Mol Cell Biol, 2001 Aug, 21(15), 4868 - 74 Mutational analysis of the Cy motif from p21 reveals sequence degeneracy and specificity for different cyclin-dependent kinases; Wohlschlegel JA et al.; Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit . Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity . Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif . Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity . Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif . We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional . Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2 . Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2. Parasitol Int, 2001 Jul, 50(2), 73 - 80 Purification and identification of major soluble 40-kDa antigenic protein from Entamoeba histolytica: its application for serodiagnosis of asymptomatic amebiasis; Sanuki J et al.; One of the major soluble antigenic proteins of Entamoeba histolytica was purified to homogeneity and identified on a molecular basis . Its recombinant protein was expressed in Escherichia coli as a fusion protein with Shistosoma japonicum glutathione S-transferase . Apparent molecular weight of the purified antigenic protein was estimated to be 40-kDa and molecular-based analysis indicated that the purified protein was NADP+-dependent alcohol dehydrogenase (EhADH1) . The application of the purified protein for the serodiagnosis of amebiasis was evaluated using an enzyme-linked immunosorbent assay applied to sera obtained from patients with amebiasis and healthy human controls . The purified protein was well recognized by the sera from asymptomatic amebiasis humans (22/22, 100%), whereas, it was less recognized by the sera from symptomatic amebiasis patients (5/16, 31%) with amebic colitis or liver abscess . To confirm the antigenicity of EhADH1, the recombinant glutathione S-transferase-EhADH1 fusion protein was also evaluated by the enzyme-linked immunosorbent assay using the same sera . The recombinant protein was also recognized by the sera from asymptomatic amebiasis humans (14/22, 64%) and less recognized by the sera from symptomatic amebiasis patients (2/16, 13%) . These results suggest that the purified protein is applicable antigen for serodiagnostic screening of asymptomatic amebiasis humans. Acta Physiol Scand, 2001 May, 172(1), 17 - 25 Prolactin and lipopolysaccharide treatment increased apoptosis and atresia in rat ovarian follicles; Besnard N et al.; Follicular atresia is associated with the presence of increased macrophages within the follicle . What is not known is whether, in the adult rat, macrophages are instrumental in inducing apoptosis and/or atresia or whether they are simply secondary to a hormonally mediated event . As prolactin is an immunoreactive hormone and stimulates the expression of monocyte chemoattractant, the present experiments compared the effects of prolactin treatment with that of an immune challenge with lipopolysaccharide (LPS) on the invasion of macrophages into the follicular and luteal compartments of the ovary and the occurrence of apoptosis/atresia in relation to macrophage invasion . Rats were treated for 3 days with either prolactin or LPS and ovaries obtained at pro-oestrus or oestrus . Prolactin and LPS increased the number of atretic vs . healthy follicles (P < 0.008, chi2) in pro-oestrus ovaries and increased the mean number of apoptotic cells and macrophages (P < 0.05 for some groups) . Macrophages were typically observed in the thecal layer, apoptotic cells in the granulosa cell layer, although 84% follicles which had macrophages within the granulosa cell layer also contained relatively high numbers of apoptotic nuclei . Prolactin and LPS treatment in vivo reduced the progesterone response to follicle stimulating hormone (FSH) (P < 0.001) in cultures of ovarian dispersates but did not inhibit the response to forskolin . In contrast, prolactin or LPS added in vitro to the cultures inhibited the progesterone response to forskolin . Results show that both prolactin and LPS increase follicular apoptosis and atresia and reduce the progesterone response to FSH. Virology, 2001 Jul 5, 285(2), 302 - 12 Structure of the fiber head of Ad3, a non-CAR-binding serotype of adenovirus; Durmort C et al.; Adenoviruses of serotype Ad3 (subgenus B) use a still-unknown host cell receptor for viral attachment, whereas viruses from all other known subgenera use the coxsackie and adenovirus receptor (CAR) . The receptor binding domain (head) of the Ad3 fiber protein has been expressed in Escherichia coli inclusion bodies . After denaturation and renaturation using a rapid dilution method, crystals of trimeric head were obtained . The 1.6 A resolution X-ray structure shows a strict conservation of the beta-sheet scaffold of the protein very similar to the head structures of the CAR-binding serotypes Ad2, Ad5, and Ad12 . The conformation of the loops is different, with the exception of the AB loop, which forms the center of the interface in the Ad12-CAR complex structure . The structure explains why a mutation in Ad5 of one residue in the AB loop to glutamic acid, as in Ad3, abrogates binding to CAR . It is possible that the Ad3 receptor binding site is nevertheless situated similar to the CAR binding site, although it cannot be excluded that other regions of the relatively hydrophobic head surface may be used . Toxicol Appl Pharmacol, 2001 Jun 15, 173(3), 176 - 87 Mercuric ion attenuates nuclear factor-kappaB activation and DNA binding in normal rat kidney epithelial cells: implications for mercury-induced nephrotoxicity; Dieguez-Acuna FJ et al.; Mercuric ion (Hg(2+)), one of the strongest thiol-binding agents known, mediates the toxicity associated with elemental, inorganic, and organic mercurial compounds . Studies of cellular events associated with Hg(2+) toxicity have focused largely on disruption of cell membranes and impairment of mitochondrial functions . In contrast, few studies have sought to define the specific molecular mechanisms through which Hg(2+) might affect toxicity via alteration of thiol-dependent signal transduction pathways that regulate cell proliferation and survival . Of particular interest in this regard is the effect of Hg(2+) on nuclear factor-kappaB (NF-kappaB), a pleiotropic transcriptional factor that is known to require reduced cysteine moieties at critical steps of activation and DNA binding . Here, we evaluated the effects of Hg(2+) on the expression of NF-kappaB in normal rat kidney epithelial (NRK52E) cells, a principal target of Hg(2+) toxicity . The lipopolysaccharide (LPS)-inducible form of NF-kappaB was readily detected in kidney cells and has been characterized as the p50p65 heterodimer . NF-kappaB-DNA binding was prevented in a dose-related manner by Hg(2+) (0-55 microM) in vitro when added to DNA binding reactions containing the nonthiol reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) . Similarly, Hg(2+) at the same concentrations prevented DNA binding of a human recombinant wild-type p50p50 homodimer in binding reactions, and this effect was attenuated using a mutant form of the p50 protein containing a cys(62)-->ser(62) mutation . The inhibition of p50-DNA binding by Hg(2+) was reversible in a dose-related manner in vitro by competitive thiols DTT, GSH, and l-cysteine in binding reactions . In contrast, competitive thiols added to nuclear binding reactions were unable to reverse attenuation of LPS-mediated NF-kappaB-DNA binding affinity when cells were pretreated in vivo with Hg(2+) at concentrations as low as 2 microM prior to LPS administration . Immunoblot analyses indicted that Hg(2+) pretreatment of kidney cells substantially diminished, in a dose-related manner, the concentration of p65 translocated into the nucleus following LPS administration . Additionally, Hg(2+) pretreatment impaired both the phosphorylation and degradation of IkappaBalpha, suggesting a specific effect on NF-kappaB activation at the level of IkappaBalpha proteolysis . Finally, Hg(2+) at concentrations as low as 5 microM significantly diminished NF-kappaB-mediated transcriptional activity when administered to kidney cells transiently transfected with an NF-kappaB-driven luciferase reporter gene (pLuc-4xNF-kappaB) prior to LPS treatment . These findings demonstrate that Hg(2+), at low cellular concentrations, attenuates NF-kappaB activation at sites associated with IkappaBalpha phosphorylation and degradation, nuclear translocation of the p50p65 heterodimer, and association of p50-cys(62) with the DNA kappaB binding site . Attenuation of NF-kappaB activation by Hg(2+) through these mechanisms may underlie apoptotic or other cytotoxic responses that are known to be associated with low level Hg(2+) exposure in kidney epithelial cells . Protein Expr Purif, 2001 Jul, 22(2), 349 - 58 Expression of active human C1 inhibitor serpin domain in Escherichia coli; Lamark T et al.; Human C1 inhibitor is a highly glycosylated serine protease inhibitor of the serpin family . The protein contains two disulfide bonds . In this study, an N-terminally truncated form of recombinant C1 inhibitor was overexpressed in Escherichia coli strains BL21(DE3) and AD494(DE3), the latter enabling the formation of disulfide bonds within the cytoplasm . With both strains, a major fraction of the recombinant protein produced appeared to be insoluble . However, the soluble fraction of lysates from strain AD494(DE3) inhibited the C1s target protease in functional assays . Recombinant C1 inhibitor produced in this strain also displayed the ability to complex with C1s in vitro . In contrast, lysates from strain BL21(DE3) displayed no C1 inhibitor activity . These data support the notion that glycosylation is not important, whereas disulfide bond formation appears to be essential for the production of an active recombinant C1 inhibitor . Thus, bacterial strains that permit the formation of disulfide bonds may represent a reliable system for the production of recombinant C1 inhibitor . However, a major obstacle to large-scale production will be to produce the protein in a soluble form . Attempts to increase the yield of soluble protein by coexpression of the GroEL/ES chaperonins resulted in an increase in solubility . Protein Expr Purif, 2001 Jul, 22(2), 307 - 17 High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins; Holloway DE et al.; Angiogenin (Ang) is a small basic protein which belongs to the pancreatic ribonuclease superfamily . It potently induces the formation of new blood vessels and has emerged as a promising anticancer target . Mice possess genes encoding one ortholog (mAng) and three homologs of Ang, designated angiogenin-related protein (mAngrp), angiogenin-3 (mAng-3), and angiogenin-4 (mAng-4) . Structural and functional study of these homologs has been hampered by the low yield of protein from the existing heterologous expression system . In the experiments described, we used a pET expression vector to express these proteins in the cytoplasm of Escherichia coli BL21-CodonPlus(DE3)-RIL cells, whereupon substantial amounts of each accumulated in the form of insoluble aggregates . The proteins were renatured using an arginine-assisted procedure and subsequently purified by cation-exchange chromatography and reversed-phase HPLC; each purified protein was shown to be enzymatically active toward tRNA . The yields of pure mAngrp and mAng-3 were 7.6 and 12 mg/liter culture, respectively, representing substantial increases over previously reported experiments . This is also the first report of the expression and purification of mAng-4, obtained here in a yield of 30 mg/liter culture . The ready availability of milligram quantities of these proteins will enable further functional studies and high-resolution structural analyses to be conducted . Protein Expr Purif, 2001 Jul, 22(2), 276 - 85 Purification of a bacterially expressed herpes simplex virus type 1 origin binding protein for use in posttranslational processing studies; Bronstein JC et al.; The origin binding protein (OBP) encoded by the UL9 open reading frame of herpes simplex virus type 1 (HSV-1) plays an essential role in productive infection by promoting the initiation of viral DNA synthesis . In this study, OBP was inducibly expressed in Escherichia coli and purified to homogeneity using a two-step chromatographic separation procedure . The properties of this recombinant OBP (rOBP) were found to be indistinguishable from those of the virus-encoded protein . Since rOBP was synthesized in bacterial cells, it lacked the posttranslational processing which normally occurs in OBP produced in HSV-1-infected mammalian cells and could therefore be exploited in experiments which addressed the effects of protein modification on OBP function . As an initial study, the impact of phosphorylation on enzymatic activity was examined using rOBP which had been treated with a panel of purified cellular kinases . rOBP was found to act as a substrate for nearly all of the kinases tested in (32)P-labeled phosphate transfer assays . However, only phosphorylation by protein kinase A (PKA, or cAMP-dependent protein kinase) was shown to significantly alter the enzymatic properties of rOBP, as it increased by five- to eightfold the ATPase activity associated with this protein . Activation of this critical viral DNA replication enzyme by a cAMP-dependent kinase such as PKA may be of some relevance in the natural course of HSV-1 infections, since reactivation of latent virus is thought to involve both signal transduction events and the induction of viral DNA synthesis . Thus, the expression and purification strategy outlined in this work provides an economical source of unmodified HSV-1 OBP that should prove useful in future in vitro studies . Protein Expr Purif, 2001 Jul, 22(2), 267 - 75 Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B; Nakajima H et al.; A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier . This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density . Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli . The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis . Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells . This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin . Protein Expr Purif, 2001 Jul, 22(2), 258 - 66 {W206R}-procaspase 3: an inactivatable substrate for caspase 8; Rank KB et al.; We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag . The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels . Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive . Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment . The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R) . This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis . Protein Expr Purif, 2001 Jul, 22(2), 225 - 33 Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: a component of the P1/P2/P0 complex; Juri Ayub M et al.; The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis . P0 is the minimal portion of the stalk that is able to support accurate protein synthesis . The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite . The T . cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag . The identity of the protein was confirmed by immunoblotting . Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions . TcP0 showed high tendency to aggregation during refolding assays . However, TcP0 could be efficiently folded in the presence of a low concentration of SDS . The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence . Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS . The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation . Protein Expr Purif, 2001 Jul, 22(2), 220 - 4 Characterization of horse cytochrome c expressed in Escherichia coli; Patel CN et al.; We have expressed horse cytochrome c in Escherichia coli . The gene was designed with E . coli codon bias and assembled by using a recursive polymerase chain reaction method . The far-ultraviolet and near-ultraviolet/Soret circular dichroism (CD) spectra show that the structure of recombinant horse cytochrome c is the same as that of the authentic protein . CD-detected thermal denaturation studies were used to measure the thermodynamic parameters associated with two-state denaturation . The free energy of denaturation for the recombinant protein is 10.0 +/- 2.3 kcal mol(-1) at pH 4.6 and 25 degrees C, which agrees with the value for the authentic protein . The expression system will help advance our understanding of the roles of cytochrome c in electron transfer, oxidative stress, and apoptosis by allowing the production of protein variants . Protein Expr Purif, 2001 Jul, 22(2), 180 - 8 Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli; Lee J et al.; Recombinant purine nucleoside phosphorylase (PNPase) from Escherichia coli was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate-liberating enzymes . The E . coli enzyme was overexpressed in E . coli by inserting the genomic fragment containing the deoD gene downstream of the isopropyl beta-d-thiogalactoside-inducible promotor of pSE380 expression vector . The recombinant protein was purified to approximately 90% homogeneity and with a yield of approximately 9000 units of activity/L of culture, using an efficient one-column procedure . A continuous spectrophotometric assay coupling P(i) release to the phosphorolysis of the nucleoside analogue 7-methylinosine (m(7)Ino) was recently described . Here, we report the steady-state kinetic parameters of the recombinant E . coli PNPase catalyzed reaction with m(7)Ino and P(i) as substrates and compare these parameters with those of a bacterial PNPase commercially available for use in coupled assays . Under the assay conditions described, the recombinant E . coli protein is active at higher pH values and is stable up to a temperature of approximately 55 degrees C and following multiple freeze-thaw cycles . It is activated by high ionic strength but loses some activity following dialysis or concentration under pressure . Finally, a new procedure for the synthesis of m(7)Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase-coupled P(i) assays more attractive . Protein Expr Purif, 2001 Jul, 22(2), 174 - 9 High-level soluble production and characterization of porcine ribonuclease inhibitor; Klink TA et al.; Ribonucleases can be cytotoxic if they retain their ribonucleolytic activity in the cytosol . The cytosolic ribonucleolytic activity of ribonuclease A (RNase A) and other pancreatic-type ribonucleases is limited by the presence of excess ribonuclease inhibitor (RI) . RI is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases that competitively inhibits their ribonucleolytic activity . RI had been overproduced as inclusion bodies, but its folding in vitro is inefficient . Here, porcine RI (pRI) was overproduced in Escherichia coli using the trp promoter and minimal medium . This expression system maintains pRI in the soluble fraction of the cytosol . pRI was purified by affinity chromatography using immobilized RNase A and by anion-exchange chromatography . The resulting yield of 15 mg of purified RI per liter of culture represents a 60-fold increase relative to previously reported recombinant DNA systems . Differential scanning calorimetry was used to study the thermal denaturation of pRI, RNase A, and the pRI-RNase A complex . The conformational stability of the complex is greater than that of the individual components . J Comp Pathol, 2001 Jul, 125(1), 15 - 24 Effects of lipopolysaccharide on a macrophage-like cell line (HS-P) from a rat histiocytic sarcoma; Yamate J et al.; Lipopolysaccharide (LPS) is a major modulator of macrophage functions . To characterize a newly established rat histiocytic sarcoma-derived cell line (HS-P), immunophenotypic changes and cellular growth responses of HS-P cells exposed to LPS were investigated and compared with those of MT-9 cells isolated from a rat malignant fibrous histiocytoma . MT-9 cells have somewhat histiocytic features, because occasional cells react to rat macrophage-specific antibodies . Addition of LPS to cultured HS-P cells increased the numbers of cells immunopositive to ED1 (rat macrophage-specific antibody) and ED2 (rat histiocyte-specific antibody) and stimulated the phagocytosis of latex beads, whereas LPS-treated MT-9 cells did not show such immunophenotypic changes . LPS-treated HS-P cells showed enhanced immunolabelling of alpha-smooth muscle actin, suggesting a possible modulation of macrophages towards myofibroblastic cells . To evaluate cellular growth after the addition of LPS or fetal bovine serum, DNA synthesis was examined by measuring tritiated thymidine incorporation, and the mRNA expression of c- jun and c- myc (immediate early genes in the cell cycle) was examined by Northern blot analysis . In HS-P cells, the addition of serum greatly increased DNA synthesis and induced high expression of c- jun and c- myc; in contrast, LPS markedly depressed DNA synthesis and reduced the expression of c- jun and c- myc . HS-P cells were more sensitive than MT-9 cells to the growth-promoting effect of serum and the growth-inhibiting effect of LPS . The study demonstrated that HS-P cells are highly LPS-responsive, indicating that they would be useful for studies of macrophage functions . Copyright Harcourt Publishers Ltd. Growth Horm IGF Res, 2001 Feb, 11(1), 49 - 57 A new radioimmunoassay to measure rat insulin-like growth factor binding protein-4 (IGFBP-4) in serum, wound fluid and conditioned media; Chelius D et al.; Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor-I (IGF-I) and IGF-II . Because of the possible role of IGFBP-4 in multiple systems including reproduction, pregnancy, bone formation and wound healing, we developed a radioimmunoassay (RIA) to measure the concentration of IGFBP-4 in the serum, extracellular fluid and conditioned media of cells of rodents so that these animal models could be better exploited . Rat IGFBP-4 was expressed in Escherichia coli and purified to homogeneity . The recombinant material was used to raise a rabbit polyclonal antibody against rat IGFBP-4 and for iodination and standards . The RIA developed was sensitive to less than 1 ng/mL of rat IGFBP-4 and IGF-I did not interfere . There was no cross-reactivity with other rat IGFBPs on immuno-Western analysis of serum and wound fluid . IGFBP-4 from mouse serum did cross-react in our assay; however, serum from horse, pig or human showed no cross-reaction . Human IGFBP-1 and IGFBP-3 showed a very weak cross-reactivity . Serum IGFBP-4 levels showed no gender difference but did reveal a significant 66% increase in older rats . During the course of wound healing, which is IGF-I dependent, IGFBP-4 showed no changes . In conclusion an RIA for rat IGFBP-4 has been developed that specifically measures the concentration of IGFBP-4 in the sera, extracellular fluid and conditioned media of rodents . With this assay, the role of IGFBP-4 and IGFs in growth, development and the function of multiple systems can be further investigated using rodent models . Br J Cancer, 2001 Jul 6, 85(1), 129 - 36 Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen; Forsberg G et al.; Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule . Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy . A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas . We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen . The recombinant product 5T4FabV13-SEA(D227A)bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a Kd of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II . 5T4FabV13-SEA(D227A)tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate . 5T4FabV13-SEA(D227A)induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10(-10)M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective . Immunotherapy of 5T4FabV13-SEA(D227A)against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells . Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA(D227A) . Thus, 5T4FabV13-SEA(D227A)has highly attractive properties for therapy of human NSCLC . Bioorg Chem, 2001 Jun, 29(3), 146 - 55 Effect of an E461G mutation of beta-galactosidase (Escherichia coli, lac Z) on pL rate profiles and solvent deuterium isotope effects; Richard JP et al.; An E461G mutation of beta-galactosidase results in the disappearance of the high pL (L = H, D) downward break in the rate profiles for k(cat)/K(m) for wild-type enzyme-catalyzed hydrolysis of 4-nitrophenyl beta-D-galactopyranoside (Gal-OPNP) and a decrease from (k(cat))(HOH)/(k(cat))(DOD) = 1.7 to (k(cat))(HOH)/(k(cat))(DOD) = 1.2 in the solvent deuterium isotope effect . These observations provide evidence that the propionic acid side chain of Glu 461 is protonated at catalytically active free beta-galactosidase and they are consistent with a role for this residue in Bronsted acid catalysis at the leaving group . The earlier observation that this same E461G mutation results in the loss of a downward break at high pH in the rate profile for k(s) for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to water cannot be simply explained by a mechanism in which the single side chain of Glu 461 functions to provide general acid catalysis in the rate limiting step for formation of the beta-D-galactopyranosyl intermediate and general base catalysis of breakdown of this intermediate . Evidence is presented that there may be different catalytic mechanisms, with different roles for the side chain for Glu-461, for nucleophilic addition of water and of small alkyl alcohols to the beta-D-galactopyranosyl reaction intermediate . Bioorg Chem, 2001 Jun, 29(3), 140 - 5 Inhibition of beta-lactamases by 6,6-bis(hydroxylmethyl)penicillanate; Nagase T et al.; beta-Lactamases of classes A and C are the two most prevalent resistant determinants to beta-lactam antibiotics among bacterial pathogens . Both these enzymes pursue different mechanisms for their catalytic processes, highlighted by the fact that the hydrolytic water molecule in each approaches the ester of the intermediary acyl-enzyme species from the opposite ends . 6,6-Bis(hydroxylmethyl)penicillanate was designed as an inhibitor that would impair the approach of the hydrolytic water molecule in either of these enzymes upon formation of the acyl-enzyme species . The design, synthesis, and kinetic evaluation of this inhibitor are disclosed herein . Arch Biochem Biophys, 2001 Jul 15, 391(2), 255 - 64 Molecular recognition in the p450cam monooxygenase system: direct monitoring of protein-protein interactions by using optical biosensor; Ivanov YD et al.; A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted . The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized . The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated . It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system . Evidence for PdR/P450cam complex formation was obtained . It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes . A ternary PdR/Pd/P450cam complex was also registered . Its lifetime was sufficient to permit up to 60 turnovers to occur . The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins . Arch Biochem Biophys, 2001 Jul 15, 391(2), 188 - 96 UDP-galactose 4-epimerase from Escherichia coli: formation of catalytic site during reversible folding; Barat B et al.; UDP-galactose 4-epimerase from Escherichia coli is a homodimer of molecular weight 39 kDa/subunit having noncovalently bound NAD acting as cofactor . Denaturation by 8 M urea at pH 7.0 causes 85% loss of its secondary structure and dissociation of its constituent molecules . Dilution of the denaturant by buffer at pH 8.5 leads to functional reconstitution of the dimeric holoenzyme . The refolding process is biphasic: after 2 min an equilibrium conformer is formed having 72% of its native secondary structure and by 60 min reactivation becomes complete . The early intermediate has lower energy of activation against thermal denaturation than the reactivated state . Patterns of trypsin digestion suggests a native like structure of this intermediate . Variation of solvent viscosity and ionic strength and inclusion of proline cis-trans isomerase in the refolding process do not alter kinetics of reactivation . Moreover, unaltered kinetics of reactivation against variation of temperature, pH, and duration of denaturation strongly suggests absence of proline cis/trans isomerization . Measurement of kinetics of (i) recovery of tertiary structure by protein fluorescence; (ii) incorporation of NAD from quantitation of bound cofactor; (iii) formation of substrate binding site by specific interaction with extrinsic fluorophore 1-anilino-8-naphthalene sulfonic acid and quenching by 5'-UMP, a competitive inhibitor; and (iv) recovery of activity indicate that they are all comparable . It appears that internal rearrangement of the protein during refolding, shielded from solvent, is the rate-limiting step of generation of cofactor binding site which ultimately leads to maturation of the holoenzyme structure . J Gene Med, 2001 May-Jun, 3(3), 260 - 70 Factors influencing cross-presentation of non-self antigens expressed from recombinant adeno-associated virus vectors; Sarukhan A et al.; BACKGROUND: We have previously demonstrated that recombinant adeno-associated virus vectors expressing the influenza virus hemagglutinin (rAAV-HA) in skeletal muscle results in T-cell priming and muscle fiber destruction due to cross-presentation of HA by dendritic cells (DC) . Based on controversial observations concerning the stability of non-self proteins expressed from rAAV vectors it is important to understand the factors influencing cross-presentation of transgene products following rAAV mediated gene transfer, in order to be able to use this vector safely in the clinic . METHODS: In order to understand the factors influencing in vivo cross-presentation of non-self proteins, we have retargeted the immunogenic lacZ protein in the context of rAAV from the cytoplasm to the cell surface and studied the activation of LacZ specific immune responses following intramuscular mediated gene transfer . In addition, using tools available for studying in vitro HA-specific T-cell activation, our aim was to identify the cell types involved in class I and class II restricted cross-presentation as well as the nature of the cross-presented material . RESULTS: By retargeting the lacZ protein in the context of rAAV to the cell membrane, we found that one of the factors influencing the efficiency of cross-presentation of non-self antigens is the localization of the transgene product within the target cells . Following rAAV-LacZ mediated gene transfer to the muscle we demonstrated that the membrane-bound form of LacZ resulted in target cell destruction, which is in stark contrast to the stability observed with rAAV-LacZ vectors expressing the cytoplasmic form of LacZ . Using an in vitro assay, we were able to show that dendritic cells (DC) in addition to B-cells cross-presented HA to class II restricted T-cells whereas only the former were able to activate class I restricted CD8+ T-cells . High-dose antigens were needed for efficient class I restricted T-cell priming, whereas class II restricted T-cells were activated by less antigen . CONCLUSION: The present results indicate that immune responses to non-self antigens expressed from rAAV vectors depend on the accessibility of such antigens to different local antigen-presenting cells. Genetika, 2001 May, 37(5), 591 - 601 {Homologous recombination and chromosomal rearrangements in Escherichia coli strains carrying a heterozygous tandem duplication}; Sukhodolets VV et al.; Heterozygous tandem duplications formed in conjugational matings in Escherichia coli provides a convenient model system for studying the evolution of bacterial chromosome . Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes . In this work, an extended tandem duplication in the deo operon of E . coli carrying deoA deoB::Tn5/deoC deoD thr::Tn9 alleles was examined . Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3 mutations . The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ (or recF) genotype . It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of "adaptive" recombination. Int Arch Allergy Immunol, 2001 Jun, 125(2), 144 - 51 Recombinant Dirofilaria immitis-derived antigen can suppress passive cutaneous anaphylaxis reactions; Furuhashi Y et al.; BACKGROUND: High levels of antigen-nonspecific IgE are produced in animals infected with helminth parasites . Generally, the increase in IgE is thought to exacerbate allergic reactions . However, high levels of antigen-nonspecific IgE may alter some features of anaphylactic reactions . To investigate the molecular mechanisms of antigen-nonspecific IgE production induced during filarial infections, we previously constructed rDiAg (recombinant Dirofilaria immitis-derived antigen) in Escherichia coli . In the present study, we examined the effect of rDiAg on the production of antigen-nonspecific IgE and on allergic cutaneous reactions in rats . METHODS: Osmotic pumps filled with 200 microg of rDiAg or with 200 microl of PBS (control) were subcutaneously implanted in Wistar rats, and plasma samples were collected weekly thereafter . IgE levels were determined by ELISA . Homologous passive cutaneous anaphylaxis (PCA) reactions with anti-DNP-As IgE were examined 21 days after implantation . (125)I-IgE binding assays were examined on peritoneal mast cells from rDiAg-infused rats and control rats . RESULTS: Antigen-nonspecific IgE production was induced in rDiAg-infused rats . PCA reactions were suppressed in rDiAg-infused rats in spite of high levels of IgE and a markedly increased expression of Fc epsilon RI . (125)I-IgE did not bind to mast cells derived from rDiAg-infused rats, but it bound dose dependently to mast cells derived from control rats . CONCLUSION: The present data support the hypothesis that antigen-nonspecific IgE might protect against antigen-specific IgE by means of competition for mast cell receptors . rDiAg is an essential factor to induce antigen-nonspecific IgE in helminth infections . Mol Endocrinol, 2001 Jul, 15(7), 1140 - 53 Reconstruction of ligand-dependent transactivation of Choristoneura fumiferana ecdysone receptor in yeast; Tran HT et al.; Ecdysteroids play an important role in regulating development and reproduction in insects . Interaction of 20-hydroxyecdysone (20E) with ecdysone receptor (EcR) as a heterodimer with ultraspiracle (USP) protein triggers the activation of 20E-responsive genes . In this paper we describe a ligand-mediated transactivation system in yeast using the spruce budworm Choristoneura fumiferana ecdysone receptor (CfEcR) . Coexpression of C . fumiferana USP (CfUSP) with CfEcR in yeast led to constitutive transcription of the reporter gene . However, deletion of the A/B domain of CfUSP abolished constitutive activity observed for the CfUSP:CfEcR complex . Replacement of USP with its mammalian homolog retinoid X receptors (RXRs) abolished the constitutive activity of the heterodimer but it did not restore EcR ligand-mediated transactivation . These data suggest that USP and its A/B domain play a role in the constitutive function of CfEcR:USP in yeast . A ligand-mediated transactivation was observed when GRIP1, a mouse coactivator gene, was added to EcR:RXR or EcR:DeltaA/BUSP complexes . Deletion of the A/B domain of EcR in the context of DeltaA/BEcR:RXR:GRIP1 or DeltaA/BEcR:DeltaA/BUSP:GRIP1 dramatically improved the ligand-dependent transactivation . This is the first example of highly efficient ligand-dependent transactivation of insect EcR in yeast . Analysis of transactivation activity of different ecdysteroidal compounds showed that the yeast system remarkably mimics the response observed in insect tissue culture cells and whole insect systems . The results open the way to develop assays that can be used to screen novel species-specific ecdysone agonist/antagonist insecticides. Mol Endocrinol, 2001 Jul, 15(7), 1049 - 61 The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains; Cohen RN et al.; The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors {NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)} in the absence of ligand to silence transcription . NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box) . We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT . Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains . An analogous domain is not present in SMRT . This domain is specific for TR and interacts poorly with RAR . Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells . Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions . In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1 . Additional regions distal to the CoRNR box sequence are needed for optimal binding . Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1 . These preferences are likely to be important in corepressor action in vivo. J Virol, 2001 Aug, 75(15), 7018 - 29 The vaccinia virus superoxide dismutase-like protein (A45R) is a virion component that is nonessential for virus replication; Almazan F et al.; A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented . The open reading frame is predicted to encode a 125-amino-acid protein (M(r), of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD) . Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus . In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity . The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected . Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions . A monoclonal antibody raised against the A45R protein expressed in E . coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection . Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories . Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core . A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized . The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages . Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection . A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence. J Virol, 2001 Aug, 75(15), 6786 - 99 DNA binding by Kaposi's sarcoma-associated herpesvirus lytic switch protein is necessary for transcriptional activation of two viral delayed early promoters; Lukac DM et al.; Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types . The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency . Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP) . Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation . The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3' to it . Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required . The 3' flanking sequences also play a critical role in DNA binding and transactivation . The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element . Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide. J Virol, 2001 Aug, 75(15), 6758 - 68 African swine fever virus structural protein pE120R is essential for virus transport from assembly sites to plasma membrane but not for infectivity; Andres G et al.; This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication . Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions . Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72 . Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression . Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system . In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions . One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections . Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas . Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity . The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane. J Biol Chem, 2001 Sep 7, 276(36), 33681 - 8 Epub 2001 Jul 02. Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB; Panse VG et al.; SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli . We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods . BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51 . Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide . The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy . The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB . Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues . In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein . The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate . This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein. J Biol Chem, 2001 Sep 7, 276(36), 33930 - 7 Epub 2001 Jul 02. Bisphosphonates are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase; Montalvetti A et al.; We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase of Trypanosoma cruzi . The protein (T . cruzi farnesyl pyrophosphate synthase, TcFPPS) is an attractive target for drug development, since the growth of T . cruzi is inhibited by carbocation transition state/reactive intermediate analogs of its substrates, the nitrogen-containing bisphosphonates currently in use in bone resorption therapy . The protein predicted from the nucleotide sequence of the gene has 362 amino acids and a molecular mass of 41.2 kDa . Several sequence motifs found in other FPPSs are present in TcFPPS . Heterologous expression of TcFPPS in Escherichia coli produced a functional enzyme that was inhibited by the nitrogen-containing bisphosphonates alendronate, pamidronate, homorisedronate, and risedronate but was less sensitive to the non-nitrogen-containing bisphosphonate etidronate, which, unlike the nitrogen-containing bisphosphonates, does not affect parasite growth . The protein contains a unique 11-mer insertion located near the active site, together with other sequence differences that may facilitate the development of novel anti-Chagasic agents. J Biol Chem, 2001 Sep 7, 276(36), 34252 - 8 Epub 2001 Jul 02. The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon; James CM et al.; Each of the genes encoding the methyltransferases initiating methanogenesis from trimethylamine, dimethylamine, or monomethylamine by various Methanosarcina species possesses one naturally occurring in-frame amber codon that does not appear to act as a translation stop during synthesis of the biochemically characterized methyltransferase . To investigate the means by which suppression of the amber codon within these genes occurs, MtmB, a methyltransferase initiating metabolism of monomethylamine, was examined . The C-terminal sequence of MtmB indicated that synthesis of this mtmB1 gene product did not cease at the internal amber codon, but at the following ochre codon . Antibody raised against MtmB revealed that Escherichia coli transformed with mtmB1 produced the amber termination product . The same antibody detected primarily a 50-kDa protein in Methanosarcina barkeri, which is the mass predicted for the amber readthrough product of the mtmB1 gene . Sequencing of peptide fragments from MtmB by Edman degradation and mass spectrometry revealed no change in the reading frame during mtmB1 expression . The amber codon position corresponded to a lysyl residue using either sequencing technique . The amber codon is thus read through during translation at apparently high efficiency and corresponds to lysine in tryptic fragments of MtmB even though canonical lysine codon usage is encountered in other Methanosarcina genes. Trends Plant Sci, 2001 Jul, 6(7), 281 - 4 CREam of cytokinin signalling: receptor identified; Schmulling T; Cytokinins play a central role in the regulation of plant cell division and numerous developmental processes . Pleiotropic effects have made studies of this hormone difficult, and cytokinin signalling pathways have long remained elusive . The recent identification of CRE1 (a histidine kinase identical to AHK4 and WOL) as the cytokinin receptor of Arabidopsis thaliana is a landmark in cytokinin research . Mutations have been identified in CRE1, and the phenotype of loss-of-function mutations sheds new light on the role of cytokinins in plant development . This article describes the experimental paths leading to receptor identification and the current interpretation of its function. Biochemistry, 2001 Jul 10, 40(27), 8169 - 79 Kinetic characterization of the pH-dependent oligomerization of R67 dihydrofolate reductase; Mejean A et al.; Protein-protein recognition results from the assembly of complementary surfaces on two molecules that form a stable, noncovalent, specific complex . Our interest was to describe kinetic aspects of the recognition in order to understand the subtle molecular mechanism of association . R67 dihydrofolate reductase (DHFR) provides an ideal model to investigate kinetic parameters of protein-protein association since it is a homotetramer resulting from the pH-dependent dimerization of homodimers . We took advantage of the presence of a tryptophan residue at the dimer-dimer interface to monitor pH-dependent oligomerization of R67 DHFR using stopped-flow fluorescence techniques . Except for pH near neutrality where dissociation exhibited biphasic kinetics, association and dissociation followed monophasic kinetics fitted on a two-state model . Apparent rate constants of association k(on) and dissociation k(off) were determined at various pHs and pointed to the key role of a histidine located at the dimer-dimer interface in the pH control of tetramerization . The values of the tetramer-dimer equilibrium dissociation constant were calculated from the ratio k(off) /k(on) and correlated well with those previously measured at equilibrium . The thermodynamic parameters and the activation energies of both the association and dissociation were determined and indicated that the association is enthalpy driven and suggested that the formation of four hydrogen bonds (one per monomer) is responsible for the thermodynamic stability of the tetramer . Detailed analysis of the biphasic kinetics led to an original model, in which protonation of the tetramer is the triggering event for the dissociation process while the association involves primarily the unprotonated dimers. Biochemistry, 2001 Jul 10, 40(27), 8118 - 25 Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites; Tardif KD et al.; Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids . To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val) . Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids . The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction . Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS . As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine . Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine . Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS . We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine . Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme . Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule . Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS. Biochemistry, 2001 Jul 10, 40(27), 8109 - 17 Ion concentration and temperature dependence of DNA binding: comparison of PurR and LacI repressor proteins; Moraitis MI et al.; Purine repressor (PurR) binding to specific DNA is enhanced by complexing with purines, whereas lactose repressor (LacI) binding is diminished by interaction with inducer sugars despite 30% identity in their protein sequences and highly homologous tertiary structures . Nonetheless, in switching from low- to high-affinity DNA binding, these proteins undergo a similar structural change in which the hinge region connecting the DNA and effector binding domains folds into an alpha-helix and contacts the DNA minor groove . The differences in response to effector for these proteins should be manifest in the polyelectrolyte effect which arises from cations displaced from DNA by interaction with positively charged side chains on a protein and is quantitated by measurement of DNA binding affinity as a function of ion concentration . Consistent with structural data for these proteins, high-affinity operator DNA binding by the PurR-purine complex involved approximately 15 ion pairs, a value significantly greater than that for the corresponding state of LacI (approximately 6 ion pairs) . For both proteins, however, conversion to the low-affinity state results in a decrease of approximately 2-fold in the number of cations released per dimeric DNA binding site . Heat capacity changes (DeltaC(p)) that accompany DNA binding, derived from buried apolar surface area, coupled folding, and restriction of motional freedom of polar groups in the interface, also reflect the differences between these homologous repressor proteins . DNA binding of the PurR-guanine complex is accompanied by a DeltaC(p) (-2.8 kcal mol(-1) K(-1)) more negative than that observed previously for LacI (-0.9 to -1.5 kcal mol(-1) K(-1)), suggesting that more extensive protein folding and/or enhanced structural rigidity may occur upon DNA binding for PurR compared to DNA binding for LacI . The differences between these proteins illustrate plasticity of function despite high-level sequence and structural homology and undermine efforts to predict protein behavior on the basis of such similarities. Mol Biotechnol, 2001 Mar, 17(3), 193 - 9 A plasmid cloning vector with precisely regulatable copy number in Escherichia coli; Herman-Antosiewicz A et al.; We have developed a genetic system allowing for precise regulation of plasmid copy number in Escherichia coli cells . A cloning vector based on this system is described in this article . The pTC lambda 3 plasmid is a lambda replicon, but transcription controlling initiation of plasmid DNA replication starts from the PtetA promoter instead of phage lambda PR promoter . Additionally, activity of PtetA promoter is negatively controlled by the TetR repressor whose gene is located on the same plasmid vector and is induced by an analog of tetracycline, autoclaved chlortetracycline (aCT) . Using different concentrations of the inducer it is possible to strictly regulate the copy number of pTC lambda 3 and thus the copy number of a cloned gene . The usefulness of the system for the regulatable production of a protein encoded by a gene inserted into pTC lambda 3 plasmid is demonstrated by dependence of beta-galactosidase activity on the lacZ gene dosage. Nat Rev Genet, 2001 Jul, 2(7), 504 - 15 Evolving responsively: adaptive mutation; Rosenberg SM; A basic principle of genetics is that the likelihood that a particular mutation occurs is independent of its phenotypic consequences . The concept of adaptive mutation seemed to challenge this principle with the discoveries of mutations stimulated by stress, some of which allow adaptation to the stress . The emerging mechanisms of adaptive genetic change cast evolution, development and heredity into a new perspective, indicating new models for the genetic changes that fuel these processes. Nucleic Acids Res, 2001 Jul 1, 29(13), 2725 - 32 Translesional synthesis on DNA templates containing the 2'-deoxyribonolactone lesion; Berthet N et al.; A site-specifically modified oligonucleotide containing a single 2'-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(-)) in exonuclease activity . Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2'-deoxyribonolactone . KF exo(-) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP approximately dCMP and thus appeared to obey the 'A rule' for preferential incorporation as has been shown previously for the 2'-deoxyribose abasic site . In the sequence context examined, the primer extension by KF exo(-) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP . These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations . We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(-) and thus the miscoding and mutational potential of the 2'-deoxyribonolactone in E.coli. J Exp Bot, 2001 Jun, 52(359), 1375 - 80 Arabidopsis coactivator ALY-like proteins, DIP1 and DIP2, interact physically with the DNA-binding domain of the Zn-finger poly(ADP-ribose) polymerase; Storozhenko S et al.; Two novel homologous Arabidopsis proteins, DIP1 and DIP2, interact with the DNA-binding domain of plant poly(ADP-ribose) polymerase (PARP) in the yeast two-hybrid system and in vitro . Their homology to the transcriptional coactivator ALY suggests that plant PARP may play a role in the regulation of transcription. J Biol Chem, 2001 Sep 7, 276(36), 33906 - 14 Epub 2001 Jun 29. Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine; Mizoue LS et al.; Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1 . To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken . The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling . The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger . This result also shows that signaling residues are not confined to chemokine N termini, as generally thought . F50A showed no detectable binding, underscoring its importance to the stability of the complex . K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration . Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1 . Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors. J Biol Chem, 2001 Sep 14, 276(37), 34784 - 91 Epub 2001 Jun 29. ATP utilization by yeast replication factor C . III . The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading; Schmidt SL et al.; The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid . Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis . All of the mutant RFC complexes were capable of interacting with PCNA . Complexes containing a rfc1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis . In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity . In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding . A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations . Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations. EMBO J, 2001 Jul 2, 20(13), 3587 - 95 Expression of the glucose transporter gene, ptsG, is regulated at the mRNA degradation step in response to glycolytic flux in Escherichia coli; Kimata K et al.; We report a novel post-transcriptional control of the ptsG gene encoding the major glucose transporter IICB(Glc) . We demonstrate that the level of IICB(Glc) is markedly reduced when the glycolytic pathway is blocked by a mutation in either the pgi or pfkA gene encoding phosphoglucose isomerase or phosphofructokinase, respectively . This down-regulation of ptsG is not exerted at the transcriptional level . Both northern blot and S1 analyses demonstrate that the mutation dramatically accelerates the degradation of ptsG mRNA . The degradation of ptsG mRNA occurs in wild-type cells when alpha-methylglucoside, a non- metabolizable analog of glucose, is present in the medium . The addition of any one of the glycolytic intermediates downstream of the block prevents the degradation of ptsG mRNA . The rapid degradation of ptsG mRNA is eliminated when RNase E is thermally inactivated . We conclude that the glycolytic pathway controls ptsG expression by modulating RNase E-mediated mRNA degradation . This is the first instance in which the glycolytic flux has been shown to affect the expression of a specific gene through mRNA stability. EMBO J, 2001 Jul 2, 20(13), 3554 - 64 Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair; Missura M et al.; The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system . Although XPA-RPA has been implicated in damage recognition, its activity in the DNA repair pathway remains controversial . By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak preference of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations . Further screening with artificially distorted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA . Conversely, RPA recognizes single-stranded sites but not backbone bending . Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA . The affinity of XPA for sharply bent duplexes, characteristic of architectural proteins, is not compatible with a direct function during recognition of nucleotide lesions . Instead, XPA in conjunction with RPA may constitute a regulatory factor that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly. J Biochem (Tokyo), 2001 Jul, 130(1), 63 - 71 Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron-sulfur proteins; Tokumoto U et al.; The iron-sulfur (Fe-S) cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into target proteins with the aid of complex machinery . In bacteria, several proteins encoded by the iscRSUA-hscBA-fdx-ORF3 cluster (isc operon) have been proposed to execute crucial tasks in the assembly of Fe-S clusters . To elucidate the in vivo function, we have undertaken a systematic mutational analysis of the genes in the Escherichia coli isc operon . In all functional tests, i.e . growth rate, nutritional requirements and activities of Fe-S enzymes, the inactivation of the iscS gene elicited the most drastic alteration . Strains with mutations in the iscU, hscB, hscA, and fdx genes also exhibited conspicuous phenotypical consequences almost identical to one another . The effect of the inactivation of iscA was small but appreciable on Fe-S enzymes . In contrast, mutants with inactivated iscR or ORF3 showed virtually no differences from wild-type cells . The requirement of iscSUA-hscBA-fdx for the assembly of Fe-S clusters was further confirmed by complementation experiments using a mutant strain in which the entire isc operon was deleted . Our findings support the conclusion that IscS, via cysteine desulfurase activity, provides the sulfur that is subsequently incorporated into Fe-S clusters by assembler machinery comprising of the iscUA-hscBA-fdx gene products . The results presented here indicate crucial roles for IscU, HscB, HscA, and Fdx as central components of the assembler machinery and also provide evidence for interactions among them. J Biochem (Tokyo), 2001 Jul, 130(1), 23 - 31 PKNbeta interacts with the SH3 domains of Graf and a novel Graf related protein, Graf2, which are GTPase activating proteins for Rho family; Shibata H et al.; PKNbeta is a novel isoform of PKNalpha, which is one of the target protein kinases for the small GTPase Rho . By yeast two-hybrid screening of a human embryonic kidney 293 cell cDNA library with the PKNbeta linker region containing proline-rich motifs as a bait, clones encoding Graf (GAP for Rho Associated with Focal adhesion kinase) and a novel Graf-related protein, termed Graf2, were isolated . The full length of Graf2 contains a putative PH domain, a RhoGAP domain, and an SH3 domain as well as Graf . Northern and Western blot analyses demonstrated that Graf2 is expressed in several tissues, with the highest expression in skeletal muscle . Recombinant Graf2 exhibited GTPase-activating activity toward the small GTPase RhoA and Cdc42Hs, but not toward Rac1, in vitro . The SH3 domains of Graf and Graf2 purified from Escherichia coli bound directly to PKNbeta . Graf or Graf2 was co-immunoprecipitated with PKNbeta in COS-7 cells transiently transfected with Graf or Graf2 and PKNbeta expression constructs . The catalytically active form of PKNbeta phosphorylated Graf and Graf2 in vitro . The interplay of PKNbeta and the GTPase-activating proteins, Graf and Graf2, may offer a novel mechanism regulating the Rho-mediated signaling. J Biochem (Tokyo), 2001 Jul, 130(1), 13 - 8 On the effect of sodium dodecyl sulfate on the structure of beta-galactosidase from Escherichia coli . A fluorescence study; D'Auria S et al.; An understanding of the structure-function relationship of proteins under different chemical-physical conditions is of fundamental importance for an understanding of their structure and function in cells . In this paper we report the effects of sodium dodecyl sulfate and temperature on the structure of beta-galactosidase from Escherichia coli, as monitored by fluorescence spectroscopy . The structure of the protein was studied in the temperature range of 10-60 degrees C in the absence and presence of sodium dodecyl sulfate by frequency-domain measurement of the intrinsic fluorescence intensity and anisotropy decays . The time-resolved fluorescence data in the absence of SDS indicated that at 10 degrees C the tryptophanyl emission decays were well described by a three exponential decays model, and that the temperature increase resulted in shortening of the long-lived component with little change in the short- and middle-lived components . The addition of SDS to the protein solution also affected the long-lived component . The effects of the detergent and temperature on the enzyme structure were also investigated by means of quenching experiments and anisotropy decays . The obtained results showed that the presence of SDS confers more flexibility to the protein structure, and suggest a strict relation between enzyme activity and protein flexibility. Eur J Biochem, 2001 Jul, 268(13), 3851 - 7 The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning; Rodina EV et al.; Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases) . In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42-->Asn is three times more active than native and retains its activity at high Mg2+ concentration . In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis . pH-independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated . It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration . All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme . These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase . Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site . Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition. Eur J Biochem, 2001 Jul, 268(13), 3744 - 50 Overproduction of a functional A1 ATPase from the archaeon Methanosarcina mazei Gö1 in Escherichia coli; Lemker T et al.; Single subunits of the A1 ATPase from the archaeon Methanosarcina mazei Go1 were produced in E . coli as MalE fusions and purified, and polyclonal antibodies were raised against the fusion proteins . A DNA fragment containing the genes ahaE, ahaC, ahaF, ahaA, ahaB, ahaD, and ahaG, encoding the hydrophilic A1 domain and part of the stalk of the A1AO ATPase of M . mazei Go1, was constructed, cloned into an expression vector and transformed into different strains of Escherichia coli . In any case, a functional, ATP-hydrolysing A1 ATPase was produced . Western blots demonstrated the production of subunits A, B, C, and F in E . coli, and minicell analyses suggested that subunits D, E, and G were produced as well . This is the first demonstration of a heterologous production of a functional ATPase from an archaeon . The A1 ATPase was sensitive to freezing but lost only about 50% of its activity within 18 days on ice . Inhibitor studies revealed that the heterologously produced A1 ATPase is insensitive to azide, dicyclohexylcarbodiimide and bafilomycin A1, but sensitive to diethylstilbestrol and its analogues dienestrol and hexestrol . The expression system described here will open new avenues towards the functional and structural analyses of this unique class of enzymes. Eur J Biochem, 2001 Jul, 268(13), 3728 - 35 Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3; Weers PM et al.; Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family . Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain . This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface . Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C . Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min . ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength . In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength . Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH . Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0 . Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH . These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure. Yi Chuan Xue Bao, 2001, 28(6), 575 - 82 {Cloning of gene related to salt tolerance from Sinorhizobium fredii RT19 and its expression in Escherichia coli}; Ge SC et al.; A 4.4 kb DNA fragment related to salt tolerance containing three open reading frames was isolated from the gene library of S . fredii strain RT19 . By subcloning and functional analysis, only ORF2 related to salt tolerance was obtained . The ORF2 was ligated to expression vectors pThioHisA, B and C, respectively, and recombinant expression vectors pGA, pGB and pGC containing 1.5 kb DNA fragment related to salt tolerance were constructed . These recombinant expression vectors were transformed into E . coli DH5 alpha . Inducing by IPTG and analyzing with SDS-PAGE, it was found that the fusion protein encoded by pGC was expressed, and its molecular weight was equal to the sum of thioredoxin encoded by trxA and ORF2 putative protein molecular weight . The Western blot demonstrated that the target gene was successfully expressed in E . coli. Cancer Res, 2001 Jul 1, 61(13), 5116 - 25 Retrovirus-mediated expression of the base excision repair proteins, formamidopyrimidine DNA glycosylase or human oxoguanine DNA glycosylase, protects hematopoietic cells from N,N',N"-triethylenethiophosphoramide (thioTEPA)-induced toxicity in vitro and in vivo; Kobune M et al.; Modulation of DNA damage repair activity could lead to new approaches to reduce cytotoxic side effects of chemotherapy . N,N',N"-Triethylenethiophosphoramide (thioTEPA) induces the formation of amino-ethyl adducts of guanine, resulting in imidazole ring opening {formamidopyrimidine (Fapy)} and is associated with significant myelosuppression in dose-intensive therapies . In Escherichia coli, Fapy lesions are repaired by the Fapy-DNA glycosylase (Fpg) protein . We hypothesized that the expression of the Fpg could increase resistance of hematopoietic cells to thioTEPA-induced cytotoxicity . Expression of Fpg in bone marrow (BM) cells via a retrovirus vector was associated with demonstrable 8-oxodeoxyguanosine DNA glycosylase activity . BM cells were infected with a recombinant retrovirus, SF91, containing the Fpg gene and expressing the enhanced green fluorescence protein (EGFP) via an internal ribosomal entry site element . Control mice received BM transduced with the backbone containing IRES-EGFP alone . Fpg-transduced and GFP+ BM hematopoietic cells were resistant in vitro to thioTEPA at multiple concentrations . Mice transplanted with transduced cells were treated with four doses of thioTEPA (10 mg/kg) given over 7 weeks . Despite low transduction efficiency, peripheral blood leukocytes, hemoglobin, and platelet counts of thioTEPA-treated Fpg mice were significantly higher than treated control mice (P < 0.05) . In addition, after treatment, the BM, spleen, and thymic cellularity as well as the number of GFP+ progenitor cells in the BM of treated mice were significantly higher than those of control group . Selection of Fpg-transduced cells in vivo was demonstrated by an increase in the mean fluorescence intensity of peripheral mononuclear cells of Fpg mice compared with pretreatment value . In addition, a significant increase in the EGFP-bright cells was demonstrated, suggesting preferential survival of high-expressing hematopoietic cells . Similar results were demonstrated in vitro with primary BM expressing the human functional counterpart of Fpg, OGG1 . These results show that expression of the Fpg or hOGG1 protein protects hematopoietic cells from thioTEPA-induced DNA damage and suggest that a high level of expression of these repair proteins is required to establish resistance to this drug . Expression of Fpg and/or OGG1 may provide an novel approach to preventing thioTEPA-induced toxicity of primary hematopoietic cells. J Biomol NMR, 2001 May, 20(1), 83 - 8 Domain orientation in beta-cyclodextrin-loaded maltose binding protein: diffusion anisotropy measurements confirm the results of a dipolar coupling study; Hwang PM et al.; Maltose binding protein (MBP) is a 370-residue two-domain molecule involved in bacterial chemotaxis and sugar uptake . Rotational diffusion tensors were calculated for a complex between MBP and beta-cyclodextrin using backbone 15N T1 and T1rho relaxation times and steady state 1H-15N NOE values . The tensors obtained for each of the two domains in the protein were subsequently used to determine the relative domain orientation in the molecule . The average domain orientation determined using this approach agrees well with results from dipolar coupling data, but differs significantly from the domain orientation deduced from X-ray studies of the complex. J Biomol NMR, 2001 May, 20(1), 71 - 5 A method for efficient isotopic labeling of recombinant proteins; Marley J et al.; A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented . The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56 . The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density . Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced . The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths. J Biomol NMR, 2001 May, 20(1), 1 - 10 Side chain mobility as monitored by CH-CH cross correlation: the example of cytochrome b5; Banci L et al.; The mobility of betaCH2 moieties in oxidized and reduced cytochrome b5 was studied by analyzing the 13C relaxation of the J-split components, in terms of C-H dipole-C-H dipole cross correlation rates . A 2D 13C-1H experiment is proposed to measure these rates that provide the internal effective reorientation correlation time for each CH2 moiety . It is found that higher mobility is present in the alpha helices forming the heme pocket . On the contrary, the beta strands, which form the hydrophobic core of the molecule, have the lowest mobility . The general pattern is the same for the oxidized and reduced species, indicating that any oxidation-dependent property detected for backbone NH moieties does not affect the CH2 mobility. Evolution Int J Org Evolution, 2001 May, 55(5), 897 - 908 Variation of enzyme activities at a branched pathway involved in the utilization of gluconate in Escherichia coli; Wang IN et al.; Twenty-four strains of Escherichia coli from the ECOR collection were characterized for growth rate in gluconate minimal salts medium and for Vmax and Km of the three enzymes (gluconokinase, 6-phosphogluconate dehydrogenase, and 6-phosphogluconate dehydratase) that form a branch point for the utilization of gluconate . A total of 11 characters--growth rate, three Vmax values, four Km values, and three Vmax/Km values--were determined for these 24 ECOR strains . Most of the characters were normally distributed . Statistical tests showed that growth rate is significantly less variable than enzyme activities . Also, analyses of variance showed significant differences among strains and among the extant five genetic groups of E . coli for the characters measured . A Mantel test showed that, for some characters, closely related strains shared similar character values . Two hypotheses regarding the relationships between growth rate and enzyme activity and between various enzyme activities were tested . None of the expected correlations between growth rate and enzyme activity or between enzyme activities was detected . The results were discussed in terms of metabolic control analysis and neutral theory. FEMS Microbiol Lett, 2001 May 1, 198(2), 151 - 7 CbbR, a LysR-type transcriptional regulator from Hydrogenophilus thermoluteolus, binds two cbb promoter regions; Terazono K et al.; The cbbR encoding the LysR-type transcriptional regulator is located downstream of cbbLSQOYA and this gene is located upstream of cbbFPT in divergent transcription . The two promoter regions with LysR-binding sites are located in the cbbL upstream region and in the cbbR-cbbF intergenic region . Electrophoretic mobility shift assays using a cell extract of Escherichia coli harboring a plasmid containing cbbR and the DNA fragments of promoter regions indicated that CbbR binds in both regions . NADPH caused differences in the complex of CbbR and DNA. Oncogene, 2001 Jun 14, 20(27), 3590 - 5 HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes; Jager AC et al.; Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes . MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1) . We report here novel HNPCC-hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients . We demonstrate that these mutant HNPCC-hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo . The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1-hEXO1 complex formation and hMutLalpha heterodimer assembly essential for MMR activity. Nature, 2001 Jun 28, 411(6841), 1077 - 81 Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule; Changela A et al.; A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA . Such enzymes-DNA topoisomerases-alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone . DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate . Here we report the crystal structure, at 2.05 A resolution, of an inactive mutant of E . coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule . The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site . We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme . The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis . These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements. Microbiology, 2001 Jul, 147(Pt 7), 1875 - 85 Characterization of an autostimulatory substance produced by Escherichia coli; Weichart DH et al.; The recovery of dilute populations of stationary phase cells of Escherichia coli was studied using an automatic growth analyser . The addition of 30% supernatant from 2-d-old stationary phase cells of the organism reproducibly shortened the apparent lag times by 22-57.5%, depending on the age of the inoculum . True lag times, as determined by colony counts, of stationary phase cells were reduced by supernatant addition by 41-62% . The growth-stimulating substance was characterized and partly purified from supernatants: the active material was shown to be dialysable, heat-stable, acid- and alkali-stable and protease-resistant . Extraction with ethyl acetate or ion-exchange resins was not successful, but the active material could be quantitatively extracted with ethanol after saturation with salt . It is concluded that the active substance is a small, non-proteinaceous, non-ionic organic molecule . Separation of extracts by HPLC indicated that the stimulatory substance is weakly hydrophobic and has retention times similar to those of uracil . So far, however, the exact chemical identity of the active substance has not been elucidated. J Biol Chem, 2001 Aug 10, 276(32), 30106 - 10 Epub 2001 Jun 27. Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen; Trent JT 3rd et al.; Neuroglobin is a newly discovered mammalian hemoglobin that is expressed predominately in the brain (Burmester, T., Welch, B., Reinhardt, S., and Hankeln, T . (2000) Nature 407, 520-523) . Neuroglobin has less than 25% identity with other vertebrate globins and shares less than 30% identity with the annelid nerve myoglobin it most closely resembles among known hemoglobins . Spectroscopic and kinetic experiments with the recombinant protein indicate that human neuroglobin is the first example of a hexacoordinate hemoglobin in vertebrates and is similar to plant and bacterial hexacoordinate hemoglobins in several respects . The ramifications of hexacoordination and potential physiological roles are explored in light of the determination of an O(2) affinity that precludes neuroglobin from functioning in traditional O(2) storage and transport. J Med Chem, 2001 Jul 5, 44(14), 2366 - 9 Synthesis and biological activity of 7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF) and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF); Borrell JI et al.; We recently described the syntheses of 12a-c, 4-amino-7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF), and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), in six steps from commercially available p-substituted methyl benzoates in 20-27% overall yields . Such analogues were tested in vitro against CCRF-CEM leukemia cells and showed that they are completely devoid of any activity, the IC(50) being higher than 20 microg/mL for all cases . To clarify if the presence of the carbonyl group in position C7, the distinctive feature of our synthetic methodology, is the reason for this lack of activity, we have now obtained the 7-oxo substituted analogues of 5-DATHF and DDATHF, 18a-c, in 10-30% overall yield . Testing of 18a-c in vitro against CCRF-CEM leukemia cells revealed that these compounds are totally inactive . A molecular modeling study of 18b inside the active site of the complex E . coliGARTFase-5-DATHF-GAR pointed to an electronic repulsion between the atoms of the 7-oxo group and the carbonyl group of Arg90 as a possible explanation for the inactivity of 18a-c. J Mol Biol, 2001 Jul 6, 310(2), 449 - 70 Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: comparison with functionally related proteins; Foloppe N et al.; The chemistry of active-site cysteine residues is central to the activity of thiol-disulfide oxidoreductases of the thioredoxin superfamily . In these reactions, a nucleophilic thiolate is required, but the associated pK(a) values differ vastly in the superfamily, from less than 4 in DsbA to greater than 7 in Trx . The factors that stabilize this thiolate are, however, not clearly established . The glutaredoxins (Grxs), which are members of this superfamily, contain a Cys-Pro-Tyr-Cys motif in their active site . In reduced Grxs, the pK(a) of the N-terminal active-site nucleophilic cysteine residue is lowered significantly, and the stabilization of the corresponding thiolate is expected to influence the redox potential of these enzymes . Here, we use a combination of long molecular dynamics (MD) simulations, pK(a) calculations, and experimental investigations to derive the structure and dynamics of the reduced active site from Escherichia coli Grx3, and investigate the factors that stabilize the thiolate . Several different MD simulations converged toward a consensus conformation for the active-site cysteine residues (Cys11 and Cys14), after a number of local conformational changes . Key features of the model were tested experimentally by measurement of NMR scalar coupling constants, and determination of pK(a) values of selected residues . The pK(a) values of the Grx3 active-site residues were calculated during the MD simulations, and support the underlying structural model . The structure of Grx3, in combination with the pK(a) calculations, indicate that the pK(a) of the N-terminal active-site cysteine residue in Grx3 is intermediate between that of its counterpart in DsbA and Trx . The pK(a) values in best agreement with experiment are obtained with a low (<4) protein dielectric constant . The calculated pK(a) values fluctuate significantly in response to protein dynamics, which underscores the importance of the details of the underlying structures when calculating pK(a) values . The thiolate of Cys11 is stabilized primarily by direct hydrogen bonding with the amide protons of Tyr13 and Cys14 and the thiol proton of Cys14, rather than by long-range interactions from charged groups or from a helix macrodipole . From the comparison of reduced Grx3 with other members of the thioredoxin superfamily, a unifying theme for the structural basis of thiol pK(a) differences in this superfamily begins to emerge . J Mol Biol, 2001 Jul 6, 310(2), 379 - 401 Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding; Holbrook JA et al.; Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid . Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H' site from lambda-phage DNA . At low to moderate {K(+)} (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (approximately 10(2)) and a very small non-specific site size . In this {K(+)} range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to {K(+)} . Above 100 mM K(+), only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing {K(+)} . When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity . We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 A of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs) . These salt-bridges increase in stability with increasing temperature and decreasing {K(+)} . To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low {K(+)} require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA . From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB . J Mol Biol, 2001 Jul 6, 310(2), 283 - 90 Rational design and molecular characterization of a chimaeric response regulator protein; Bock A et al.; BvgA and EvgA are closely related response regulators from Bordetella pertussis and Escherichia coli . To analyze the domain borders and linker sequences of these proteins, we used limited proteolysis and matrix-assisted laser desorption/ionization-mass spectrometry analysis of the in-gel-digested proteolytic fragments . The thermolysin-sensitive linker regions were found to extend from Leu130 to Thr144 for BvgA and from Leu127 to Ser133 for EvgA . These data provided the rationale for the construction of the chimaeric protein HA . HA carries the EvgA receiver and BvgA output domains, fused in the central part of the linker sequences of the parent proteins . Thermolysin-sensitive sites of HA were found at positions identical with those in the EvgA and BvgA linker sequences, indicating intact folding of its receiver and output domains . Consistent with this, the chimaera showed virtually unchanged phosphorylation and dimerization properties . However, BvgA and HA differed in the effect of phosphorylation on their DNA-binding activities . In the case of BvgA, phosphorylation resulted in an increased affinity and specificity in DNA binding, whereas the DNA-binding properties of HA were not affected by phosphorylation . The chimaera HA was unable to activate transcription of the BvgA-dependent fha promoter, either in vivo or in vitro . These results indicate that the phosphorylation-induced activation of BvgA requires specific interactions between the receiver and output domains that are disturbed in the chimaera . Exp Eye Res, 2001 Jul, 73(1), 1 - 7 Efficiency and toxicity of liposome-mediated gene transfer to corneal endothelial cells; Pleyer U et al.; Gene transfer to corneal endothelial cells could be an important advance to modulate functions of these critical cells and is a field of current investigations . The development of gene transfer methods is a prerequisite for gene therapy to realize its full potential . We attempted to investigate and optimize the efficacy and safety of cationic liposome mediated gene transfer into corneal endothelial cells using different lipid formulations . Mono- and polycationic lipids and the neutral helper lipid dioleolphosphotidyl-ethanolamine (DOPE) were used for preparation of cationic liposomes . Six liposomal formulations containing DAC/DOPE 30/70 (DAC 30), DOSGA/DOPE 30/70 (DOSGA 30), DOSGA 100, DMRIE/DOPE 50/50 (DMRIE 50) and SP/DOPE 20/80 (SP 20) were complexed with the pUT 651-plasmid, encoding the E . coli beta-galactosidase gene . Subconfluent primary and passaged bovine corneal endothelial cells (BCEC) were transfected with different amounts of liposomes and DNA or uncomplexed free DNA as control . Quantitative expression of beta-galactosidase was measured using a colorimetric assay . In order to assess the effects on cell viability and growth, a modified acidic phosphatase assay was employed . Differences were detected using these various liposome preparations . Transfection experiments demonstrated the highest gene expression using SP 20> DMRIE 50 ranging at approximately 3 mU per beta-gal per well . Low expression of beta-galactosidase was achieved using DAC 30, DOSGA 30 and DOSGA 100 . No beta-galactosidase expression was found in control dishes . There was no difference seen following transfection of primary or subsequent passages of BCEC . As indicated by the acid phosphatase assay, no significant toxicity was detected for the most efficient lipids used . Of the preparations studied, SP 20 appeared as the optimal vehicle for plasmid-mediated transfection of BCEC . The ability to deliver genes to BCEC via liposomes could be valuable, since the use of other vectors for transfection may be limited by undesired effects . Neurol Res, 2001 Jun, 23(4), 410 - 6 3-Aminobenzamide, a poly (ADP-ribose) synthetase inhibitor, attenuates the acute inflammatory responses and brain injury in experimental Escherichia coli meningitis in the newborn piglet; Park WS et al.; The aim of the present study was to evaluate the anti-inflammatory and neuroprotective effects of a poly (ADP-ribose) synthetase inhibitor 3-aminobenzamide during the early phase of experimental bacterial meningitis in the newborn piglet . Meningitis was induced by intracisternal injection of 10(8) colony forming units of Escherichia coli in 100 microl of saline . 3-Aminobenzamide, given 30 mg kg(-1) as a bolus i.v . injection 30 min before induction of meningitis, significantly attenuated the meningitis-induced acute inflammatory responses such as increased cerebrospinal fluid (CSF) lactate concentration, CSF leukocytosis and increased CSF tumor necrosis factor-alpha level . However, meningitis-induced increase in intracranial pressure and decrease in CSF glucose level were not significantly improved . Increased cerebral cortical cell membrane lipid peroxidation products (conjugated dienes) and decreased brain ATP/phosphocreatine levels observed in the meningitis group were also significantly improved with 3-aminobenzamide treatment . However, the improvement of reduced Na+, K+-ATPase activity did not reach a statistical significance (p = 0.06) . In summary, 3-aminobenzamide significantly attenuated the acute inflammatory responses and the ensuing brain injury during the early phase of neonatal bacterial meningitis . These findings suggest that poly (ADP-ribose) synthetase inhibitors such as 3-aminobenzamide might be a promising novel anti-inflammatory and neuroprotective adjuvant therapy in neonatal bacterial meningitis. J Zoo Wildl Med, 2000 Dec, 31(4), 532 - 8 Chorionic villus sampling for sex determination in a western lowland gorilla (Gorilla gorilla gorilla); Ramsay PA et al.; Chorionic villus sampling was undertaken on an anesthetized gorilla (Gorilla gorilla gorilla) to determine the fetal sex in the first trimester of pregnancy . The tissue samples were subject to sex determination by polymerase chain reaction, fluorescent in situ hybridization, and cell culture with cytogenetic analysis . Polymerase chain reaction testing was found to be the most accurate and rapid method of sex determination on this tissue sample. Rev Physiol Biochem Pharmacol, 2001, 143, 81 - 136 Transport of proteins into mitochondria; Truscott KN et al.; Most mitochondrial proteins are nuclear-encoded and synthesised as preproteins on polysomes in the cytosol . They must be targeted to and translocated into mitochondria . Newly synthesised preproteins interact with cytosolic factors until their recognition by receptors on the surface of mitochondria . Import into or across the outer membrane is mediated by a dynamic protein complex coined the translocase of the outer membrane (TOM) . Preproteins that are imported into the matrix or inner membrane of mitochondria require the action of one of two translocation complexes of the inner membrane (TIMs) . The import pathway of preproteins is predetermined by their intrinsic targeting and sorting signals . Energy input in the form of ATP and the electrical gradient across the inner membrane is required for protein translocation into mitochondria . Newly imported proteins may require molecular chaperones for their correct folding. Biotechnol Bioeng, 2001 Sep 5, 74(5), 364 - 75 Probing the performance limits of the Escherichia coli metabolic network subject to gene additions or deletions; Burgard AP et al.; An optimization-based procedure for studying the response of metabolic networks after gene knockouts or additions is introduced and applied to a linear flux balance analysis (FBA) Escherichia coli model . Both the gene addition problem of optimally selecting which foreign genes to recombine into E . coli, as well as the gene deletion problem of removing a given number of existing ones, are formulated as mixed-integer optimization problems using binary 0-1 variables . The developed modeling and optimization framework is tested by investigating the effect of gene deletions on biomass production and addressing the maximum theoretical production of the 20 amino acids for aerobic growth on glucose and acetate substrates . In the gene deletion study, the smallest gene set necessary to achieve maximum biomass production in E . coli is determined for aerobic growth on glucose . The subsequent gene knockout analysis indicates that biomass production decreases monotonically, rendering the metabolic network incapable of growth after only 18 gene deletions . In the gene addition study, the E . coli flux balance model is augmented with 3,400 non-E . coli reactions from the KEGG database to form a multispecies model . This model is referred to as the Universal model . This study reveals that the maximum theoretical production of six amino acids could be improved by the addition of only one or two genes to the native amino acid production pathway of E . coli, even though the model could choose from 3,400 foreign reaction candidates . Specifically, manipulation of the arginine production pathway showed the most promise with 8.75% and 9.05% predicted increases with the addition of genes for growth on glucose and acetate, respectively . The mechanism of all suggested enhancements is either by: 1) improving the energy efficiency and/or 2) increasing the carbon conversion efficiency of the production route . Nat Struct Biol, 2001 Jul, 8(7), 597 - 601 Interhelical hydrogen bonds in the CFTR membrane domain; Therien AG et al.; Critical mutations in the membrane-spanning domains of proteins cause many human diseases . We report the expression in Escherichia coli of helix-loop-helix segments of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel domain in milligram quantities . Analysis of gel migration patterns of these constructs, in conjunction with circular dichroism spectroscopy, demonstrate that a neutral-to-charged, CF-phenotypic point mutation of a hydrophobic residue (V232D) in the CFTR transmembrane (TM) helix 4 induces a hydrogen bond with neighboring wild type Gln 207 in TM helix 3 . As an electrostatic crosslink within a hydrocarbon phase, such a hydrogen bond could alter the normal assembly and alignment of CFTR TM helices and/or impede their movement in response to substrate transport . Our results imply that membrane proteins may be vulnerable to loss of function through formation of membrane-buried interhelical hydrogen bonds by partnering of proximal polar side chains. Plant Cell Physiol, 2001 Jun, 42(6), 650 - 6 Lipoic acid metabolism in Arabidopsis thaliana: cloning and characterization of a cDNA encoding lipoyltransferase; Wada M et al.; Lipoic acid is an essential coenzyme required for activity of several key enzyme complexes, such as the pyruvate dehydrogenase complex, in the central metabolism . In these complexes, lipoic acid must be covalently attached to one of the component proteins for it to have biological activity . We report the cloning and characterization of Arabidopsis thaliana LIP2 cDNA for lipoyltransferase that catalyzes the transfer of the lipoyl group from lipoyl-acyl carrier protein to lipoate-dependent enzymes . This cDNA was shown to code for lipoyltransferase by its ability to complement an Escherichia coli lipB null mutant lacking lipoyltransferase activity . The expressed enzyme in the E . coli mutant efficiently complemented the activity of pyruvate dehydrogenase complex, but less efficiently than that of 2-oxoglutarate dehydrogenase complex . Comparison of the deduced amino acid sequence of LIP2 with those of E . coli and yeast lipoyltransferases showed a marked sequence similarity and the presence of a leader sequence presumably required for import into mitochondria . Southern and northern hybridization analyses suggest that LIP2 is a single-copy gene and is expressed as an mRNA of 860 nt in leaves . Western blot analysis with an antibody against lipoyltransferase demonstrated that a 29 kDa form of lipoyltransferase is located in the mitochondrial compartment of A . thaliana. Plant Cell Physiol, 2001 Jun, 42(6), 627 - 34 Serine acetyltransferase involved in cysteine biosynthesis from spinach: molecular cloning, characterization and expression analysis of cDNA encoding a plastidic isoform; Noji M et al.; A cDNA clone that encodes a chloroplast-localizing isoform of serine acetyltransferase (SATase) (EC 2.3.1.30) was isolated from spinach (Spinacia oleracea L.) . The cDNA encodes a polypeptide of 347 amino acids containing a putative transit peptide of ca . 60-70 amino acids at the N-terminal . Deduced amino acid sequence of SATase from spinach exhibited homology with other SATases from plants . DNA blot hybridization analysis showed the presence of 2-3 copies of Sat gene in the genome of spinach . RNA blot hybridization analysis indicated the constitutive expression of Sat gene in green and etiolated seedlings of spinach . Bacterial expression of the cDNA could directly rescue the cysteine auxotrophy of Escherchia coli caused by a lack of SATase locus (cysE) . Catalytically active SATase protein was produced in E . coli cells . L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the activity of recombinant spinach SATase, indicating the regulatory function of SATase in this metabolic pathway . A chloroplastic localization of this spinach SATase was revealed by the analyses of transgenic plant expressing transit peptide of SATase-beta-glucuronidase (GUS) fusion protein, and transient expression using the transit peptide-green fluorescent protein (GFP) fusion protein . The result from in vitro translation analysis suggests that this cDNA may encode both plastidic and cytosolic SATases. J Clin Microbiol, 2001 Jul, 39(7), 2418 - 24 Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis; Landowski CP et al.; Laboratory diagnosis of tuberculosis is often difficult . Immunodetection of circulating Mycobacterium tuberculosis proteins shed during active infection would not depend on an intact host immune response and could take advantage of the speed and low costs afforded by antibody-based assays . We previously showed that patients with active tuberculosis had increased levels of circulating antigen 85 (Ag85) proteins independent of their tuberculin skin test status (S . I . Bentley-Hibbert, X . Quan, T . Newman, K . Huygen, and H . P . Godfrey, Infect . Immun . 67:581-588, 1999) . To extend these observations to a Mycobacterium bovis BCG-vaccinated population and t