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