Eur J Biochem, 2000 Jan, 267(1), 145 - 54 Characterization of a sulfurtransferase from Arabidopsis thaliana; Papenbrock J et al.; A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase-like proteins . One gene and several homologous EST clones were identified . One of the EST clones was used for screening an Arabidopsis cDNA library . The isolated full-length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa . Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions . Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes in Arabidopsis . Recombinant proteins with and without the putative transit peptide were expressed in E . coli with an N-terminal His6-tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors . Both recombinant proteins catalyzed the formation of SCN- from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3-mercaptopyruvate to thiosulfate . A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size . A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts . The function of the protein is still speculative . Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants. Int J Mol Med, 2000 Jan, 5(1), 15 - 20 Molecular approach in cancer epidemiology: early detection of carcinogen-induced mutations in a whole genome (Review); Burnouf D et al.; Chronic exposure of organisms to endo- or exogenous genotoxic products results in the accumulation of mutations in the genome and eventually to the development of cancers . Early detection of these mutations would allow the identification of at risk individuals who present a high load of mutations either because of an occupational or environmental exposure, or because of less efficient DNA repair processes . However, highly specific and sensitive assays are required to allow the detection of point mutations in a whole genome . We review a long-term study on the mutagenesis induced in E.coli by an aromatic amide, the N-2-acetylaminofluorene . A major contribution of this work was to reveal the presence of specific mutation hot spot sequences . Taking advantage of this observation, we designed a specific, sensitive and semi-quantitative in vitro assay allowing the detection of carcinogen induced mutations . This assay has been validated in vivo and demonstrate the sensitivity of the technique in early detection of mutations and its usefullness in molecular epidemiology, early diagnostic and prognosis. Gut, 2000 Jan, 46(1), 46 - 51 Production of a panel of recombinant gliadins for the characterisation of T cell reactivity in coeliac disease; Arentz-Hansen EH et al.; BACKGROUND/AIMS: Coeliac disease is a chronic intestinal disorder most probably caused by an abnormal immune reaction to wheat gliadin . The identification of the HLA-DQ2 and HLA-DQ8 as the molecules responsible for the HLA association in coeliac disease strongly implicates a role for CD4 T cells in disease pathogenesis . Indeed, CD4 T cells specific for gliadin have been isolated from the small intestine of patients with coeliac disease . However, identification of T cell epitopes within gliadin has been hampered by the heterogeneous nature of the gliadin antigen . To aid the characterisation of gliadin T cell epitopes, multiple recombinant gliadins have been produced from a commercial Nordic wheat cultivar . METHODS: The alpha-gliadin and gamma-gliadin genes were amplified by polymerase chain reaction from cDNA and genomic DNA, cloned into a pET expression vector, and sequenced . Genes encoding mature gliadins were expressed in Escherichia coli and tested for recognition by T cells . RESULTS: In total, 16 alpha-gliadin genes with complete open reading frames were sequenced . These genes encoded 11 distinct gliadin proteins, only one of which was found in the Swiss-Prot database . Expression of these gliadin genes produced a panel of recombinant alpha-gliadin proteins of purity suitable for use as an antigen for T cell stimulation . CONCLUSION: This study provides an insight into the complexity of the gliadin antigen present in a wheat strain and has defined a panel of pure gliadin antigens that should prove invaluable for the future mapping of epitopes recognised by intestinal T cells in coeliac disease. Genes Dev, 1999 Dec 1, 13(23), 3081 - 91 The cAMP receptor protein CRP can function as an osmoregulator of transcription in Escherichia coli; Landis L et al.; Transcription of the P1 promoter of the Escherichia coli proP gene, which encodes a transporter of osmoprotectants, is strongly induced by a shift to hyperosmotic media . Unlike most other osmotically regulated promoters, the induction occurs for a brief period of time, corresponding to the replacement of intracellular K(+) glutamate with osmoprotecting compounds . This burst of proP transcription is correlated with the osmolarity-dependent binding of the cAMP receptor protein CRP to a site within the proP P1 promoter . We show that CRP-cAMP functions as an osmotically sensitive repressor of proP P1 transcription in vitro . Binding of CRP to the proP promoter in vivo is transiently destabilized after a hyperosmotic shift with kinetics that correspond to the derepression of transcription, whereas Fis and Lac repressor binding is not osmotically sensitive . Similar osmotic regulation of proP P1 transcription by the CRP* mutant implies that binding of cAMP is not responsible for the unusual osmotic sensitivity of CRP activity . Osmotic regulation of CRP activity is not limited to proP . Activation of the lac promoter by CRP is also transiently inhibited after an osmotic upshift, as is the binding of CRP to the galdelta4P1 promoter . These findings suggest that CRP functions in certain contexts to regulate gene expression in response to osmotic changes, in addition to its role in catabolite control. J Exp Biol, 2000 Jan, 203 Pt 1, 35 - 40 Rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase; Senior AE et al.; The rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase is of the order of 10(11)-fold . We present a cyclic enzyme mechanism for the reaction, relate it to known F(1) X-ray structure and speculate on the linkage between enzyme reaction intermediates and subunit rotation . Next, we describe five factors known to be important in the Escherichia coli enzyme for the rate acceleration . First, the provision of substrate binding energy by residues lining the catalytic site is substantial; beta-Lys155 and beta-Arg182 are specific examples, both of which differentially support substrate MgATP versus product MgADP binding . Second, octahedral coordination of the Mg(2+) in MgATP is crucial for both catalysis and catalytic site asymmetry . The residues involved are beta-Thr156, beta-Glu185 and beta-Asp242 . Third, there is stabilization of a pentacoordinate phosphorus catalytic transition state by residues beta-Lys155, beta-Arg182 and alpha-Arg376 . Fourth, residue beta-Glu181 binds the substrate water and stabilizes the catalytic transition state . Fifth, there is strong positive catalytic cooperativity, with binding of MgATP at all three sites yielding the maximum rate (V(max)); the molecular basis of this factor remains to be elucidated. J Exp Biol, 2000 Jan, 203 Pt 1, 29 - 33 Cross-linking and electron microscopy studies of the structure and functioning of the Escherichia coli ATP synthase; Capaldi RA et al.; ATP synthase, also called F(1)F(o)-ATPase, catalyzes the synthesis of ATP during oxidative phosphorylation . The enzyme is reversible and is able to use ATP to drive a proton gradient for transport purposes . Our work has focused on the enzyme from Escherichia coli (ECF(1)F(o)) . We have used a combination of methods to study this enzyme, including electron microscopy and chemical cross-linking . The utility of these two approaches in particular, and the important insights they give into the structure and mechanism of the ATP synthase, are reviewed. J Exp Biol, 2000 Jan, 203 Pt 1, 19 - 28 Structure and function of the F(o) complex of the ATP synthase from Escherichia coli; Altendorf K et al.; The membrane-bound ATP synthase (F(1)F(o)) from mitochondria, chloroplasts and bacteria plays a crucial role in energy-transducing reactions . In the case of Escherichia coli, the reversible, proton-translocating ATPase complex consists of two different entities, F(1) and F(o) . The water-soluble F(1) part carries the catalytic sites for ATP synthesis and hydrolysis . It is associated with the membrane-embedded F(o) complex, which functions as a proton channel and consists of subunits a, b and c present in a stoichiometry of 1:2:12.Subunit b was isolated by preparative gel electrophoresis, acetone-precipitated and renatured in a cholate-containing buffer . Reconstituted subunit b together with purified ac subcomplex is active in proton translocation and F(1) binding, thereby demonstrating that subunit b had recovered its native conformation . Circular dichroism spectroscopy of subunit b reconstituted into liposomes revealed a rather high degree of alpha -helical conformation of 80% . After addition of a His(6)-tag to the N terminus of subunit a, a stable ab(2) subcomplex was purified instead of a single subunit a, arguing in favour of a direct interaction between these subunits . After addition of subunit c and reconstitution into phospholipid vesicles, an F(o) complex was obtained exhibiting rates of proton translocation and F(1) binding comparable with those of wild-type F(o).The epitopes of monoclonal antibodies against subunit c are located in the hydrophilic loop region (cL31-Q42) as mapped by enzyme-linked immunosorbent assay using overlapping synthetic heptapeptides . Binding studies revealed that all monoclonal antibodies (mAbs) bind to everted membrane vesicles irrespective of the presence or absence of F(1) . Although the hydrophilic region of subunit c, and especially the highly conserved residues cA40, cR41, cQ42 and cP43, are known to interact with subunits gamma and epsilon of the F(1) part, the mAb molecules have no effect on the function of F(o), either in proton translocation or in F(1) binding . However, the F(1) part and the mAb molecule(s) are bound simultaneously to the F(o) complex, suggesting that not all c subunits are involved in the interaction with F(1). J Exp Biol, 2000 Jan, 203 Pt 1, 9 - 17 Coupling H(+) transport to rotary catalysis in F-type ATP synthases: structure and organization of the transmembrane rotary motor; Fillingame RH et al.; H(+)-transporting F(1)F(o)-type ATP synthases utilize a transmembrane H(+) potential to drive ATP formation by a rotary catalytic mechanism . ATP is formed in alternating beta subunits of the extramembranous F(1) sector of the enzyme, synthesis being driven by rotation of the gamma subunit in the center of the F(1) molecule between the alternating catalytic sites . The H(+) electrochemical potential is thought to drive gamma subunit rotation by first coupling H(+) transport to rotation of an oligomeric rotor of c subunits within the transmembrane F(o) sector . The gamma subunit is forced to turn with the c(12) oligomeric rotor as a result of connections between subunit c and the gamma and epsilon subunits of F(1) . In this essay, we will review recent studies on the Escherichia coli F(o) sector . The monomeric structure of subunit c, determined by nuclear magnetic resonance (NMR), is discussed first and used as a basis for the rest of the review . A model for the structural organization of the c(12) oligomer in F(o), deduced from extensive cross-linking studies and by molecular modeling, is then described . The interactions between the the a(1)b(2) 'stator' subcomplex of F(o) and the c(12) oligomer are then considered . A functional interaction between transmembrane helix 4 of subunit a (aTMH-4) and transmembrane helix 2 of subunit c (cTMH-2) during the proton-release step from Asp61 on cTMH-2 is suggested . Current a-c cross-linking data can only be explained by helix-helix swiveling or rotation during the proton transfer steps . A model that mechanically links helix rotation within a single subunit c to the incremental 30 degrees rotation of the c(12) oligomer is proposed . In the final section, the structural interactions between the surface residues of the c(12) oligomer and subunits epsilon and gamma are considered . A molecular model for the binding of subunit epsilon between the exposed, polar surfaces of two subunits c in the oligomer is proposed on the basis of cross-linking data and the NMR structures of the individual subunits. Biochem J, 2000 Jan 1, 345 Pt 1, 153 - 60 Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme; Abbadi A et al.; A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-{acyl-carrier-protein (ACP)} synthase III (KAS III), which catalyses the committing condensing reaction . Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP . It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli . We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e . formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation . Incubation of recombinant enzyme with {1-(14)C}acetyl-CoA-labelled Cys(111), and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity . The Cys(111)Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP . We replaced His(261) with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His(261) . Double mutants Cys(111)Ser/His(261)Ala and Cys(111)Ser/His(261)Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of {2-(14)C}malonyl-ACP, a reaction indicating the role of His(261) in general-acid catalysis . Finally, alanine scanning revealed the involvement of Arg(150) and Arg(306) in KAS III catalysis . The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme. Biochem J, 2000 Jan 1, 345 Pt 1, 145 - 51 Palmitoylation of the 25-kDa synaptosomal protein (SNAP-25) in vitro occurs in the absence of an enzyme, but is stimulated by binding to syntaxin; Veit M; The neuronal N-ethylmaleimide-sensitive-factor-attachment-protein receptor (SNARE) proteins 25-kDa synaptosomal protein (SNAP-25), syntaxin 1 and synaptobrevin 2 interact to form the intermembrane SNARE complex, which mediates docking and fusion of synaptic vesicles with the plasma membrane . Assembly of the SNARE complex is accompanied by conformational changes, especially in SNAP-25 . SNAP-25 is palmitoylated in vivo at cysteine residues located in the middle of the molecule . Acylation is required for membrane binding or membrane targeting of this intrinsically hydrophilic protein . Palmitoylation of recombinant SNAP-25 was studied in vitro in the absence of an enzyme source with {(3)H}palmitoyl-CoA as the lipid donor . {(3)H}Palmitate incorporation into unbound SNAP-25 was negligible, but was stimulated 100-fold when SNAP-25 was present in the SNARE complex . SNAP-25 in a binary complex with syntaxin 1 was palmitoylated with almost the same efficiency . A mutant of SNAP-25, which was not acylated in vivo, did not incorporate {(3)H}palmitate in this assay . {(3)H}Palmitate incorporation into wild-type SNAP-25 was blocked by chemical blocking of free SH groups, but slightly stimulated by reduction of disulfide-bonds . This shows that palmitoylation of SNAP-25 in vitro occurs at the same cysteine residues that are palmitoylated in vivo . This demonstrates that efficient palmitoylation of SNAP-25 depends on an interaction with a physiological binding partner . It suggests further that palmitoylation of SNAP-25 requires the alpha-helical conformation of the protein, which is induced by binding to syntaxin 1. Biochem J, 2000 Jan 1, 345 Pt 1, 53 - 60 Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies; Abou Hachem M et al.; The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography . Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose . Binding to insoluble substrates was significantly enhanced by the presence of Na(+) and Ca(2+) ions . The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-glucan . CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities . Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities . The binding of the modules was shown to be dominated by enthalpic forces . The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module . This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose. Electrophoresis, 1999 Oct, 20(15-16), 3145 - 55 Applications of capillary electrophoresis in biotechnology; Lagu AL; Capillary electrophoresis (CE)-related techniques are increasingly being used as a matter of routine practice in the biotechnology discipline . Since recombinant DNA-derived proteins and the antisense oligonucleotides constitute a large portion of the applications of these techniques, they have been emphasized in this review . Analyses by CE of Escherichia coli-derived proteins and glycosylated proteins derived from mammalian cell cultures are summarized, as well as those of the carbohydrate chains that have been enzymatically removed from the protein . Applications of CE in the analysis of the antisense oligonucleotides for the determination of purity and the analytical studies on the metabolism of these modified oligonucleotides, by CE are reviewed . The literature mainly covers the period from 1996. Scand J Immunol, 1999 Dec, 50(6), 619 - 25 Expression and analysis of recombinant salmon parvalbumin, the major allergen in Atlantic salmon (Salmo salar); Van Do T et al.; The parvalbumin from white muscle of Atlantic salmon was previously found to be a major allergen, and designated Sal s1 . Two distinct cDNAs, 14.1 and 24.1, which comprise the entire parvalbumin-encoding regions, were cloned, revealing transcripts from two different parvalbumin genes . In the present study, the protein-coding regions of these cDNAs were subcloned into an Escherichia coli expression vector (pET-19b) . Both proteins were expressed and the generated target proteins were localized in both soluble and insoluble fractions of the expression host . The recombinant products in the soluble fraction were purified using the His tag-purification system and analysed on Western blots with anti-salmon parvalbumin polyclonal rabbit sera and sera from patients allergic to fish . Both recombinant products (His10-14.1 and His10-24.1) reacted positively with salmon parvalbumin-specific immunoglobulin G (IgG) from rabbits, and with specific immunoglobulin E (IgE) from the sera of six fish-allergic patients . The allergenicity of His10-14.1 was confirmed using enzyme-linked immunosorbent assay (ELISA) . The 14.1 cDNA of salmon parvalbumin was shown to be the dominant type represented in a muscle cDNA library. Scand J Immunol, 1999 Dec, 50(6), 580 - 7 Ribosomal protein L7 included in tuberculin purified protein derivative (PPD) is a major heat-resistant protein inducing strong delayed-type hypersensitivity; Kitaura H et al.; The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis . It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis . In two-dimensional electrophoretic analysis of PPDs from M . tuberculosis and M . bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance . Two such proteins corresponded to ribosomal proteins L7 and L12 . The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction . Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity . There were a few other unidentified proteins . It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli . L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG . We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes . L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity . The activity of L7/L12 was heat resistant . Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected . The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity. J Sleep Res, 1995 Mar, 4(1), 30 - 40 Somnogenic effects of sleep deprivation and Escherichia coli inoculation in rabbits; Toth LA et al.; To evaluate the impact of sleep deprivation (SD) on microbially induced alterations in sleep, we used gentle handling to deprive rabbits of sleep for 4 h before or after intravenous inoculation with Escherichia coli (EC) . Sleep was monitored for the next 20 h . EC inoculation alone increased slow-wave sleep (SWS) time, delta-wave amplitude (DWA) during sleep and SWS bout length during the initial 2-4 h after inoculation . During the following 8-20 h, DWA during SWS was reduced relative to control values . SD alone increased SWS time and SWS bout length for 2 h after the end of the SD period . Rabbits subjected to SD for 4 h prior to EC inoculation demonstrated increases in SWS time, DWA during SWS and SWS bout length 2-4 h postinoculation . At some time points, these effects were greater in magnitude than those induced by either manipulation alone, but they were generally equivalent to the additive effects of the individual treatments . Rabbits subjected to SD after EC inoculation (i.e . during the period in which EC increases sleep) demonstrated increases in SWS time and DWA during SWS for only 2 h after the end of the SD period, suggesting that these animals maintained a sleep deficit as compared to rabbits inoculated with EC alone . SD alone elicited hyperthermia in rabbits, and EC-inoculated rabbits subjected to SD developed fevers greater than those induced by either treatment alone . Other clinical indices were not significantly affected by the combined treatments . These data indicate that the sleep changes that occur subsequent to bacterial inoculation are altered in sleep-deprived rabbits, but that SD does not exacerbate clinical illness in this model. Mutat Res, 1999 Dec 7, 435(3), 245 - 54 Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid; Schouten KA et al.; Endonuclease V (deoxyinosine 3'-endonuclease) of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA's containing hypoxanthine, uracil, or mismatched bases . An endonuclease V (nfi) mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles . No marked differences were seen in frequencies of spontaneous reversion . However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfi(+) strains, they displayed a high frequency of A:T-->G:C, and G:C-->A:T transition mutations . Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine . The nitrous acid-induced A:T-->G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues . A confirmatory finding was that the mutagenesis was depressed at a locus containing N(6)-methyladenine, which is known to be relatively resistant to nitrosative deamination . An alkA mutation did not significantly enhance the frequency of A:T-->G:C mutations in an nfi mutant, even though AlkA (3-methyladenine-DNA glycosylase II) has hypoxanthine-DNA glycosylase activity . The nfi mutants also displayed high frequencies of nitrous acid-induced G:C-->A:T transitions . These mutations could not be explained by cytosine deamination because an ung (uracil-DNA N-glycosylase) mutant was not similarly affected . However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA . The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination. Mutat Res, 1999 Dec 7, 435(3), 171 - 213 A phylogenomic study of DNA repair genes, proteins, and processes; Eisen JA et al.; The ability to recognize and repair abnormal DNA structures is common to all forms of life . Studies in a variety of species have identified an incredible diversity of DNA repair pathways . Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments) . Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited . Complete genome sequences can provide potential sources of new information about repair in different species . In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences . We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics . We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence. FEBS Lett, 1999 Dec 17, 463(3), 371 - 4 Interdomain interactions within the gene 3 protein of filamentous phage; Chatellier J et al.; Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p) . The g3p consists of three domains (g3p-D1, D2 and D3) linked by flexible glycine-rich linkers . All three domains are indispensable for phage infectivity; the g3p-D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p-D2 domain to the F-pilus and the g3p-D3 domain anchors g3p to the phage coat . The N-terminal domains g3p-D1 and D2 interact with each other; this interaction is abrogated by binding of g3p-D2 to the F-pilus leading to the release of g3p-D1 to bind to TolA . Here, using phages with deletions in g3p, we have discovered a specific interaction between the two N-terminal domains and g3p-D3, the C-terminal domain of g3p . We propose that these interdomain interactions within g3p lead to a compact and stable organisation when displayed on the phage tip, but that during infection, this compact state must be unraveled. FEBS Lett, 1999 Dec 17, 463(3), 331 - 5 Requirement for phospholipids of the translocation of the trimethylamine N-oxide reductase through the Tat pathway in Escherichia coli; Mikhaleva NI et al.; Trimethylamine N-oxide reductase (TorA) is an anaerobically synthesized molybdoenzyme . It is translocated across the cytoplasmic membrane in a folded conformation via the Tat pathway of Escherichia coli . The requirement for phospholipids for the export of this enzyme was analyzed in the pgsA and pss mutants lacking anionic phospholipids and phosphatidylethanolamine, respectively . Anaerobic growth did not influence phospholipid composition of the pgsA and pss mutants . Interestingly, both pgsA and pss mutations severely retarded the translocation of TorA into the periplasm . Therefore, translocation of proteins through the Tat pathway is dependent on the anionic phospholipids and on lipid polymorphism. FEBS Lett, 1999 Dec 17, 463(3), 211 - 5 Effective expression and purification of recombinant onconase, an antitumor protein; Notomista E et al.; Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines . One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials . We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields . The recombinant protein has biological and catalytic properties identical to those of the natural enzyme. FEBS Lett, 1999 Dec 17, 463(3), 205 - 10 Substitution of a conserved amino acid residue alters the ligand binding properties of peroxisome proliferator activated receptors; Causevic M et al.; Mutation of glutamic acid 282 of PPARalpha to glycine has been shown to result in an increased EC(50) for a wide variety of PPAR activating compounds . This has suggested that mutant receptor has a reduced ability to bind ligand . In this study we show that this mutation reduces the affinity of mPPARalpha and hPPARgamma for the fluorescent fatty acid, cis-parinaric acid and that the mutant hPPARgamma protein has a reduced affinity for the radiolabelled compound, SB236636 . These data confirm the role of this glutamic acid in ligand binding and support recent crystal structure observations regarding a proposed novel mode of ligand entry into the PPAR ligand binding cavities. Nucleic Acids Res . 2000 Jan 15;28(2):e4. The specificity of protein-DNA crosslinking by formaldehyde: in vitro and in drosophila embryos; Toth J et al.; Formaldehyde crosslinking has been widely used to study binding of specific proteins to DNA elements in intact cells . However, previous studies have not determined if this crosslinker preserves the bona fide pattern of DNA binding . Here we show that formaldehyde crosslinking of Drosophila embryos maps an interaction of the transcription factor Zeste to a known target element in the Ultrabithorax promoter . This data agrees broadly with previous mapping of the same Zeste binding sites by in vivo UV crosslinking, though the formaldehyde method does give a low, possibly artifactual signal on other DNA fragments that is not detected by the UV method . We also demonstrate, using an in vitro assay, that formaldehyde crosslinking accurately reflects the DNA binding specificities of both Zeste and a second transcription factor, Eve . The crosslinking reagent methylene blue is shown to preserve DNA binding specificity in vitro as well . Our results suggest that crosslinking by formaldehyde, and possibly also by methylene blue, provide an accurate guide to the interaction of proteins with their high affinity target sites in cells. Nucleic Acids Res . 2000 Jan 15;28(2):e3. UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-ligand interactions; Ge H; Protein-protein interactions have been widely used to study gene expression pathways and may be considered as a new approach to drug discovery . Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative, multi-purpose, effective and easy technology to determine not only specific protein-protein interactions, but also specific interactions of proteins with DNA, RNA, ligands and other small chemicals . (i) Since purified proteins are used, the results can be easily interpreted . (ii) UPA can be used multiple times for different targets, making it economically affordable for most laboratories, hospitals and biotechnology companies . (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation is required . (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared with most existing methods . (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases. Biochemistry, 1999 Dec 21, 38(51), 16882 - 8 ATP and other nucleotides stabilize the Rho-mRNA complex; Gan E et al.; Transcription termination factor Rho from Escherichia coli is a protein that consists of a single 47 kDa protomeric unit that can form a hexameric structure . To determine whether active hexamers can form on an RNA by assembly of subunits, we measured the dependence of complex formation on the concentration of Rho protein in the presence and absence of various nucleotides and related the binding properties to association states determined from sedimentation properties . The results show that the presence of adenine nucleotides converts RNA binding from a multimeric process to a largely monomeric process and that the change correlates with the stabilization of multimers of Rho by the nucleotides . The experimental evidence also indicates that the hexameric form of Rho is stabilized slightly by binding to a transcript but that the protein on RNA is in equilibrium with nonhexameric forms . These results suggest that a Rho hexamer can form on a transcript by addition of subunits to a partial assembly, which means that the complex can consist of six subunits surrounding an RNA transcript as proposed in recent models for Rho action. Biochemistry, 1999 Dec 21, 38(51), 16847 - 56 Coupled oxidation of heme covalently attached to cytochrome b562 yields a novel biliprotein; Rice JK et al.; A variant of Escherichia coli cytochrome b(562) with covalently attached heme can be converted to a biliverdin-containing protein in two distinct stages by coupled oxidation and acid hydrolysis . The first stage of coupled oxidation yields a stable verdoheme-containing protein . This verdoheme protein is unusual in three respects . First, the verdoheme group is covalently bound to the protein through a c-type thioether linkage . Second, the oxidation stops at the verdoheme stage, and finally, this is the first report of verdoheme generated from a heme protein with exclusive methionine ligation to the heme iron . In addition, the oxidation process does not require denaturation of the protein . The product has been characterized by optical spectroscopy, ESI mass spectrometry, and (1)H NMR . The NMR data show that the predominant product is the result of oxidation at the alpha-meso carbon . A collective evaluation of data on the topic suggests that the electronic structure of the heme, not protein steric effects, is the main factor in controlling the regiospecificity of the oxidation site . In the second stage of conversion to a biliprotein, we demonstrate that the verdoheme ring can be opened by treatment with aqueous formic acid to give alpha-biliverdin covalently attached to the folded protein . This product, a protein-bound linear tetrapyrrole as characterized by optical spectroscopy and mass spectrometry, is an example of a phycobilin chromophore that has not been observed previously. Biochemistry, 1999 Dec 21, 38(51), 16783 - 93 New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with beta-GAR and 10-formyl-5,8,10-trideazafolic acid; Greasley SE et al.; The crystal structure of Escherichia coli GAR Tfase at 2.1 A resolution in complex with 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, K(i) = 260 nM), an inhibitor designed to form an enzyme-assembled multisubstrate adduct with the substrate, beta-GAR, was studied to determine the exact nature of its inhibitory properties . Rather than forming the expected covalent adduct, the folate inhibitor binds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedral intermediate of the formyl transfer reaction . In this hydrated form, the inhibitor not only provides unexpected insights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between 10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lack the alcohol . The presence of the hydrated aldehyde was confirmed in solution by (13)C-(1)H NMR spectroscopy of the ternary GAR Tfase-beta-GAR-10-formyl-TDAF complex using the (13)C-labeled 10-formyl-TDAF . This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors, provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplastic agents. Biochemistry, 1999 Dec 21, 38(51), 16777 - 82 Location of helix III in the lactose permease of Escherichia coli as determined by site-directed thiol cross-linking; Wang Q et al.; The six N-terminal transmembrane helices (N(6)) and the six C-terminal transmembrane helices (C(6)) in the lactose permease of Escherichia coli, each containing a single Cys residue, were coexpressed, and cross-linking was studied . The proximity of paired Cys residues in helices III (position 78, 81, 84, 86, 87, 88, 90, 93, or 96) and VII (position 227, 228, 231, 232, 235, 238, 239, 241, 243, 245, or 246) was examined by using iodine or two rigid homobifunctional thiol-specific cross-linking reagents with different lengths {N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N, N'-p-phenylenedimaleimide (p-PDM; 10 A)} . Cys residues in the periplasmic half of helix III (position 87, 93, or 96) cross-link to Cys residues in the periplasmic half of helix VII (position 235, 238, 239, 241, or 245) . In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices III (position 78 or 81) and VII (position 227, 228, 213, 232, or 235) . Therefore, the periplasmic halves of helices III and VII are in close proximity, and the helices tilt away from each other toward the cytoplasmic face of the membrane . On the basis of the findings, a modified helix packing model for the permease is presented. Biochemistry, 1999 Dec 21, 38(51), 16740 - 8 EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase; Kay CW et al.; Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer . Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase . The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD . In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz . From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted . Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5) . To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied . All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD . The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety . Two further hfc's have been detected and assigned to the inequivalent protons at C(1') . Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees. RNA, 1999 Dec, 5(12), 1656 - 64 Proteins neighboring 18S rRNA conserved sequences 609-618 and 1047-1061 within the 40S human ribosomal subunit; Malygin AA et al.; The structure of human 40S ribosomal subunits has been probed by a cross-linking strategy based on the use of oligonucleotide derivatives that modify proteins in the vicinity of specific 18S rRNA sequences . The oligonucleotide derivatives carried a p-azidoperfluorobenzamide group at the 5' ends and were complementary to 18S rRNA sequences 609-618 and 1047-1061, homologous to the highly conserved regions designated as the "530 stem-loop" and "790 stem-loop", respectively, in Escherichia coli 16S rRNA . Ribosomal proteins surrounding these sequences were the main targets of the cross-linking . Proteins S3 and S5 were cross-linked to the derivative complementary to the sequence 609-618, and proteins S2 and S3 were modified by the derivative complementary to the sequence 1047-1061 . Cross-linking was not affected by binding of 40S subunits to either poly(U) or poly(U) and Phe-tRNA(Phe). Int J Biochem Cell Biol, 1999 Nov, 31(11), 1315 - 26 Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I; Nakashima Y et al.; Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography . The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0 . It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin . A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing . The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity . The mature hen DNase I protein was found to consist of 262 amino acids . In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively . A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression . The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes . This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I. J Antibiot (Tokyo), 1999 Oct, 52(10), 895 - 9 A mammalian vector carrying the bleomycin N-acetyltransferase gene from bleomycin-producing Streptomyces verticillus as a selective marker; Sugiyama M et al.; A gene, blmB, encodes a bleomycin (Bm) N-acetyltransferase, designated BAT, from Bm-producing Streptomyces verticillus and confers resistance to Bm in Streptomyces and Escherichia coli . COS-1 cells transfected with a plasmid designated pEF-BOS/blmB, in which blmB is under the control of a strong promoter from the human polypeptide chain elongation factor 1alpha, transiently produced BAT . Immuno-cytochemical analysis using an anti-BAT monoclonal antibody revealed that BAT was localized in the nucleus of the blmB-carrying COS-1 cells . NIH/3T3 cells, transfected with pEF-BOS/blmB, stably expressed BAT at least for one month . The stable transformants of blmB showed specific resistance to the Bm family of antibiotics, suggesting that blmB has potential as a selective marker in gene transfer studies with mammalian cells. Pharmazie, 1999 Nov, 54(11), 847 - 50 Evaluation of in vitro toxicity of N,N-dimethyl-2-propen-1-amines isomers; Oliveira DA et al.; The trypanocidal activities of cis-3-(4'-bromo{1,1'-biphenyl}-4-yl)- 3-(phenyl)-N,N-dimethyl-2-propen-1-amine (Vb) and cis-3-(4'-bromo{1,1'-biphenyl}-4-yl)-3-(4-bromophenyl)-N,N-dimethyl-2- propen-1-anine (Vg) appeared 6.3 and 3.5 fold more active than the trans-isomers, respectively . Multi-endpoints for toxicity were also applied . Neutral red uptake (NRU), tetrazolium salt reduction (MTT), DNA content on V79 fibroblast cell culture and acute toxicity von E . coli were measured . The IC50 through DNA contents was lower for the cis-isomers in both series of compounds 5b: 7.8 microM and 5g: 5.2 microM) . NRU values for derivative 5b in isomeric mixture shows the same value as the isolated isomers however, in the case of 5g a more significant toxicity of the cis-isomer was found . MTT values show that 5g is more toxic than 5b . In both cases, the acute toxicity of the trans-isomers was higher than that of the cis-isomers. Infect Immun, 2000 Jan, 68(1), 233 - 8 Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons; Lakey DL et al.; A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins . To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E . coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase . Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E . coli 54-fold . The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes . Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, suggesting that the enhanced protein production was due primarily to enhanced efficiency of translation . Codon replacement in the genes encoding antigen 85A and superoxide dismutase yielded four- to sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E . coli. Infect Immun, 2000 Jan, 68(1), 64 - 71 Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant; Oswald E et al.; Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E . coli (EHEC) produce the characteristic "attaching and effacing" (A/E) lesion of the brush border . Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell . Several eae have been cloned from different EPEC and EHEC strains isolated from humans and animals . These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized . Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3' region by using PCR amplification . We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species . The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplifying the conserved 5' end were used . We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3' region of this new intimin type . This intimin, referred to as "epsilon," was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165 . Intimin epsilon is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin beta, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins alpha and gamma. Virology, 1999 Dec 5, 265(1), 96 - 109 The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo; Wang Y et al.; The nucleocapsid (N) protein of mouse hepatitis virus (MHV) and the cellular heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) are RNA-binding proteins, binding to the leader RNA and the intergenic sequence of MHV negative-strand template RNAs, respectively . Previous studies have suggested a role for both N and hnRNP-A1 proteins in MHV RNA synthesis . However, it is not known whether the two proteins can interact with each other . Here we employed a series of methods to determine their interactions both in vitro and in vivo . Both N and hnRNP-A1 genes were cloned and expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and their interactions were determined with a GST-binding assay . Results showed that N protein directly and specifically interacted with hnRNP-A1 in vitro . To dissect the protein-binding domain on the N protein, 15 deletion constructs were made by PCR and expressed as GST fusion proteins . Two hnRNP-A1-binding sites were identified on N protein: site A is located at amino acids 1 to 292 and site B at amino acids 392 to 455 . In addition, we found that N protein interacted with itself and that the self-interacting domain coincided with site A but not with site B . Using a fluorescence double-staining technique, we showed that N protein colocalized with hnRNP-A1 in the cytoplasm, particularly in the perinuclear region, of MHV-infected cells, where viral RNA replication/transcription occurs . The N protein and hnRNP-A1 were coimmunoprecipitated from the lysates of MHV-infected cells either by an N- or by an hnRNP-A1-specific monoclonal antibody, indicating a physical interaction between N and hnRNP-A1 proteins . Furthermore, using the yeast two-hybrid system, we showed that N protein interacted with hnRNP-A1 in vivo . These results thus establish that MHV N protein interacts with hnRNP-A1 both in vitro and in vivo . Mol Med, 1999 Oct, 5(10), 664 - 71 Identification and expression of mutations in the hydroxymethylbilane synthase gene causing acute intermittent porphyria (AIP); Solis C et al.; BACKGROUND: Acute intermittent porphyria (AIP), an autosomal dominant inborn error, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (EC 4.3.1.8; HMB-synthase) . This disease is characterized by acute, life-threatening neurologic attacks that are precipitated by various drugs, hormones, and other factors . The enzymatic and/or biochemical diagnosis of AIP heterozygotes is problematic; therefore, efforts have focused on the identification of HMB-synthase mutations so that heterozygotes can be identified and educated to avoid the precipitating factors . In Spain, the occurrence of AIP has been reported, but the nature of the HMB-synthase mutations causing AIP in Spanish families has not been investigated . Molecular analysis was therefore undertaken in nine unrelated Spanish AIP patients . MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and family members of nine unrelated Spanish families with AIP . The HMB-synthase gene was amplified by long-range PCR and the nucleotide sequence of each exon was determined by cycle sequencing . RESULTS: Three new mutations, a missense, M212V; a single base insertion, g4715insT; and a deletion/insertion, g7902ACT-->G, as well as five previously reported mutations (G111R, R116W, R149X R167W, and R173W) were detected in the Spanish probands . Expression of the novel missense mutation M212V in E . coli revealed that the mutation was causative, having <2% residual activity . CONCLUSIONS: These studies identified the first mutations in the HMB-synthase gene causing AIP in Spanish patients . Three of the mutations were novel, while five previously reported lesions were found in six Spanish families . These findings enable accurate identification and counseling of presymptomatic carriers in these nine unrelated Spanish AIP families and further demonstrate the genetic heterogeneity of mutations causing AIP. Oncogene, 1999 Dec 2, 18(51), 7265 - 73 Interaction partners of Dlk/ZIP kinase: co-expression of Dlk/ZIP kinase and Par-4 results in cytoplasmic retention and apoptosis; Page G et al.; Dlk/ZIP kinase is a newly discovered serine/threonine kinase which, due to its homology to DAP kinase, was named DAP like kinase, Dlk . This kinase is tightly associated with nuclear structures, it undergoes extensive autophosphorylation and phosphorylates myosin light chain and core histones H3, H2A and H4 in vitro . Moreover, it possesses a leucine zipper which mediates interaction with transcription factor ATF-4, therefore it was called ZIP kinase . We employed the yeast two-hybrid system to identify interaction partners of Dlk that might serve as regulators or targets . Besides ATF-4 and others we found Par-4, a modulator of transcription factor WT1 and mediator of apoptosis . Complex formation between Dlk and Par-4 was confirmed by GST pull-down experiments and kinase reactions in vitro and coexpression experiments in vivo . The interaction domain within Dlk was mapped to an arginine-rich region between residues 338 - 417, rather than to the leucine zipper . Strikingly, coexpression of Dlk and Par-4 lead to relocation of Dlk from the nucleus to the cytoplasm, particularly to actin filaments . These interactions provoked a dramatic reorganization of the cytoskeleton and morphological symptoms of apoptosis, thus suggesting a functional relationship between Dlk and Par-4 in the control of apoptosis. Eur J Immunol, 1999 Dec, 29(12), 3877 - 86 Escherichia coli infection induces only fetal thymus-derived gamma delta T cells at the infected site; Matsuzaki G et al.; Intraperitoneal infection of mice with Escherichia coli induced activated TCR gamma delta T cells in the peritoneal cavity . We provide evidence that the E . coli-induced gamma delta T cells are derived only from the fetal thymus on the following grounds . The gamma delta T cells were not induced in athymic nude mice and irradiated bone marrow-transferred mice which lack fetal thymus-derived T cells . However, E . coli infection of fetal thymus-grafted nude mice did induce fetal thymus-derived gamma delta T cells . These results suggest that the fetal thymus-derived gamma delta T cells colonize the periphery during early ontogeny, and are maintained until adult age . The E . coli-induced gamma delta T cells express only the Vdelta1 gene . Vgamma6 was predominantly expressed whereas anti-Vgamma1 and anti-Vgamma4 monoclonal antibodies stained less than 3 % of the cells . Direct sequencing of PCR products revealed that Vgamma6 and Vdelta1 genes expressed by the E . coli-induced gamma delta T cells were invariant sequences identical to those expressed in the fetal thymus . The antigen (Ag) specificity of a T cell hybridoma expressing the fetal type Vgamma6 / Vdelta1(+) TCR could not be identified as the cells failed to respond to lipopolysaccharide, E . coli Ag, mycobacterial heat shock protein 65, or isopentenyl pyrophosphate . These results suggest that the Vgamma6 / Vdelta1(+) gamma delta T cells derived from fetal thymus can participate in immune responses against bacterial infection through recognition of a novel class of Ag which is not yet identified. FEBS Lett, 1999 Dec 10, 463(1-2), 146 - 50 Smooth muscle alpha-tropomyosin crosslinks to caldesmon, to actin and to myosin subfragment 1 on the muscle thin filament; Golitsina NL et al.; To obtain proximity information between tropomyosin (Tm) and caldesmon (CaD) on the muscle thin filament, we cloned gizzard alphaTm and created two single Cys mutants S56C/C190S (56Tm) and D100C/C190S (100Tm) . They were labeled with benzophenone maleimide (BPM) and UV-irradiated on thin filaments . One chain of BPM-56Tm and two chains of BPM-100Tm crosslinked to CaD . Only BPM-100Tm crosslinked to actin in the absence and presence of CaD and binding of low ratios of myosin subfragment 1 (S1) prevented the crosslinking . Tm-S1 crosslinks were produced when actin.Tm was saturated with S1 . Thus, CaD on the actin.Tm filament is located <10 A away from Tm amino acids 56 and 100; in the closed state of the actin.Tm filament, Tm residue 100 is located close to the actin surface and is moved further away in the S1-induced open state; in the open state, S1 binds close to Tm. FEBS Lett, 1999 Dec 10, 463(1-2), 24 - 8 The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures; Flohr F et al.; To define domains of the human MxA GTPase involved in GTP hydrolysis and antiviral activity, we used two monoclonal antibodies (mAb) directed against different regions of the molecule . mAb 2C12 recognizes an epitope in the central interactive region of MxA, whereas mAb M143 is directed against the N-terminal G domain . mAb 2C12 greatly stimulated MxA GTPase activity, suggesting that antibody-mediated crosslinking enhances GTP hydrolysis . In contrast, monovalent Fab fragments of 2C12 abolished GTPase activity, most likely by blocking intramolecular interactions required for GTPase activation . Interestingly, intact IgG molecules and Fab fragments of 2C12 both prevented association of MxA with viral nucleocapsids and neutralized MxA antiviral activity in vivo . mAb M143 had no effect on MxA function, indicating that this antibody binds outside functional regions . These data demonstrate that the central region recognized by 2C12 is critical for regulation of GTPase activity and viral target recognition. J Biol Chem, 1999 Dec 24, 274(52), 37093 - 6 Identification of discriminator base atomic groups that modulate the alanine aminoacylation reaction; Fischer AE et al.; Specific aminoacylation of tRNAs involves activation of an amino acid with ATP followed by amino acid transfer to the tRNA . Previous work showed that the transfer of alanine from Escherichia coli alanyl-tRNA synthetase to a cognate RNA minihelix involves a transition state sensitive to changes in the tRNA acceptor stem . Specifically, the "discriminator" base at position 73 of minihelix(Ala) is a critical determinant of the transfer step of aminoacylation . This single-stranded nucleotide has previously been shown by solution NMR to be stacked predominantly onto G(1) of the first base pair of the alanine acceptor stem helix . In this work, RNA duplex(Ala) variants were prepared to investigate the role of specific discriminator base atomic groups in aminoacylation catalytic efficiency . Results indicate that the purine structure appears to be important for stabilization of the transition state and that major groove elements are more critical than those located in the minor groove . This result is in accordance with the predicted orientation of a class II synthetase at the end of the acceptor helix . In particular, substitution of the exocyclic amino group of A(73) with a keto-oxygen resulted in negative discrimination at this site . Taken together, these new results are consistent with the involvement of major groove atomic groups of the discriminator base in the formation of the transition state for the amino acid transfer step. J Biol Chem, 1999 Dec 24, 274(52), 36935 - 43 Substitution of pyridoxal 5'-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis; Schnackerz KD et al.; D-Serine dehydratase (DSD) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of D-serine to pyruvate and ammonia . Spectral studies of enzyme species where the natural cofactor was substituted by pyridoxal 5'-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and pyridoxal 5'-phosphate monomethyl ester (PLPMe) were used to gain insight into the structural basis for binding of cofactor and substrate analogues . PDMP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD and PLPMe-DSD are catalytically inactive . The emission spectrum of native DSD when excited at 280 nm shows maxima at 335 and 530 nm . The energy transfer band at 530 nm is very likely generated as a result of the proximity of Trp-197 to the protonated internal Schiff base . The cofactor analogue-reconstituted DSD species exhibit emission intensities decreasing from PLS-DSD, to PLPMe-DSD, and PDMP-DSD, when excited at 415 nm . Large increases in fluorescence intensity at 530 (540) nm can be observed for cofactor analogue-reconstituted DSD in the presence of substrate analogues when excited at 415 nm . In the absence and presence of substrate analogues, virtually identical far UV CD spectra were obtained for all DSD species . The visible CD spectra of native DSD, PDMP-DSD, and PLS-DSD exhibit a band centered on the visible absorption maximum with nearly identical intensity . Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species results in most cases in a decrease or elimination of ellipticity . The results are interpreted in terms of local conformational changes and/or changes in the orientation of the bound cofactor (analogue). J Bacteriol, 1999 Dec, 181(24), 7621 - 5 RNase E enzymes from rhodobacter capsulatus and Escherichia coli differ in context- and sequence-dependent in vivo cleavage within the polycistronic puf mRNA; Heck C et al.; The 5' pufQ mRNA segment and the pufLMX mRNA segment of Rhodobacter capsulatus exhibit different stabilities . Degradation of both mRNA segments is initiated by RNase E-mediated endonucleolytic cleavage . While Rhodobacter RNase E does not discriminate between the different sequences present around the cleavage sites within pufQ and pufL, Escherichia coli RNase E shows preference for the sequence harboring more A and U residues. J Bacteriol, 1999 Dec, 181(24), 7608 - 13 Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides; Mortimer MW et al.; The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant . The S . meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type gltA with the gltA::kan allele . The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate . CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose . CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14 . High concentrations of arabinose (0.4%) or succinate (0 . 2%) restored the green fluorescence to CSDX1 . High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan . CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen . The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle. J Bacteriol, 1999 Dec, 181(24), 7580 - 7 The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn(2+) flavoprotein; Reed DW et al.; Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize D-lactate as a sole source for both carbon and electrons . The A . fulgidus open reading frame, AF0394, which is predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP) . The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E . coli when coexpressed with the E . coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG . When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A . fulgidus Dld enzyme . Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen . The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity. J Bacteriol, 1999 Dec, 181(24), 7552 - 7 Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA; Miller C et al.; We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication . Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division . As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number . The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions . The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins . Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis. J Bacteriol, 1999 Dec, 181(24), 7531 - 44 Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ; Ma X et al.; In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring . Several C-terminal deletions of E . coli FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal ftsZ mutants when expressed on a plasmid . To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines . All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant . Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function . In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization . Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins . Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA . Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA . In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion. J Bacteriol, 1999 Dec, 181(24), 7500 - 8 Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon; Soutourina O et al.; Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility . In the present paper, we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation . This results from a reduced expression in vivo of fliA and fliC, which encode the specific flagellar sigma factor and flagellin, respectively . Overexpression of the flhDC master operon restored, at least in part, motility in crp and hns mutant strains, suggesting that this operon is the main target for both regulators . Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays . In vitro transcription experiments showed that CAP activates flhDC expression while H-NS represses it . In agreement with this observation, the activity of a transcriptional fusion carrying the flhDC promoter was decreased in the crp strain and increased in the hns mutant . In contrast, the activity of a transcriptional fusion encompassing the entire flhDC regulatory region extending to the ATG translational start codon was strongly reduced in both hns and crp mutants . These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo . Finally, the lack of complementation of the nonmotile phenotype in a crp mutant by activation-deficient CAP mutated proteins and characterization of cfs, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates flhDC transcription by binding to its promoter and interacting with RNA polymerase. J Bacteriol, 1999 Dec, 181(24), 7479 - 84 Genetic analysis of a chromosomal region containing genes required for assimilation of allantoin nitrogen and linked glyoxylate metabolism in Escherichia coli; Cusa E et al.; Growth experiments with Escherichia coli have shown that this organism is able to use allantoin as a sole nitrogen source but not as a sole carbon source . Nitrogen assimilation from this compound was possible only under anaerobic conditions, in which all the enzyme activities involved in allantoin metabolism were detected . Of the nine genes encoding proteins required for allantoin degradation, only the one encoding glyoxylate carboligase (gcl), the first enzyme of the pathway leading to glycerate, had been identified and mapped at centisome 12 on the chromosome map . Phenotypic complementation of mutations in the other two genes of the glycerate pathway, encoding tartronic semialdehyde reductase (glxR) and glycerate kinase (glxK), allowed us to clone and map them closely linked to gcl . Complete sequencing of a 15.8-kb fragment encompassing these genes defined a regulon with 12 open reading frames (ORFs) . Due to the high similarity of the products of two of these ORFs with yeast allantoinase and yeast allantoate amidohydrolase, a systematic analysis of the gene cluster was undertaken to identify genes involved in allantoin utilization . A BLASTP search predicted four of the genes that we sequenced to encode allantoinase (allB), allantoate amidohydrolase (allC), ureidoglycolate hydrolase (allA), and ureidoglycolate dehydrogenase (allD) . The products of these genes were overexpressed and shown to have the predicted corresponding enzyme activities . Transcriptional fusions to lacZ permitted the identification of three functional promoters corresponding to three transcriptional units for the structural genes and another promoter for the regulatory gene allR . Deletion of this regulatory gene led to constitutive expression of the regulon, indicating a negatively acting function. J Bacteriol, 1999 Dec, 181(24), 7470 - 8 Type 1 fimbriation and phase switching in a natural Escherichia coli fimB null strain, Nissle 1917; Stentebjerg-Olesen B et al.; Escherichia coli Nissle 1917 has been used as a probiotic against intestinal disorders for many decades . It is a good colonizer of the human gut and has been reported to be able to express type 1 fimbriae . Type 1 fimbriae are surface organelles which mediate alpha-D-mannose-sensitive binding to various host cell surfaces . The expression is phase variable, and two tyrosine recombinases, FimB and FimE, mediate the inversion of the fimbrial phase switch . Current evidence suggests that FimB can carry out recombination in both directions, whereas FimE-catalyzed switching is on to off only . We show here that under liquid shaking growth conditions, Nissle 1917 did not express type 1 fimbriae, due to a truncation of the fimB gene by an 1,885-bp insertion element . Despite its fimB null status, Nissle 1917 was still capable of off-to-on switching of the phase switch and expressing type 1 fimbriae when grown under static conditions . This phase switching was not catalyzed by FimE, by truncated FimB, or by information residing within the insertion element . No further copies of fimB seemed to be present on the chromosome of Nissle 1917, suggesting that another tyrosine recombinase in Nissle 1917 is responsible for the low-frequency off-to-on inversion of the phase switch that is strongly favored under static growth conditions . This is the first report documenting the non-FimB- or non-FimE-catalyzed inversion of the fim switch. J Bacteriol, 1999 Dec, 181(24), 7457 - 63 A cyclic AMP receptor protein mutant that constitutively activates an Escherichia coli promoter disrupted by an IS5 insertion; Podolny V et al.; Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated . This phenotype is the result of the constitutive expression of the fucO gene (in the fucAO operon), which encodes one of the enzymes in the fucose metabolic pathway . The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently oriented fucAO and fucPIK operons . Though expression of the fucAO operon was constitutive, the fucPIK operon became noninducible such that the mutant could no longer grow on fucose . A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected . Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP) . We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIK promoters, and we identify the CRP-binding site that is required for this activity . Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase . For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences. J Bacteriol, 1999 Dec, 181(24), 7449 - 56 Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic; Duret S et al.; To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1 . An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S . citri, and into the S . citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E . coli . Electrotransformation of S . citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants . In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32 . During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene . In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype . Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S . citri strain . These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S . citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S . citri, a mollicute which probably lacks a functional RecA protein. EMBO J, 1999 Dec 15, 18(24), 7077 - 85 Aphid transmission of cauliflower mosaic virus requires the viral PIII protein; Leh V et al.; The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood . We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor . Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission . Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively . A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions . Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins . Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids. EMBO J, 1999 Dec 15, 18(24), 7063 - 76 The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties; Cabedo H et al.; The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding motif . Here we show that 50K is a GTPase that differs extensively from regulatory GTPases such as p21 . Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinity for GTP, and extremely low affinity for GDP . Moreover, it can form self-assemblies . Strikingly, the 17 kDa GTPase domain of 50K conserves the guanine nucleotide-binding and GTPase activities of the intact 50K molecule . Therefore, the structural requirements for GTP binding and GTP hydrolysis by 50K are without precedent and justify a separate classification in the GTPase superfamily . Immunoelectron microscopy reveals that 50K is a cytoplasmic protein partially associated with the inner membrane . We prove that o454 is allelic with trmE, a gene involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, which is found in the wobble position of some tRNAs . Our results demonstrate that 50K is essential for viability depending on the genetic background . We propose that combination of mutations affecting the decoding process, which separately do not reveal an obvious defect in growth, can give rise to lethal phenotypes, most likely due to synergism. EMBO J, 1999 Dec 15, 18(24), 6934 - 49 Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB; Mogk A et al.; We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts . Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins . Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (</=30 kDa) cytosolic proteins and include essential proteins . The DnaK system in addition acts with ClpB to form a bi-chaperone system that quantitatively solubilizes aggregates of most of these proteins . Efficient solubilization also occurred in an in vivo order-of-addition experiment in which aggregates were formed prior to induction of synthesis of the bi-chaperone system . Our data indicate that large-sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation . Keywords: chaperones/heat-shock response/Hsp70/protein denaturation/thermotolerance Science, 1999 Dec 17, 286(5448), 2337 - 9 Binding of transcription termination protein nun to nascent RNA and template DNA; Watnick RS et al.; The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation . RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA . To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker . The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts . When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template . Nun may arrest transcription by anchoring RNAP to DNA. J Struct Biol, 1999 Dec 1, 128(1), 15 - 8 Animation of the dynamical events of the elongation cycle based on cryoelectron microscopy of functional complexes of the ribosome; Frank J et al.; Using three-dimensional cryoelectron microscopy, the binding positions of tRNA and elongation factors EF-G and EF-Tu (the latter complexed with aminoacyl tRNA and GTP) on the ribosome were determined in previous studies . On the basis of these studies, the dynamical events that take place in the course of the elongation cycle of protein synthesis have been animated . The resulting 3-min movie is accessible on the website of this journal . The following article provides a brief annotation of those frames of the movie for which experimental support is available . Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 579 - 83 Negative regulatory role of the Escherichia coli hfq gene in cell division; Takada A et al.; We found that the hfq::cat mutant strain produced minicells at high frequency . Minicell production by the mutant strain was more prominent in poor media and in the stationary phase than in rich media and in the exponentially growing phase . The amount of the cell division protein FtsZ increased up to two- to threefold of the wild-type cells in the hfq::cat mutant in the stationary phase, while such differences were not observed in the exponentially growing phase . Increased ftsZ mRNA levels were also observed in the hfq::cat mutant in the stationary phase . These results suggest a negative regulatory role of the DNA-, RNA-binding protein Hfq in cell division in the stationary phase . Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 466 - 71 Inhibition of biotin carboxylase by a reaction intermediate analog: implications for the kinetic mechanism; Blanchard CZ et al.; The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA carboxylase . One component of the acetyl CoA carboxylase complex is biotin carboxylase which catalyzes the ATP-dependent carboxylation of biotin . The Escherichia coli form of biotin carboxylase can be isolated from the other components of the acetyl CoA carboxylase complex such that enzymatic activity is retained . The synthesis of a reaction intermediate analog inhibitor of biotin carboxylase has been described recently (Organic Lett . 1, 99-102, 1999) . The inhibitor is formed by coupling phosphonoacetic acid to the 1'-N of biotin . In this paper the characterization of the inhibition of biotin carboxylase by this reaction-intermediate analog is described . The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM . Noncompetitive inhibition was found for the analog versus biotin . Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate . Biotin was found to be a noncompetitive substrate inhibitor of biotin carboxylase . These data suggested that biotin carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin . Biochem Biophys Res Commun, 1999 Nov 30, 265(3), 625 - 9 Two interaction modes of the gp41-derived peptides with gp41 and their correlation with antimembrane fusion activity; Ryu JR et al.; Peptides derived from gp41 effectively block the gp41-mediated cell fusion or HIV infection . A 36-mer (naDP178), 51-mer (C51) and 27-mer peptide (C27) from the membrane proximal region of gp41 have been examined their interaction modes with the coiled-coil motif of gp41 presented in thioredoxin (Trx-N) or the bacterially expressed ectodomain of gp41 (Ec-gp41ec) . All of these peptides effectively inhibited the gp41-mediated membrane fusion, however, they showed distinct interaction modes with Ec-gp41ec or Trx-N . C51 peptide bound tightly to Trx-N, and it increased the solubility of Ec-gp41ec . naDP178 showed very weak binding affinity to Trx-N, however, it effectively solubilized Ec-gp41ec . In contrast, C27 peptide showed significant binding to Trx-N; however, it did not affect the solubility of Ec-gp41ec . These interaction modes of C-peptides were assumed to be related to their different inhibitory mechanism against gp41-mediated cell fusion . Protein Expr Purif, 1999 Dec, 17(3), 449 - 55 Vector engineering anomalies: impact on fusion protein purification performance; Pasquinelli RS et al.; Recombinant protein purification using IMAC is often carried out by protein fusion to affinity tags . We have identified several tags useful for protein purification on Zn(II)-IDA columns . These tags were fused to the green fluorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA . Each fusion protein exhibited elution heterogeneity (elution in two distinct pHs) from Zn(II)-IDA columns This led us to believe that two populations of fluorescent proteins were being expressed: one without the tag coeluting with Escherichia coli proteins at pH 7.5 and one bearing the tag eluting at a pH lower than pH 7.5 . Assessment of the constructs revealed the possibility of a ribosomal binding site and start codon between the fusion tag and the rGFPuv sequence which might be used as a secondary translation start site . This hypothesis was confirmed by changing the second ATG (methionine) codon to an ACG (threonine) codon . The protein produced from this new construct eluted in a single fraction from a Zn(II)-IDA column . Thus, vector irregularities (along with other possibilities) should be examined when searching for the cause of elution heterogeneity of a target protein . Protein Expr Purif, 1999 Dec, 17(3), 443 - 8 Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli; Chang M et al.; The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed . The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b . The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture . The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography . The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography . It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column . Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme . Protein Expr Purif, 1999 Dec, 17(3), 428 - 34 Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli; Oh GH et al.; The carboxypeptidase Y (CPY) propeptide from Saccharomyces cerevisiae was developed as a fusion partner for the efficient expression of small polypeptides in Escherichia coli . Six consecutive histidine residues (6xHis) were fused to the N-terminus of the CPY propeptide for the facilitated purification of fusion proteins using immobilized metal ion affinity chromatography . In addition, a methionine or the pentapeptide (Asp)(4)-Lys linker was inserted at the junction between the CPY propeptide and the target polypeptide to release the target polypeptide by digestion with cyanogen bromide or enterokinase . Therapeutically valuable peptide hormones, such as salmon calcitonin precursor (sCAL-Gly), a fragment of human parathyroid hormone (hPTH(1-34)), and human glucagon were successfully expressed in E . coli as fusion polypeptides with the fusion partner . SDS-PAGE analyses showed that the majority of the expressed fusion sCAL-Gly and fusion hPTH(1-34) were present in the form of inclusion bodies, whereas about 66% of the expressed human glucagon was in a soluble form . Almost complete cleavage of the fusion polypeptides was obtained by digestion with enterokinase . Reverse-phase HPLC analyses showed that the target polypeptides released from the fusion proteins were identical to their native forms . Protein Expr Purif, 1999 Dec, 17(3), 421 - 7 Use of thiophilic adsorption chromatography for the one-step purification of a bacterially produced antibody F(ab) fragment without the need for an affinity tag; Fiedler M et al.; Thiophilic adsorption chromatography (TAC) was employed for the purification of a recombinant F(ab) fragment of the antibody IN-1 from the periplasmic protein fraction of Escherichia coli . Adsorption of the F(ab) fragment to the T-gel was achieved at a high concentration of ammonium sulfate and turned out to be independent of the presence of a His(6) tag or Strep tag or of the human or murine nature of the C(H)1 and C(L) domains (subclass IgG1/kappa) . Elution was effected by means of a decreasing salt gradient, yielding fractions with the correctly assembled, heterodimeric F(ab) fragment at high purity . Interestingly, the single substitution of an alanine residue with phenylalanine in the CDR-L1 of the F(ab) fragment significantly enhanced the retention on the column so that quantitative elution necessitated prolonged application of a low-salt buffer . Our findings suggest that TAC is generally suitable for the isolation of bacterially produced F(ab) fragments and support the notion that aromatic side chains play an important role in the interaction with the affinity matrix . This method should prove valuable in the production of proteins for in vivo applications as might be the case for the F(ab) fragment of the antibody IN-1, which promotes axonal regeneration in the central nervous system . Protein Expr Purif, 1999 Dec, 17(3), 373 - 86 Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors; Halling BP et al.; Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila . The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains . The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists . USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein . Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM . Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate . In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer . The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites . A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer . Protein Expr Purif, 1999 Dec, 17(3), 351 - 7 Expression and purification of the transcription factor NtcA from the cyanobacterium Anabaena PCC 7120; Wisen S et al.; The transcription factor NtcA from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli . In order to optimize the expression of NtcA, random silent mutations were introduced at the 5' end of the DNA encoding the protein . To get as high a yield of pure protein as possible, different strategies of expression as well as purification conditions were used . Under optimal expression conditions, a high-level expression clone of NtcA was coexpressed with GroEL-ES at 37 degrees C . A hexahistidine tag was added to the N-terminus of the protein in order to allow purification on an IMAC affinity column . Expression followed by one purification step using IMAC affinity chromatography gave a yield of 30-40 mg pure NtcA protein per liter of bacterial culture . Gel-shift experiments showed that the recombinant NtcA was active in binding a DNA sequence containing an NtcA-specific site . Protein Expr Purif, 1999 Dec, 17(3), 345 - 50 Expression, isolation, and characterization of a chloroplast targeting peptide; Wienk HL et al.; For the first time a method is described in which an N-terminal targeting peptide is isolated from Escherichia coli . After overexpression, purification, and cleavage of a fusion protein the protease-sensitive transit peptide from the chloroplast precursor protein preferredoxin could be isolated by HPLC . It was characterized by N-terminal amino acid sequencing and electrospray mass spectrometry . Its functionality was suggested by in vitro import competition experiments with isolated pea chloroplasts, in which the isolated peptide inhibited the import of radioactively labeled preferredoxin . Results from import competition experiments performed with a transit peptide deletion mutant suggested that the four extreme C-terminal amino acids lack information to interact with the chloroplast import machinery . J Mol Biol, 1999 Dec 17, 294(5), 1363 - 74 Selective degradation of unfolded proteins by the self-compartmentalizing HtrA protease, a periplasmic heat shock protein in Escherichia coli; Kim KI et al.; HtrA, which has a high molecular mass of about 500 kDa, is a periplasmic heat shock protein whose proteolytic activity is essential for the survival of Escherichia coli at high temperatures . To determine the structural organization of HtrA, we have used electron microscopy and chemical cross-linking analysis . The averaged image of HtrA with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity, and its side-on view showed a two-layered structure . Thus, HtrA behaves as a dodecamer consisting of two stacks of hexameric ring . HtrA can degrade thermally unfolded citrate synthase and malate dehydrogenase but cannot when in their native form . HtrA degraded partially unfolded casein more rapidly upon increasing the incubation temperature . However, it hydrolyzed oxidized insulin B-chain, which is fully unfolded, at nearly the same rate at all of the temperatures tested . HtrA also rapidly degraded reduced insulin B-chain generated by treatment of insulin with dithiothreitol but not A-chain or intact insulin . Moreover, HtrA degraded fully unfolded alpha-lactalbumin, of which all four disulfide bonds were reduced, but not the native alpha-lactalbumin and its unfolded intermediates containing two or three disulfide bonds . These results indicate that unfolding of the protein substrates, such as by exposure to high temperatures or reduction of disulfide bonds, is essential for their access into the inner chamber of the double ring-shaped HtrA, where cleavage of peptide bonds may occur . Thus, HtrA with a self-compartmentalizing structure may play an important role in elimination of unfolded proteins in the periplasm of Escherichia coli . J Mol Biol, 1999 Dec 17, 294(5), 1287 - 97 Crystal structure of Escherichia coli methionyl-tRNA synthetase highlights species-specific features; Mechulam Y et al.; The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution . Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only . Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles . Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion . J Mol Biol, 1999 Dec 17, 294(5), 1231 - 8 Structural analysis of an RNase T1 variant with an altered guanine binding segment; Hoschler K et al.; The ribonuclease T1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific inhibitor 2'-GMP . The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 A resolution . Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A, 2'-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions . The phosphate group of 2'-GMP is located about 4.2 A apart from its position in wild-type ribonuclease T1-2'-GMP complexes, allowing a Ca(2+), coordinating this phosphate group, to enter the binding pocket . The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate . The presence of Ca(2+) was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent . J Mol Biol, 1999 Dec 17, 294(5), 1073 - 86 A sequence in the Escherichia coli fdhF "selenocysteine insertion Sequence" (SECIS) operates in the absence of selenium; Liu Z et al.; The UGA codon context of the Escherichia coli fdhF mRNA includes an element called the selenocysteine insertion sequence (SECIS) that is responsible for the UGA-directed incorporation of the amino acid selenocysteine into a protein . Here, we describe an extended fdhF SECIS that includes the information for an additional function: the prevention of UGA readthrough under conditions of selenium deficiency . This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it . These two regions act independently and additively, and probably through different mechanisms . The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes . These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites . Here, we present a model for the E . coli fdhF SECIS as a multifunctional RNA structure containing three functional elements . Depending on the availability of selenium, the SECIS enables one of two alternatives for the translational machinery: either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain . J Surg Res, 1999 Dec, 87(2), 217 - 24 Blockade by polyunsaturated n-3 fatty acids of endotoxin-induced monocytic tissue factor activation is mediated by the depressed receptor expression in THP-1 cells; Chu AJ et al.; BACKGROUND: Monocytic hypercoagulation often occurs in inflammatory conditions . We have previously reported that polyunsaturated n-3 fatty acids (n-3 FA) including eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) prevent the activation of monocytic tissue factor (TF) induced by bacterial endotoxin {lipopolysaccharide (LPS)} in cell cultures and animals . HYPOTHESIS: We herein explore the mode of inhibitory action of n-3 FA to determine if LPS transmembrane signaling is blocked, exerting such antagonism . RESULTS: Exposure of human leukemia monocytic THP-1 cells to bacterial endotoxin (Escherichia coli 0111:B04, 1.5 microg/ml) for 6 h significantly activated TF activity and the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL)-1beta in conditioned medium . Pretreatment with n-3 FA, 20:5 and 22:6 at 10 microM, resulted in time-dependent suppression of not only TF activation but also the elicitation of NO, TNF-alpha, and IL-1beta . These LPS responses were substantially depressed by more than 50% after a 72-h pretreatment . FACScan analysis showed that n-3 FA readily prevented fluorescein isothiocyanate (FITC)-conjugated LPS from binding to THP-1 cells by approximately 70% . The observation that anti-CD14 mAb diminished FITC-LPS binding in a dose-dependent fashion has revealed CD14 dependency in LPS recognition . LPS upregulated CD14 expression, which was significantly arrested by n-3 FA . Similarly, the upregulation of the expression of CD11b, another proposed LPS receptor, was also minimally but significantly depressed by n-3 FA . CONCLUSION: The present study demonstrates that n-3 FA are able to block LPS transmembrane signaling via suppression of the receptor upregulation, mediating a variety of significant antagonisms against LPS action . Brain Behav Immun, 1999 Dec, 13(4), 303 - 14 Increased sensitivity of prediabetic nonobese diabetic mouse to the behavioral effects of IL-1; Bluth et al.; The nonobese diabetic (NOD) mouse is a model of spontaneous insulin-dependent diabetes mellitus (IDDM) or type I diabetes . In humans, and in animal models of IDDM, the progression of the disease is modulated by various environmental factors, particularly infectious agents . Interleukin-1 (IL-1) plays a pivotal role in the development of IDDM, and modulation of its synthesis may be a mechanism by which environmental modulation of disease progression occurs . Since various alterations at the level of the gene, number, and sensitivity of IL-1 receptors have been described in different animal models of autoimmune disease, we investigated, in the prediabetic NOD mouse, the presence of IL-1 receptors and their functional behavioral characteristics . Here we present evidence that prediabetic NOD mice exhibit a normal distribution and density of functional brain IL-1 receptors, but are more sensitive to the behavioral effects of IL-1 than the control ICR strain . Arch Biochem Biophys, 1999 Dec 15, 372(2), 340 - 6 Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum; Takaya N et al.; Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen . In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide . We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol . Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F . oxysporum and acts as nitric oxide reductase . Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria . Arch Biochem Biophys, 1999 Dec 15, 372(2), 280 - 4 One- and two-photon-induced fluorescence from recombinant green fluorescent protein; Xia AD et al.; The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser . Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon . The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra . A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation . Arch Biochem Biophys, 1999 Dec 15, 372(2), 271 - 9 Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase; di Salvo ML et al.; Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively . Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp . To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period . About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form . Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability . Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms . In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence . The t(1/2) for deamidation of Asn(220) was at least 200 h . This residue may become deamidated only after some unfolding of the enzyme . The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme . There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme . Arch Biochem Biophys, 1999 Dec 15, 372(2), 248 - 60 Characterization of recombinant human brain-derived neurotrophic factor variants; Sunasara KM et al.; The purpose of this work was the chemical characterization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli . This paper also addresses the question of the in vitro activity of these variants . Chemical characterization of the variants employed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing . The BDNF variants in this work have been designated by the order of their elution as observed from the high temperature RPLC assay . It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at positions 31 and 61, respectively . Peak 4, which is chromatographically a single peak, contained three variants . Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methionine at position 92 . Peak 5 had norleucine at position 31 instead of methionine . All of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB receptor-ligand binding domain of the variants . Arch Biochem Biophys, 1999 Dec 15, 372(2), 230 - 7 Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway; Loida PJ et al.; The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation . In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase . Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid . These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as