Biochim Biophys Acta, 2002 Dec 16, 1601(2), 215 - 21 Characterization of recombinant horse cytochrome c synthesized with the assistance of Escherichia coli cytochrome c maturation factors; Kellogg JA et al.; Cytochromes c are characterized by the presence of a protoporphyrin IX group covalently attached to the polypeptide via one or two thioether bonds to Cys side chains . The heme attachment process, known as cytochrome c maturation, occurs posttranslationally in the periplasm (for bacterial cytochromes c) or in the mitochondrial intermembrane space (for eukaryotic cytochromes c) through a pathway dependent on the organism . It is demonstrated in this work that a mitochondrial cytochrome c expressed in Escherichia coli that undergoes maturation under control of the E . coli cytochrome c maturation factors achieves a native-like structure and stability . The recombinant protein is characterized spectroscopically (by circular dichroism (CD), absorption, and nuclear magnetic resonance (NMR) spectroscopy) and it is verified that the heme and its environment are indistinguishable from authentic horse cytochrome c . Mass spectrometry reveals that the recombinant protein is not acetylated at the N terminus, however, no significant effect on protein structure or stability is detected as a result. Biochim Biophys Acta, 2002 Dec 16, 1601(2), 172 - 7 Isolation, crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes; Karring H et al.; Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E . coli elongation factor Tu (EF-Tu) . In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E . coli EF-Tu . The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E . coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised . The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees . The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees. Biochim Biophys Acta, 2002 Nov 4, 1600(1-2), 111 - 7 The myristoylation of the neuronal Ca2+ -sensors guanylate cyclase-activating protein 1 and 2; Hwang JY et al.; Guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins with a fatty acid (mainly myristic acid) that is covalently attached at the N terminus . Myristoylated forms of GCAP were produced in E . coli by coexpression of yeast N-myristoyl-transferase . Proteins with nearly 100% degree of myristoylation were obtained after chromatography on a reversed phase column . Although proteins were denatured by this step, they could be successfully refolded . Nonmyristoylated GCAPs activated bovine photoreceptor guanylate cyclase 1 less efficiently than the myristoylated forms . Maximal activity of guanylate cyclase at low Ca(2+)-concentration decreased about twofold, when GCAPs lacked myristoylation . In addition, the x-fold activation of cyclase was lower with nonmyristoylated GCAPs . Myristoylation of GCAP-2 had no influence on the apparent affinity for photoreceptor guanylate cyclase 1, but GCAP-1 has an about sevenfold higher affinity for cyclase, when it is myristoylated . We conclude that myristoylation of GCAP-1 and GCAP-2 is important for fine tuning of guanylate cyclase activity. Plant J, 2002 Nov, 32(4), 481 - 93 Differential expression and functional analysis of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds; Duval FD et al.; Implication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated . Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N-terminal alpha-helix . CaM was shown to be encoded by a small multigenic family, and full-length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5' and 3' untranslated regions . Expression studies, performed by semiquantitative RT-PCR, demonstrated differential expression patterns of the three transcripts during germination . PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion . In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues . To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep-tagged proteins (CaM1-ST, CaM2-ST and CaM3-ST) expressed in Escherichia coli . Gel mobility shift assays revealed that CaM1-ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2-ST and CaM3-ST affinities for the same peptide were reduced . Affinity differences were also observed for CaM isoform binding to Trp-3, an idealised helical CaM-binding peptide . However, the three proteins activated in the same way the CaM-dependent pea NAD kinase . Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context. Eur J Biochem, 2002 Dec, 269(23), 5982 - 91 Structural analysis of deacylated lipopolysaccharide of Escherichia coli strains 2513 (R4 core-type) and F653 (R3 core-type); Muller-Loennies S et al.; Lipopolysaccharide (LPS) of Escherichia coli strain 2513 (R4 core-type) yielded after alkaline deacylation one major oligosaccharide by high-performance anion-exchange chromatography (HPAEC) which had a molecular mass of 2486.59 Da as determined by electrospray ionization mass spectrometry . This was in accordance with the calculated molecular mass of a tetraphosphorylated dodecasaccharide of the composition shown below . NMR-analyses identified the chemical structure as where l-alpha-d-Hep is l-glycero-alpha-d-manno-heptopyranose and Kdo is 3-deoxy-alpha-d-manno-oct-2-ulopyranosylonic acid and all hexoses are present as d-pyranoses . We have also isolated the complete core-oligosaccharides of E . coli F653 LPS for which only preliminary data were available and investigated the deacylated LPS by NMR and MS . The proposed structure determined previously by methylation analysis was confirmed and is shown below . In addition we have quantified the side-chain heptose substitution of the inner core with GlcpN ( approximately 30%) and confirmed that this sugar is only present when the phosphate at the second l,d-Hepp residue is absent. Biol Reprod, 2002 Dec, 67(6), 1897 - 906 Overexpression of monkey oviductal protein: purification and characterization of recombinant protein and its antibodies; Natraj U et al.; The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP) . Molecular cDNA cloning of most of the mammalian OGP has been accomplished . The nucleotide and deduced amino acid sequences show a remarkable homology across species and also to chitinase protein . Even though OGP has been shown to interact with gametes and the early embryo, the protein's direct function has not yet been established . A prerequisite for such studies is the availability of well-characterized protein in bulk . We used recombinant DNA technology to obtain OGP (rOGP) . An authentic partial cDNA clone encoding bonnet monkey (Macaca radiata) OGP (accession number AF132 215) was recloned into expression vector pET20b . Overexpression of the protein could be demonstrated after induction with isopropylthio-beta-galactopyranoside . Recombinant protein was purified by gel filtration of Escherichia coli lysate through Sephadex G75 . The protein migrated with a molecular weight of approximately 14 kDa on SDS-PAGE . The molecular weight as assessed by matrix-assisted laser adsorption time-of-flight was 14 439 daltons . With Western blot procedures the protein could be immunostained with antibodies to human OGP, baboon OGP, and antipeptide antibodies generated against a well-conserved region of mammalian OGP . The monospecificity of rabbit antibodies generated against rOGP was established by its ability to immunostain human OGP (100-110 kDa) isolated from hydrosalpinx by Western blot analysis, and the antibody immunostained epithelial cells that secrete OGP in human fallopian tubes . OGP binding sites on the head and tail region of monkey sperm could be demonstrated by using antibody against rOGP. J Biotechnol, 2003 Feb 13, 100(3), 181 - 91 Evaluation of different promoters and host strains for the high-level expression of collagen-like polymer in Escherichia coli; Yin J et al.; The increased expression of collagen-like polymer, CLP3.1-his which consists of 52 repeating peptide (GAPGAPGSQGAPGLQ), in Escherichia coli was investigated . The effects of three promoters, thermally inducible promoter, T7 promoter and T7lac promoter, and three Escherichia coli host strains, BL21, BL21(DE3) and BL21(DE3){pLysS} which differ in stringency of suppressing basal transcription, were compared . Based on the CLP3.1-his expression level, solubility of CLP3.1-his in cells and basal transcription that occurred in the absence of induction, two expression systems, BL21(DE3) containing plasmid pJY-2 with T7lac promoter and BL21(DE3){pLysS} containing plasmid pJY-1 with T7 promoter, were selected . With these two expression systems, CLP3.1-his expression levels greater than 40% (g/g) of total cellular proteins and CLP3.1-his concentrations of 0.1-0.2 gl(-1) can be achieved by using Luria-Bertani medium in shake flask batch cultures . The CLP3.1-his accumulated in the cells is totally soluble and no basal transcription was found before induction . These two high-level expression systems are promising for use in scale-up production. J Surg Res, 2002 Nov, 108(1), 69 - 76 G-protein coupled receptor kinase 2 is altered during septic shock in rats; Kadoi Y et al.; BACKGROUND: One of the key mechanisms leading to beta-adrenergic receptor-specific desensitization is the phosphorylation of agonist-occupied receptors by the specific beta-adrenergic receptor kinase (GRK2) . The present study examines whether GRK2 is altered during septic shock in rats . MATERIALS AND METHODS: Male Wistar rats (7 weeks) weighing between 250 and 300 g were anesthetized with pentobarbital (10 mg/kg ip) . Escherichia coli endotoxin (10 mg/kg in 0.3 mL of saline) or saline (0.3 ml) was injected intravenously via the dorsal vein . Hemodynamic parameters and humoral mediators were measured at 2 h after the administration of endotoxin . The hearts were immediately excised to examine beta-adrenergic receptor density and GRK2 level . We also studied the inotropic response to isoproterenol at the same time in other animals . RESULTS: Myocardial beta-adrenergic receptor density in the membrane fraction was decreased after an intravenous administration of 10 mg/kg LPS (LPS group: baseline value; 82 +/- 11 fmol/mg protein; 120 min after LPS; 58 +/- 11 fmol/mg protein, P < 0.05) . GRK2 levels in the membrane and cytosolic fraction of the control group did not change . In the LPS group, GRK2 levels in the membrane fraction were increased at 60 and 120 min after the treatment (60 min; control, 4.5 +/- 0.4; pithed control, 4.4 +/- 0.6; LPS group, 6.2 +/- 0.3; pithed LPS group, 5.5 +/- 0.4; 120 min: control, 4.4 +/- 0.3; pithed control, 4.9 +/- 0.7; LPS group, 7.1 +/- 0.3; pithed LPS group, 5.9 +/- 0.4; densitometric unit, respectively: P < 0.05) . CONCLUSIONS: GRK2 levels in the membrane fraction are increased during septic shock in rats . GRK2 might play a role in the impairment of the beta-adrenergic receptor signal transduction system. Res Vet Sci, 2002 Dec, 73(3), 297 - 306 A family of activation associated secreted protein (ASP) homologues of Cooperia punctata; Yatsuda AP et al.; Activation-associated secreted proteins (ASP) of nematodes have been studied as potential vaccine components . In this study we report the cloning and analysis of cDNA and genomic sequences of Cooperia punctata and establish the presence of two 75% identical ASP-1 genes in C . punctata . Additional C . punctata ASP paralogues were shown to be present . Analysis of PCR products amplified from genomic DNA from a pool of worms revealed extensive sequence diversity within this family of proteins, reflecting the presence of different ASP paralogues in a single worm as well as extensive polymorphisms between different worms . ASP proteins contain a conserved region called the sperm-coating protein (SCP) domain of unknown function, which is present as a single copy in proteins from yeast and a wide range of multi-cellular organisms . Only in three nematodes has a protein composed of duplicated SCP-domains been identified . C . punctata is the first organism in which at least two such genes are found . Database searches identified similarity of the C-terminal cysteine-rich domain of ASP proteins to a nematode metallothionein motif . Cp-asp-1b was expressed in Escherichia coli and both the N-terminal and C-terminal domain were shown to be recognized by sera of C . punctata infected bovines . The description of the asp gene family of C . punctata provides the basis for more detailed studies into the extent of variation and immunological recognition of this family that may assist in rational vaccine design. Vaccine, 2002 Nov 22, 21(1-2), 78 - 88 Evaluation of the immunocontraceptive potential of Escherichia coli expressed recombinant non-human primate zona pellucida glycoproteins in homologous animal model; Govind CK et al.; In order to evaluate the immunocontraceptive potential of zona pellucida (ZP) glycoproteins, recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-1 (r-bmZP1) and -2 (r-bmZP2) were expressed as polyhistidine fusion proteins in Escherichia coli . Female bonnet monkeys were immunized with the purified r-bmZP1 (n=5) and r-bmZP2 (n=4) conjugated to diphtheria toxoid (DT) . Immunization led to generation of antibodies against r-bmZP1, r-bmZP2 and DT as determined by enzyme linked immunosorbent assay . The immunized animals exhibited normal menstrual cyclicity and progesterone profile, except during the summer amenorrhoea . Immunized animals, when mated with males of proven fertility, showed protection from conceiving for cumulative 45 ovulatory cycles in r-bmZP1-DT immunized group and 32 ovulatory cycles in r-bmZP2-DT immunized group . Ovarian histopathology of both the immunized groups revealed the presence of atretic follicles with degenerated oocytes, which may have been the principle cause for the failure of immunized animals to conceive in spite of the decline in either anti-r-bmZP1 or anti-r-bmZP2 antibody titres to background levels . These studies demonstrate, for the first time, that the block of fertility subsequent to immunization with r-bmZP1 and r-bmZP2, in a homologous non-human primate model, may be mediated due to ovarian dysfunction. BMC Evol Biol . 2002 Oct 30;2(1):19 {Epub ahead of print} Long-term experimental evolution in Escherichia coli . XI . Rejection of non-transitive interactions as cause of declining rate of adaptation; De Visser JA et al.; BACKGROUND: Experimental populations of Escherichia coli have evolved for 20,000 generations in a uniform environment . Their rate of improvement, as measured in competitions with the ancestor in that environment, has declined substantially over this period . This deceleration has been interpreted as the bacteria approaching a peak or plateau in a fitness landscape . Alternatively, this deceleration might be caused by non-transitive competitive interactions, in particular such that the measured advantage of later genotypes relative to earlier ones would be greater if they competed directly . RESULTS: To distinguish these two hypotheses, we performed a large set of competitions using one of the evolved lines . Twenty-one samples obtained at 1,000-generation intervals each competed against five genetically marked clones isolated at 5,000-generation intervals, with three-fold replication . The pattern of relative fitness values for these 315 pairwise competitions was compared with expectations under transitive and non-transitive models, the latter structured to produce the observed deceleration in fitness relative to the ancestor . In general, the relative fitness of later and earlier generations measured by direct competition agrees well with the fitness inferred from separately competing each against the ancestor . These data thus support the transitive model . CONCLUSION: Non-transitive competitive interactions were not a major feature of evolution in this population . Instead, the pronounced deceleration in its rate of fitness improvement indicates that the population early on incorporated most of those mutations that provided the greatest gains, and subsequently relied on beneficial mutations that were fewer in number, smaller in effect, or both. Biochem J, 2003 Mar 1, 370(Pt 2), 661 - 9 Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class; Sawicki R et al.; From the fruitfly, Drosophila melanogaster, ten members of the cluster of Delta-class glutathione S-transferases (GSTs; formerly denoted as Class I GSTs) and one member of the Epsilon-class cluster (formerly GST-3) have been cloned, expressed in Escherichia coli, and their catalytic properties have been determined . In addition, nine more members of the Epsilon cluster have been identified through bioinformatic analysis but not further characterized . Of the 11 expressed enzymes, seven accepted the lipid peroxidation product 4-hydroxynonenal as substrate, and nine were active in glutathione conjugation of 1-chloro-2,4-dinitrobenzene . Since the enzymically active proteins included the gene products of DmGSTD3 and DmGSTD7 which were previously deemed to be pseudogenes, we investigated them further and determined that both genes are transcribed in Drosophila . Thus our present results indicate that DmGSTD3 and DmGSTD7 are probably functional genes . The existence and multiplicity of insect GSTs capable of conjugating 4-hydroxynonenal, in some cases with catalytic efficiencies approaching those of mammalian GSTs highly specialized for this function, indicates that metabolism of products of lipid peroxidation is a highly conserved biochemical pathway with probable detoxification as well as regulatory functions. J Basic Microbiol, 2002, 42(6), 367 - 72 Immunocytochemical localization of the stress-induced DpsA protein in the cyanobacterium Synechococcus sp . strain PCC 7942; Durham KA et al.; Proteins of the Dps family are divergent ferritins that have been shown to bind DNA with high affinity during periods of nutrient and oxidative stress . Such binding protects the chromosome from peroxide attack . Surprisingly, we show by immunocytochemistry that the cyanobacterial Dps homolog, DpsA, localizes preferentially to the thylakoid membrane in Synechococcus sp . strain PCC7942 . We propose that two DpsA pools are functioning in this species--an insoluble fraction bound to the chromosome, and a soluble fraction acting as a ferritin involved metal homeostasis of the photosynthetic apparatus . This model is presented in light of recent work on the E . coli Dps protein showing that DNA binding is regulated by the metal-binding capacity of the Dps complex (Frenkiel-Krispin et al . 2001) . Additionally, the pattern of DpsA localization in cells as they progress through the growth curve suggests that the DpsA complex may be involved in metal ion transport across the cell envelope. Crit Care Med, 2002 Nov, 30(11), 2493 - 500 Interaction between hemoglobin and glutathione in the regulation of blood flow in normal and septic pigs; Tejedor C et al.; BACKGROUND: Hemoglobin (Hb) induces vasoconstriction by heme group binding nitric oxide in an irreversible fashion . Recent in vitro studies indicate that the thiol groups in Hb reversibly bind nitric oxide and participate in trans-nitrosylation reactions with other thiols . Sepsis is a pathophysiologic state characterized by vasodilation mediated, at least in part, by an excessive release of nitric oxide . The role of nitrosothiols (RSNOs) in these changes is unknown . OBJECTIVES: We tested the following in a porcine model of sepsis: (i) whether glutathione (GSH) reverses the hemodynamic effects of Hb; (ii) whether GSH induces an increase in blood flow in sepsis; (iii) whether RSNO plasma concentration increases in sepsis and is related to hypotension . DESIGN: Nonrandomized animal controlled study . SETTING: Animal research facility in a university hospital . SUBJECTS: Anesthetized pigs were monitored with a pulmonary artery catheter and ultrasonic blood flow probes in the mesenteric artery and the portal vein for measurement of systemic, mesenteric, and portal blood flows (Q(TOT), Q(MES), and Q(POR), respectively) . Four groups of pigs were studied: nonseptic, septic, nonseptic treated with Hb (stroma-free purified porcine hemoglobin), and septic treated with Hb (n = 6 in each group) . INTERVENTIONS: Sepsis was induced at 0 min by the administration of live Escherichia coli . Hb (400 mg/kg/hr) was administered at 240 mins, followed by glutathione (1 g iv) . MEASUREMENTS AND MAIN RESULTS: Hb induced a pressor response and a decrease in Q(TOT), Q(MES), and Q(POR) . Glutathione reversed the effects of Hb on Q(MES) and Q(POR) . In septic pigs not treated with Hb, GSH induced an increase in Q(POR) . RSNO plasma concentration increased after the induction of sepsis and correlated significantly with blood pressure . CONCLUSIONS: These results indicate the reversibility of the effects of Hb by GSH, probably by interactions between nitric oxide and the reduced sulfhydryl groups in Hb, and suggest a role of RSNOs in the cardiovascular changes of sepsis. Protein Sci, 2002 Dec, 11(12), 2848 - 59 Quantitative chimeric analysis of six specificity determinants that differentiate Escherichia coli aspartate from tyrosine aminotransferase; Shaffer WA et al.; The six mutations, referred to as the Hex mutations, that together have been shown to convert Escherichia coli aspartate aminotransferase (AATase) specificity to be substantially like that of E . coli tyrosine aminotransferase (TATase) are dissected into two groups, (T109S/N297S) and (V39L/K41Y/T47I/N69L) . The letters on the left and right of the numbers designate AATase and TATase residues, respectively . The T109S/N297S pair has been investigated previously . The latter group, the "Grease" set, is now placed in the AATase framework, and the retroGrease set (L39V/Y41K/I47T/L69N) is substituted into TATase . The Grease mutations in the AATase framework were found primarily to lower K(M)s for both aromatic and dicarboxylic substrates . In contrast, retroGrease TATase exhibits lowered k(cat)s for both substrates . The six retroHex mutations, combining retroGrease and S109T/S297N, were found to invert the substrate specificity of TATase, creating an enzyme with a nearly ninefold preference (k(cat)/K(M)) for aspartate over phenylalanine . The retroHex mutations perturb the electrostatic environment of the pyridoxal phosphate cofactor, as evidenced by a spectrophotometric titration of the internal aldimine, which uniquely shows two pK(a)s, 6.1 and 9.1 . RetroHex was also found to have impaired dimer stability, with a K(D) for dimer dissociation of 350 nM compared with the wild type K(D) of 4 nM . Context dependence and additivity analyses demonstrate the importance of interactions of the Grease residues with the surrounding protein framework in both the AATase and TATase contexts, and with residues 109 and 297 in particular . Context dependence and cooperativity are particularly evident in the effects of mutations on k(cat)/K(M)(Asp) . Effects on k(cat)/K(M)(Phe) are more nearly additive and context independent. J Biol Chem, 2003 Jan 24, 278(4), 2411 - 8 Epub 2002 Nov 18. A dimeric mechanism for contextual target recognition by MutY glycosylase; Wong I et al.; MutY, an adenine glycosylase, initiates the critical repair of an adenine:8-oxo-guanine base pair in DNA arising from polymerase error at the oxidatively damaged guanine . Here we demonstrate for the first time, using presteady-state active site titrations, that MutY assembles into a dimer upon binding substrate DNA and that the dimer is the functionally active form of the enzyme . Additionally, we observed allosteric inhibition of glycosylase activity in the dimer by the concurrent binding of two lesion mispairs . Active site titration results were independently verified by gel mobility shift assays and quantitative DNA footprint titrations . A model is proposed for the potential functional role of the observed polysteric and allosteric regulation in recruiting and coordinating interactions with the methyl-directed mismatch repair system. J Biol Chem, 2003 Jan 24, 278(4), 2425 - 31 Epub 2002 Nov 18. Hairpin formation in Friedreich's ataxia triplet repeat expansion; Heidenfelder BL et al.; Triplet repeat tracts occur throughout the human genome . Expansions of a (GAA)(n)/(TTC)(n) repeat tract during its transmission from parent to child are tightly associated with the occurrence of Friedreich's ataxia . Evidence supports DNA slippage during DNA replication as the cause of the expansions . DNA slippage results in single-stranded expansion intermediates . Evidence has accumulated that predicts that hairpin structures protect from DNA repair the expansion intermediates of all of the disease-associated repeats except for those of Friedreich's ataxia . How the latter repeat expansions avoid repair remains a mystery because (GAA)(n) and (TTC)(n) repeats are reported not to self-anneal . To characterize the Friedreich's ataxia intermediates, we generated massive expansions of (GAA)(n) and (TTC)(n) during DNA replication in vitro using human polymerase beta and the Klenow fragment of Escherichia coli polymerase I . Electron microscopy, endonuclease cleavage, and DNA sequencing of the expansion products demonstrate, for the first time, the occurrence of large and growing (GAA)(n) and (TTC)(n) hairpins during DNA synthesis . The results provide unifying evidence that predicts that hairpin formation during DNA synthesis mediates all of the disease-associated, triplet repeat expansions. J Biol Chem, 2003 Jan 24, 278(4), 2767 - 72 Epub 2002 Nov 18. Holliday junction binding activity of the human Rad51B protein; Yokoyama H et al.; The human Rad51B protein is involved in the recombinational repair of damaged DNA . Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis . In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg(2+) or Mn(2+) and hydrolyzed ATP in a DNA-dependent manner . When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination . In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference . Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans. Am J Physiol Endocrinol Metab, 2003 Mar, 284(3), E574 - 82 Epub 2002 Nov 19. Infection impairs insulin-dependent hepatic glucose uptake during total parenteral nutrition; Donmoyer CM et al.; Total parenteral nutrition (TPN) markedly augments net hepatic glucose uptake (NHGU) and hepatic glycolysis in the presence of mild hyperglycemia and hyperinsulinemia . This increase is impaired by an infection . We determined whether the adaptation to TPN alters the responsiveness of the liver to insulin and whether infection impairs that response . Chronically catheterized dogs received TPN for 5 days . On day 3 of TPN, either a nonlethal hypermetabolic infection was induced (INF, n = 5) or a sham surgery was performed (SHAM, n = 5) . Forty-two hours after clot implantation, somatostatin and glucagon (34 +/- 3 vs . 84 +/- 11 pg/ml in artery, SHAM vs . INF) were infused, and a three-step (120 min each) isoglycemic (approximately 120 mg/dl) hyperinsulinemic (approximately 12, 25, and 50 microU/ml) clamp was performed to simulate levels seen in normal, infected, and exogenous insulin treatment states . In SHAM, NHGU (3.5 +/- 0.2 to 4.2 +/- 0.4 to 4.6 +/- 0.5 mg x kg(-1) x min(-1)) modestly increased . In INF, NHGU was consistently lower at each insulin step (1.1 +/- 0.5 to 2.6 +/- 0.5 to 2.8 +/- 0.7 mg x kg(-1) x min(-1)) . Although NHGU increased from the first to the second step in INF, it did not increase further with the highest dose of insulin . Despite increases in NHGU, net hepatic lactate release did not increase in SHAM and fell in INF . In summary, in the TPN-adapted state, liver glucose uptake is unresponsive to increases in insulin above the basal level . Although the infection-induced increase in insulin sustains NHGU, further increments in insulin enhance neither NHGU nor glycolysis. Anal Biochem, 2002 Dec 1, 311(1), 50 - 6 Purification of Shiga-like toxin 1 by pigeon egg white glycoproteins immobilized on Sepharose gels; Tomoda H et al.; The galabiose structure Galalpha1-4Gal is rarely found in natural glycoproteins, but is abundantly present in pigeon egg white proteins as Galalpha(1-4)Galbeta(1-4)GlcNAc termini . Pigeon ovalbumin, ovomucoid, or the whole egg white were immobilized on periodate-oxidized Sepharose CL-6B gels by reductive amination . These gels were found to bind Shiga-like toxin type 1 (SLT-1) specifically and efficiently . SLT-1 was eluted from the gel beads with 0.5 M melibiose, which was more efficient and milder than elution with 4.5 M MgCl(2) . SLT-1 was purified to homogeneity from the crude extract of Escherichia coli SLT100 expressing SLT-1 by a single affinity chromatographic step in 83-88% yield . The capacity of the gel was estimated to be ca . 1mg toxin/ml gel . Interestingly, SLT-2 was not bound by these affinity gels containing Galalpha1-4Galbeta1-4GlcNAc termini . Since SLT-2 has been shown to bind to Galalpha1-4Galbeta1-4Glc-terminating compounds, our results suggest that Glc in globotriose moiety is important for binding SLT-2, and replacing the Glc with GlcNAc in this triose renders it ineffective for binding SLT-2 . J Mol Biol, 2002 Nov 22, 324(2), 319 - 30 Secondary and tertiary structure formation of the beta-barrel membrane protein OmpA is synchronized and depends on membrane thickness; Kleinschmidt JH et al.; The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example . OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli . OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles . We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA . Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer . When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively . The formation of beta-sheet secondary structure and closure of the beta-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer . OmpA, and presumably other beta-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of alpha-helical bundle membrane proteins . These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins. J Mol Biol, 2002 Nov 22, 324(2), 227 - 36 The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases; Cheng YS et al.; The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death . In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity . Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A . Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site . There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry . Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA . Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI . A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested. Biochem Soc Trans, 2002 Nov, 30(Pt 6), 1175 - 80 Folding of the td pre-RNA with the help of the RNA chaperone StpA; Mayer O et al.; The td group I intron is inserted in the reading frame of the thymidylate synthase gene, which is mainly devoid of structural elements . In vivo, translation of the pre-mRNA is required for efficient folding of the intron into its splicing-competent structure . The ribosome probably resolves exon-intron interactions that interfere with splicing . Uncoupling splicing from translation, by introducing a non-sense codon into the upstream exon, reduces the splicing efficiency of the mutant pre-mRNA . Alternatively to the ribosome, co-expression of genes that encode proteins with RNA chaperone activity promote folding of the td pre-mRNA in vivo . These proteins also efficiently accelerate folding of the td pre-mRNA in vitro . In order to understand the mechanism of action of RNA chaperones, we probed the impact of the RNA chaperone StpA on the structure of the td intron in vivo and compared it with that of the well characterized Cyt-18 protein, which is a group-I-intron-specific splicing factor . We found that the two proteins have opposite effects on the structure of the td intron . While StpA loosens the three-dimensional structure, Cyt-18 tightens it up . Furthermore, mutations that destabilize the intron structure render the mutants sensitive to StpA, whereas splicing of these mutants is rescued by Cyt-18 . Our results provide direct evidence for protein-induced conformational changes within a catalytic RNA in vivo . Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the stabilization of the native three-dimensional structure. Biochem Soc Trans, 2002 Nov, 30(Pt 6), 1095 - 9 Acyl-CoA measurements in plants suggest a role in regulating various cellular processes; Graham IA et al.; Acyl-CoA esters have been shown to be involved in regulating metabolism and cell signalling in bacteria, yeast and mammalian cells, but little is known about their role in plants . Using a new method for the sensitive detection and quantification of acyl-CoA esters, we have recently shown that acyl-CoA pools can be dramatically altered in transgenic oilseed rape embryos, engineered to produce medium-chain fatty acids, and in mutant Arabidopsis seedlings that are unable to mobilize storage lipid . The consequences of these alterations are discussed in the context of oil yield and organelle biogenesis and the possible role of acyl-CoAs in regulating these processes. Biochem Soc Trans, 2002 Nov, 30(Pt 6), 1014 - 9 Interactions of CD55 with non-complement ligands; Lea S; Decay Accelerating Factor (or CD55) is a major regulator of the alternative and classical pathways of complement activation and is expressed on all serum-exposed cells . It is commonly hijacked by invading pathogens, including many enteroviruses and uropathogenic Escherichia coli, to promote cellular attachment prior to infection . This review will attempt to summarize our knowledge about these interactions between CD55 and various pathogens and also what is known about the non-complement interaction between CD55 and CD97. Bioconjug Chem, 2002 Nov-Dec, 13(6), 1186 - 92 Class-selective drug detection: fluorescently-labeled calmodulin as the biorecognition element for phenothiazines and tricyclic antidepressants; Douglass PM et al.; A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants (TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as the recognition element . A calmodulin mutant containing a unique cysteine residue at position 109 on the protein was expressed in Escherichia coli . Following purification, the environment-sensitive, thiol-specific fluorophores N-{2-(1-maleimidyl)ethyl}-7-(diethylamino)coumarin-3-carboxamide (MDCC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), and 4-{N-(2-(iodoacetoxy)ethyl)-N-methylamino}-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester) were coupled to the C109 site of the mutant protein . The response of labeled CaM in the presence of calcium to increasing concentrations of chlorpromazine hydrochloride (CPZ), as well as other phenothiazines and structurally related antipsychotics and antidepressants, was investigated . Fluorescence measurements were performed on benchtop and microtiter plate fluorometers . The responses were characterized as a change in the signal intensity of the labeled protein upon ligand binding, and the stability of the system was monitored over a nine-month period . The assay showed specificity for the phenothiazine and TCA classes of drugs, with limits of detection in the micromolar range . Selectivity studies indicated negligible response of the biosensing system to structurally unrelated compounds . This work represents a proof-of-concept assay for rapid, homogeneous detection of drugs employing binding proteins as the biorecognition element. Cell Mol Life Sci, 2002 Sep, 59(9), 1554 - 60 Cellulase genes from the parabasalian symbiont Pseudotrichonympha grassii in the hindgut of the wood-feeding termite Coptotermes formosanus; Nakashima KI et al.; Cellulase genes of Pseudotrichonympha grassii (Hypermastigida: Eucomonymphidae), the symbiotic flagellate in the hindgut of the wood-feeding termite Coptotermes formosanus, were isolated and characterized . The nucleotide sequences of the major cellulase component in the hindgut of C . formosanus were determined based on its N-terminal amino acid sequence . The five isolated nucleotide sequences (PgCBH-homos) had an open reading frame of 1350 bp showing similarity to catalytic domains of glycoside hydrolase family (GHF) 7 members, and primary structure comparison with GHF7 members whose tertiary structures are well-characterized revealed the overall similarity between PgCBH-homo and the catalytic domain of a processive cellulase Cel7A (formerly CBHI) from the aerobic fungus Trichoderma reesei . Functional expression of PgCBH-homos in Escherichia coli, using the carboxymethylcellulose-Congo red assay, demonstrated the actual cellulolytic activity of PgCBH-homo . RT-PCR showed that PgCBH-homos were expressed, from the three flagellates in the hindgut, specifically in P . grassii. Biochem Cell Biol, 2002, 80(5), 563 - 8 Assessing the structure of membrane proteins: combining different methods gives the full picture; Stahlberg H et al.; The rotor stoichiometry of F-ATPases has been revealed by the combined approaches of X-ray diffraction (XRD), electron crystallography, and atomic force microscopy (AFM) . XRD showed the rotor from the yeast mitochondrial F-ATPase to contain 10 subunits . AFM was used to visualize the tetradecameric chloroplast rotors, and electron crystallography and AFM together revealed the rotors from Ilyobacter tartaricus to be composed of 11 subunits . While biochemical methods had determined an approximate stoichiometric value, precise measurements and new insights into a species-dependent rotor stoichiometry became available by applying the three structural tools together . The structures of AQP1, a water channel, and G1pF, a glycerol channel, were determined by electron crystallography and XRD . The combination of both of these structural tools with molecular dynamics simulations gave a differentiated description of the mechanisms determining the selectivity of water and glycerol channels . This illustrates that the combination of different methods in structural biology reveals more than each method alone. IUBMB Life, 2002 Aug, 54(2), 85 - 8 Swapping of three-dimensional domains as a molecular mechanism of dimerization of aminoacyl-tRNA synthetases; Deniziak MA et al.; It has been shown recently that many proteins undergo oligomerization through exchange of structural elements . That process, termed a 3D domain swapping, is the replacement of a portion of the tertiary structure of a protein with an identical piece from a second polypeptide chain . When the exchange is reciprocated, domain-swapped dimers embrace with the exchange of elements of secondary structure or domains; however, if the exchange is not reciprocated but propagated along multiple polynucleotide chains, higher-order assemblies may form . In this paper we discuss swapping as a general mechanism of aminoacyl-tRNA synthetases dimerization, specifically plant methionyl-tRNA synthetase. IUBMB Life, 2002 Aug, 54(2), 67 - 72 Selection of peptide ligands binding to fibroblast growth factor receptor 1; Fan H et al.; Inappropriate expression of fibroblast growth factors (FGFs) or activation of FGF receptors (FGFRs) could contribute to several human angiogenic pathologies . In an attempt to design antagonists of FGF, we developed a screening procedure for identifying peptide ligands binding to FGFR1 . To retain the natural conformation of FGFR1 during screening, we expressed recombinant FGFR1 on the surface of Sf9 insect cells . A 6-mer phage display peptide library was then screened on the cell surface and a group of hydrophobic peptide sequences were identified . Further experiments demonstrated that the phages displaying these sequences can specifically bind to FGFR1 . The docking analysis suggests that the peptide ValTyrMetSerProPhe can specifically bind to the hydrophobic surface of FGFR1 . The synthetic peptide Ac-ValTyrMetSerProPhe-NH2 can inhibit mitogenic activity of aFGF and has the potential to become a therapeutic agent as an aFGF antagonist. Indian J Med Res, 2002 Jun, 115, 251 - 4 Multiplex PCR for detection of stx genes of Escherichia coli; Rahman H; BACKGROUND & OBJECTIVE: Shiga toxin producing Escherichia coli (STEC), which includes both enterohaemorrhagic Esch . coli (EHEC, 0157: 7H) and non-EHEC (non-0157) produces two major Shiga toxins (Stx1 & Stx2) . Detection of Stx or stx genes is the only approach to detect all the different types of STEC . A multiplex PCR is used for the detection of stx genes from EHEC and non-EHEC strains isolated from patients of enteritis . METHODS: Ten EHEC and 35 non-EHEC strains obtained from patients with diarrhoea and enteritis were studied . A single and multiplex PCR (mPCR) were used for detection of stx genes using specific primers . In single PCR, the stx 1 and stx 2 genes were amplified separately while in mPCR, the two genes were amplified together in a single reaction . The PCR products were detected by electrophoresis . RESULTS: All the EHEC strains were found to harbour one or both stx genes as detected by single and multiplex PCR . Of the non-EHEC strains tested, 14.28 per cent were positive for stx genes . Multiplex PCR gave similar results and showed 100 per cent agreement with that of single PCR . INTERPRETATION & CONCLUSION: The results indicated that stx genes are common in the EHEC strains but they are less prevalent among non-EHEC strains . Because of simplicity, rapidity and specificity, mPCR assay represents a good alternative to traditional methods for the detection of stx genes of STEC. World J Gastroenterol, 2002 Dec, 8(6), 1050 - 2 Over-expression of LPTS-L in hepatocellular carcinoma cell line SMMC-7721 induces crisis; Liao C et al.; AIM: To evaluate the function of the longer transcripts LPTS-L in hepatocellular carcinoma cell line SMMC-7721 . METHODS: SMMC-7721 cells were transfected with LPTS-L expression construct and stably transfected cells were selected by G418 . Multiple single clones formed and were checked for their phenotype . In the study of the effect on telomerase activity of LPTS-L in vitro, GST-LPTS-L fusion protein was expressed in E.coli and purified by glutathione-agarose column . Telomeric repeat amplification protocol (TRAP) assays were performed to study the influence of telomerase activity in SMMC-7721 cells . RESULTS: Over-expression of LPTS-L induced SMMC-7721 cells into crisis . LPTS-L could inhibit the telomerase activity in SMMC-7721 cells in vitro . CONCLUSION: LPTS-L is a potent telomerase inhibitor . Over-expression of LPTS-L can induce hepatoma cells into crisis due to the reduction of telomerase activity. Infect Immun, 2002 Dec, 70(12), 6976 - 86 Expression of a novel Leishmania gene encoding a histone H1-like protein in Leishmania major modulates parasite infectivity in vitro; Papageorgiou FT et al.; We describe identification and characterization of a novel two-copy gene of the parasitic protozoan Leishmania that encodes a nuclear protein designated LNP18 . This protein is highly conserved in the genus Leishmania, and it is developmentally regulated . It is an alanine- and lysine-rich protein with potential bipartite nuclear targeting sequence sites . LNP18 shows sequence similarity to H1 histones of trypanosomatids and of higher eukaryotes and in particular with histone H1 of Leishmania major . The nuclear localization of LNP18 was determined by indirect immunofluorescence and Western blot analysis of isolated nuclei by using antibodies raised against the recombinant protein as probes . The antibodies recognized predominantly a 18-kDa band or a 18-kDa-16-kDa doublet . Photochemical cross-linking of intact parasites followed by Western blot analysis provided evidence that LNP18 is indeed a DNA-binding protein . Generation of transfectants overexpressing LNP18 allowed us to determine the role of this protein in Leishmania infection of macrophages in vitro . These studies revealed that transfectants overexpressing LNP18 are significantly less infective than transfectants with the vector alone and suggested that the level of LNP18 expression modulates Leishmania infectivity, as assessed in vitro. Infect Immun, 2002 Dec, 70(12), 6658 - 64 Counteracting interactions between lipopolysaccharide molecules with differential activation of toll-like receptors; Hajishengallis G et al.; We investigated counteracting interactions between the lipopolysaccharides (LPS) from Escherichia coli (Ec-LPS) and Porphyromonas gingivalis (Pg-LPS), which induce cellular activation through Toll-like receptor 4 (TLR4) and TLR2, respectively . We found that Ec-LPS induced tolerance in THP-1 cells to subsequent tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1beta) induction by Pg-LPS, though the reverse was not true, and looked for explanatory differential effects on the signal transduction pathway . Cells exposed to Pg-LPS, but not to Ec-LPS, displayed persisting expression of IL-1 receptor-associated kinase without apparent degradation, presumably allowing prolonged relay of downstream signals . Accordingly, cells pretreated with Pg-LPS, but not with Ec-LPS, were effectively activated in response to subsequent exposure to either LPS molecule, as evidenced by assessing nuclear factor (NF)-kappaB activity . In fact, Pg-LPS primed THP-1 cells for enhanced NF-kappaB activation and TNF-alpha release upon restimulation with the same LPS . This was a dose-dependent effect and correlated with upregulation of surface TLR2 expression . Furthermore, we observed inhibition of NF-kappaB-dependent transcription in a reporter cell line pretreated with Ec-LPS and restimulated with Pg-LPS (compared to cells pretreated with medium only and restimulated with Pg-LPS), but not when the reverse treatment was made . Although Pg-LPS could not make cells tolerant to subsequent activation by Ec-LPS, Pg-LPS inhibited Ec-LPS-induced TNF-alpha and IL-6 release when the two molecules were added simultaneously into THP-1 cell cultures . Pg-LPS also suppressed P . gingivalis FimA protein-induced NF-kappaB-dependent transcription in the 3E10/huTLR4 reporter cell line, which does not express TLR2 . This rules out competition for common signaling intermediates, suggesting that Pg-LPS may block component(s) of the TLR4 receptor complex . Interactions between TLR2 and TLR4 agonists may be important in the regulation of inflammatory reactions. Ann N Y Acad Sci, 2002 Oct, 971, 608 - 14 Analysis of signaling pathways using functional proteomics; Resing KA; Advances in analytical methods for protein analysis by mass spectrometry provide new tools for global analysis of the expressed protein profile of cells (referred to as proteomics) . Currently, available methodology samples only part of the proteome . This is sufficient for analysis of signal transduction, because signaling pathways contain enzymes, which modify high-abundance proteins other than those of the pathway . Thus, modulation of the signaling through a pathway will produce a "footprint" in the proteome that is characteristic of a specific cell phenotype . Comparison of different samples to identify these differences in posttranslational modification or protein expression is referred to as functional proteomics . This review surveys the methods in widest use in functional proteomics, as well as a few promising new ones . Although proteomic analyses were first conducted 26 years ago, a renewed interest is fueled by several recent advances . Most important are the availability of public genome and protein databases and the development of high-sensitivity, easy-to-use mass spectrometers and database search engines capable of exploiting these databases . Other important advances include improved two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), computer programs for analysis of the 2D-PAGE gel images, protocols for proteolytic digestion of proteins in excised gel pieces, and low-flow chromatography methods . Despite the limitations of these methods, they can distinguish subtle changes in the phenotype of cells, providing the basis for future studies in regulation of the phenotype. Mutat Res, 2002 Nov 26, 521(1-2), 151 - 63 Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations; Atienzar FA et al.; The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations . The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands . However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes . In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles . Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo{a}pyrene (B{a}P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated . In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons . A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected . Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA . Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences . Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations. Biochem Biophys Res Commun, 2002 Nov 29, 299(2), 268 - 72 PqqC/D, which converts a biosynthetic intermediate to pyrroloquinoline quinone; Toyama H et al.; PqqC/D was purified from Escherichia coli transformant . The purified enzyme converted an intermediate that accumulated in a pqqC mutant of Methylobacterium extorquens AM1 to PQQ . The reaction did not show any dependence of NAD(P)H that was observed in the crude extract before purification . PqqC/D reacted with the intermediate stoichiometrically, but not catalytically . When partially purified proteins from the crude extract of E . coli were added to the reaction mixture, the rate of PQQ production increased dependent on the amount of NADPH added and the total amount of PQQ produced increased. Biochem Biophys Res Commun, 2002 Nov 29, 299(2), 252 - 7 Decrease in cell viability in an RMF, sigma(38), and OmpC triple mutant of Escherichia coli; Samuel Raj V et al.; In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth . There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC) . To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted . Viability of the triple mutant decreased to less than 1% of normal cells . The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability . The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability . The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase . The triple mutant had a reduced level of ribosomes and of intracellular cations. Biochem Biophys Res Commun, 2002 Nov 29, 299(2), 189 - 95 Phanerochaete chrysosporium NADPH-cytochrome P450 reductase kinetic mechanism; Warrilow AG et al.; The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and >123 cytochrome P450 (CYP) genes . How a single CPR can drive many CYPs is an important area of study . We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system . Native CPR and a NH(2)-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity . Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life . Removal of the N-terminal anchor of P . chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies. Biochem Biophys Res Commun, 2002 Nov 29, 299(2), 169 - 72 Unusual cyanide bindings to a heme-regulated phosphodiesterase from Escherichia coli: effect of Met95 mutations; Watanabe M et al.; In order to understand heme environment of a heme-regulated phosphodiesterase (Ec DOS), the binding behavior of cyanide to the Fe (III) complex was examined . Interestingly, the rate of cyanide binding to full-length Ec DOS was unusually slow with k(on)=0.0022mM(-1)s(-1), while the rate for the isolated heme domain of Ec DOS (0.045mM(-1)s(-1)) was 20-fold higher . Ala and Leu mutations at Met95, which has been suggested to be a heme axial ligand, increased the k(on) rate 11- and 8-fold, respectively, and dramatically decreased the cyanide dissociation rate from the isolated heme domain . His mutation at Met95, on the other hand, caused a 17-fold decrease in the k(on) value . We discuss the unusual cyanide binding behavior and the role of Met95 in controlling cyanide binding. Cell, 2002 Nov 15, 111(4), 543 - 51 Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation; Sauer FG et al.; Periplasmic chaperones direct the assembly of adhesive, multi-subunit pilus fibers that play critical roles in bacterial pathogenesis . Pilus assembly occurs via a donor strand exchange mechanism in which the N-terminal extension of one subunit replaces the chaperone G(1) strand that transiently occupies a groove in the neighboring subunit . Here, we show that the chaperone primes the subunit for assembly by holding the groove in an open, activated conformation . During donor strand exchange, the subunit undergoes a topological transition that triggers the closure of the groove and seals the N-terminal extension in place . It is this topological transition, made possible only by the priming action of the chaperone that drives subunit assembly into the fiber. Eur J Immunogenet, 2002 Dec, 29(6), 517 - 23 Recombinant DRB sequences produced by mismatch repair of heteroduplexes during cloning in Escherichia coli; Longeri M et al.; Recombinant chimeric sequences originating from a mixture of the sequences of two different alleles are frequently found after amplification and cloning in Escherichia coli of exon 2 of the major histocompatibility complex (MHC) DRB genes . Several authors have suggested that the recombinant molecules result from in vitro recombination during PCR; nevertheless, a clear experimental demonstration of this hypothesis is lacking . In order to understand the mechanism producing the chimeric sequences, we set up a simple experiment based on the different restriction patterns of parental and recombinant sequences . Our data demonstrate that in the analysed case most of the recombinant variants were not produced by in vitro recombination during PCR, but were the result of the mismatch repair of heteroduplex molecules during cloning in E . coli . The high mutation rate in the alpha-helix region of DRB expressed genes, both after cloning in E . coli and after the germ-line differentiation process in vertebrates, suggests that the observed mutations are the result of similar gene conversion processes, probably favoured by chi-dependent microrecombination events. Biochem J, 2003 Mar 1, 370(Pt 2), 679 - 86 Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase; Hirooka K et al.; trans -Long-chain prenyl diphosphate synthases catalyse the sequential condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate to produce the C(30)-C(50) prenyl diphosphates, which are precursors of the side chains of prenylquinones . Based on the relationship between product specificity and the region around the first aspartate-rich motif in trans -prenyl diphosphate synthases characterized so far, we have isolated the cDNA for a member of trans -long-chain prenyl diphosphate synthases from Arabidopsis thaliana . The cDNA was heterologously expressed in Escherichia coli, and the recombinant His(6)-tagged protein was purified and characterized . Product analysis revealed that the cDNA encodes solanesyl diphosphate (C(45)) synthase (At-SPS) . At-SPS utilized farnesyl diphosphate (FPP; C(15)) and geranylgeranyl diphosphate (GGPP; C(20)), but did not accept either the C(5) or the C(10) allylic diphosphate as a primer substrate . The Michaelis constants for FPP and GGPP were 5.73 microM and 1.61 microM respectively . We also performed an analysis of the side chains of prenylquinones extracted from the A . thaliana plant, and showed that its major prenylquinones, i.e . plastoquinone and ubiquinone, contain the C(45) prenyl moiety . This suggests that At-SPS might be devoted to the biosynthesis of either or both of the prenylquinone side chains . This is the first established trans -long-chain prenyl diphosphate synthase from a multicellular organism. Biochemistry, 2002 Nov 26, 41(47), 14076 - 84 Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin; Kim YH et al.; Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host . With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation . Specific modifications include deamidation and truncation . Urea-induced denaturation was studied for recombinantly expressed wild-type betaB1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (betaB1(DeltaN41)), and other truncated versions of these proteins generated by calpain II digestion . Tryptophan fluorescence was used to monitor loss of global tertiary structure . Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain . Our results indicated that the deamidated mutant was significantly destabilized relative to WT . Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea . Unfolding of WT was completely reversible, whereas Q204E failed to fully refold . Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT . Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability . These studies indicated that a single-site deamidation could significantly diminish the stability of lens betaB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation. Biochemistry, 2002 Nov 26, 41(47), 14066 - 75 Synthesis of nucleotide analogues that potently and selectively inhibit human DNA primase; Moore CL et al.; DNA primase synthesizes short RNA oligonucleotides that DNA polymerase alpha further elongates in order to initiate the synthesis of all new DNA strands during eukaryotic DNA replication . To develop potent and specific primase inhibitors, we combined 2'-modified sugars with bases incapable of normal Watson-Crick hydrogen bonding . The presence of a 2'-hydroxyl in either the ara or ribo configuration greatly enhances the ability of primase to polymerize a nucleotide . Further modifying the 2'-position by including both a hydroxyl and methyl group at this position greatly reduced the ability of primase to polymerize the resulting nucleotides . Replacing the base of the NTP with analogues incapable of normal Watson-Crick hydrogen bonding (benzimidazole, nitrobenzimidazole, and dichlorobenzimidazole) resulted in compounds that inhibited primase quite well and with similar potency . We synthesized arabinofuranosylbenzimidazole triphosphate (araBTP) and found that this sugar change increased inhibition by 2-4-fold relative to the ribofuranosyl analogue . AraBTP inhibited polymerization of both purines and pyrimidines, although primase polymerized only small amounts of the compound . Interestingly, even though araBTP was not readily polymerized by primase, it inhibited primase almost as potently as araATP, a compound that primase polymerizes extremely rapidly and that results in very strong chain termination . Importantly, this compound was a very weak inhibitor of and only slowly polymerized by DNA polymerase alpha, indicating that it is a specific primase inhibitor . The potential utility and mechanistic implications of these inhibitors are discussed. Biochemistry, 2002 Nov 26, 41(47), 13956 - 64 Structurally distinct modes of recognition of the KIX domain of CBP by Jun and CREB; Campbell KM et al.; Gene expression is coordinated in part by interactions between transcriptional activators and other transcription factors such as coactivators . The KIX domain of the coactivator and histone acetyltransferase CREB binding protein (CBP) binds numerous mammalian and viral transcriptional activators such as BRCA1, CREB, c-Jun, c-Myb, p53, papillomavirus E2, and HTLV-1 Tax . Formation of the CREB-CBP complex depends on phosphorylation of the KID region of CREB and involves induced folding of KID upon binding a hydrophobic groove of the KIX domain of CBP . Here we investigate the formation of the complex formed by human KIX and the N-terminal activation domain of human c-Jun . The c-Jun activation domain and KID do not share significant sequence similarity . Circular dichroism spectroscopy shows that the Jun N-terminal activation domain is intrinsically disordered in isolation and that KIX binding is independent of Jun phosphorylation . In contrast to the mode of binding exhibited by CREB, NMR chemical shift mapping indicates that the c-Jun activation domain binds to a distinctly different surface of KIX than used by CREB . Moreover, NMR and sedimentation equilibrium studies show that the activation domains of c-Jun and CREB can simultaneously bind the KIX domain of CBP . The results illustrate a new mode of binding and combinatorial recruitment via the KIX domain of CBP by multiple transcriptional activators. Biochemistry, 2002 Nov 26, 41(47), 13902 - 14 Solution NMR structure and backbone dynamics of the PsaE subunit of photosystem I from Synechocystis sp . PCC 6803; Barth P et al.; PsaE is a small peripheral subunit of photosystem I (PSI) that is very accessible to the surrounding medium . It plays an essential role in optimizing the interactions with the soluble electron acceptors of PSI, ferredoxin and flavodoxin . The solution structure of PsaE from the cyanobacterium Synechocystis sp . PCC 6803 has been investigated by NMR with a special emphasis on its protein dynamic properties . PsaE is characterized by a well-defined central core that consists of a five-stranded beta-sheet (+1, +1, +1, -4x) . Four loops (designated the A-B, B-C, C-D, and D-E loops) connect these beta-strands, the overall resulting structure being that of an SH3-like domain . As compared to previously determined PsaE structures, conformational differences are observed in the first three loops . The flexibility of the loops was investigated using (15)N relaxation experiments . This flexibility is small in amplitude for the A-B and B-C loops, but is large for the C-D loop, particularly in the region corresponding to the missing sequence of Nostoc sp . PCC 8009 . The plasticity of the connecting loops in the free subunit is compared to that when bound to the PSI and discussed in relation to the insertion process and the function(s) of PsaE. Biochemistry, 2002 Nov 26, 41(47), 13894 - 901 Recombinant equine cytochrome c in Escherichia coli: high-level expression, characterization, and folding and assembly mutants; Rumbley JN et al.; To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable . This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli . Starting with the yeast Cyt c plus heme lyase construct of Pollock et al . {Pollock, W . B., Rosell, F . I., Twitchett, M . B., Dumont, M . E., and Mauk, A . G . (1998) Biochemistry 37, 6124-6131}, an E . coli-based system was designed that consistently produces high yields of recombinant eucaryotic (equine) Cyt c . Systematic changes to the ribosome binding site, plasmid sequence, E . coli strain, growth temperature, and growth duration increased yields from 2 to 3 mg/L to as much as 105 mg/L . Issues related to purification, fidelity of heme insertion, equilibrium stability, and introduction and analysis of mutant forms are described . As an example, variants tailored for folding studies are discussed . These remove known pH-dependent kinetic folding barriers (His26 and His33 and N-terminus), reveal an additional kinetic trap at higher pH due to some undetermined residue(s), and show how a new barrier can be placed at different points in the folding pathway in order to trap and characterize different folding intermediates . In addition, destabilizing glycine mutants in the N-terminal helix are shown to affect the fractional yield of a heme inverted Cyt c isoform. Biochemistry, 2002 Nov 26, 41(47), 13876 - 82 Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives; Plesniak L et al.; EnvZ is a histidine protein kinase important for osmoregulation in bacteria . While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized . The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy . The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB . The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket . Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM) . The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket . The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases . Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms . Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold . Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C . The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies. Biol Chem, 2002 Sep, 383(9), 1459 - 62 An efficient method for the preparation of long heteroduplex DNA as substrate for mismatch repair by the Escherichia coli MutHLS system; Thomas E et al.; We present a method that allows preparing long DNA containing defined mismatches without the use of gel electrophoretic or chromatographic purification steps . The preparation starts with the synthesis of two PCR products, which are identical except for those positions that will later form the mismatches . One of the PCR primers must be 5'-phosphorylated, such that in two separate reactions two PCR products are obtained, which are 5'-phosphorylated in one strand . After removal of the phosphorylated strands by lambda-exonuclease, the resulting single strands are hybridized to form the mismatch-containing heteroduplex . The application of this procedure is demonstrated for the analysis of the Escherichia coli MutHLS system. Biol Chem, 2002 Sep, 383(9), 1373 - 81 The in vitro assembly of hair follicle keratins: comparison of cortex and companion layer keratins; Hofmann I et al.; The hair follicle consists of a complex system of multiple tissue compartments that are clearly distinguishable by their morphology and type of differentiation . We have synthesized hair follicle-specific keratins from the companion layer (K6hf, K17) and the hair cortex (Ha1, Hb3, Hb6) in Escherichia coli . The assembly of purified keratins in mixtures of K6hf/K17 and in mixtures of hair cortex keratins was compared in urea solutions, low ionic strength and physiological strength buffers, by urea melting gels, electron microscopy and analytical ultracentrifugation . Both types of keratin mixtures, keratins from the companion layer and keratins from the hair cortex, formed heterotypic complexes at 5 M urea . In low ionic strength buffers, the keratins from the companion layer were assembled to bona fide intermediate filaments . In contrast, mixtures of hair cortex keratins stayed in an oligomeric state with a mean s value of 9 as determined in sedimentation velocity experiments . Hair cortex keratins were, however, assembled into intermediate filaments at physiological salt conditions . A point mutated hair cortex keratin {Hb6(Glu402Lys)} formed no long filaments when mixed with Ha1; instead, the assembled structures showed a length distribution of 50.8 +/- 13.4 nm, comparable to the size distribution of assembly intermediates called 'unit-length' filaments. Biol Chem, 2002 Sep, 383(9), 1363 - 72 Enzymatic activity of the Arabidopsis sulfurtransferase resides in the C-terminal domain but is boosted by the N-terminal domain and the linker peptide in the full-length enzyme; Burow M et al.; Sulfurtransferases/rhodaneses are a group of enzymes widely distributed in plants, animals, and bacteria that catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate . Sulfurtransferases (STs) consist of two globular domains of nearly identical size and conformation connected by a short linker sequence . In plant STs this linker sequence is exceptionally longer than in sequences from other species . The Arabidopsis ST1 protein (AJ131404) contains five cysteine residues: one residue is universally conserved in all STs and considered to be catalytically essential; a second one, closely located in the primary sequence, is conserved only in sequences from eukaryotic species . Of the remaining three cysteine residues two are conserved in the so far known plant STs and one is unique to the Arabidopsis ST1 . The aim of our study was to investigate the role of the two-domain structure, of the unique plant linker sequence and of each cysteine residue . The N- and C-terminal domains of the Arabidopsis ST1, the full-length protein with a shortened linker sequence and several point-mutated proteins were overexpressed in E . coli, purified and used for enzyme activity measurements . The C-terminal domain itself displayed ST activity which could be increased by adding the separately prepared N-terminal domain . The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wild-type activity, probably because of a drastically reduced protein stability . The replacement of each cysteine residue resulted in mutant forms which differed significantly in their stability, in the specific ST activities, and in their kinetic parameters which were determined for 3-mercaptopyruvate as well as thiosulfate as sulfur substrates: mutation of the putative active site cysteine (C332) essentially abolished activity; for C339 a crucial role at least for the turnover of thiosulfate could be identified. Biol Chem, 2002 Sep, 383(9), 1335 - 42 The Hsp90 co-chaperones Cdc37 and Sti1 interact physically and genetically; Abbas-Terki T et al.; Cdc37 associates with the heat-shock protein 90 (Hsp90) molecular chaperone as one of several auxiliary proteins that are collectively referred to as Hsp90 co-chaperones . Cdc37 has been proposed to be a specificity factor for Hsp90, directing it notably towards kinases . It is not known whether Cdc37 is essential for viability in the budding yeast Saccharomyces cerevisiae because of Hsp90-dependent or -independent functions or both . Sti1 and Cpr7 are non-essential Hsp90 co-chaperones that bind to a common surface on Hsp90 through tetratricopeptide repeats (TPR) . We have found that Sti1 is specifically retained from yeast extracts by immobilized Cdc37 . Similarly, the endogenous proteins are also found in a complex . Moreover, purified recombinant Sti1 and Cdc37 interact in the complete absence of Hsp90 . Complexes between Cdc37 and Sti1 are not unique to this TPR protein since endogenous Cdc37 can be co-purified with exogenously expressed Cpr7 fused to glutathione-S-transferase . The heterogeneity of Cdc37 complexes, both with and without Hsp90, may expand the functional diversity of Cdc37 . Here we show that the combination of cdc37 and sti1 mutations is synthetically lethal, suggesting that direct contacts between Cdc37 and Sti1 may at least contribute to vital functions in yeast. Biol Chem, 2002 Sep, 383(9), 1325 - 33 A dimeric mutant of the homotetrameric single-stranded DNA binding protein from Escherichia coli; Landwehr M et al.; A single amino acid substitution (Y78R) at the dimer-dimer interface of homotetrameric single stranded DNA binding protein from E . coli (EcoSSB) renders the protein a stable dimer . This dimer can bind single-stranded DNA albeit with greatly reduced affinity . In vivo this dimeric SSB cannot replace homotetrameric EcoSSB . Amino acid changes at the rim of the dimer-dimer interface nearby (Q76K, Q76E) show an electrostatic interaction between a charged amino acid at position 76 and bound nucleic acid . In conclusion, nucleic acid binding to homotetrameric SSB must take place across both dimers to achieve functionally correct binding. Transgenic Res, 2002 Oct, 11(5), 521 - 31 Biological activity of human granulocyte-macrophage colony stimulating factor is maintained in a fusion with seed glutelin peptide; Sardana RK et al.; Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants . Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression . Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5' end . With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein . Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active . Furthermore, native tobacco seed extracts inhibited the activity of E . coli-derived GM-CSF in this cytokine-dependent cell line . The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited . The feasibility of oral delivery of such stable seed-produced cytokines is discussed. Appl Microbiol Biotechnol, 2002 Nov, 60(3), 336 - 41 Epub 2002 Oct 05. Construction and characterization of phage-displayed leukocyte surface molecule CD99; Tayapiwatana C et al.; The phage display technique has been described for the production of various recombinant molecules . In the present report, we used this technique to display a leukocyte surface molecule, CD99 . PCR subcloning of CD99 cDNA from the mammalian expression vector pCDM8 to the phagemid expression vector pComb3HSS was performed . The resulting phagemid, pComb3H-CD99, was transformed into Escherichia coli XL-1 Blue . CD99 was displayed on the phage particles following infection of the transformed E . coli with the filamentous phage VCSM13 . Using sandwich ELISA, the filamentous phage-displayed CD99 was captured by a CD99 monoclonal antibody (mAb) then detected with anti-M13 conjugated to horseradish peroxidase, confirming that the CD99 molecule was displayed on the phage particles . The CD99-phages inhibited induction of Jurkat cell aggregation by CD99 mAb MT99/1 . Proper folding of the displayed CD99 bioactive domain was inferred from this finding . Our results demonstrate that the phage display technique can be applied to the generation of full-length CD99 molecules . The phage carrying this cell surface protein will be useful for identification of its counter receptor or ligand. Mol Genet Genomics, 2002 Nov, 268(3), 287 - 97 Epub 2002 Oct 17. Identification of a single nuclear localization signal in the C-terminal domain of an Aspergillus DNA topoisomerase II; Kim KH et al.; DNA topoisomerase II (topo II) is a major nuclear protein that plays an important role in DNA metabolism . We have isolated the gene for topo II ( TOP2) from the filamentous fungus Aspergillus terreus . The deduced amino acid sequence revealed that topo II consists of 1,587 amino acids and has a calculated molecular weight of 180 kDa; the protein expressed in Escherichia coli has an estimated molecular weight of 185 kDa . Expression of topo II polypeptides tagged with yellow fluorescent protein (YFP) in budding yeast suggests that the C-terminal region of the topo II is essential for transport of the fusion protein into the nucleus . The nuclear localization signal (NLS) sequence of topo II is a non-classical bipartite type containing two interdependent, positively charged clusters separated by 15 amino acids . Alanine scanning mutagenesis and deletion analyses showed further that a stretch of 23 amino acid residues (positions 1,234-1,256) is necessary for nuclear import . In addition, we confirmed, using co-immunoprecipitation and two-hybrid analysis, that this non-classical NLS interacts with importin alpha in budding yeast . These results suggest that the fungal topo II NLS is functional in yeast cells. J Biol Chem, 2003 Jan 31, 278(5), 3235 - 40 Epub 2002 Nov 14. Cellular polyamines promote the aggregation of alpha-synuclein; Antony T et al.; The cellular polyamines putrescine, spermidine, and spermine accelerate the aggregation and fibrillization of alpha-synuclein, the major protein component of Lewy bodies associated with Parkinson's disease . Circular dichroism and fluorometric thioflavin T kinetic studies showed a transition of alpha-synuclein from unaggregated to highly aggregated states, characterized by lag and transition phases . In the presence of polyamines, both the lag and transition times were significantly shorter . All three polyamines accelerated the aggregation and fibrillization of alpha-synuclein to a degree that increased with the total charge, length, and concentration of the polyamine . Electron and scanning force microscopy of the reaction products after the lag phase revealed the presence of aggregated particles (protofibrils) and small fibrils . At the end of the transition phase, alpha-synuclein formed long fibrils in all cases, although some morphological variations were apparent . In the presence of polyamines, fibrils formed large networks leading ultimately to condensed aggregates . In the absence of polyamines, fibrils were mostly isolated . We conclude that the polyamines at physiological concentrations can modulate the propensity of alpha-synuclein to form fibrils and may hence play a role in the formation of cytosolic alpha-synuclein aggregates. J Biol Chem, 2003 Jan 17, 278(3), 1708 - 12 Epub 2002 Nov 14. Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis; Piotrowski M et al.; A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography . The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria . N-carbamoylputrescine amidohydrolase activity was confirmed by identification of two of the three occurring products, namely putrescine and ammonia . In contrast, no enzymatic activity could be detected when applying various compounds including nitriles, amines, and amides as well as other N-carbamoyl compounds, indicating the specificity of the enzyme for N-carbamoylputrescine . Like the homologous beta-alanine synthases, NLP1 showed positive cooperativity toward its substrate . The native enzyme had a molecular mass of 279 kDa as shown by blue-native polyacrylamide gel electrophoresis, indicating a complex of eight monomers . Expression of the NLP1 gene was found in all organs investigated, but it was not induced upon osmotic stress, which is known to induce biosynthesis of putrescine . This is the first report of cloning and expression of a plant N-carbamoylputrescine amidohydrolase and the first time that N-carbamoylputrescine amidohydrolase activity of a recombinant protein could be shown in vitro . NLP1 is one of the two missing links in the arginine decarboxylase pathway of putrescine biosynthesis in higher plants. J Biol Chem, 2003 Jan 24, 278(4), 2141 - 6 Epub 2002 Nov 14. The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone . A novel mechanism for defense against oxidative stress; Xia L et al.; The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate . Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation . The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute . TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors . The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms . In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium . These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage. Antimicrob Agents Chemother, 2002 Dec, 46(12), 4022 - 5 Postantibiotic effect and delay of regrowth in strains carrying mutations that save proteins or RNA; Dolcino M et al.; The postantibiotic effect (PAE) values found for proteinase-defective (Lon(-)) Escherichia coli and RNase-defective E . coli exposed to antibiotics were reduced (31 to 60% and 35 to 50%, respectively) in comparison with the control (AB1157), and in the recA13 mutant these values were about 0.4 h with all drugs . Nalidixic acid, under anaerobic conditions, induced no PAE (0 to 0.1 h) in AB1157 . A delay in regrowth (0.2 to 0.26 h) was noted with dnaA46(Ts), gyrA43(Ts), and gyrB41(Ts) mutants cultured for 2 h at 43 degrees C . These findings suggest that when proteins and RNA are saved, the cell rapidly resumes the original growth rate. Antimicrob Agents Chemother, 2002 Dec, 46(12), 3940 - 6 Emergence of tetracycline resistance in Helicobacter pylori: multiple mutational changes in 16S ribosomal DNA and other genetic loci; Dailidiene D et al.; Tetracycline is useful in combination therapies against the gastric pathogen Helicobacter pylori . We found 6 tetracycline-resistant (Tet(r)) strains among 159 clinical isolates (from El Salvador, Lithuania, and India) and obtained the following four results: (i) 5 of 6 Tet(r) isolates contained one or two nucleotide substitutions in one part of the primary tetracycline binding site in 16S rRNA (AGA(965-967) {Escherichia coli coordinates} changed to gGA, AGc, guA, or gGc {lowercase letters are used to represent the base changes}), whereas the sixth (isolate Ind75) retained AGA(965-967); (ii) PCR products containing mutant 16S ribosomal DNA (rDNA) alleles transformed recipient strains to Tet(r) phenotypes, but transformants containing alleles with single substitutions (gGA and AGc) were less resistant than their Tet(r) parents; (iii) each of 10 Tet(r) mutants of reference strain 26695 (in which mutations were induced with metronidazole, a mutagenic anti-H . pylori agent) contained the normal AGA(965-967) sequence; and (iv) transformant derivatives of Ind75 and of one of the Tet(r) 26695 mutants that had acquired mutant rDNA alleles were resistant to tetracycline at levels higher than those to which either parent strain was resistant . Thus, tetracycline resistance in H . pylori results from an accumulation of changes that may affect tetracycline-ribosome affinity and/or other functions (perhaps porins or efflux pumps) . We suggest that the rarity of tetracycline resistance among clinical isolates reflects this need for multiple mutations and perhaps also the deleterious effects of such mutations on fitness . Formally equivalent mutations with small but additive effects are postulated to contribute importantly to traits such as host specificity and virulence and to H . pylori's great genetic diversity. Eur J Med Res, 2002 Oct 29, 7(10), 447 - 52 Effects of Porphyromonas gingivalis on the activation of mouse macrophages by lipopolysaccharide; Schaumann R et al.; Porphyromonas gingivalis (PG) is a micro-organism that is suggested to play an etiologic role in acute and chronic periodontitis . The present study was undertaken to evaluate the question whether PG is capable of inducing interleukin (IL)-1beta, IL-6, macrophage inflammatory protein (MIP)-2, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in macrophages . Furthermore, the effect of PG on the activation of macrophages by Escherichia coli-lipopolysaccharide (LPS) was studied . The cytokines were analyzed by detection of specific mRNA . The mRNA was amplified by RT-PCR and semi-quantitatively analyzed by high performance liquid chromatography and densitometrically, respectively . These studies demonstrate that LPS was more active than PG in inducing mRNA expression of IL-1beta, IL-6, MIP-2 and GM-CSF . Moreover, PG reduced the mRNA expression of the macrophages stimulated with LPS, especially the IL-1beta and IL-6 mRNA expression was decreased. FEMS Microbiol Lett, 2002 Nov 5, 216(2), 255 - 62 Cellular localisation of the clamp protein during DNA replication; Kongsuwan K et al.; The beta subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP . The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome . The formation of GFP-beta fluorescent foci in GFP-beta expressing cells required DNA replication and their number per cell was dependent on cell growth . Examination of GFP-beta foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-beta foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication. Biochem Biophys Res Commun, 2002 Nov 22, 299(1), 42 - 8 Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain; Obita T et al.; DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA . The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein . We overproduced and purified a protein containing only this domain plus a methionine residue . This protein was stable as a monomer and maintained DnaA box-specific binding activity . We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments . We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six alpha-helices and no beta-sheets, consistent with results of CD analysis . Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the alpha-helices . These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins. J Parasitol, 2002 Oct, 88(5), 1000 - 6 Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis; Na BK et al.; A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli . The cysteine proteinase cDNA fragment was amplified by reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases . The 5' and 3' regions of the gene were amplified using rapid amplification of cDNA ends . The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232 . Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the catalytic triad . Analysis of the expressed protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa . Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin . The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites . The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls . These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis. Dev Biol (Basel), 2002, 109, 107 - 18 The use of an animal immunogenicity model in the development of Protropin somatrem (methionyl human growth hormone); Jones AJ; The clinical development of methionyl human growth hormone, with particular emphasis on immunogenicity and the effects of antibody development, are summarized . In an animal model in rhesus monkeys, the immunogenicity of dinical preparations was reduced by the inclusion of additional purification steps in the manufacturing process . The immunogenic response in patients was also decreased by these improvements . No safety consequences related to antibody formation were observed and the occurrence of growth attenuation due to antibodies was found to be extremely low (<0.1%) . The data suggest that the immunogenicity was not due to the N-terminal methionine or E . coli protein impurities: rather it was probably caused by small amounts of growth hormone with subtle structural alterations whose nature remains unknown. Proc Natl Acad Sci U S A, 2002 Nov 26, 99(24), 15398 - 403 Epub 2002 Nov 14. The substrate binding domain of DnaK facilitates slow protein refolding; Tanaka N et al.; We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states . The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution . The effects of DnaK384-638 on the reactivation of beta-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions . In the presence of DnaK384-638, the maximum yield of active beta-galactosidase was improved from 45% to 65% after a 48-h refolding reaction . Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of beta-galactosidase and consequently suppressed aggregation . DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h . On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins . These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding. Nucleic Acids Res, 2002 Nov 15, 30(22), 4975 - 84 Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid; Terato H et al.; Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions . In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa . The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA {in combination with endonuclease (Endo) IV} and Endo VIII recognized Xan in the tested enzymes . The activity (V(max)/K(m)) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol . The activity of AlkA and Endo VIII for Xan was further substantiated by the release of {(3)H}Xan from the substrate . The treatment of E.coli with N-methyl-N'-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA(+) but not alkA(-) strains . The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain . AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan. Nucleic Acids Res, 2002 Nov 15, 30(22), 4864 - 71 Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing; Wu W et al.; An intein-mediated approach was developed for expression and affinity purification of a protein that is lethal to Escherichia coli . The protein, I-TevI, is an intron-encoded endonuclease . The approach involved the insertional inactivation of I-TevI with a controllable mini-intein placed in front of a cysteine required for splicing (an I-TevI::intein fusion) . The purification was facilitated by a chitin-binding domain inserted into the mini-intein . Affinity purification of the I-TevI::intein fusion precursor on a chitin column was followed by pH-controllable splicing to restore the structure and function of I-TevI . To study the impact of the insertion context on I-TevI inactivation, the chimeric intein was inserted independently in front of seven cysteines of I-TevI . One of the seven intein integrants yielded I-TevI of high activity . This technique is, in principle, generalizable to the expression and purification of other cytotoxic proteins and is amenable to scale-up. J Biol Chem, 2003 Jan 24, 278(4), 2452 - 60 Epub 2002 Nov 13. High resolution crystal structures of human Rab5a and five mutants with substitutions in the catalytically important phosphate-binding loop; Zhu G et al.; GTPase domain crystal structures of Rab5a wild type and five variants with mutations in the phosphate-binding loop are reported here at resolutions up to 1.5 A . Of particular interest, the A30P mutant was crystallized in complexes with GDP, GDP+AlF(3), and authentic GTP, respectively . The other variant crystals were obtained in complexes with a non-hydrolyzable GTP analog, GppNHp . All structures were solved in the same crystal form, providing an unusual opportunity to compare structures of small GTPases with different catalytic rates . The A30P mutant exhibits dramatically reduced GTPase activity and forms a GTP-bound complex stable enough for crystallographic analysis . Importantly, the A30P structure with bound GDP plus AlF(3) has been solved in the absence of a GTPase-activating protein, and it may resemble that of a transition state intermediate . Conformational changes are observed between the GTP-bound form and the transition state intermediate, mainly in the switch II region containing the catalytic Gln(79) residue and independent of A30P mutation-induced local alterations in the P-loop . The structures suggest an important catalytic role for a P-loop backbone amide group, which is eliminated in the A30P mutant, and support the notion that the transition state of GTPase-mediated GTP hydrolysis is of considerable dissociative character. J Mol Microbiol Biotechnol, 2002 Nov, 4(6), 519 - 24 Enhancement of the solubility of proteins overexpressed in Escherichia coli by heat shock; Chen J et al.; Protein misfolding resulting in the formation of inclusion bodies is one of the major problems during protein overexpression in Escherichia coil . In this paper, we introduce a new method, which is simply to heat shock a cell culture prior to protein induction, allowing effective enhancement of the solubility and thereby the yield of overexpressed proteins in E . coli . Using this method, we show that the solubility of the E . coli protein KsgA-AN is significantly increased when overexpressed from a T7 promoter . In addition, we also show that the solubility of several Caenorhabditis elegans proteins are also enhanced after heat-shock treatment when expressed in E . coli . Taken together, these results suggest that the "heat-shock protocol" is a generalizable and useful method for increasing the solubility of many proteins overexpressed in E . coli. Eur J Immunol, 2002 Dec, 32(12), 3366 - 75 Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability, and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells; Cunliffe SL et al.; Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive . We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P . and Kappler, J., Immunity 1998 . 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes . Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)) . The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation . The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells . It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide . This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios. Curr Microbiol, 2003 Jan, 46(1), 70 - 6 Role for the cyanobacterial HtpG in protection from oxidative stress; Hossain MM et al.; The heat shock protein HtpG, which is a homolog of HSP90, is essential for basal and acquired thermotolerances in cyanobacteria . Recently we demonstrated that HtpG was involved in the acclimation to low temperatures in cyanobacteria . In this study, we elucidated a role of HtpG in the cyanobacterium Synechococcus sp . PCC 7942, in the acclimation to oxidative stress that was caused by high irradiance and/or methyl viologen . The inactivation of the htpG gene resulted in a decrease in the survival rate and an increase in the photoinhibition of photosystem II when cells in a liquid medium were incubated under high light conditions . The htpG mutant was highly sensitive to methyl viologen when it was grown on an agar plate . High irradiance and/or methyl viologen greatly increased the expression of the htpG gene as well as the groEL gene in the wild-type strain . Taken together, our results suggest that HtpG may play a role by itself or with other molecular chaperones in the acclimation to oxidative stress. Nature, 2002 Nov 14, 420(6912), 190 - 3 Metabolic network structure determines key aspects of functionality and regulation; Stelling J et al.; The relationship between structure, function and regulation in complex cellular networks is a still largely open question . Systems biology aims to explain this relationship by combining experimental and theoretical approaches . Current theories have various strengths and shortcomings in providing an integrated, predictive description of cellular networks . Specifically, dynamic mathematical modelling of large-scale networks meets difficulties because the necessary mechanistic detail and kinetic parameters are rarely available . In contrast, structure-oriented analyses only require network topology, which is well known in many cases . Previous approaches of this type focus on network robustness or metabolic phenotype, but do not give predictions on cellular regulation . Here, we devise a theoretical method for simultaneously predicting key aspects of network functionality, robustness and gene regulation from network structure alone . This is achieved by determining and analysing the non-decomposable pathways able to operate coherently at steady state (elementary flux modes) . We use the example of Escherichia coli central metabolism to illustrate the method. Nature, 2002 Nov 14, 420(6912), 186 - 9 Escherichia coli K-12 undergoes adaptive evolution to achieve in silico predicted optimal growth; Ibarra RU et al.; Annotated genome sequences can be used to reconstruct whole-cell metabolic networks . These metabolic networks can be modelled and analysed (computed) to study complex biological functions . In particular, constraints-based in silico models have been used to calculate optimal growth rates on common carbon substrates, and the results were found to be consistent with experimental data under many but not all conditions . Optimal biological functions are acquired through an evolutionary process . Thus, incorrect predictions of in silico models based on optimal performance criteria may be due to incomplete adaptive evolution under the conditions examined . Escherichia coli K-12 MG1655 grows sub-optimally on glycerol as the sole carbon source . Here we show that when placed under growth selection pressure, the growth rate of E . coli on glycerol reproducibly evolved over 40 days, or about 700 generations, from a sub-optimal value to the optimal growth rate predicted from a whole-cell in silico model . These results open the possibility of using adaptive evolution of entire metabolic networks to realize metabolic states that have been determined a priori based on in silico analysis. J Exp Bot, 2002 Dec, 53(379), 2453 - 4 Isolation and expression analysis of the isopropylmalate synthase gene family of Arabidopsis thaliana; Junk DJ et al.; Isopropylmalate synthase (IPMS) is the first enzyme in the leucine biosynthetic pathway . It is the branch point in the biosynthesis of leucine and the other branched-chain amino acids . IPMS is also regulated by negative feedback inhibition by the end-product leucine . There are four highly homologous loci within the Arabidopsis thaliana genome, which contain sequences that code for IPMS . Through library screening and RT-PCR the expression patterns of three of these loci namely IMS1, IMS2, and IMS3 have been isolated and then characterized . cDNAs of IMS2 and IMS3 lacking the 5' chloroplast leader sequence were able to complement a leucine auxotroph of E . coli. J Biol Chem, 2003 Feb 21, 278(8), 6128 - 35 Epub 2002 Nov 12. ZIP3, a new splice variant of the PKC-zeta-interacting protein family, binds to GABAC receptors, PKC-zeta, and Kv beta 2; Croci C et al.; The correct targeting of modifying enzymes to ion channels and neurotransmitter receptors represents an important biological mechanism to control neuronal excitability . The recent cloning of protein kinase C-zeta interacting proteins (ZIP1, ZIP2) identified new scaffolds linking the atypical protein kinase PKC-zeta to target proteins . GABA(C) receptors are composed of three rho subunits (rho 1-3) that are highly expressed in the retina, where they are clustered at synaptic terminals of bipolar cells . A yeast two-hybrid screen for the GABA(C) receptor rho 3 subunit identified ZIP3, a new C-terminal splice variant of the ZIP protein family . ZIP3 was ubiquitously expressed in non-neuronal and neuronal tissues, including the retina . The rho 3-binding region of ZIP3 contained a ZZ-zinc finger domain, which interacted with 10 amino acids conserved in rho 1-3 but not in GABA(A) receptors . Consistently, only rho 1-3 subunits bound to ZIP3 . ZIP3 formed dimers with ZIP1-3 and interacted with PKC-zeta and the shaker-type potassium channel subunit Kv beta 2 . Different domains of ZIP3 interacted with PKC-zeta and the rho 3 subunit, and simultaneous assembly of ZIP3, PKC-zeta and rho 3 was demonstrated in vitro . Subcellular co-expression of ZIP3 binding partners in the retina supported the proposed protein interactions . Our results indicate the formation of a ternary postsynaptic complex containing PKC-zeta, ZIP3, and GABA(C) receptors. J Biol Chem, 2003 Jan 24, 278(4), 2515 - 21 Epub 2002 Nov 12. Encapsulation of an 86-kDa assembly intermediate inside the cavities of GroEL and its single-ring variant SR1 by GroES; Song JL et al.; We described previously that during the assembly of the alpha(2)beta(2) heterotetramer of human mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD), chaperonins GroEL/GroES interact with the kinetically trapped heterodimeric (alphabeta) intermediate to facilitate conversion of the latter to the native BCKD heterotetramer . Here, we show that the 86-kDa heterodimeric intermediate possesses a native-like conformation as judged by its binding to a fluorescent probe 1-anilino-8-naphthalenesulfonate . This large heterodimeric intermediate is accommodated as an entity inside cavities of GroEL and its single-ring variant SR1 and is encapsulated by GroES as indicated by the resistance of the heterodimer to tryptic digestion . The SR1-alphabeta-GroES complex is isolated as a stable single species by gel filtration in the presence of Mg-ATP . In contrast, an unfolded BCKD fusion protein of similar size, which also resides in the GroEL or SR1 cavity, is too large to be capped by GroES . The cis-capping mechanism is consistent with the high level of BCKD activity recovered with the GroEL-alphabeta complex, GroES, and Mg-ATP . The 86-kDa native-like heterodimeric intermediate in the BCKD assembly pathway represents the largest protein substrate known to fit inside the GroEL cis cavity underneath GroES, which significantly exceeds the current size limit of 57 kDa established for unfolded proteins. Brain Res Bull, 2002 Nov 30, 59(3), 213 - 6 Biological effects of stannous chloride, a substance that can produce stimulation or depression of the central nervous system; Silva CR et al.; It was demonstrated that tin, as stannous chloride (SnCl(2)), can facilitate the neuromuscular transmission by accelerating the transmitter release from the nerve terminals in the mouse . When this salt is injected into laboratory animals, it can produce stimulation or depression of the central nervous system . Because calcium (Ca(2+)) influx into the cytoplasm is indispensable to release the transmitter, it would be possible that SnCl(2) increases the Ca(2+) influx at the nerve terminals but not by blocking the K(+) channels . SnCl(2) is known to inhibit the immune response in rodents and to induce tumor generation in thyroid gland . There is no general agreement regarding its genotoxicity and it was discussed that the effects of this salt might depend on the physicochemical conditions and the route of its administration . SnCl(2) has been used in many sectors of human interest, such as food industry and nuclear medicine . This salt is directly administered to human beings endovenously, when it is used as a reducing agent to prepare 99mTc-radiopharmaceuticals which are also used for cerebral studies . SnCl(2) is capable to promote the generation of reactive oxygen species (ROS) that are responsible for the oxidative stress . Oxidative stress has been related with aging and other neurological diseases . So, it is relevant to evaluate other biological effects of SnCl(2) . We decided to stu