein probed by site-directed mutagenesis; Chen VC et al.; Kallistatin is a serine proteinase inhibitor that forms complexes with tissue kallikrein and inhibits its activity . In this study, we compared the inhibitory activity of recombinant human kallistatin and two mutants, Phe388Arg (P1) and Phe387Gly (P2), toward human tissue kallikrein . Recombinant kallistatins were expressed in Escherichia coli and purified to apparent homogeneity using metal-affinity and heparin-affinity chromatography . The complexes formed between recombinant kallistatins and tissue kallikrein were stable for at least 150 h . Wild-type kallistatin as well as both Phe388Arg and Phe387Gly mutants act as inhibitors and substrates to tissue kallikrein as analyzed by complex formation . Kinetic analyses showed that the inhibitory activity of Phe388Arg variant toward tissue kallikrein is two-fold higher than that of wild type (P1Phe), whereas Phe387Gly had only 7% of the inhibitory activity toward tissue kallikrein as compared to wild type . The Phe388Arg variant but not wild type inhibited plasma kallikrein's activity . These results indicate that P1Arg variant exhibits more potent inhibitory activity toward tissue kallikrein while wild type (P1Phe) is a more selective inhibitor of tissue kallikrein . The P2 phenylalanine is essential for retaining the hydrophobic environment for the interaction of kallistatin and kallikrein.

J Biol Chem, 2000 Sep 8, 275(36), 27762 - 7
Characterization of a novel 8-oxoguanine-DNA glycosylase activity in Escherichia coli and identification of the enzyme as endonuclease VIII; Hazra TK et al.; 8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A . The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic . However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A . This could be carried out by a second OGG, OGG2, identified in yeast and human cells . We have characterized a new OGG activity in E . coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase . Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G) . Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil . The presence of OGG2 type enzyme in both E . coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand.

J Biol Chem, 2000 Sep 8, 275(36), 28093 - 9
Structural basis for the feedback regulation of Escherichia coli pantothenate kinase by coenzyme A; Yun M et al.; Pantothenate kinase (PanK) is a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the phosphorylation of pantothenic acid to form phosphopantothenate . CoA is a feedback inhibitor of PanK activity by competitive binding to the ATP site . The structures of the Escherichia coli enzyme, in complex with a nonhydrolyzable analogue of ATP, 5'-adenylimido-diphosphate (AMPPNP), or with CoA, were determined at 2.6 and 2.5 A, respectively . Both structures show that two dimers occupy an asymmetric unit; each subunit has a alpha/beta mononucleotide-binding fold with an extensive antiparallel coiled coil formed by two long helices along the dimerization interface . The two ligands, AMPPNP and CoA, associate with PanK in very different ways, but their phosphate binding sites overlap, explaining the kinetic competition between CoA and ATP . Residues Asp(127), His(177), and Arg(243) are proposed to be involved in catalysis, based on modeling of the pentacoordinate transition state . The more potent inhibition by CoA, compared with the CoA thioesters, is explained by a tight interaction of the CoA thiol group with the side chains of aromatic residues, which is predicted to discriminate against the CoA thioesters . The PanK structure provides the framework for a more detailed understanding of the mechanism of catalysis and feedback regulation of PanK.

J Biol Chem, 2000 Sep 8, 275(36), 28157 - 66
GATA zinc finger interactions modulate DNA binding and transactivation; Trainor CD et al.; GATA-1 and other vertebrate GATA factors contain a DNA binding domain composed of two adjacent homologous zinc fingers . Whereas only the C-terminal finger of GATA-1 is capable of independent binding to the GATA recognition sequence, double GATA sites that require both fingers for high affinity interaction are found in several genes . We propose a mechanism whereby adjacent zinc fingers interact to influence the binding and transactivation properties of GATA-1 at a subset of DNA-binding sites . By using two such double GATA sites we demonstrate that the N-terminal finger and adjacent linker region can alter the binding specificity of the C-terminal finger sufficiently to prevent it from recognizing some consensus GATA sequences . Therefore, the two zinc fingers form a composite binding domain having a different DNA binding specificity from that shown by the constituent single C-terminal finger . Furthermore, we compare two of these double sites and show that high affinity binding of GATA-1 to a reporter gene does not necessarily induce transactivation, namely the sequence of the DNA-binding site can alter the ability of GATA-1 to stimulate transcription.

Enzyme Microb Technol, 2000 Jul 1, 27(1-2), 19 - 25
Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli; Chao Y et al.; With L-aspartate (L-Asp) as the amino donor, L-phenylalanine (L-Phe) can be prepared from phenylpyruvate (PPA) via an amination reaction mediated by aminotransferase (encoded by aspC) . On the other hand, L-Asp can be produced by an aspartase (encoded by aspA) -catalyzed reaction using fumaric acid as substrate . To overproduce aspartase in Escherichia coli, the aspA gene was cloned and overexpressed 180 times over the wild-type level . The use of AspA-overproducing E . coli strain for L-Asp production exhibited an 83% conversion, approaching to the theoretical yield, whereas the wild-type strain obtained scarcely L-Asp . Furthermore, the recombinant strain overproducing both AspA and AspC was able to produce L-Asp and L-Phe simultaneously by using fumaric acid and PPA as substrates . As a result, the conversion yields obtained for L-Asp and L-Phe were 78% and 85%, respectively . In sharp contrast, the wild-type strain attained a conversion of L-Phe less than 15% and an undetectable level of L-Asp . This result illustrates a potential and attractive process to yield both L-Asp and L-Phe by coupling AspA and AspC . A further study on the repeated use of the recombinant strain immobilized with calcium alginate showed that after eight batch runs L-Asp conversion maintained roughly constant (around 75%), whereas L-Phe conversion dropped to 65% from 81% . This result indicates the stability of AspA being superior to AspC.

J Clin Invest, 2000 Jun, 105(12), 1769 - 77
Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells; Steiner TS et al.; Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide . EAEC infections are associated with intestinal inflammation and growth impairment in infected children, even in the absence of diarrhea . We previously reported that prototype EAEC strains rapidly induce IL-8 production by Caco-2 intestinal epithelial cells, and that this effect is mediated by a soluble, heat-stable factor released by these bacteria in culture . We herein report the cloning, sequencing, and expression of this biologically active IL-8-releasing factor from EAEC, and its identification as a flagellin that is unique among known expressed proteins . Flagella purified from EAEC 042 and several other EAEC isolates potently release IL-8 from Caco-2 cells; an engineered aflagellar mutant of 042 does not release IL-8 . Finally, cloned EAEC flagellin expressed in nonpathogenic E . coli as a polyhistidine-tagged fusion protein maintains its proinflammatory activity . These findings demonstrate a major new means by which EAEC may cause intestinal inflammation, persistent diarrhea, and growth impairment that characterize human infection with these organisms . Furthermore, they open new approaches for diagnosis and vaccine development . This novel pathogenic mechanism of EAEC extends an emerging paradigm of bacterial flagella as inflammatory stimuli.

Biotechnol Bioeng, 2000 Aug 20, 69(4), 461 - 7
A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration; Sarney DB et al.; A new easily scalable approach to the recovery of biologically active oligosaccharides from milk has been developed which relies on the combination of enzymatic treatment of defatted milk using beta-galactosidase and nanofiltration . It was shown that enzymatic hydrolysis of lactose significantly improves the efficiency and selectivity of membrane-based separations . With the best membrane, as much as 6.7 g of oligosaccharides (containing very little contaminating lactose) could be obtained from one liter of defatted human milk in just four nanofiltration cycles . The human milk oligosaccharides recovered by this method were shown to inhibit binding of intimin, an adhesion molecule of enteropathogenic Escherichia coli, to epithelial cells in vitro . No significant difference in the oligosaccharide profile between samples prepared by this method and conventional gel-permeation chromatography was found . The developed approach is also suitable for the recovery of substantial quantities of tri- and tetra-saccharides from caprine milk .

Biotechnol Bioeng, 2000 Aug 20, 69(4), 418 - 28
Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor; Kim DY et al.; The specific molecular interactions of alpha-helical peptide, human glucagon (i.e., intermolecular self-association and specific receptor-binding affinity) provided a rationale for using the glucagon as the fusion expression partner to achieve high productivity of foreign proteins both in vivo (in bacterial fusion-expression system) and in vitro (in affinity column chromatography) . The fusion of glucagon peptide(s) effectively promoted homogeneous aggregate formation of recombinant proteins while avoiding intermolecular crosslinking by disulfide bridges . High sensitivity of the self-aggregation to sequence effects resulted from two distinct nonpolar domains of glucagon, determining specificity of molecular interaction and aggregate size of recombinant proteins . An N-terminal domain of glucagon molecule (Phe6-Tyr10-Tyr13) could be a certain hydrophobic moiety involved in intermolecular self-association (probably, via helix-helix docking), while a C-terminal domain (Phe22-Trp25-Leu26) seems to critically affect the oligomer size in the off-pathway aggregation of synthesized fusion proteins . An N-terminal extracellular domain of human glucagon receptor was recombinantly expressed in Escherichia coli, immobilized to a chromatography column, and efficiently renatured to a conformation that attains high specificity in interaction with N-terminus glucagon molecules of recombinant fusion proteins . Through column chromatography employing the receptor fragment as affinity ligand, the recombinant proteins were efficiently purified from total intracellular proteins, and the long-term ligand stability was evidently proven through multiple cyclic-purification experiments . Major scaffolds for using protein ligands are large-scale production in a low-cost expression system and long-term stable operation with selective-binding affinity . From this point of view, the extracellular fragment of human glucagon receptor used in this study seems to be a new potent ligand for fusion protein-based affinity chromatography .

J Biol Chem, 2000 Sep 8, 275(36), 27957 - 63
Conformational requirements of collagenous peptides for recognition by the chaperone protein HSP47; Koide T et al.; The collagen binding chaperone HSP47 interacts with procollagen in the endoplasmic reticulum and plays a crucial role in the biosynthesis of collagen . We recently demonstrated that typical collagen model peptides, (Pro-Pro-Gly)(n), possess sufficient structural information for interaction with HSP47 (Koide, T., Asada, S., and Nagata, K . (1999) J . Biol . Chem . 274, 34523-34526) . Here we show that binding of (Gly-Pro-Pro)(n) peptides to HSP47 can be detected using the two-hybrid system in yeast if a trimerizing domain is fused to the C termini of the peptides . Some peptides interacted with HSP47 at a lowered assay temperature at 24 degrees C but not at 30 degrees C, indicating the importance of conformational change of the substrate peptides . To analyze the spectrum of HSP47 substrate sequences, we performed two-hybrid screening of collagen-like peptides in designed random peptide libraries using HSP47 as a bait . In selected peptides, the enrichment ratio calculated for each amino acid residue correlated strongly with the contribution of the residue to triple-helix stability independently determined using synthetic collagen model peptides . Taken together, our results suggest that HSP47 preferentially recognizes collagenous Gly-X-Y repeats in triple-helical conformation . We also demonstrated that screening of combinatorial peptide libraries is a powerful strategy to determine conformational requirements as well as the elucidation of binding motifs in primary structure.

Pharmacogenetics, 2000 Jun, 10(4), 343 - 53
Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol; Li DN et al.; Most drug metabolizing cytochrome P450s (P450) are predominantly expressed in the liver . In contrast, human CYP1B1 is an extrahepatic P450 which is overexpressed in many tumours and has been strongly implicated in the activation of carcinogens . Rare allelic variants of the CYP1B1 gene which encode an inactive protein have been identified . However, four polymorphisms which most likely do not abolish functionality have been described . In this report, we have characterized the functional consequences of these . A CYP1B1 cDNA, identical to a cDNA published previously, served as a template to introduce allelic changes either separately or in combination . The resulting effects on CYP1B1 activity were determined in membranes isolated from Escherichia coli which coexpressed CYP1B1 together with P450 reductase . None of the allelic changes affected the CYP1B1 expression level . The allelic changes Arg48 to Gly, Ala19 to Ser and Asn453 to Ser had little influence on the Vmax and the Km of the CYP1B1 mediated 2- and 4-hydroxylation of estradiol . In contrast, the Km of these metabolic pathways was increased at least three-fold by the allelic change Va432 to Leu or by simultaneously changing Val432 to Leu and Asn453 to Ser . However, these alterations had little effect on the kinetic parameters of other CYP1B1 mediated reactions such as the epoxidation of (-)-trans-(7R,8R)-benzo{a}pyrene 7,8-dihydrodiol as determined by (r-7,t-8,t-9,c-10)-benzo{a}pyrene tetraol formation, or such as the O-dealkylation of ethoxyresorufin and the 1'-hydroxylation of bufuralol . Molecular modelling suggests that amino acid residue 432 of CYP1B1 may be involved in the interaction between CYP1B1 and P450 reductase . Since 4-hydroxyestradiol has been implicated in hormonal carcinogenesis and CYP1B1 is expressed in target tissues, the data presented demonstrate that polymorphisms in CYP1B1 have the potential to affect disease susceptibility.

Mol Reprod Dev, 2000 Jul, 56(3), 424 - 35
Cloning, sequencing, and expression analysis of mouse glucosamine-6-phosphate deaminase (GNPDA/oscillin); Amireault P et al.; It was reported that a hamster protein, called "oscillin," with a sequence related to that of an Escherichia coli GNPDA triggered Ca(2+) oscillations in mammalian oocytes when introduced into their cytoplasm upon fertilization . Recently, it was shown that GNPDA/oscillin is ubiquitously expressed in rat tissues and that a recombinant hamster GNPDA/oscillin protein does not exhibit oscillin activity when injected into oocytes . In the mouse, the nature and role of such a GNPDA/oscillin is not known, but another candidate protein, tr-kit, has been proposed as a sperm factor causing oocyte activation . In order to clarify this issue, we have characterized the mouse homolog of hamster and human GNPDA/oscillin, and examined its expression along with that of tr-kit, in parallel . We report here the molecular cloning and sequencing of mouse GNPDA/oscillin, which shows over 96% identity with the hamster and human homologs . Using specific primers, we performed an RT-PCR analysis to determine the tissue distribution of mouse GNPDA/oscillin mRNA . Unlike tr-kit mRNA which is expressed solely in mouse testis, GNPDA/oscillin mRNA is detected in unfertilized oocytes and in all tissues examined including testis, heart, thymus, liver, ovary, uterus, kidney, spleen, and lung . The protein itself is also detected in all tissues examined by Western blots . Indirect immunofluorescence studies, using an antibody raised against hamster GNPDA, demonstrate that GNPDA is lost with the acrosome reaction of mouse spermatozoa, is localized in the equatorial and neck regions of the human spermatozoa and the post-acrosomal region of the hamster spermatozoa . Our results thus indicate that mouse GNPDA/oscillin, the homolog of hamster oscillin, unlike tr-kit, does not exhibit some of the required characteristics expected from a putative sperm-derived oocyte-activating factor .

Environ Mol Mutagen, 2000, 35(4), 328 - 35
Activation of MeIQ (2-amino-3,4-dimethylimidazo- {4,5-f}quinoline) by sequence variants of recombinant human cytochrome P450 1A2; Josephy PD et al.; Understanding the relationships between the sequences and catalytic activities of P450 enzymes that catalyze the bioactivation of mutagens and carcinogens is an important goal in mutation research . Escherichia coli strain DJ4309 expresses recombinant human P450 1A2 and activates promutagens such as MeIQ (2-amino-3, 4-dimethylimidazo{4,5-f}quinoline), as measured by induction of reverse mutations detected as lacZ(+) colonies on minimal lactose (ML) plates . Pools of P450 1A2 mutants were constructed by polymerase chain reaction (PCR) mutagenesis of putative substrate recognition sites (SRSs) . Cultures of individual clones were patched onto MeIQ/ML plates and the growth of revertant microcolonies within each patch was inspected after 2 days of incubation . Beginning with a pool of several thousand clones, we identified 25 distinct P450 1A2 SRS variants with altered activities . In this study, the MeIQ dose-responses of all the variants are reported . The implications of the results are considered with reference to published models of the protein's structure .

Biotechnol Bioeng, 2000 Aug 5, 69(3), 275 - 85
Framework for online optimization of recombinant protein expression in high-cell-density Escherichia coli cultures using GFP-fusion monitoring; Chae HJ et al.; A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-cell-density cultivation of Escherichia coli . A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E . coli JM105 was developed . A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity . The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-beta-D-thiogalactopyranoside (IPTG) induction system . Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay) . Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized .

Biochem J, 2000 Jul 1, 349(Pt 1), 141 - 51
Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity; Dobransky T et al.; Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms . In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases . Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme . Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively . Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h . By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus . Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity . The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity . Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity . These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.

J Immunol, 2000 Jul 1, 165(1), 473 - 82
Differential involvement of Src family kinases in Fc gamma receptor-mediated phagocytosis; Suzuki T et al.; The tyrosine phosphorylation cascade originated from Fc gamma receptors (Fc gamma Rs) is essential for macrophage functions including phagocytosis . Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined . In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc gamma R-mediated signaling that leads to phagocytosis in a kinase-dependent manner . We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope . Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc gamma R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis . In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling . Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc gamma RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck . These findings suggest that Src family kinases are differentially involved in Fc gamma R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.

J Mol Biol, 2000 Jun 2, 299(2), 431 - 46
Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases; Sherlin LD et al.; The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops . E . coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair . To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases . The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition . Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair . Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition . By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48 . Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS . To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS . These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15 . The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases .

J Mol Biol, 2000 Jun 2, 299(2), 391 - 403
Coordinated control of XerC and XerD catalytic activities during Holliday junction resolution; Arciszewska LK et al.; Site-specific recombinases XerC and XerD function in the segregation of circular bacterial replicons . In a recombining nucleoprotein complex containing two molecules each of XerC and XerD, coordinated reciprocal switches in recombinase activity ensure that only XerC or XerD is active at any one time . Mutated recombinases that carry sub?stitutions of a catalytic arginine residue stimulate cleavage and strand exchange mediated by the partner recombinase on DNA substrates that are normally recombined poorly by the partner . This is associated with a reciprocal impairment of the recombinase's own ability to initiate catalysis . The extent of this switch in catalysis is modulated by changes in recombination site sequence and is not a direct consequence of any catalytic defect . We propose that altered interactions between the mutated proteins and their wild-type partners lead to an increased level of an alternative Holliday junction intermediate that has a conformation appropriate for resolution by the partner recombinase . The results indicate how subtle changes in protein-DNA architecture at a Holliday junction can redirect recombination outcome .

J Mol Biol, 2000 Jun 2, 299(2), 311 - 24
Interactions between activating region 3 of the Escherichia coli cyclic AMP receptor protein and region 4 of the RNA polymerase sigma(70) subunit: application of suppression genetics; Rhodius VA et al.; The Escherichia coli cyclic AMP receptor protein, CRP, induces transcription at Class II CRP-dependent promoters by making three different activatory contacts with different surfaces of holo RNA polymerase . One contact surface of CRP, known as Activating Region 3 (AR3), is functional in the downstream subunit of the CRP dimer and is predicted to interact with region 4 of the RNAP sigma(70) subunit . We have previously shown that a mutant CRP derivative that activates transcription primarily via AR3, CRP HL159 KE101 KN52, requires the positively charged residues K593, K597 and R599 in sigma(70) for activation . Here, we have used the positive control substitution, EK58, to disrupt AR3-dependent activation by CRP HL159 KE101 KN52 . We then screened random mutant libraries and an alanine scan library of sigma(70) for candidates that restore activation by CRP HL159 KE101 KN52 EK58 . We found that changes at R596 and R599 in sigma(70) can restore activation by CRP HL159 KE101 KN52 EK58 . This suggests that the side-chains of both R596 and R599 in sigma(70) clash with K58 in CRP . Maximal activation by CRP HL159 KE101 KN52 EK58 is achieved with the substitutions RE596 or RD596 in sigma(70) . We propose that there are specific charge-charge interactions between E596 or D596 in sigma(70) and K58 in AR3 . Thus, no increase in activation is observed in the presence of another positive control substitution, EG58 (CRP HL159 KE101 KN52 EG58) . Similarly, both sigma(70) RE596 and sigma(70) RD596 can restore activation by CRP EK58 but not CRP EG58, and they both decrease activation by wild-type CRP . We suggest that E596 and D596 in sigma(70) can positively interact with K58 in AR3, thereby enhancing activation, but negatively interact with E58, thereby decreasing activation . The substitution, KA52 in AR3 increases Class II CRP-dependent activation by removing an inhibitory lysine residue . However, this increase is not observed in the presence of either sigma(70) RE596 or sigma(70) RD596 . We conclude that the inhibitory side-chain, K52 in AR3, clashes with R596 in sigma(70) . Finally, we show that the sigma(70) RE596 and RD596 substitutions affect CRP-dependent activation from Class II, but not Class I, promoters .

J Mol Biol, 2000 Jun 2, 299(2), 295 - 310
Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3; Rhodius VA et al.; At Class II CRP-dependent promoters, the Escherichia coli cyclic AMP receptor protein (CRP) activates transcription by making multiple interactions with RNA polymerase (RNAP) . Two discrete surfaces of CRP, known as Activating Region 1 (AR1) and Activating Region 2 (AR2), interact with the C-terminal and N-terminal domains, respectively, of the alpha subunit of RNAP . Activating Region 3 (AR3) is a third separate surface of CRP, which is thought to interact with a target in the C-terminal region of the RNAP sigma(70) subunit . We have used a CRP mutant that functions primarily via AR3, CRP HL159 KE101 KN52, as a tool to identify residues within AR3 that are important for activation . This was achieved by screening a random mutant library of the gene encoding CRP HL159 KE101 KN52 for positive control mutants at Class II CRP-dependent promoters, and also by performing alanine scanning mutagenesis . Using both in vivo reporter assays and in vitro transcription assays, we measured the effects of key substitutions within AR3 on transcription activation in both CRP HL159 KE101 KN52 and wild-type CRP . We show that a cluster of negatively charged surface-exposed residues at positions 53, 54, 55 and 58 is required for optimal activation at a Class II, but not at a Class I, CRP-dependent promoter . We conclude that these residues in AR3 of CRP form an activatory determinant for Class II transcription activation . Abortive initiation assays were used to show that this activatory determinant accelerates the rate of isomerisation from the closed to open complex at a Class II CRP-dependent promoter . AR3 of CRP also contains an inhibitory determinant: the lysine residue at position 52 of CRP is inhibitory to maximal levels of transcription activation from Class II promoters . We show that the negative effects of K52 are not simply due to "masking" of the negatively charged residues at positions 53, 54, 55 and 58 . Our results suggest that, during activation by wild-type CRP, the activatory and inhibitory determinants of AR3 balance each other . Thus, activation is predominantly determined by AR1 and AR2 .

J Mol Biol, 2000 May 26, 299(1), 199 - 212
Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module; Gruez A et al.; Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide . Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups . The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction . We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein . The structure can be divided into three domains . The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH . These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family . By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module . In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein . Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase . The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer . Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.

J Mol Biol, 2000 May 26, 299(1), 91 - 101
Mutations in the N-terminal region of RecA that disrupt the stability of free protein oligomers but not RecA-DNA complexes; Eldin S et al.; We have introduced targeted mutations in two areas that make up part of the RecA subunit interface . In the RecA crystal structure, cross-subunit interactions are observed between the Lys6 and Asp139 side-chains, and between the Arg28 and Asn113 side-chains . Unexpectedly, we find that mutations at Lys6 and Arg28 impose sever defects on the oligomeric stability of free RecA protein, whereas mutations at Asn113 or Asp139 do not . However, Lys6 and Arg28 mutant proteins showed an apparent normal formation of RecA-DNA complexes . These results suggest that cross-subunit contacts in this region of the protein are different for free RecA protein filaments versus RecA-DNA nucleoprotein filaments . Mutant proteins with substitutions at either Lys6 or Arg28 show partial inhibition of DNA strand exchange activity, yet the mechanistic reasons for this inhibition appear to be distinct . Although Lys6 and Arg28 appear to be more important to the stability of free RecA protein, as opposed to the stability of the catalytically active nucleoprotein filament, our results support the idea that the cross-subunit interactions made by each residue play an important role in optimizing the catalytic organization of the active RecA oligomer.

J Mol Biol, 2000 May 26, 299(1), 75 - 89
DNA melting and promoter clearance by eukaryotic RNA polymerase I; Kahl BF et al.; Ribosomal RNA transcription initiation requires the melting of DNA to form an open complex, formation of the first few phosphodiester bonds, commencement of RNA polymerase I movement along the DNA, clearance of the promoter, and the formation of a steady-state ternary elongation complex . We examined DNA melting and promoter clearance by using potassium permanganate, diethylpyrocarbonate and methidiumpropylEDTA.Fe(II) footprinting . In combination, these methods demonstrated: (1) TIF-IB and RNA polymerase I are the only proteins required for formation of an initial approximately 9 base-pair open promoter region . This finding contradicts earlier results using diethylpyrocarbonate alone, which suggested an RNA synthesis requirement for stable melting . (2) DNA melting is temperature-dependent, with a tm between 15 and 20 degrees C . (3) Temperature-dependency of melting, as well as stalling the polymerase at sites close to the transcription start site revealed that the melted DNA region initially opens upstream of the transcription initiation site, and enlarges in a downstream direction coordinate with initiation, eventually attaining a steady-state transcription bubble of approximately 19 base-pairs . (4) The RNA-DNA hybrid protects the template DNA from single-strand footprinting reagents . The hybrid is 9 bp in length, consistent with the longer hybrid estimated by some for the Escherichia coli polymerase and with the hybrids estimated for eukaryotic polymerases II and III.

Arch Biochem Biophys, 2000 Jun 15, 378(2), 349 - 55
Localization of C-terminal domains required for the maximal activity or for determination of substrate preference of maize branching enzymes; Hong S et al.; Previous analysis of a chimeric enzyme mBEII-IBspHI, in which the C-terminal 229 amino acids of maize endosperm branching enzyme isoform II (mBEII) are replaced by the corresponding 284 amino acids of isoform I (mBEI), suggested that the carboxyl terminus of maize branching enzymes may be involved in catalytic efficiency and substrate preference . In the present study, additional hybrids of mBEI and mBEII were generated and expressed in Escherichia coli BL21 (DE3) to dissect the structure/function relationships of the C-terminal regions of maize branching enzymes . A truncated form of purified mBEII-IBspHI, which lacks the C-terminal 58 amino acids, retained similar levels of V(max) in branching activity, K(m) for reduced amylose AS 320, and substrate preference for amylose than amylopectin when compared to mBEII-IBspHI . This indicates that the C-terminal extension derived from mBEI is not required for either catalysis or substrate preference . However, deletion of an additional 87 amino acids from the carboxyl terminus resulted in complete loss of activity . Replacement of the deleted C-terminal additional 87 amino acids with the corresponding 79 amino acids from mBEII restored 25% of the mBEII-IBspHI branching activity without altering substrate preference . It thus appears that a C-terminal region encompassing Leu649-Asp735 of mBEII-IBspHI is required for maximum catalytic efficiency . Another C-terminal region, residues Gln510-Asp648, of mBEII-IBspHI (Gln476-Asp614 of mBEI) may be involved in substrate-preference determination .

Arch Biochem Biophys, 2000 Jun 15, 378(2), 333 - 9
Human fatty acid omega-hydroxylase, CYP4A11: determination of complete genomic sequence and characterization of purified recombinant protein; Kawashima H et al.; The gene of the human fatty acid omega-hydroxylase, CYP4A11, has been isolated from a human BAC library, and its complete genomic sequence has been determined . The CYP4A11 gene spanned 12,568 bp and contained 12 exons . The known PPAR recognition elements (PPRE), which were reported to be involved in the induction of CYP4A6 by clofibric acid, were not observed within the 5'-flanking region of the CYP4A11 gene . The recombinant CYP4A11 protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 6.4 nmol of P450/mg of protein . This P450 exhibited omega-hydroxylation activity toward laurate, with a turnover number of 14.7 nmol/min/nmol of P450 . The apparent K(m) and V(max) values were 56.7 microM and 15.2 nmol/min/nmol of P450, respectively . It also showed omega-hydroxylation activity toward palmitate, with a turnover number of 0.78 nmol/min/nmol of P450 . Although several reports from other groups described that CYP4A11 preparations catalyzed omega-hydroxylation of arachidonic acid, our purified recombinant protein exhibited no activity toward arachidonic acid nor prostaglandin A(1) .

Arch Biochem Biophys, 2000 Jun 15, 378(2), 216 - 23
Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts; Wolff DJ et al.; C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution . The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively . As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene . This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0 . C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM . The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin . C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor . As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers . These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface . Copyright 2000 Academic Press.

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