J Mol Med, 2002 Dec, 80(12), 782 - 90 Epub 2002 Oct 01. Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46,XY pure gonadal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes; Assumpcao JG et al.; The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians . In humans mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases . We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites . One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis . The Escherichia coli expressed SRY(N65H) protein did not present DNA-binding activity in vitro . The other mutation, a G to T transversion, causes the R30I substitution . This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings . The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR . The R30I mutation is located upstream to the HMG box, within the (29)RRSSS(33) phosphorylation site . The E . coli expressed SRY(R30I) protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro. J Mol Med, 2002 Dec, 80(12), 770 - 81 Epub 2002 Nov 22. Mechanisms underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides; Andersen MS et al.; Chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides have been developed for site-specific correction of episomal and chromosomal target genes . The gene repair approach relies on specific hybridization of the oligonucleotides to the target gene generating a mismatch with the targeted point mutation . Restored gene function is anticipated to occur through activation of endogenous repair systems that recognize the created mismatch . We present an overview of the gene correction results obtained in several target genes by employing various oligonucleotide designs and a discussion of the possible mechanisms underlying the gene correction techniques . Experimental data suggest that modified single-stranded oligonucleotides form intermediate three-stranded heteroduplexes involving the human RecA homologue, hRad51, whereas chimeric RNA/DNA oligonucleotides may participate in three or four-stranded intermediate structures . Protein factors such as hRad52, hRad54, hRPA, and p53 may modulate the heteroduplex formation and participate in the activation of the endogenous mismatch repair and/or nucleotide excision repair pathway(s) . The efficiency of the gene correction process may furthermore be influenced by the differential recognition of mismatches by repair enzymes and possible sequence context effects. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16776 - 81 Epub 2002 Dec 13. Dynamic assembly of MinD into filament bundles modulated by ATP, phospholipids, and MinE; Suefuji K et al.; Accurate positioning of the division septum at the equator of Escherichia coli cells requires a rapid oscillation of MinD ATPase between the polar halves of the cell membrane, together with the division inhibitor MinC, under MinE control . The mechanism underlying MinD oscillation remains poorly understood . Here, we demonstrate that purified MinD assembles into protein filaments in the presence of ATP . Incubation with phospholipid vesicles further stimulates MinD polymerization . Addition of purified MinE in the presence of lipids promotes bundling of MinD filaments as well as their disassembly through activation of MinD ATPase . MinE thus provokes a net decay in the steady-state MinD polymer mass . Taken together, our results suggest that reversible MinD assembly modulated by MinE underlies the dynamic processing of positional information in E . coli to identify precisely the nascent site for cell division. J Biol Chem, 2003 Feb 28, 278(9), 6673 - 9 Epub 2002 Dec 12. Variations in the response of mouse isozymes of adenylosuccinate synthetase to inhibitors of physiological relevance; Borza T et al.; Vertebrates have acidic and basic isozymes of adenylosuccinate synthetase, which participate in the first committed step of de novo AMP biosynthesis and/or the purine nucleotide cycle . These isozymes differ in their kinetic properties and N-leader sequences, and their regulation may vary with tissue type . Recombinant acidic and basic synthetases from mouse, in the presence of active site ligands, behave in analytical ultracentrifugation as dimers . Active site ligands enhance thermal stability of both isozymes . Truncated forms of both isozymes retain the kinetic parameters and the oligomerization status of the full-length proteins . AMP potently inhibits the acidic isozyme competitively with respect to IMP . In contrast, AMP weakly inhibits the basic isozyme noncompetitively with respect to all substrates . IMP inhibition of the acidic isozyme is competitive, and that of the basic isozyme noncompetitive, with respect to GTP . Fructose 1,6-bisphosphate potently inhibits both isozymes competitively with respect to IMP but becomes noncompetitive at saturating substrate concentrations . The above, coupled with structural information, suggests antagonistic interactions between the active sites of the basic isozyme, whereas active sites of the acidic isozyme seem functionally independent . Fructose 1,6-bisphosphate and IMP together may be dynamic regulators of the basic isozyme in muscle, causing potent inhibition of the synthetase under conditions of high AMP deaminase activity. J Biol Chem, 2003 Feb 21, 278(8), 6490 - 4 Epub 2002 Dec 12. Identification of a novel protein with guanylyl cyclase activity in Arabidopsis thaliana; Ludidi N et al.; Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) from guanosine 5'-triphosphate (GTP) . While many cGMP-mediated processes in plants have been reported, no plant molecule with GC activity has been identified . When the Arabidopsis thaliana genome is queried with GC sequences from cyanobacteria, lower and higher eukaryotes no unassigned proteins with significant similarity are found . However, a motif search of the A . thaliana genome based on conserved and functionally assigned amino acids in the catalytic center of annotated GCs returns one candidate that also contains the adjacent glycine-rich domain typical for GCs . In this molecule, termed AtGC1, the catalytic domain is in the N-terminal part . AtGC1 contains the arginine or lysine that participates in hydrogen bonding with guanine and the cysteine that confers substrate specificity for GTP . When AtGC1 is expressed in Escherichia coli, cell extracts yield >2.5 times more cGMP than control extracts and this increase is not nitric oxide dependent . Furthermore, purified recombinant AtGC1 has Mg(2+)-dependent GC activity in vitro and >3 times less adenylyl cyclase activity when assayed with ATP as substrate in the absence of GTP . Catalytic activity in vitro proves that AtGC1 can function either as a monomer or homo-oligomer . AtGC1 is thus not only the first functional plant GC but also, due to its unusual domain organization, a member of a new class of GCs. J Biol Chem, 2003 Feb 28, 278(9), 6642 - 50 Epub 2002 Dec 12. Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function; Salsi V et al.; The CCAAT box is one of the most common elements in eukaryotic promoters and is activated by NF-Y, a conserved trimeric transcription factor with histone-like subunits . Usually one CCAAT element is present in promoters at positions between -60 and -100, but an emerging class of promoters harbor multiple NF-Y sites . In the triple CCAAT-containing cyclin B2 cell-cycle promoter, all CCAAT boxes, independently from their NF-Y affinities, are important for function . We investigated the relationships between NF-Y and p300 . Chromatin immunoprecipitation analysis found that NF-Y and p300 are bound to the cyclin B2 promoter in vivo and that their binding is regulated during the cell cycle, positively correlating with promoter function . Cotransfection experiments determined that the coactivator acts on all CCAAT boxes and requires a precise spacing between the three elements . We established the order of in vitro binding of the three NF-Y complexes and find decreasing affinities from the most distal Y1 to the proximal Y3 site . Binding of two or three NF-Y trimers with or without p300 is not cooperative, but association with the Y1 and Y2 sites is extremely stable . p300 favors the binding of NF-Y to the weak Y3 proximal site, provided that a correct distance between the three CCAAT is respected . Our data indicate that the precise spacing of multiple CCAAT boxes is crucial for coactivator function . Transient association to a weak site might be a point of regulation during the cell cycle and a general theme of multiple CCAAT box promoters. FEBS Lett, 2002 Dec 18, 532(3), 465 - 8 The PLP-dependent biotin synthase from Escherichia coli: mechanistic studies; Ollagnier-de-Choudens S et al.; Biotin synthase (BioB), an iron-sulfur enzyme, catalyzes the last step of the biotin biosynthesis pathway . The reaction consists in the introduction of a sulfur atom into two non-activated C-H bonds of dethiobiotin . Substrate radical activation is initiated by the reductive cleavage of S-adenosylmethionine (AdoMet) into a 5'-deoxyadenosyl radical . The recently described pyridoxal 5'-phosphate-bound enzyme was used to show that only one molecule of AdoMet, and not two, is required for the formation of one molecule of biotin . Furthermore 5'-deoxyadenosine, a product of the reaction, strongly inhibited biotin formation, an observation that may explain why BioB is not able to make more than one turnover . However this enzyme inactivation is not irreversible. FEBS Lett, 2002 Dec 18, 532(3), 427 - 31 Identification and characterization of single-domain thiosulfate sulfurtransferases from Arabidopsis thaliana; Bauer M et al.; Sulfurtransferases/rhodaneses (ST) are a group of enzymes widely distributed in all three phyla that catalyze the transfer of sulfur from a donor to a thiophilic acceptor substrate . All ST contain distinct structural domains, and can exist as single-domain proteins, as tandemly repeated modules in which the C-terminal domain bears the active site, or as members of multi-domain proteins . We identified several ST in Arabidopsis resembling the C-terminus of the Arabidopsis two-domain ST1 and the single-domain GlpE protein from Escherichia coli . Two of them (accession numbers BAB10422 and BAB10409) were expressed in E . coli and purified . Both proteins showed thiosulfate-specific ST enzyme activity. FEBS Lett, 2002 Dec 18, 532(3), 415 - 8 Reliability of transmembrane predictions in whole-genome data; Kall L et al.; Transmembrane prediction methods are generally benchmarked on a set of proteins with experimentally verified topology . We have investigated if the accuracy measured on such datasets can be expected in an unbiased genomic analysis, or if there is a bias towards 'easily predictable' proteins in the benchmark datasets . As a measurement of accuracy, the concordance of the results from five different prediction methods was used (TMHMM, PHD, HMMTOP, MEMSAT, and TOPPRED) . The benchmark dataset showed significantly higher levels (up to five times) of agreement between different methods than in 10 tested genomes . We have also analyzed which programs are most prone to make mispredictions by measuring the frequency of one-out-of-five disagreeing predictions. FEBS Lett, 2002 Dec 18, 532(3), 387 - 90 A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding; Zuniga R et al.; The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic . CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA . No function has been assigned to CP2 . The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence . Protein sequence alignments revealed that A . ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS . However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs . A . ferrooxidans TrpRS is functional in vivo in Escherichia coli . A deletion mutant of A . ferrooxidans trpS lacking the coding region of CP2 was constructed . The in vivo activity of the mutant TrpRS in E . coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion . However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation . Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA. Phytochemistry, 2003 Jan, 62(2), 159 - 63 The biochemical origin of pentenol emissions from wounded leaves; Fisher AJ et al.; Large releases of 1-penten-3-ol (pentenol) and 1-penten-3-one (pentenone) were recently observed from a variety of leaves subjected to freeze-thaw damage in the presence of oxygen . In order to understand the biochemical origins of these volatiles, soybean leaf extracts were used to determine if the formation of pentenol and pentenone can be explained by known O(2)-dependent lipoxygenase (LOX) reactions . Enzymatic formation of these C5 volatiles was found to be dependent on alpha-linolenic acid or the 13(S)-hydroperoxide of alpha-linolenic acid {13(S)-HPOT} and blocked by LOX inhibitors . Five soybean leaf LOX isozyme genes (VLXA, VLXB, VLXC, VLXD, and VLXE) were then expressed in Escherichia coli and used in in vitro incubations with 13(S)-HPOT to test for volatile formation . Each of the LOX isozymes catalyzed the formation of low levels of pentenol, but not pentenone . It therefore seems likely that the C5,13-cleavage activity of LOX is the direct source of abundant pentenol and the indirect source of pentenone observed upon leaf wounding. Zhonghua Xue Ye Xue Za Zhi, 2002 Sep, 23(9), 480 - 2 {Preparation and characterization of a single chain antibody fragment of mAb SZ-21 against platelets GPIIIa}; An G et al.; OBJECTIVE: To prepare a single chain antibody (ScFv) of mAb SZ-21 against platelet GPIIIa for its future clinical application . METHODS: The expression vector pET20b-SZ-21ScFv was constructed and the fusion protein was expressed in E . coli BL21 (DE3) PlysS . The activated fusion protein was obtained after a series of purification steps, including cell breakage, inclusion body solubilization, His-bind resin affinity chromatography and protein refolding . RESULTS: The fusion protein yields were up to 21% of the total amount of bacteria protein . The ScFv fragment could inhibit ADP-induced platelets aggregation in a dose-dependent manner in vitro and the maximal inhibition rate was obtained at a concentration of 20 micro g/ml . It also reacted with endothelial cells as detected by flow cytometry . Moreover, the ScFv fragment was able to inhibit the binding of fibrinogen to platelet . CONCLUSION: The SZ-21ScFv fragment had the activity to inhibit platelets aggregation and the binding of fibrinogen to platelet, being potentially useful for the treatment of thrombotic diseases. Chem Res Toxicol, 2002 Dec, 15(12), 1619 - 26 Site-specific mutagenesis in Escherichia coli by N2-deoxyguanosine adducts derived from the highly carcinogenic fjord-region benzo{c}phenanthrene 3,4-diol 1,2-epoxides; Ramos LA et al.; Although there have been numerous studies of site-specific mutagenesis by dGuo adducts of benzo{a}pyrene diol epoxides (B{a}P DEs), the present study represents the first example of site-specific mutagenesis by dGuo adducts of the highly carcinogenic benzo{c}phenanthrene 3,4-diol 1,2-epoxides (B{c}Ph DEs) . The eight adducts that would result from cis- and trans-opening at C-1 of four optically active isomers of B{c}Ph DEs by the N(2)-amino group of dGuo were incorporated into 5'-TTCGAATCCTTCCCCC (context III) and 5'-GGGGTTCCCGAGCGGC (context IV) at the underlined site . These modified oligonucleotides along with unmodified controls were ligated into single-stranded M13mp7L2, which were then used to transfect SOS-induced Escherichia coli . Upon replication of the lesions in each of the two sequence contexts, mutational analysis of the progeny was performed by differential hybridization . For the 16 adducts, the mutation frequencies varied over 2 orders of magnitude with a reasonably even distribution (0.4-1% for three adducts, 1-2% for six adducts, 3-7.4% for five adducts, and one adduct each at 11 and 39%) . For all but this last adduct, the mutation frequency for a given B{c}Ph DE adduct was less than for its B{a}P analogue with the same stereochemistry in the same sequence . For the vectors containing adducts with S configuration at the site of attachment of the hydrocarbon to the dGuo base, the main base substitution was G --> T followed by G --> A . In contrast, for the vectors containing adducts with R configuration, the main base substitution was G --> A . The most notable observation in the present study is the low frequency of mutations induced by the B{c}Ph DE-dGuo adducts relative to their B{a}P counterparts . A possible structural basis for this difference is proposed. Chem Res Toxicol, 2002 Dec, 15(12), 1595 - 601 Influence of local duplex stability and N6-methyladenine on uracil recognition by mismatch-specific uracil-DNA glycosylase (Mug); Valinluck V et al.; To maintain genomic integrity, DNA repair enzymes continually remove damaged bases and lesions resulting from endogenous and exogenous processes . These repair enzymes must distinguish damaged bases from normal bases to prevent the inadvertent removal of normal bases, which would promote genomic instability . The mechanisms by which this high level of specificity is accomplished are as yet unresolved . One member of the uracil-DNA glycosylase family of repair enzymes, Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug), is reported to distinguish U:G mispairs from U:A base pairs based upon specific contacts with the mispaired guanine after flipping the target uracil out of the duplex . However, recent studies suggest other mechanisms for base selection, including local duplex stability . In this study, we used the modified base N6-methyladenine to probe the effect of local helix perturbation on Mug recognition of uracil . N6-Methyladenine is found in E . coli as part of both the mismatch repair and restriction-modification systems . In its cis isomer, N6-methyladenine destabilizes hydrogen bonding by interfering with pseudo-Watson-Crick base pairing . It is observed that the selection of uracil by Mug is sequence dependent and that uracil residues in sequences of reduced thermostability are preferentially removed . The replacement of adenine by N6-methyladenine increases the frequency of removal of the uracil residue paired opposite the modified adenine . These results are in accord with suggestions that local helix stability is an important determinant of base recognition by some DNA repair enzymes and provide a potential strategy for identifying the sequence location of modified bases in DNA. Chem Res Toxicol, 2002 Dec, 15(12), 1572 - 80 Mutagenic spectrum of butadiene-derived N1-deoxyinosine adducts and N6,N6-deoxyadenosine intrastrand cross-links in mammalian cells; Kanuri M et al.; Reactive metabolites of 1,3-butadiene, including 1,2-epoxy-3-butene (BDO), 1,2:3,4-diepoxybutane (BDO(2)), and 3,4-epoxy-1,2-butanediol (BDE), form both stable and unstable base adducts in DNA and have been implicated in producing genotoxic effects in rodents and human cells . N1 deoxyadenosine adducts are unstable and can undergo either hydrolytic deamination to yield N1 deoxyinosine adducts or Dimroth rearrangement to yield N(6) adducts . The dominant point mutation observed at AT sites in both in vivo and in vitro mutagenesis studies using BD and its epoxides has been A --> T transversions followed by A --> G transitions . To understand which of the butadiene adducts are responsible for mutations at AT sites, the present study focuses on the N1 deoxyinosine adduct at C2 of BDO and N(6),N(6)-deoxyadenosine intrastrand cross-links derived from BDO(2) . These lesions were incorporated site-specifically and stereospecifically into oligodeoxynucleotides which were engineered into mammalian shuttle vectors for replication bypass and mutational analyses in COS-7 cells . Replication of DNAs containing the R,R-BDO(2) intrastrand cross-link between N(6) positions of deoxyadenosine yielded a high frequency (59%) of single base substitutions at the 3' adducted base, while 19% mutagenesis was detected using the S,S-diastereomer . Comparable studies using the R- and S-diastereomers of the N1 deoxyinosine adduct gave rise to approximately 50 and 80% A --> G transitions with overall mutagenic frequencies of 59 and 90%, respectively . Collectively, these data establish a molecular basis for A --> G transitions that are observed following in vivo and in vitro exposures to BD and its epoxides, but fail to reveal the source of the A --> T transversions that are the dominant point mutation. Vitam Horm, 2002, 65, 313 - 31 Function and regulation of cytosolic molecular chaperone CCT; Kubota H; Molecular chaperones are a group of proteins that assists in the folding of newly synthesized proteins or in the refolding of denatured proteins . The cytosolic chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone that plays an important role in the folding of proteins in the eukaryotic cytosol . Actin, tubulin, and several other proteins are known to be folded by CCT, and an estimated 15% of newly translated proteins in mammalian cells are folded with the assistance of CCT . CCT differs from other chaperonin family proteins in its subunit composition, which consists of eight subunit species comprising the CCT 16-mer double-ring-like complex . CCT preferentially recognizes quasinative (or partially folded) intermediates, whereas its Escherichia coli homologue GroEL recognizes more unfolded intermediates, especially those displaying hydrophobic surfaces . Molecular evolutionary analyses have suggested that each subunit species has a specific function in addition to contributing to a common ATPase activity . Consistent with this view, it has been suggested that each subunit recognizes specific substrate proteins (or their parts) and that they collectively modulate the ATPase activity of the complex . The overall expression of CCT in mammalian cells is primarily dependent on cell growth, but each subunit exhibits an individual patterns of expression . Recent progress in CCT research is reviewed, focusing particularly on CCT function and expression . From these observations, the possible roles of the distinct subunits in CCT-assisted folding in the eukaryotic cytosol are discussed. Mol Cancer Ther, 2002 Oct, 1(12), 1129 - 37 Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors; Mulryan K et al.; The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression . Such properties make 5T4 an excellent putative target for cancer immunotherapy . The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models . We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ) . Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively . C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4 . MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS . In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c . tumors . Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals . Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity . These data support the use of MVA-5T4 for tumor immunotherapy. Electrophoresis, 2002 Dec, 23(24), 4060 - 6 Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All; Volpi N et al.; A sensitive method has been developed for the visualization of nonradiolabelled glycosaminoglycans resolved by agarose gel electrophoresis using staining with toluidine blue followed by Stains-All procedure . This method, which can detect as little as 10 ng of a single species, can be used to stain a few micrograms of a complex polysaccharide mixture . The combination of agarose gel electrophoresis and sequential toluidine blue/Stains-All staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate) and nonsulfated polyanions (i.e., hyaluronate, defructosylated capsular polysaccharide K4) as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes and the identification and quantification of the contaminations of other polysaccharides within glycosaminoglycan preparations with great sensitivity (about 0.1%) . Furthermore, this method can be used to stain low-molecular-mass fractions and oligosaccharides derived from the natural polyanions, such as heparin . This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited. Science, 2002 Dec 13, 298(5601), 2191 - 5 Experimental identification of downhill protein folding; Garcia-Mira MM et al.; Theory predicts the existence of barrierless protein folding . Without barriers, folding should be noncooperative and the degree of native structure should be coupled to overall protein stability . We investigated the thermal unfolding of the peripheral subunit binding domain from Escherichia coli's 2-oxoglutarate dehydrogenase multienzyme complex (termed BBL) with a combination of spectroscopic techniques and calorimetry . Each technique probed a different feature of protein structure . BBL has a defined three-dimensional structure at low temperatures . However, each technique showed a distinct unfolding transition . Global analysis with a statistical mechanical model identified BBL as a downhill-folding protein . Because of BBL's biological function, we propose that downhill folders may be molecular rheostats, in which effects could be modulated by altering the distribution of an ensemble of structures. Plant Physiol, 2002 Dec, 130(4), 2188 - 98 Biosynthesis of UDP-xylose . Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms; Harper AD et al.; UDP-xylose (Xyl) is an important sugar donor for the synthesis of glycoproteins, polysaccharides, various metabolites, and oligosaccharides in animals, plants, fungi, and bacteria . UDP-Xyl also feedback inhibits upstream enzymes (UDP-glucose {Glc} dehydrogenase, UDP-Glc pyrophosphorylase, and UDP-GlcA decarboxylase) and is involved in its own synthesis and the synthesis of UDP-arabinose . In plants, biosynthesis of UDP-Xyl is catalyzed by different membrane-bound and soluble UDP-GlcA decarboxylase (UDP-GlcA-DC) isozymes, all of which convert UDP-GlcA to UDP-Xyl . Because synthesis of UDP-Xyl occurs both in the cytosol and in membranes, it is not known which source of UDP-Xyl the different Golgi-localized xylosyltransferases are utilizing . Here, we describe the identification of several distinct Arabidopsis genes (named AtUXS for UDP-Xyl synthase) that encode functional UDP-GlcA-DC isoforms . The Arabidopsis genome contains five UXS genes and their protein products can be subdivided into three isozyme classes (A-C), one soluble and two distinct putative membrane bound . AtUxs from each class, when expressed in Escherichia coli, generate active UDP-GlcA-DC that converts UDP-GlcA to UDP-Xyl . Members of this gene family have a large conserved C-terminal catalytic domain (approximately 300 amino acids long) and an N-terminal variable domain differing in sequence and size (30-120 amino acids long) . Isoforms of class A and B appear to encode putative type II membrane proteins with their catalytic domains facing the lumen (like Golgi-glycosyltransferases) and their N-terminal variable domain facing the cytosol . Uxs class C is likely a cytosolic isoform . The characteristics of the plant Uxs support the hypothesis that unique UDP-GlcA-DCs with distinct subcellular localizations are required for specific xylosylation events. Plant Physiol, 2002 Dec, 130(4), 2164 - 76 Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation; Mizutani M et al.; A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process . The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C . sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined . The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers . The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein . The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase . The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside . These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons . The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism . The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed. Plant Physiol, 2002 Dec, 130(4), 2142 - 51 cDNA cloning, heterologous expressions, and functional characterization of malonyl-coenzyme a:anthocyanidin 3-o-glucoside-6"-o-malonyltransferase from dahlia flowers; Suzuki H et al.; In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety . In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e . malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers . We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family . Dv3MaT coded for a protein of 460 amino acids . Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant . The Dv3MaT cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized . The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (K(m), 18.8 microM) to pelargonidin 3-O-glucoside (K(m), 46.7 microM) to produce pelargonidin 3-O-6"-O-malonylglucoside with a k(cat) value of 7.3 s(-1) . The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers . Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside . Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11-63 mol % of total anthocyanins in the flower) . The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside . Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of beta-glucosidase . These results suggest that malonylation should serve as a strategy for pigment stabilization in the flowers. Plant Physiol, 2002 Dec, 130(4), 2085 - 94 Differential regulation of RNA levels of gibberellin dioxygenases by photoperiod in spinach; Lee DJ et al.; Previous work with spinach (Spinacia oleracea) has shown that the level of gibberellin (GA) 20-oxidase is strongly up-regulated by long days (LD) . In the present work, the effect of photoperiod on expression of other GA dioxygenases was investigated and compared with that of GA 20-oxidase . Two GA 2-oxidases and one GA 3-oxidase were isolated from spinach by reverse transcription-polymerase chain reaction with degenerate primers and by 5'- and 3'-rapid amplification of cDNA ends . As determined by high-performance liquid chromatography with on-line radioactivity detection, the SoGA3ox1 gene product catalyzed 3beta-hydroxylation of GA(9) to GA(4) and GA(20) to GA(1) . The SoGA2ox1 and the SoGA2ox2 gene products catalyzed 2beta-hydroxylation of GA(9) to GA(51) and GA(20) to GA(29) . The product of GA(20) metabolism by SoGA3ox1 was identified as GA(1) by gas chromatography-mass spectrometry, whereas the products of GA(1) and GA(20) metabolism by SoGA2ox1 and SoGA2ox2 were identified as GA(8) and GA(29), respectively . SoGA2ox1 also metabolized GA(53) to GA(97) . The levels of SoGA20ox1 transcripts were greatly increased in all organs tested in LD conditions, but the levels of SoGA3ox1 transcripts were only slightly increased in blades and petioles . A decrease in the levels of the SoGA2ox1 transcripts in young leaves and tips in LD conditions is opposite to the expression pattern of the SoGA20ox1 . Expression of SoGA20ox1 in petioles and young leaves was strongly up-regulated by a supplementary 16 h of light, but the levels of SoGA3ox1 and SoGA2ox1 transcripts did not change . It is concluded that regulation and maintenance of GA(1) concentration in spinach are primarily attributable to changes in expression of SoGA20ox1. Plant Physiol, 2002 Dec, 130(4), 2061 - 8 Cloning and expression of the gene for soybean hydroxyisourate hydrolase . Localization and implications for function and mechanism; Raychaudhuri A et al.; The gene encoding hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme, has been cloned from soybean (Glycine max) . The gene encodes a protein that is 560 amino acids in length and contains a 31-amino acid signal sequence at the N terminus that is not present in the mature protein . The presence of two SKL motifs near the C terminus suggests that the protein resides in the peroxisome . This expectation is borne out by results from immunogold electron microscopy, which revealed that hydroxyisourate hydrolase was localized in the peroxisomes of uninfected root nodules . The gene encoding hydroxyisourate hydrolase was expressed in Escherichia coli, and soluble, catalytically active enzyme was purified to homogeneity . Sequence analysis revealed considerable homology with members of the beta-glucosidase family of enzymes . Two glutamate residues, E199 and E408, align with the conserved glutamates that play catalytic roles in the beta-glucosidases . However, the other residues that have been identified by crystallography to interact directly with the substrates in beta-glucosidases are not conserved in hydroxyisourate hydrolase . The E199A and E408A hydroxyisourate hydrolase mutants were devoid of detectable catalytic activity . Analysis of transcripts for hydroxyisourate hydrolase demonstrated that its level of expression was highest in the nodule; mRNA was detectable 12 d after infection and increased until 21 d postinfection, then declined . In a similar manner, immunodetection of hydroxyisourate hydrolase indicated preferential localization in the nodule; the amount of protein detected was maximal at 21 d postinfection . The pattern of expression of hydroxyisourate hydrolase matched that of urate oxidase, and supports the hypothesis that hydroxyisourate hydrolase plays a role in ureide metabolism. Plant Physiol, 2002 Dec, 130(4), 1958 - 66 Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis; Muramoto T et al.; The HY1 gene of Arabidopsis encodes a plastid heme oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors . To determine the enzymatic properties of plant heme oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase . The fusion protein was soluble and expressed at high levels . Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme . In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXalpha from heme with the concomitant production of carbon monoxide . Heme oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo . In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity . These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme oxygenases differ substantially. J Biol Chem, 2003 Jan 31, 278(5), 2777 - 80 Epub 2002 Dec 11. Inhibition of HIV-1 ribonuclease H by a novel diketo acid, 4-{5-(benzoylamino)thien-2-yl}-2,4-dioxobutanoic acid; Shaw-Reid CA et al.; Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein . We have identified a novel thiophene diketo acid, 4-{5-(benzoylamino)thien-2-yl}-2,4-dioxobutanoic acid, that selectively inhibits polymerase-independent RNase H cleavage (IC(50) = 3.2 microm) but has no effect on DNA polymerization (IC(50) > 50 microm) . The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone . Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the alpha-C helix from Escherichia coli RNase HI, suggesting these compounds affect RNase H indirectly . In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation . In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn(2+) implying a direct interaction between the inhibitor and the RNase H active site . These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-{5-(benzoylamino)thien- 2-yl}-2,4-dioxobutanoic acid that specifically targets RNase H. J Biol Chem, 2003 Feb 21, 278(8), 6136 - 44 Epub 2002 Dec 11. Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span; Fei YJ et al.; We have cloned and functionally characterized two Na(+)-coupled dicarboxylate transporters, namely ceNaDC1 and ceNaDC2, from Caenorhabditis elegans . These two transporters show significant sequence homology with the product of the Indy gene identified in Drosophila melanogaster and with the Na(+)-coupled dicarboxylate transporters NaDC1 and NaDC3 identified in mammals . In a mammalian cell heterologous expression system, the cloned ceNaDC1 and ceNaDC2 mediate Na(+)-coupled transport of various dicarboxylates . With succinate as the substrate, ceNaDC1 exhibits much lower affinity compared with ceNaDC2 . Thus, ceNaDC1 and ceNaDC2 correspond at the functional level to the mammalian NaDC1 and NaDC3, respectively . The nadc1 and nadc2 genes are not expressed at the embryonic stage, but the expression is detectable all through the early larva stage to the adult stage . Tissue-specific expression pattern studies using a reporter gene fusion approach in transgenic C . elegans show that both genes are coexpressed in the intestinal tract, an organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism . Independent knockdown of the function of these two transporters in C . elegans using the strategy of RNA interference suggests that NaDC1 is not associated with the regulation of average life span in this organism, whereas the knockdown of NaDC2 function leads to a significant increase in the average life span . Disruption of the function of the high affinity Na(+)-coupled dicarboxylate transporter NaDC2 in C . elegans may lead to decreased availability of dicarboxylates for cellular production of metabolic energy, thus creating a biological state similar to that of caloric restriction, and consequently leading to life span extension. J Biol Chem, 2003 Feb 14, 278(7), 4491 - 9 Epub 2002 Dec 11. Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture; Davey MJ et al.; The Mcm2-7p heterohexamer is the presumed replicative helicase in eukaryotic cells . Each of the six subunits is required for replication . We have purified the six Saccharomyces cerevisiae MCM proteins as recombinant proteins in Escherichia coli and have reconstituted the Mcm2-7p complex from individual subunits . Study of MCM ATPase activity demonstrates that no MCM protein hydrolyzes ATP efficiently . ATP hydrolysis requires a combination of two MCM proteins . The fifteen possible pairwise mixtures of MCM proteins yield only three pairs of MCM proteins that produce ATPase activity . Study of the Mcm3/7p ATPase shows that an essential arginine in Mcm3p is required for hydrolysis of the ATP bound to Mcm7p . Study of the pairwise interactions between MCM proteins connects the remaining MCM proteins to the Mcm3/7p pair . The data predict which subunits in the ATPase pairs bind the ATP that is hydrolyzed and indicate the arrangement of subunits in the Mcm2-7p heterohexamer. J Biol Chem, 2003 Feb 14, 278(7), 4919 - 25 Epub 2002 Dec 11. Nitric oxide binding properties of neuroglobin . A characterization by EPR and flash photolysis; Van Doorslaer S et al.; Neuroglobin is a recently discovered member of the globin superfamily . Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O(2) concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe(2+)-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO . Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form . After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation . Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k(on)) and low dissociation rates (k(off)) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins . Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants . Structural and kinetic characteristics of neuroglobin and its mutants are analyzed . NO production in rapidly growing E . coli cell cultures is discussed. Microbiology, 2002 Dec, 148(Pt 12), 3865 - 72 Involvement of a putative molybdenum enzyme in the reduction of selenate by Escherichia coli; Bebien M et al.; Selenium oxyanions, particularly selenite, can be highly toxic to living organisms . Few bacteria reduce both selenate and selenite into the less toxic elemental selenium . Insights into the mechanisms of the transport and the reduction of selenium oxyanions in Escherichia coli were provided by a genetic analysis based on transposon mutagenesis . Ten mutants impaired in selenate reduction were analysed . Three of them were altered in genes encoding transport proteins including a porin, an inner-membrane protein and a sulfate carrier . Two mutants were altered in genes required for molybdopterin biosynthesis, strongly suggesting that the selenate reductase of E . coli is a molybdoenzyme . However, mutants deleted in various oxomolybdenum enzymes described so far in this species still reduced selenate . Finally, a mutant in the gene ygfK encoding a putative oxidoreductase was obtained . This gene is located upstream of ygfN and ygfM in the ygfKLMN putative operon . YgfN and YgfM code for a molybdopterin-containing enzyme and a polypeptide carrying a FAD domain, respectively . It is therefore proposed that the selenate reductase of E . coli is a structural complex including the proteins YgfK, YgfM and YgfN . In addition, all the various mutants were still able to reduce selenite into elemental selenium . This implies that the transport and reduction of this compound are clearly distinct from those of selenate. Microbiology, 2002 Dec, 148(Pt 12), 3801 - 11 Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli; Puskarova A et al.; Bacterial accommodation to moderate concentrations of cadmium is accompanied by transient activation of general stress proteins as well as a sustained induction of other proteins of hitherto unknown functions . One of the latter proteins was previously identified as the product of the Escherichia coli yodA ORF . The yodA ORF encodes 216 aa residues (the YodA protein) and the increased synthesis of YodA during cadmium stress was found probably to be a result of transcriptional activation from one single promoter upstream of the structural yodA gene . Analysis of a transcriptional gene fusion, P(yodA)-lacZ, demonstrated that basal expression of yodA is low during exponential growth and expression is increased greater than 50-fold by addition of cadmium to growing cells . However, challenging cells with additional metals such as zinc, copper, cobalt and nickel did not increase the level of yodA expression . In addition, hydrogen peroxide also increased yodA expression whereas the superoxide-generating agent paraquat failed to do so . Surprisingly, cadmium-induced transcription of yodA is dependent on soxS and fur, but independent of oxyR . Moreover, a double relA spoT mutation abolished induction of yodA during cadmium exposure but ppGpp is not sufficient to induce yodA since expression of the gene is not elevated during stationary phase . After 45 min of cadmium exposure the YodA protein was primarily detected in the cytoplasmic fraction but was later (150 min) found in both the cytoplasmic and periplasmic compartments. Microbiology, 2002 Dec, 148(Pt 12), 3779 - 87 An unexpected absence of queuosine modification in the tRNAs of an Escherichia coli B strain; Dineshkumar TK et al.; The post-transcriptional processing of tRNAs decorates them with a number of modified bases important for their biological functions . Queuosine, found in the tRNAs with GUN anticodons (Asp, Asn, His, Tyr), is an extensively modified base whose biosynthetic pathway is still unclear . In this study, it was observed that the tRNA(Tyr) from Escherichia coli B105 (a B strain) migrated faster than that from E . coli CA274 (a K-12 strain) on acid urea gels . The organization of tRNA(Tyr) genes in E . coli B105 was found to be typical of the B strains . Subsequent analysis of tRNA(Tyr) and tRNA(His) from several strains of E . coli on acid urea gels, and modified base analysis of tRNA preparations enriched for tRNA(Tyr), showed that E . coli B105 lacked queuosine in its tRNAs . However, the lack of queuosine in tRNAs was not a common feature of all E . coli B strains . The tgt and queA genes in B105 were shown to be functional by their ability to complement tgt and queA mutant strains . These observations suggested a block at the step of the biosynthesis of preQ(1) (or preQ(0)) in the B105 strain . Interestingly, a multicopy vector harbouring a functional tgt gene was toxic to E . coli B105 but not to CA274 . Also, in mixed cultures, E . coli B105 was readily competed out by the CA274 strain . The importance of these observations and this novel strain (E . coli B105) in unravelling the mechanism of preQ(1) or preQ(0) biosynthesis is discussed. Am J Respir Crit Care Med, 2003 Feb 15, 167(4), 570 - 9 Epub 2002 Dec 12. Postlipopolysaccharide oxidative damage of mitochondrial DNA; Suliman HB et al.; Selected structural and functional alterations of mitochondria induced by bacterial lipopolysaccharide (LPS) were investigated on the basis of the hypothesis that LPS initiates hepatic mitochondrial DNA (mtDNA) damage by oxidative mechanisms . After a single intraperitoneal injection of Escherichia coli LPS, liver mtDNA copy number decreased, as determined by Southern analysis, within 24 hours relative to nuclear 18S rRNA (p < 0.05) . LPS induced a novel oxidant-dependent 3.8-kb mtDNA deletion in the region encoding NADH dehydrogenase subunits 1 and 2 and cytochrome c oxidase subunit I, which correlated with mitochondrial glutathione depletion . Expression of mitochondrial mRNA and transcription of mitochondrial RNA were suppressed, whereas mRNA expression increased for selected nuclear-encoded mitochondrial proteins . Resolution of mtDNA damage was mediated by importation of mitochondrial transcription factor A protein, a central regulator of mtDNA copy number, accompanied by binding of mitochondrial protein extract to the mitochondrial transcription factor A DNA-binding site . Hence, mtDNA integrity and transcriptional capacity after LPS administration appeared to be reinstated by mitochondrial biogenesis . These data provide the first link between LPS-mediated hepatic injury and a specific oxidative mtDNA deletion, which inhibits mitochondrial transcription and is restored by activation of mechanisms that lead to biogenesis. Biochem Biophys Res Commun, 2003 Jan 3, 300(1), 93 - 101 Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35; Lim WJ et al.; The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli . The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da) . The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX . The enzyme was 43-69% sequence identical with other glycogen branching enzymes . The enzyme is the most similar to GlgB of E . coli and contained the four regions conserved among the alpha-amylase family . The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE . The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C. Biochem Biophys Res Commun, 2003 Jan 3, 300(1), 29 - 35 Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli; Jeong JK et al.; The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan . Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed . The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175) . Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively . These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs . Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different . This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures. Lancet, 2002 Dec 7, 360(9348), 1831 - 7 Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Cheung CY, Poon LL, Lau AS, Luk W, Lau YL, Shortridge KF, Gordon S, Guan Y, Peiris JS. BACKGROUND: In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33% . The biological basis for the severity of human H5N1 disease has remained unclear . We tested the hypothesis that virus-induced cytokine dysregulation has a role . METHODS: We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro . Secretion of tumour necrosis factor alpha (TNF alpha) from macrophages infected with the viruses was compared by ELISA . By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-alpha response . FINDINGS: The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF alpha and interferon beta . The concentration of TNF-alpha protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide . The non-structural (NS) gene-segment of H5N1/97 viruses contributed to the increase in TNF alpha induced by the virus . INTERPRETATION: The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF alpha . This characteristic may contribute to the unusual severity of human H5N1 disease. J Control Release, 2002 Dec 13, 85(1-3), 263 - 70 Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxin from Escherichia coli (LTK63); Pine S et al.; Groups of 10 Balb/c mice were immunized intranasally (IN) with influenza haemagglutinin (HA), and a genetically detoxified mutant of heat-labile enterotoxin from Escherichia coli (LTK63) at several different doses . IN immunization at the optimal dose combination for HA and LTK63 induced equivalent levels of serum IgG antibodies to intramuscular (IM) immunization with HA alone, and induced significantly enhanced IgA titers in nasal wash . However, haemagglutination inhibition (HI) assays showed that the IM vaccine induced approximately 10-fold higher HI titers than IN immunization with HA and LTK63 . In a second study, HA and LTK63 was compared to a licensed emulsion adjuvant MF59 by the IN route . LTK63 was shown to be significantly more potent than MF59 when evaluated at the optimal dose combination with HA . Hence, the LTK63 and HA combination represents an attractive candidate for evaluation as an IN vaccine in larger animal models, or humans. J Control Release, 2002 Dec 13, 85(1-3), 169 - 80 Delivery of subunit vaccines in maize seed; Lamphear BJ et al.; The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens . Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms . Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans . Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain . We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses . Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed. Biochem J, 2003 Mar 15, 370(Pt 3), 867 - 71 Degradation of mutant initiator protein DnaA204 by proteases ClpP, ClpQ and Lon is prevented when DNA is SeqA-free; Slominska M et al.; A mutant form of the Escherichia coli replication initiator protein, DnaA204, is unstable . At low growth rates, the dnaA204 mutant cells experience a limitation of initiator protein and grow with reduced initiation frequency and DNA concentration . The mutant DnaA protein is stabilized by the lack of SeqA protein . This stabilization was also observed in a dam mutant where the chromosome remains unmethylated . Since unmethylated DNA is not bound by SeqA, this indicates that DnaA204 is not stabilized by the lack of SeqA protein by itself, but rather by lack of SeqA complexed with DNA . Thus the destabilization of DnaA204 may be due either to interaction with SeqA-DNA complexes or changes in nucleoid organization and superhelicity caused by SeqA . The DnaA204 protein was processed through several chaperone/protease pathways . The protein was stabilized by the presence of the chaperones ClpA and ClpX and degraded by their cognate protease ClpP . The dnaA204 mutant was not viable in the absence of ClpY, indicating that this chaperone is essential for DnaA204 stability or function . Its cognate protease ClpQ, as well as Lon protease, degraded DnaA204 to the same degree as ClpP . The chaperones GroES, GroEL and DnaK contributed to stabilization of DnaA204 protein. Mol Cell Biochem, 2002 Oct, 239(1-2), 61 - 8 Solution structure of fatty acid-binding protein from human brain; Rademacher M et al.; Human brain-type fatty acid-binding protein (B-FABP) has been recombinantly expressed in Escherichia coli both unlabelled and 15N-enriched for structure investigation in solution using high-resolution NMR spectroscopy . The sequential assignments of the 1H and 15N resonances were achieved by applying multidimensional homo- and heteronuclear NMR experiments . The ensemble of the 20 final energy-minimized structures, representing human B-FABP in solution, have been calculated based on a total of 2490 meaningful distance constraints . The overall B-FABP structure exhibits the typical backbone conformation described for other members of the FABP family, consisting often antiparallel beta-strands (betaA to betaJ) that form two almost orthogonal beta-sheets, a helix-turn-helix motif that closes the beta-barrel on one side, and a short N-terminal helical loop . A comparison with the crystal structure of the same protein complexed with docosahexaenoic acid reveals only minor differences in both secondary structure and overall topology . Moreover, the NMR data indicate a close structural relationship between human B-FABP and heart-type FABP with respect to fatty acid binding inside the protein cavity. Ai Zheng, 2002 Jul, 21(7), 751 - 6 {Cloning, sequencing, expression, and primary identification of recombinant mouse protein kinase CK2 alpha subunit}; Chen XW et al.; BACKGROUND & OBJECTIVE: Protein kinase CK2 is a highly conserved and ubiquitous eukaryotic serine/threonine kinase that is elevated and can serve as an oncogene in many tumor cells . To further research the structure and function of CK2, this study was designed to construct, express, and preliminarily identify a recombinant expression plasmid which contains the cDNA encoding mouse protein kinase CK2 alpha subunit . METHODS: The aimed cDNA was obtained from NIH 3T3 mouse fibroblasts by RT-PCR . Nde I/BamH I-digested PCR product was directly cloned into pT7-7 expression vector which had been digested by Nde I/BamH I and dephosphorylated by calf intestinal alkaline phosphatase in advance . After E . coli DH5 alpha was transformed with the recombinant DNA by CaCl2 method, transformants were obtained . The positive clones were screened out primarily by gel electrophoresis, and then analyzed by digesting with restriction enzyme . Four positive clones were selected at random and sequenced respectively . The correct recombinant plasmid was transformed into E . coli BL21(DE3) and then expressed by inducing with IPTG . The products were identified with Western blotting . RESULTS: The positive rate of transformants was 100% . The results of restriction analysis indicated that DNA band size of the insert fragment and recombinant plasmid were consistent with theoretically predicated values . The sequencing results showed one of the four clones possessed the cDNA sequence which has no mutation in the processing of PCR, which was termed as pTMCKA . One protein with molecular mass of 42 kDa was overexpressed by inducing with IPTG . The Western blot results confirmed that the recombinant product could specially react with antibody against human CK2 alpha subunit . CONCLUSIONS: The authors have successfully cloned and expressed recombinant mouse protein kinase CK2 alpha subunit in this experiment. Ai Zheng, 2002 Jul, 21(7), 740 - 4 {Cloning and expression of single chain Fv gene against human colorectal carcinoma}; Fang J et al.; BACKGROUND & OBJECTIVE: Single chain Fv(scFv) has been employed as a favorable targeting carrier in the therapy and diagnosis of tumors due to its advantages in relatively low immunogenity and stronger penetrance to tumor tissues over intact mAb . This study was designed to recombine the genes from the variable regions of light chain and heavy chain of ND-1, a monoclonal antibody against human colorectal carcinoma, by a short peptide (Gly4Ser)3 to construct the ND-1scFv gene . The ND-1scFv protein was expressed in Escherichia coli . METHODS: VH and VL gene were amplified from hybridoma cell IC-2, secreting monoclonal antibody ND-1, by RT-PCR, and then were connected to each other by a linker peptide using extension overlap splicing PCR to obtain the ND-1scFv gene . The latter was cloned into the expression vector PET-28a(+) and induced by IPTG to express a fusion protein scFv and His-tag in E . coli BL-21 . The expressed product was purified by affinity chromatography using Ni-NTA resin and its immunoactivity was analyzed using ELISA . RESULTS: Sequence analysis showed that scFv gene consisted of 732 bp, among them, 354 bp for heavy chain gene, located upstream of scFv gene, and 330 bp for the light chain gene, located donstream . SDS-PAGE analysis showed that the relative molecular weight of fusion protein is 30 kDa which was consistent with the theoretically predicted value . scFv expression was in the form of an inclusion body, and SDS-PAGE analysis of the purified scFv showed 94% purity . ELISA analysis revealed that scFv had equal immunoreactivity to the parent ND-1 antibody . CONCLUSIONS: ND-1scFv gene against human colorectal carcinoma was successfully constructed, and functionally expressed in E . coli. Ai Zheng, 2002 Aug, 21(8), 838 - 42 {In situ gene therapy for murine gastric carcinoma with UPRT/5-FU enzyme/prodrug system mediated by retrovirus}; Ji SR et al.; BACKGROUND & OBJECTIVE: 5-Fluorouracil(5-FU), a widely used chemotherapeutic drug, has a limited overall effect in the treatment of human solid tumors due to resistance . This study was designed to investigate if antitumor activation of 5-FU could be enhanced by transfection of uracil phosphoribosyltransferase(UPRT) gene . METHODS: The UPRT gene encoding uracil phosphoribosyltransferase was amplified from Escherichia Coli K12 genome and subcloned into retrovirus expression vector pLXSN, Recombinant retrovirus was packaged and used further to infect murine gastric cancer cell line MFC . The sensitivity of MFC transfected with UPRT gene to 5-FU was determined by MTT method . In situ gene therapy was performed by regional repeated injections of concentrated and purified recombinant retrovirus carrying UPRT gene intratumorally and followed by administration of 5-FU intraperitoneally(i.p.) . RESULTS: The 5-FU sensitivity in MFC transfected with the UPRT gene increased 17.26-fold compared to the control cells . In situ transfection of the UPRT gene mediated by retrovirus vector followed by the administration of 5-FU (10 mg/kg) significantly inhibited the tumor growth (P < 0.005) with an inhibition rate of 87.18% and prolonged the survival . CONCLUSION: Transfection of UPRT gene can render the murine gastric cancer cell line MFC be more sensitive to low concentration of 5-FU and significantly improve the antitumor effect of 5-FU both in vitro and in vivo. J Virol, 2003 Jan, 77(1), 328 - 39 Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, is a viral pathogenicity factor; Lung OY et al.; Viral envelope fusion proteins are important structural proteins that mediate viral entry and may affect or determine the host range of a virus . The acquisition, exchange, and evolution of such envelope proteins may dramatically affect the success and evolutionary divergence of viruses . In the family Baculoviridae, two very different envelope fusion proteins have been identified . Budded virions of group I nucleopolyhedroviruses (NPVs) such as the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), contain the essential GP64 envelope fusion protein . In contrast group II NPVs and granuloviruses have no gp64 gene but instead encode a different envelope protein called F . F proteins from group II NPVs can functionally substitute for GP64 in gp64null AcMNPV viruses, indicating that GP64 and these F proteins serve a similar functional role . Interestingly, AcMNPV (and other gp64-containing group I NPVs) also contain an F gene homolog (Ac23) but the AcMNPV F homolog cannot compensate for the loss of gp64 . In the present study, we show that Ac23 is expressed and is found in budded virions . To examine the function of F protein homologs from the gp64-containing baculoviruses, we generated an Ac23null AcMNPV genome by homologous recombination in E . coli . We found that Ac23 was not required for viral replication or pathogenesis in cell culture or infected animals . However, Ac23 accelerated the mortality of infected insect hosts by approximately 28% or 26 h . Thus, Ac23 represents an important viral pathogenicity factor in larvae infected with AcMNPV. J Biol Chem, 2003 Feb 21, 278(8), 6145 - 52 Epub 2002 Dec 10. Mutation of interfacial residues disrupts subunit folding and particle assembly of Physalis mottle tymovirus; Umashankar M et al.; Virus-like particles (VLPs) serve as excellent model systems to identify the pathways of virus assembly . To gain insights into the assembly mechanisms of the Physalis mottle tymovirus (PhMV), six interfacial residues, identified based on the crystal structure of the native and recombinant capsids, were targeted for mutagenesis . The Q37E, Y67A, R68Q, D83A, I123A, and S145A mutants of the PhMV recombinant coat protein (rCP) expressed in Escherichia coli were soluble . However, except for the S145A mutant, which assembled into VLPs similar to that of wild type rCP capsids, all the other mutants failed to assemble into VLPs . Furthermore, the purified Q37E, Y67A, R68Q, D83A, and I123A rCP mutants existed essentially as partially folded monomers as revealed by sucrose density gradient analysis, circular dichroism, fluorescence, thermal, and urea denaturation studies . The rCP mutants locked into such conformations probably lack the structural signals/features that would allow them to assemble into capsids . Thus, the mutation of residues involved in inter-subunit interactions in PhMV disrupts both subunit folding and particle assembly. J Biol Chem, 2003 Feb 21, 278(8), 5539 - 47 Epub 2002 Dec 10. Changes in conserved region 3 of Escherichia coli sigma 70 reduce abortive transcription and enhance promoter escape; Cashel M et al.; Mutations within the Escherichia coli rpoD gene encoding amino acid substitutions in conserved region 3 of the sigma(70) subunit of E . coli RNA polymerase restore normal stress responsiveness to strains devoid of the stress alarmone, guanosine-3',5'-(bis)pyrophosphate (ppGpp) . The presence of a mutant protein, either sigma(70)(P504L) or sigma(70)(S506F), suppresses the physiological defects in strains devoid of ppGpp . In vitro, when reconstituted into RNA polymerase holoenzyme, these sigma mutants confer unique transcriptional properties, namely they reduce the probabilities of forming abortive RNAs . Here we investigated the behavior of these mutant enzymes during transcription of the highly abortive cellular promoter, gal P2 . No differences between mutant and wild-type enzymes were observed prior to and including open complex formation . Remarkably, the mutant enzymes produced drastically reduced levels of gal P2 abortive RNAs and increased production of full-length gal P2 RNAs relative to the wild-type enzyme, leading to greatly reduced ratios of abortive to productive RNAs . These results are attributed mainly to a decreased formation of unproductive initial transcribing complexes with the mutant polymerases and increased rates of promoter escape . Altered transcription properties of these mutant polymerases arise from an alternative structure of the sigma(70) region 3.2 segment that permits efficient positioning of the nascent RNA into the RNA exit channel displacing sigma and facilitating sigma release. Vet Microbiol, 2003 Feb 25, 91(4), 309 - 23 Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli; Pai WC et al.; Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli . MAbs were characterized and used to develop a diagnostic test . Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase . The results indicated that these MAbs delineated two epitopes I and II of esigmaA . An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes . All MAbs bound to esigmaA in its native form . After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished . However, the reactivity of MAbs recognizing epitope II was not affected . MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains . The results suggest that the two epitopes are highly conserved among these virus strains . A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures . Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually . A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained . Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers . These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections. Mol Cell Probes, 2002 Oct, 16(5), 371 - 8 Virulence typing of Escherichia coli using microarrays; van Ijperen C et al.; We describe a microarray based broad-range screening technique for Escherichia coli virulence typing . Gene probes were amplified by PCR from a plasmid bank of characterised E . coli virulence genes and were spotted onto a glass slide to form an array of capture probes . Genomic DNA from E . coli strains which were to be tested for the presence of these virulence gene sequences was labelled with fluorescent cyanine dyes by random amplification and then hybridised against the array of probes . The hybridisation, washing and data analysis conditions were optimised for glass slides, and the applicability of the method for identifying the presence of the virulence genes was determined using reference strains and clinical isolates . It was found to be a sensitive screening method for detecting virulence genes, and a powerful tool for determining the pathotype of E . coli . It will be possible to expand and automate this microarray technique to make it suitable for rapid and reliable diagnostic screening of bacterial isolates. J Med Chem, 2002 Dec 19, 45(26), 5797 - 801 Immobilization of aminothiols on poly(oxyalkylene phosphates) . Formation of poly(oxyethylene phosphates)/cysteamine complexes and their radioprotective efficiency; Georgieva R et al.; The necessity to apply near-toxic amounts of radioprotective drugs to achieve adequate protection during radiation treatments represents a major problem in human medicine . One of the promising strategies to suppress the toxicity of these drugs involves their incorporation into biocompatible polymers . In this study cysteamine (Cy) was attached to poly(oxyethylene phosphate), POEP, via an ionic bond . Radioprotection of E . coli B cells by this substance and its acute toxicity on male C57 BL mice were measured . The toxicity of Cy immobilized within the poly(oxyethylene phosphate) was significantly lower in comparison to pure Cy while its radioprotective efficiency remained high at half the maximum tolerable dose . The high radioprotective efficiency of the Cy/POEP complexes was further confirmed on mice at different polymer molecular weight characteristics, drug immobilization degrees, application times, and doses . It was found that POEP with molecular weight 4700 Da and containing 24% repeating units with attached Cy has the highest protection potential combined with a depot effect. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2002 Dec, 19(6), 475 - 8 {Cloning of human brain-derived neurotrophin-6 gene and its expression in procaryotic cell}; Zhang C et al.; OBJECTIVE: To clone human brain-derived neurotrophin-6(NT-6) gene and to observe its expression in the procaryotic cell . METHODS: Total RNA was extracted from aborted antenatal cerebral cortex, and cDNA fragment of NT-6 was amplified through reverse transcript-polymerase chain reaction . After being incised and recovered, the NT-6 gene was cloned into pBK-CMV plasmid to construct a NT-6 gene expression vector . Expression of NT-6 gene in Escherichia coli was studied after being induced by isopropyl beta-D-thiogalactoside(IPTG) . RESULTS: The NT-6 gene expression vector was constructed and Escherichia coli with recombinant vector expressed specific protein after induction by IPTG . CONCLUSION: The cloning of human brain-derived NT-6 gene provides a basis for further studying the structure, function and clinical application of NT-6. Mol Biol Cell, 2002 Dec, 13(12), 4497 - 507 Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1; Liu H et al.; The multipotent cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in particular in the physiological response to infection and in inflammatory responses . GM-CSF is produced by many cell types, including T lymphocytes responding to T-cell receptor activation and mantle zone B lymphocytes . B-cell receptor and T-cell receptor activation generates two major signals: an increase in intracellular Ca(2+) concentration and a protein kinase cascade . Previous studies have shown that the Ca(2+)/calmodulin-dependent phosphatase calcineurin mediates stimulation of GM-CSF transcription in response to Ca(2+) . In this study, we show that Ca(2+) signaling also regulates GM-CSF transcription negatively through Ca(2+)/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1 . Wild-type Ets1 negatively affects GM-CSF transcription on Ca(2+) stimulation in the presence of cyclosporin A, which inhibits calcineurin . Conversely, Ets1 with mutated CaMK II target serines showed an increase in transactivation of the GM-CSF promoter/enhancer . Moreover, constitutively active CaMK II inhibited transactivation of GM-CSF by wild-type Ets1 but not by Ets1 with mutated CaMK II sites . Mutation of CaMK II target serines in Ets1 also relieves inhibition of cooperative transactivation of GM-CSF with the Runx1/AML1 transcription factor . In addition, the Ca(2+)-dependent phosphorylation of Ets1 reduces the binding of Ets1 to the GM-CSF promoter in vivo. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16642 - 7 Epub 2002 Dec 10. The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter; Borths EL et al.; Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients . The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD . B12 is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF . A stable complex between BtuF and BtuCD (with the stoichiometry BtuC2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures . Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter . These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes. Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16654 - 9 Epub 2002 Dec 10. Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts; Rydberg B et al.; Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes . Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and Escherichia coli cell-free extracts that nicks 5' of the ribose . The human and yeast extracts also make a subsequent cut 3' of the ribonucleotide releasing a ribonucleotide monophosphate . The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose . Screening of yeast deletion mutant cells reveals that the initial nick is made by RNase H(35), a RNase H type 2 enzyme, and the second cut is made by Rad27p, the yeast homologue of human FEN-1 protein . RNase H type 2 enzymes are present in all kingdoms of life and are evolutionarily well conserved . We knocked out the corresponding rnhb gene in E . coli and show that extracts from this strain lack the nicking activity . Conversely, a highly purified archaeal RNase HII type 2 protein has a pronounced activity . To study substrate specificity, extracts were made from a yeast double mutant lacking the other main RNase H enzymes {RNase H1 and RNase H(70)}, while maintaining RNase H(35) . It was found that a single ribose is preferred as substrate over a stretch of riboses, further strengthening a proposed role of this enzyme in the repair of misincorporated ribonucleotides rather than (or in addition to) processing RNADNA hybrid molecules. FASEB J, 2003 Jan, 17(1), 106 - 8 Epub 2002 Nov 15. Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain; Himly M et al.; In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide . Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen . Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats . Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation . The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan . A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure . Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients . However, posttranslational modifications did not influence T-cell recognition . Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy. Phytochemistry, 2003 Jan, 62(1), 47 - 52 Isolation and characterization of two fructokinase cDNA clones from rice; Jiang H et al.; Two cDNA clones, OsFKI and OsFKII, encoding fructokinase (EC 2.7.1.4) were isolated from immature seeds of rice (Oryza sativa L.) by PCR . OsFKI cDNA encoded a deduced protein of 323 amino acids that was 59-71% identical to previously characterized plant fructokinases . In contrast, OsFKII cDNA encoded a deduced protein of 336 amino acids that shared only 64% amino acid identity with OsFKI . The deduced proteins both possessed an ATP-binding motif and putative substrate recognition site sequences that were previously identified in bacterial fructokinases . Genomic DNA blot analysis also revealed that each fructokinase gene exists as a single copy in the rice genome . The identity of OsFKI and OsFKII as fructokinases was confirmed by the expression of enzyme activity in E . coli . Although both OsFKI and OsFKII utilized fructose as substrate, only OsFKII activity was strongly inhibited at a high fructose concentration . The mRNA corresponding to OsFKII accumulated at high levels in developing rice grains, whereas there were only low levels of OsFKI transcripts in immature seeds . These results indicate that fructokinase in rice endosperm is encoded by two divergent genes, which play different roles in rice grains for starch storage based on their sensitivity to substrate inhibition and level of transcripts in endosperm. J Am Chem Soc, 2002 Dec 18, 124(50), 15064 - 75 Comparison and contrasts between the active site PKs of Mn-superoxide dismutase and those of Fe-superoxide dismutase; Maliekal J et al.; The Fe- and Mn-containing superoxide dismutases catalize the same reaction and have almost superimposable active sites . Therefore, the details of their mechanisms have been assumed to be similar . However, we now show that the pH dependence of Escherichia coli MnSOD activity reflects a different active site proton equilibrium in (oxidized) Mn(3+)SOD than the event that affects the active site pK of oxidized FeSOD . We find that the universally conserved Tyr34 that has a pK above 11.5 in Fe(3+)SOD is responsible for the pK near 9.5 of Mn(3+)SOD and, thus, that the oxidized state pK of Mn(3+)SOD corresponds to an outer-sphere event whereas that of Fe(3+)SOD corresponds to an inner sphere event {Bull, C.; Fee, J . A . J . Am . Chem . Soc . 1985, 107, 3295-3304} . We also present the first description of a reduced-state pK for MnSOD . Mn(2+)SOD's pK involves deprotonation of Tyr34, as does Fe(2+)SOD's pK {Sorkin, D . L.; Miller A.-F . Biochemistry 1997, 36, 4916-4924} . However, the values of the pKs, 10.5 and 8.5 respectively, are quite different and Mn(2+)SOD's pK affects the coordination geometry of Mn(2+), most likely via polarization of the conserved Gln146 that hydrogen bonds to axially coordinated H(2)O . Our findings are consistent with the different electronic configurations of Mn(2+/3+) vs Fe(2+/3+), such as the stronger hydrogen bonding between Gln146 and coordinated solvent in MnSOD than that between the analogous Gln69 and coordinated solvent in FeSOD, and the existence of weakly localized H(2)O near the sixth coordination site of Mn(2+) in Mn(2+)SOD {Borgstahl et al . J . Mol . Biol . 2000, 296, 951-959}. Biochemistry, 2002 Dec 17, 41(50), 15000 - 6 Iron-sulfur clusters of biotin synthase in vivo: a Mössbauer study; Benda R et al.; Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only {2Fe-2S}(2+) clusters when isolated under aerobic conditions . Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either {4Fe-4S}(2+), {4Fe-4S}(+), or a mixture of {4Fe-4S}(2+) and {2Fe-2S}(2+) clusters . To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe Mossbauer spectroscopy to investigate the clusters of biotin synthase in whole cells . The results show that, in aerobically grown cells, biotin synthase contains a mixture of {4Fe-4S}(2+) and {2Fe-2S}(2+) clusters . A mixed {4Fe-4S}(2+):{2Fe-2S}(2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase . Their presence in vivo is now another argument in favor of this mixed cluster form. Biochemistry, 2002 Dec 17, 41(50), 14988 - 99 Kinetic analysis of R67 dihydrofolate reductase folding: from the unfolded monomer to the native tetramer; Bodenreider C et al.; R67 dihydrofolate reductase (DHFR) is a homotetrameric enzyme . Its subunit has a core structure consisting of five antiparallel beta-strands that form a compact beta-barrel . Our interest was to describe the molecular mechanism of the complete folding pathway of this beta-sheet protein, focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structures all along the folding process . The folding kinetics of R67 dihydrofolate reductase into dimers at pH 5.0 were first examined by intrinsic tryptophan fluorescence, fluorescence energy transfer, and circular dichroism spectroscopy . The process was shown to consist of at least four steps, including a burst, a rapid, a medium, and a slow phase . Measurements of the ellipticity at 222 nm indicated that about 50% of the total change associated with refolding occurred during the 4 ms dead time of the stopped-flow instrument, indicating a substantial burst of secondary structure . The bimolecular association step was detected using fluorescence energy transfer and corresponded to the rapid phase . The slow phase was attributed to a rate-limiting isomerization of peptidyl-prolyl bonds involving 15% of the unfolded population . A complete folding pathway from the unfolded monomer to the native tetramer was proposed and an original model based upon the existence of early partially folded monomeric intermediates, rapidly stabilized in a dimeric form able to self-associate into the native homotetramer was formulated . The rate constants of these various steps were determined by fitting the kinetic traces to this model and supported our mechanistic assumptions. Biochemistry, 2002 Dec 17, 41(50), 14935 - 43 Linkage of multiequilibria in DNA recognition by the D53H Escherichia coli cAMP receptor protein; Lin SH et al.; The transcription factor cyclic AMP receptor protein, CRP, regulates the operons that encode proteins involved in translocation and metabolism of carbohydrates in Escherichia coli . The structure of the CRP-cAMP complex reveals the presence of two sets of cAMP binding sites . Solution biophysical studies show that there are two high-affinity and two low-affinity binding sites, to which the binding of cAMP is characterized by varying degrees of cooperativity . A stoichiometry of four implies that potentially CRP can exist in five conformers with different numbers of bound cAMP . These conformers may exhibit differential affinities for specific DNA sequences . In this study, the affinity between DNA and each conformer of D53H CRP was defined through a dissection of the thermodynamic linkage scheme that included all the conformers . Loading of the high- and low-affinity sites with cAMP leads to high and low affinity for DNA, respectively . The specific magnitude of the binding constants of these conformers is DNA sequence dependent . The various association constants defined by the present study provide a solution to address an enigma of the CRP system, namely, the 3 orders of magnitude difference between the cAMP binding constants determined by in vitro studies and the cAMP concentration regime to which the bacteria respond . Under physiological conditions, the apo-CRP-DNA complex is the dominant species . As a consequence of the 1000-fold stronger affinity of cAMP to the apo-CRP-DNA complex than that to CRP, the relevant reaction is the binding of cAMP to this DNA-protein complex . The binding constant is of the order of 10(7) M(-)(1), the same concentration regime as that of cellular concentration of cAMP . In addition, under physiological conditions the species that binds to the lac and gal operons is predicted to be CRP-(cAMP)(1) . A comparison of parameters between the wild type and the mutant CRP shows that the mutation apparently shifts the various thermodynamically linked equilibria without a change in the basic mechanism that governs CRP activities . Thus, the conclusions derived from a study of the mutant are relevant to wild-type CRP . A dissection of the individual binding constants in this multiequilibria reaction scheme leads to a definition of the mechanism of action of this transcription factor. Biochemistry, 2002 Dec 17, 41(50), 14897 - 905 Proximity of cytoplasmic and periplasmic loops in NhaA Na+/H+ antiporter of Escherichia coli as determined by site-directed thiol cross-linking; Rimon A et al.; The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis . Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249) . The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM) . The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly . In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C . In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C . The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein . The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA. Biochemistry, 2002 Dec 17, 41(50), 14866 - 78 Trp repressor-operator binding: NMR and electrophoretic mobility shift studies of the effect of DNA sequence and corepressor binding on two Trp repressor-operator complexes; Jaseja M et al.; In Trp repressor-DNA complexes, most interactions either occur with phosphate groups or are water-mediated hydrogen bonds to bases . To examine the factors involved in DNA selectivity, we have studied Trp repressor binding to two operator sequences, trpR(S)() and trpO(M)(), with L-tryptophan or 5-methyltryptophan as corepressor . These operators contain all the consensus bases but differ at base pairs contacted by their phosphate groups . In electrophoretic mobility shift assays (EMSAs) the trpR(S)() sequence gives solely 1:1 protein-DNA complexes with either corepressor . The trpO(M )()sequence binds more weakly than trpR(S)() . It gives dissociating 2:1 complexes in EMSAs with L-tryptophan, but both 1:1 and 2:1 complexes are observed with 5-methyltryptophan or if glycerol is present in the gel . The backbone resonances of the TrpR-L-tryptophan-DNA complexes were assigned using triple-resonance experiments and selectively (15)N labeled protein . On changing the DNA sequence, the largest differences in the NMR spectra are at residues 78-81, at the turn of the helix-turn-helix motif and the tip of the recognition helix . I79 and A80 interact with the conserved bases of the operators, while G78 and T81 interact with phosphate groups at bases that differ between the two sequences . Changing the corepressor from L-tryptophan to 5-methyltryptophan causes effects at residues 52, 60, 61, and 85, which do not interact with the DNA . The spectra suggest that there is mutual induced fit between protein and DNA so that sequence changes at bases contacted only by the phosphate groups affect the environment of the protein at residues that bind to conserved bases elsewhere in the DNA. Biochemistry, 2002 Dec 17, 41(50), 14856 - 65 Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase; Hountondji C et al.; Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself . ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase . MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine . Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified . Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938 . Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant . Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases . Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS . The role of lysine 277 was evaluated by site-directed mutagenesis . The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme . In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val) . Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS . Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS. Biochemistry, 2002 Dec 17, 41(50), 14771 - 8 Structure and dynamics of the modular halves of Escherichia coli cyclic AMP receptor protein; Li J et al.; E . coli cyclic AMP receptor protein, CRP, is a modular protein that consists of a covalent linkage of two common structural domains . To probe the mechanism for intramolecular communications and to define the unique properties acquired by covalent linkage, the structural, and functional properties of the cAMP- and DNA-binding domains of CRP were studied separately as two independent polypeptides . The N-terminal cAMP-binding domain (alpha-CRP), including S-CRP and CH-CRP, which were generated by digestion of CRP by subtilisin and chymotrypsin, respectively, are mainly populated by beta-sheets . The C-terminal DNA-binding domain, designated as beta-CRP, consists of mostly alpha-helices . The residues of S-CRP and CH-CRP are from 1 to 116 and 1 to 136 of intact wild-type CRP, and those of beta-CRP are from 108 to 209 . The secondary structures of alpha-CRP and beta-CRP were monitored by FT-IR, and they are similar to those of the corresponding parts in intact wild-type CRP . Results from hydrogen-deuterium exchange experiments indicated that beta-CRP is more dynamic than alpha-CRP . In an earlier study, it was shown that alpha-CRP retains the function of binding cAMP {Heyduk, E., et al . (1992) Biochemistry 31, 3682-3688} . beta-CRP was able to bind to DNA, although only weakly, and was not sequence specific . Thus, a covalent linkage between the two domains is essential for the realization of the intramolecular signal transmission between the domains triggered by ligand binding . The acquisition of this unique property is intimately associated with the dynamics of the molecule. Cell Mol Life Sci, 2002 Oct, 59(10), 1658 - 65 A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers; Craig EA et al.; Mitochondria contain a specialized system of molecular chaperones that plays a critical role in the biogenesis of Fe/S centers . This Hsp70:J-protein system shows many similarities to the system found in bacteria, but the precise role of neither chaperone system has been defined . However, evidence to date suggests an interaction with the scaffold protein on which a transient Fe/S center is assembled, and thus implies a role in either assembly of the center or its transfer to recipient proteins. Cell Mol Life Sci, 2002 Oct, 59(10), 1624 - 31 Redox-regulated molecular chaperones; Graf PC et al.; The conserved heat shock protein Hsp33 functions as a potent molecular chaperone with a highly sophisticated regulation . On transcriptional level, the Hsp33 gene is under heat shock control; on posttranslational level, the Hsp33 protein is under oxidative stress control . This dual regulation appears to reflect the close but rather neglected connection between heat shock and oxidative stress . The redox sensor in Hsp33 is a cysteine center that coordinates zinc under reducing, inactivating conditions and that forms two intramolecular disulfide bonds under oxidizing, activating conditions . Hsp33's redox-regulated chaperone activity appears to specifically protect proteins and cells from the otherwise deleterious effects of reactive oxygen species . That redox regulation of chaperone activity is not restricted to Hsp33 became evident when the chaperone activity of protein disulfide isomerase was recently also shown to cycle between a low- and high-affinity substrate binding state, depending on the redox state of its cysteines. Cell Mol Life Sci, 2002 Oct, 59(10), 1617 - 23 SecB, one small chaperone in the complex milieu of the cell; Randall LL et al.; SecB is only one of a plethora of cytosolic chaperones in E . coli whose common property is that they bind nonnative proteins . It plays a crucial role during protein export via the general secretory pathway by modulating the partitioning of precursors between folding or aggregation and delivery to the membrane-bound translocation apparatus . In this latter role SecB demonstrates specific binding to a unique partner, SecA . SecB has the potential to participate in functions outside of export acting as a general nonspecific chaperone to provide buffering capacity of the nonnative state of proteins in the cytosolic pool . We discuss the interactions of SecB with its many binding partners in light of its recently determined structure, emphasizing both kinetic and thermodynamic parameters. Cell Mol Life Sci, 2002 Oct, 59(10), 1589 - 97 Structure and function of the GroE chaperone; Walter S; The Escherichia coli proteins GroEL and GroES were the first chaperones to be studied in detail and have thus become a role model for assisted protein folding in general . A wealth of both structural and functional data on the GroE system has been accumulated over the past years, enabling us now to understand the basic principles of how this fascinating protein-folding machine accomplishes its task . According to the current model, GroE processes a nonnative polypeptide in a cycle consisting of three steps . First, the polypeptide substrate is captured by GroEL . Upon binding of the co-chaperone GroES and ATP, the substrate is then discharged into a unique microenvironment inside of the chaperone, which promotes productive folding . After hydrolysis of ATP, the polypeptide is released into solution . Moreover, GroE may actively increase the folding efficiency, e.g . by unfolding of misfolded protein molecules . The mechanisms underlying these features, however, are yet not well characterized. Neurology, 2002 Dec 10, 59(11), 1776 - 9 GNE mutations in an American family with quadriceps-sparing IBM and lack of mutations in s-IBM; Vasconcelos OM et al.; Analysis for GNE mutations was performed in an American, non-Iranian Jewish, family with quadriceps-sparing inclusion body myopathy (QS-IBM) and in 11 patients with sporadic IBM (s-IBM) . Two novel nonallosteric site missense mutations were found in the QS-IBM kinship . No mutations were identified in s-IBM patients . After 8 years of follow-up and severe disease progression, the quadriceps muscle in the QS-IBM patient remains strong despite subclinical involvement documented with repeat MRI and muscle biopsy. Neurology, 2002 Dec 10, 59(11), 1689 - 93 Distal myopathy with rimmed vacuoles is allelic to hereditary inclusion body myopathy; Nishino I et al.; BACKGROUND: Distal myopathy with rimmed vacuoles (DMRV) is an autosomal-recessive disorder with preferential involvement of the tibialis anterior muscle that starts in young adulthood and spares quadriceps muscles . The disease locus has been mapped to chromosome 9p1-q1, the same region as the hereditary inclusion body myopathy (HIBM) locus . HIBM was originally described as rimmed vacuole myopathy sparing the quadriceps; therefore, the two diseases have been suspected to be allelic . Recently, HIBM was shown to be associated with the mutations in the gene encoding the bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) . OBJECTIVE: To determine whether DMRV and HIBM are allelic . METHODS: The GNE gene was sequenced in 34 patients with DMRV . The epimerase activity in lymphocytes from eight DMRV patients was also measured . RESULTS: The authors identified 27 unrelated DMRV patients with homozygous or compound-heterozygous mutations in the GNE gene . DMRV patients had markedly decreased epimerase activity . CONCLUSIONS: DMRV is allelic to HIBM . Various mutations are associated with DMRV in Japan . The loss-of-function mutations in the GNE gene appear to cause DMRV/HIBM. J Cell Biol, 2002 Dec 9, 159(5), 833 - 43 Epub 2002 Dec 09. The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP; Smith MD et al.; The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts . Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation . Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined . A recent study indicates that Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, raising the possibility that it might cycle between the cytoplasm and chloroplast as a soluble preprotein receptor . In the present study, we examined the mechanism of targeting and insertion of the Arabidopsis thaliana orthologue of Toc159, atToc159, to chloroplasts . Targeting of atToc159 to the outer envelope membrane is strictly dependent only on guanine nucleotides . Although GTP is not required for initial binding, the productive insertion and assembly of atToc159 into the Toc complex requires its intrinsic GTPase activity . Targeting is mediated by direct binding between the GTPase domain of atToc159 and the homologous GTPase domain of atToc33, the Arabidopsis Toc33/34 orthologue . Our findings demonstrate a role for the coordinate action of the Toc GTPases in assembly of the functional Toc complex at the chloroplast outer envelope membrane. J Biol Chem, 2003 Feb 28, 278(9), 6928 - 35 Epub 2002 Dec 08. Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4; Whitby MC et al.; The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis . Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks . Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses . Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all . However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well . Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point . For most substrates the leading strand template is cleaved . The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template. J Biol Chem, 2003 Feb 21, 278(8), 6066 - 74 Epub 2002 Dec 06. Aqueous access channels in subunit a of rotary ATP synthase; Angevine CM et al.; The role of subunit a in proton translocation by the Escherichia coli F(1)F(o) ATP synthase is poorly understood . In the membrane-bound F(o) sector of the enzyme, H(+) binding and release occurs at Asp(61) in the middle of the second transmembrane helix (TMH) of subunit c . Protons are thought to reach Asp(61) via an aqueous access pathway formed at least in part by one or more of the five TMHs of subunit a . In this report, we have substituted Cys into a 19-residue span of the fourth TMH of subunit a and used chemical modification to obtain information about the aqueous accessibility of residues along this helix . Residues 206, 210, and 214 are N-ethylmaleimide-accessible from the cytoplasmic side of the membrane and may lie on the H(+) transport route . Residues 215 and 218 on TMH4, as well as residue 245 on TMH5, are Ag(+)-accessible but N-ethylmaleimide-inaccessible and may form part of an aqueous pocket extending from Asp(61) of subunit c to the periplasmic surface. J Biol Chem, 2003 Apr 18, 278(16), 14053 - 8 Epub 2002 Dec 07. The COOH terminus of GATE-16, an intra-Golgi transport modulator, is cleaved by the human cysteine protease HsApg4A; Scherz-Shouval R et al.; Docking of a vesicle at the appropriate target membrane involves an interaction between integral membrane proteins located on the vesicle (v-SNAREs) and those located on the target membrane (t-SNAREs) . GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) was shown to modulate the activity of SNAREs in the Golgi apparatus and is therefore an essential component of intra-Golgi transport and post-mitotic Golgi re-assembly . GATE-16 contains a ubiquitin fold subdomain, which is terminated at the carboxyl end by an additional amino acid after a conserved glycine residue . In the present study we tested whether the COOH terminus of GATE-16 undergoes post-translational cleavage by a protease which exposes the glycine 116 residue . We describe the isolation and characterization of HsApg4A as a human protease of GATE-16 . We show that GATE-16 undergoes COOH-terminal cleavage both in vivo and in vitro, only when the conserved glycine 116 is present . We then utilize an in vitro assay to show that pure HsApg4A is sufficient to cleave GATE-16 . The characterization of this protease may give new insights into the mechanism of action of GATE-16 and its other family members. J Mol Biol, 2003 Jan 3, 325(1), 163 - 74 De novo backbone and sequence design of an idealized alpha/beta-barrel protein: evidence of stable tertiary structure; Offredi F et al.; We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein . The design was elaborated in two steps . First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel . An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure . A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized . Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model . A pronounced absorption band in the near-UV C