Escherichia coli

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 CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution . Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol . Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C . Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels . Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein . Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution . These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.

J Mol Biol, 2003 Jan 3, 325(1), 111 - 22
Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences; Micaloni C et al.; We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme . This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches . The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates . There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi . With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme . Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties . The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible . Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH) . These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate . The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme.

J Biochem (Tokyo), 2002 Dec, 132(6), 983 - 9
The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli; Yoshida H et al.; During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost . This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes . In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively . We show that (i) the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2 . The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site) . These observations are consistent with the translational inactivation of the ribosome by RMF binding . After the "Recycling" stage, ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S ribosomes are stored as 100S ribosomes and are translationally inactive . We suggest that this conversion of 70S to 100S ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage.

J Biochem (Tokyo), 2002 Dec, 132(6), 903 - 9
Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment; Asano R et al.; Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications . However, it can be difficult to obtain these materials in soluble form after their expression in bacteria . Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins . Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)) . All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E . coli (Fv(sol)) and the parent IgG . The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG . The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system . This refolded diabody could bind to lymphokine-activated killer cells . In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent . Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).

Eur J Biochem, 2002 Dec, 269(24), 6302 - 7
Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenes; Zhang J et al.; A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100 . The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organisms . The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3) . The protein was then purified, and its characteristics were studied . Experimental results showed that NaoA can convert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds . The optimal pH and temperature for NaoA was found to be pH 7-8 and 48-56 degrees C, respectively . The Km of NaoA for nitroethane is approximately 26.8 mm . NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity . Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA.

Eur J Biochem, 2002 Dec, 269(24), 6271 - 7
Heterologous expression and folding analysis of a beta-tubulin isotype from the Antarctic ciliate Euplotes focardii; Pucciarelli S et al.; Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1) . To study the beta-tubulin folding in lower eukaryotes, an isotype of beta-tubulin (beta-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli . Folding analysis was performed by incubation of the 35S-labeled, denatured beta-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric beta-tubulin . We show for the first time in protozoa that beta-tubulin folding is assisted by CCT and requires cofactor A . In addition, we observed that E . focardiibeta-T1 competes with human beta5 tubulin isotype for binding to CCT . The affinity of CCT to E . focardiibeta-T1 and beta5 tubulin are compared . Finally, the mitochondrial chaperonin mt-cpn60 binds to beta-T1 but is unable to release it in a native or quasi-native state.

Eur J Biochem, 2002 Dec, 269(24), 6195 - 203
The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains; Shimada Y et al.; There is much evidence to indicate that cholesterol forms lateral membrane microdomains (rafts), and to suggest their important role in cellular signaling . However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time . To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin, whose chemically modified form has been recently shown to bind selectively to rafts . BIAcore and structural analyses demonstrate that the C-terminal domain (domain 4) of the toxin is the smallest functional unit that has the same cholesterol-binding activity as the full-size toxin with structural stability . Cell membrane-bound recombinant domain 4 was detected in the floating low-density fractions and was found to be cofractionated with the raft-associated protein Lck, indicating that recombinant domain 4 also binds selectively to cholesterol-rich rafts . Furthermore, an enhanced green fluorescent protein-domain 4 fusion protein stains membrane surfaces in a cholesterol-dependent manner in living cells . Therefore, domain 4 of theta-toxin is an essential cholesterol-binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells.

Eur J Biochem, 2002 Dec, 269(24), 6091 - 100
Trehalose-phosphate synthase of Mycobacterium tuberculosis . Cloning, expression and properties of the recombinant enzyme; Pan YT et al.; The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced . Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus . The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE . Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E . coli ( approximately 2.3 g cell paste) . The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein . Like the enzyme originally purified from M . smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e . ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates . These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives . Although the original enzyme from M . smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin . The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm . The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose . TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+ . This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies . TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.

J Neurochem, 2002 Dec, 83(6), 1471 - 80
Rat MYH, a glycosylase for repair of oxidatively damaged DNA, has brain-specific isoforms that localize to neuronal mitochondria; Englander EW et al.; Mitochondrial genomes are exposed to a heavy load of reactive oxygen species (ROS) that damage DNA . Since in neurons, mitochondrial DNA integrity must be maintained over the entire mammalian life span, neuronal mitochondria most likely repair oxidatively damaged DNA . We show that the Escherichia coli MutY DNA glycosylase homolog (MYH) in rat (rMYH) involved in repair of oxidative damage is abundantly expressed in the rat brain, with isoforms that are exclusive to brain tissue . Confocal microscopy and western analyses reveal localization of rMYH in neuronal mitochondria . To assess involvement of MYH in the neuronal response to oxidative DNA damage, we used a rat model of respiratory hypoxia, in which acutely reduced blood oxygenation leads to generation of superoxide, and formation and subsequent removal of 8-hydroxy-2'-deoxyguanosine (8OHdG) . Removal of 8OHdG is accompanied by a spatial increase in rMYH immunoreactivity in the brain and an increase in levels of one of the three mitochondrial MYH isoforms, suggesting that inducible and non-inducible MYH isoforms exist in the brain . The mitochondrial localization of oxidative DNA damage repair enzymes in neurons may represent a specialized neuronal mechanism that safeguards mitochondrial genomes in the face of routine and accidental exposures to heavy loads of injurious ROS.

Scand J Immunol, 2002 Dec, 56(6), 580 - 7
Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis; Harboe M et al.; In addition to the previously cloned Mce1A and Mce1E genes of the Mce1 operon of Mycobacterium tuberculosis (Ahmad et al . Scand J Immunol 1999;50:510-8), Mce1B, Mce1D and Mce1F were cloned and expressed as glutathione-S-transferase (GST) fusion proteins in recombinant Escherichia coli . Polyclonal antibodies against a predicted B-cell epitope of each of the Mce1 proteins of M . tuberculosis were produced by immunizing rabbits with synthetic peptides coupled to keyhole limpet haemocyanin . These antibodies reacted specifically with the corresponding fusion protein, except for GST-Mce1F . A mouse monoclonal antibody, TB1-5 76C, raised against a synthetic 60-mer peptide corresponding to the residues 106-165 in the N-terminal part of Mce1A, reacted strongly with GST-Mce1A . The antibody cross-reacted with GST-Mce1F, but not with the other recombinant GST-Mce1 fusion proteins or free GST . Bioinformatic analysis revealed only slight homology between Mce1A and Mce1F, along the length of the polypeptide chains . Higher homology was found between the residues 106-165 of Mce1A and the residues 347-406, further into the mature Mce1F polypeptide chain . There was a striking, localized homology, indicating that the epitope reacting with the monoclonal antibody TB1-5 76C may be narrowed to the KRRITPKD region, the residues 131-138 in Mce1A corresponding to the residues 372-379 in Mce1F . This was confirmed in enzyme-linked immunosorbent assay, showing binding of TB1-5 76C to a 17-mer synthetic peptide containing the KRRITPKD sequence.

Arch Microbiol, 2002 Dec, 179(1), 50 - 6 Epub 2002 Nov 09.
Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413; Thiel T et al.; Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase . A . variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M . Competition experiments with other oxyanions suggested that the molybdate-transport system of A . variabilis also transported tungstate but not vanadate or sulfate . Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase . Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate . Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase . These data provide evidence for a molybdate storage system in A . variabilis . NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate . The closely related strain Anabaena sp . PCC 7120 transported (99)Mo in a very similar way to A . variabilis.

Pediatr Surg Int, 2002 Oct, 18(7), 595 - 9 Epub 2002 Sep 25.
In-vivo retroviral gene transfer to the liver is cancelled by an immune response against the corrected cells . Can it be avoided?
Podevin G, Pichard V, Durand S, Aubert D, Heloury Y, Ferry N.
Highly efficient retroviral-mediated gene transfer into hepatocytes in vivo has been previously reported in rats, but some reports described transient expression of the transgene that may be related to induction of an immune response against the transgene product . To devise a surgical approach to circumvent this drawback, two-thirds partial hepatectomy was performed in Wistar male rats to induce the hepatocyte division required to achieve retrovirus integration . Delivery of amphotrophic retroviral vectors (RVV) encoding Escherichia coli beta-galactosidase was performed 24 h after partial hepatectomy . In a first group (n = 11), gene delivery was performed by peripheral injection of 2 ml retrovirus-containing medium . For the second group (n = 11), asanguineous perfusion of the regenerating liver after complete vascular exclusion was carried out with 20 ml viral solution . Liver biopsies were performed sequentially in each group . In the first group, beta-galactosidase was expressed at day 7 in 7 +/- 6.3% of hepatocytes and the labeled hepatocytes had disappeared in less than 4 weeks . Polymerase chain reaction experiments demonstrated the elimination of the transduced cells and the appearance of antibodies against beta-galactosidase . Of the 11 rats in the second group, 8 were still able to express beta-galactosidase more than 6 weeks after asanguineous perfusion with no detectable antibody response . Asanguineous perfusion of the regenerating liver with RVV after complete vascular exclusion enabled long-term expression in rats and avoided the immune response present after peripheral delivery in most animals . These results suggest that the immune reaction is secondary to viral infection of antigen presenting cells . Asanguineous perfusion could thus be a way to perform gene therapy for inherited liver diseases without immunosuppressive therapy.

Mol Genet Genomics, 2002 Dec, 268(4), 518 - 24 Epub 2002 Oct 29.
Transcriptional analysis of the pst operon of Escherichia coli; Aguena M et al.; The pst operon of Escherichia coli, which encodes the phosphate-specific transport system, is composed of five genes, pstS, pstC, pstA, pstB and phoU, whose transcription is induced by phosphate starvation . A phosphate-regulated promoter located upstream of the most proximal gene ( pstS) controls the transcription of the entire operon . Though the full-length pst mRNA could be detected by an improved RT-PCR protocol, Northern analysis using several pst-specific probes failed to reveal this transcript . Instead, smaller but distinct pst mRNA species were evident . Primer-extension experiments localized the 5' ends of pst mRNAs within the operon . The data suggest that the full-length mRNA is rapidly processed post-transcriptionally.

J Biol Chem, 2003 Mar 21, 278(12), 10641 - 8 Epub 2002 Dec 05.
Probing the mechanism of a membrane transport protein with affinity inactivators; Guan L et al.; Affinity inactivators are useful for probing catalytic mechanisms . Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli . The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala(122)-->Cys in a background otherwise devoid of Cys residues . A proton electrochemical gradient (Deltamu(H(+))) markedly increases the rate of reaction between Cys(122) and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected . When the Ala(122)-->Cys mutation is combined with a mutation (Cys(154)-->Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and Deltamu(H(+)) has no effect . The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala(122) in helix IV . In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism . Thus, these inactivators behave as unique suicide substrates.

Neurobiol Aging, 2002 Nov-Dec, 23(6), 1023 - 30
gamma-Secretase: characterization and implication for Alzheimer disease therapy; Xu M et al.; gamma-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Abeta peptides which are believed to play a central role in the neuropathology of Alzheimer's disease . An in vitro gamma-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease . Biochemical studies establish that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex . Moreover, the fact that the photoreactive active gamma-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease . Presenilin/gamma-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.

Neurobiol Aging, 2002 Nov-Dec, 23(6), 991 - 1000
Intranasal immunotherapy for the treatment of Alzheimer's disease: Escherichia coli LT and LT(R192G) as mucosal adjuvants; Lemere CA et al.; Alzheimer's disease (AD) is the most common form of dementia worldwide, yet there is currently no effective treatment or cure . Extracellular deposition of amyloid-beta protein (Abeta) in brain is a key neuropathological characteristic of AD . In 1999, Schenk et al . first reported that an injected Abeta vaccine given to PDAPP mice, an AD mouse model displaying Abeta deposition in brain, led to the lowering of Abeta levels in brain . In 2000, we demonstrated that intranasal (i.n.) immunization with human synthetic Abeta1-40 peptide for 7 months led to a 50-60% reduction in cerebral Abeta burden in PDAPP mice; serum Abeta antibody titers were low (approximately 26 microg/ml) . More recently, we have optimized our i.n . Abeta immunization protocol in wild-type (WT) mice . When low doses Escherichia coli heat-labile enterotoxin (LT) were given as a mucosal adjuvant with Abeta i.n., there was a dramatic 12-fold increase in Abeta antibody titers in WT B6D2F1 mice treated two times per week for 8 weeks compared to those of mice receiving i.n . Abeta without adjuvant . A non-toxic form of LT, designated LT(R192G), showed even better adjuvanticity; anti-Abeta antibody titers were 16-fold higher than those seen in mice given i.n . Abeta without adjuvant . In both cases, the serum Abeta antibodies recognized epitopes within Abeta1-15 and were of the immunoglobulin (Ig) isotypes IgG2b, IgG1, IgG2a and low levels of IgA . This new and improved Abeta vaccine protocol is now being tested in AD mouse models with the expectation that higher Abeta antibody titers may be more effective in reducing cerebral Abeta levels.

Hybrid Hybridomics, 2002 Oct, 21(5), 385 - 92
Selection and characterization of human antibodies against hepatitis B virus surface antigen (HBsAg) by phage-display; Kim SH et al.; Hepatitis B virus (HBV) is one of the main pathogens of hepatitis and hepatocarcinoma . Human plasma-derived antibody to HBV is being used as a prophylactic for postexposure to HBV and liver transplantation currently . However, it is required to replace the plasma-derived anti-HBs antibody (Ab) to a recombinant antibody because of limited availability of human plasma with high anti-HBs Ab titer and possible contamination of human pathogens . We constructed an anti-HBs Ab-enriched phage-display library from peripheral blood B cells of vaccinated volunteers and the size of library was approximately 1.0 x 10(7) . The library was panned against hepatitis B surface antigen (HBsAg) and five different clones were isolated . All five clones exhibited the same heavy chain sequence; in contrast, light-chain exhibited one lambda and four different kappa sequences . The Fabs were expressed soluble in E . coli and exhibited affinities of 2.1 x 10(8) approximately 7.7 x 10(8) M(-1).

Hybrid Hybridomics, 2002 Oct, 21(5), 333 - 8
Antibody responses to HPV6b E polyproteins and production of monoclonal antibodies; Pietrzykowski E et al.; A range of fusion constructs (expressed in Escherichia coli) were produced that contained two or more HPV6b E proteins, producing a single continuous amino acid sequence corresponding to the sequences of the individual E proteins . The constructs also included a C-terminal hexahistidine tag fused in-frame to aid purification . The fusion proteins (polyproteins) were semipurified by Ni(++) metal affinity chromatography under denaturing conditions . Immunization of BALB/c mice with these polyproteins resulted in the production of specific E protein antibodies . The draining lymph nodes from these mice were used to produce monoclonal antibodies (MAbs) . The specificity of the polyclonal and MAbs was confirmed by immunoblotting and by screening for reaction with a series of synthetic peptides of E proteins . HPV E polyproteins were found to be immunogenic and immunization with the polyproteins resulted in specific antibody responses to the component E proteins.

Comb Chem High Throughput Screen, 2002 Dec, 5(8), 605 - 11
Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures; Dalvit C et al.; High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection . Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target . A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands . Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits . Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened . Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.

Physiol Res, 2002, 51(5), 523 - 8
Escherichia coli administered into pig amniotic cavity appear in fetal airways and attract macrophages into fetal lungs; Splichal I et al.; Escherichia coli (2 x 10(4) bacteria) of the non-pathogenic O86 strain or enteropathogenic O55 strain were administered into the pig amniotic cavity at 79 to 86 days of gestation for six or ten hours . Translocation of bacteria into fetal lungs was confirmed by cultivation as well as by light and electron microscopy . Infection caused an influx of macrophages that were immunostained in cryostat sections by monoclonal antibody recognizing calprotectin.

J Endod, 2002 Nov, 28(11), 754 - 7
Production of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by human polymorphonuclear neutrophils stimulated with Porphyromonas endodontalis lipopolysaccharide; Ko HJ et al.; This study was undertaken to investigate the capacity of polymorphonuclear neutrophils (PMNs) to secrete Macrophage Inflammatory Protein (MIP)-1alpha and MIP-1beta after stimulation with Porphyromonas endodontalis lipopolysaccharide (LPS) . Escherichia coli LPS was used as a positive control . Venous blood was collected and PMNs were isolated from healthy volunteers . Cells were cultured with various concentrations of LPS for different periods of time . Cell supernatants were assayed by enzyme-linked immunosorbent assay . The levels of chemokine secretion in PMNs stimulated with each LPS were found to be significantly higher than in the unstimulated control cells (p < 0.05), and this expression occurred in a time- and dose-dependent manner . E . coli LPS induced higher levels of cytokines than P . endodontalis LPS . These findings demonstrated that P . endodontalis LPS is capable of stimulating PMNs to produce chemotactic cytokines and suggested that PMNs stimulated with P . endodontalis LPS may play a crucial role in the inflammatory and immunopathological reactions of pulpal and periapical diseases.

Proteomics, 2002 Dec, 2(12), 1682 - 98
Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli; Yan JX et al.; Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics . Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been applied to a model system study of the Escherichia coli proteome after benzoic acid treatment . The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labelling of control and treated samples which are then mixed and run in the same gel . Pooled control and treated samples labelled with Cy trade mark 3 were used as an internal standard for both Cy5 labelled control and treated E . coli samples . Together with DeCyder trade mark imaging analysis software, more accurate quantitative analysis than conventional two-dimensional polyacrylamide gel electrophoresis was achieved . Using matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight mass spectrometry a total of 179 differentially expressed protein spots were identified . These included enzymes, stress related and substrate (e.g . amino acids, maltose, ribose and TRP repressor) binding proteins . Of the spots analysed, 77% contained only one protein species per spot, hence the change in protein expression measured was solely attributed to the identified protein . Many membrane proteins and protein isoforms were identified indicating both adequate solubilization of E . coli samples and potential post-translational modification . The results indicate that the regulatory mechanisms following benzoic acid treatment of E . coli are far more complicated than hitherto expected.

Nat Biotechnol, 2003 Jan, 21(1), 86 - 9 Epub 2002 Dec 09.
A general method for the covalent labeling of fusion proteins with small molecules in vivo; Keppler A et al.; Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function . Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein . Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin . However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function . These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains . Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

J Biol Chem, 2003 Feb 28, 278(9), 7366 - 73 Epub 2002 Dec 04.
Co-repressor release but not ligand binding is a prerequisite for transcription activation by human retinoid acid receptor alpha ligand-binding domain; Kao HY et al.; Nuclear hormone receptors coordinately regulate the activity of genetic networks through the recruitment of transcriptional co-regulators, including co-repressors and co-activators . Allosteric modulation of the ligand-binding domain by hormonal activators shifts the co-factor binding preference by defined structural changes in overlapping docking sites . We report here that mutations at conserved residues within the docking motif of the retinoic acid receptor alpha cause defects in dimerization, co-regulator association, and transcriptional regulation . Furthermore, although a minimal co-repressor receptor interaction domain is sufficient for receptor binding, flanking sequences appear to stabilize this interaction without interfering with ligand sensitivity . However, ligand sensitivity is changed by the K262A mutation, which requires much higher concentrations of all-trans-retinoic acid to promote co-repressor dissociation . Consequently, K262A functions as a dominant-negative mutant at low concentrations of all-trans-retinoic acid . As a result, transcriptional activation is mechanistically linked to co-repressor release.

J Biol Chem, 2003 Jan 31, 278(5), 2773 - 6 Epub 2002 Dec 04.
Leptomycin B, an inhibitor of the nuclear export receptor CRM1, inhibits COX-2 expression; Jang BC et al.; Cyclooxygenase (COX)-2, the inducible prostaglandin synthase, is overexpressed in cancer and chronic inflammatory diseases . Post-transcriptional regulation of COX-2 mRNA is important in controlling the expression of the COX-2 gene . Here, we report that leptomycin B (LMB), a specific inhibitor of the nuclear export factor CRM1 potently inhibits the stabilization of COX-2 mRNA in MDA-MB-231 human mammary cancer cells . However, COX-2 promoter-driven reporter gene expression is not inhibited by LMB, suggesting that LMB acts at the post-transcriptional level . Subcellular fractionation experiments indicate that LMB inhibited the time-dependent export of COX-2 mRNA into the membrane-bound polysomal compartment at the endoplasmic reticulum . LMB suppressed COX-2 expression by interleukin-1beta in HT-29 human colon cancer cells and in human umbilical vein endothelial cells but had no effect on COX-2 expression induced by Escherichia coli lipopolysaccharide in monocytic THP-1 cells . These data suggest that the nuclear export of COX-2 mRNA may be rate-liming in a cell-specific manner . LMB may be useful to control COX-2 expression in various human diseases in which COX-2 plays a pathogenetic role.

J Biol Chem, 2003 Feb 14, 278(7), 4668 - 74 Epub 2002 Dec 04.
The role of C-terminal tyrosine phosphorylation in the regulation of SHP-1 explored via expressed protein ligation; Zhang Z et al.; The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the "negative" regulation of cell signaling . The molecular basis for the regulation of SHP-1 is incompletely understood . Whereas SHP-1 has previously been shown to be phosphorylated on two tail tyrosine residues (Tyr(536) and Tyr(564)) by several protein-tyrosine kinases, the effects of these phosphorylation events have been difficult to address because of the intrinsic instability of the linkages within a protein-tyrosine phosphatase . Using expressed protein ligation, we have generated semisynthetic SHP-1 proteins containing phosphotyrosine mimetics at the Tyr(536) and Tyr(564) sites . Two phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F(2)Pmp) . Incorporation of Pmp at the 536 site led to 4-fold stimulation of the SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect . Incorporation of F(2)Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity and 1.6-fold at the 564 site . A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition . In contrast, tyrosine phosphorylation at the 564 site has the potential to engage the C-Src homology 2 domain intramolecularly, which can modestly and indirectly influence catalytic activity . The finding that phosphonate modification at each of the 536 and 564 sites can promote interaction with the Grb2 adaptor protein indicates that the intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.

J Biol Chem, 2003 Mar 7, 278(10), 8429 - 34 Epub 2002 Dec 04.
Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding; Karpowich NK et al.; BtuF is the periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD, a member of the ATP-binding cassette (ABC) transporter superfamily of transmembrane pumps . We have determined crystal structures of Escherichia coli BtuF in the apo state at 3.0 A resolution and with vitamin B12 bound at 2.0 A resolution . The structure of BtuF is similar to that of the FhuD and TroA PBPs and is composed of two alpha/beta domains linked by a rigid alpha-helix . B12 is bound in the "base-on" or vitamin conformation in a wide acidic cleft located between these domains . The C-terminal domain shares structural homology to a B12-binding domain found in a variety of enzymes . The same surface of this domain interacts with opposite surfaces of B12 when comparing ligand-bound structures of BtuF and the homologous enzymes, a change that is probably caused by the obstruction of the face that typically interacts with this domain by the base-on conformation of vitamin B12 bound to BtuF . There is no apparent pseudo-symmetry in the surface properties of the BtuF domains flanking its B12 binding site even though the presumed transport site in the previously reported crystal structure of BtuCD is located in an intersubunit interface with 2-fold symmetry . Unwinding of an alpha-helix in the C-terminal domain of BtuF appears to be part of conformational change involving a general increase in the mobility of this domain in the apo structure compared with the B12-bound structure . As this helix is located on the surface likely to interact with BtuC, unwinding of the helix upon binding to BtuC could play a role in triggering release of B12 into the transport cavity . Furthermore, the high mobility of this domain in free BtuF could provide an entropic driving force for the subsequent release of BtuF required to complete the transport cycle.

FASEB J, 2002 Dec, 16(14), 1895 - 902
Base excision repair capacity in mitochondria and nuclei: tissue-specific variations; Karahalil B et al.; Base excision repair is the main pathway for repair of oxidative base lesions in DNA . Mammalian cells must maintain genomic stability in their nuclear and mitochondrial genomes, which have different degrees of vulnerability to DNA damage . This study quantifies DNA glycosylase activity in mitochondria and nucleus from C57/BL 6 mouse tissues including brain, liver, heart, muscle, kidney, and testis . The activities of oxoguanine DNA glycosylase (OGG1), uracil DNA glycosylase, and endonuclease III homologue 1 (NTH1) were measured using oligonucleotide substrates with DNA lesions specific for each glycosylase . Mitochondrial content was normalized to citrate synthase activity and mitochondrial function was assessed by measuring cytochrome c oxidase (COX) activity . In nuclear and mitochondrial extracts, the highest DNA glycosylase activities were in testis . Brain and heart, tissues with the highest oxidative load, did not have higher levels of OGG1 or NTH1 activity than muscle or kidney, which are more glycolytic tissues . In general, mitochondrial extracts have lower DNA glycosylase activity than nuclear extracts . There was no correlation between glycosylase activities in the mitochondrial extracts and COX activity, suggesting that DNA repair enzymes may be regulated by a mechanism different from this mitochondrial enzyme.

J Theor Biol, 2003 Jan 21, 220(2), 261 - 9
The role of dimerization in noise reduction of simple genetic networks; Bundschuh R et al.; Fluctuations are an intrinsic property of genetic networks due to the small number of interacting molecules . We study the role of dimerization reactions in controlling these fluctuations in a simple genetic circuit with negative feedback . We compare two different pathways . In the dimeric pathway the proteins to be regulated form dimers in solution that afterward bind to an operator site and inhibit transcription . In the monomeric pathway monomers bind to the operator site and then recruit another monomer to form a dimer directly on the DNA . We find that while both pathways implement the same negative feedback mechanism, the protein number fluctuations in the dimeric pathway are drastically reduced compared to the monomeric pathway . This difference in the ability to reduce fluctuations may be of importance in the design of genetic networks.

Prostaglandins Leukot Essent Fatty Acids, 2002 Dec, 67(6), 365 - 72
Effect of NO synthase inhibition on cardiovascular and pulmonary dysfunction in a porcine short-term model of endotoxic shock; Albertini M et al.; In a porcine model of endotoxic shock, we evaluated the circulatory and respiratory effects of NO synthase (NOS) blockade . Twenty anaesthetised pigs were divided into three groups and studied for 240 min after induction of endotoxic shock with lipopolysaccharides of Escherichia coli (LPS) . After 180 min of endotoxic shock, one group (n = 6) received aminoguanidine, another group (n = 6) received N(G)-nitro-L -arginine methyl ester (L -NAME) and a third group (n = 8) received only LPS . A sham group (n = 3) was also studied . LPS decreased systemic arterial pressure and cardiac output (CO) and increased mean pulmonary arterial pressure (MPAP), pulmonary vascular resistance (PVR) and heart rate . Significant changes were also observed in compliance (-18.4%) and resistance (+33.6%) of the respiratory system . Aminoguanidine did not modify LPS-dependent effects, while, after L -NAME, a significant increase in MPAP, PVR and SVR and a decrease in CO were observed . In conclusion, aminoguanidine does not play a significant cardiocirculatory and pulmonary role in the short-term dysfunction of endotoxic shock, while L -NAME has a detrimental effect on haemodynamics, suggesting a protective role of constitutive NO production at vascular level during the early stages of endotoxaemia.

Gene, 2002 Oct 30, 300(1-2), 3 - 11
The dawn of gene isolation; Birnstiel ML; Ever since it became clear through the work of Watson and Crick that the gene is a stretch of double stranded helical DNA and is understandable in chemical terms, biochemists have striven to get their hands on isolated genes . The isolation of the ribosomal genes of Xenopus laevis in 1966 provided a first instance where a purified DNA of known function could be investigated, long before the advent of gene cloning technologies . The second instance was the purification of the Lac operon from Escherichia coli . Later, but still before the gene cloning days the 5S RNA genes of X . laevis and the histone genes of the sea urchin Psammechinus miliaris were isolated by physico-chemical methods, but their isolation marked the end of an era . By 1975, gene cloning technology was well established and the isolation of genes quickly became an everyday occurrence.

Mol Biochem Parasitol, 2002 Nov-Dec, 125(1-2), 47 - 57
Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA; Valadao AF et al.; A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli . In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays . SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA . The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns . Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System . The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S . mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S . mansoni p14 gene promoter . None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.

Mol Biochem Parasitol, 2002 Nov-Dec, 125(1-2), 1 - 9
Rapamycin insensitivity in Schistosoma mansoni is not due to FKBP12 functionality; Rossi A et al.; Rapamycin (RAPA) is a well-known immunosuppressant, the action of which is mediated by the immunophilin FKBP12 . Upon RAPA binding, FKBP12 forms ternary complexes with phosphatidyl inositol related kinases known as the target of RAPA (TOR), which can lead to a mitotic block at the G1-S phase transition . Such an antiproliferative effect makes RAPA an attractive anticancer, antifungal or antiparasitic compound . In this study, we found the helminth parasite Schistosoma mansoni to be insensitive to the drug . In order to elucidate the mechanism underlying RAPA resistance, the S . mansoni drug receptor FKBP12 (SmFKBP12) was cloned for functional analysis . Western blot experiments showed that the protein is constitutively expressed in all life cycle stages and in both male and female parasites . The Escherichia coli-synthesised recombinant protein possessed enzymatic activity, which was inhibitable by RAPA . Moreover, SmFKBP12 was able to complement mutant Saccharomyces cerevisiae cells lacking FKBP12 in their RAPA sensitivity phenotype, leading us to conclude that SmFKBP12 is expressed in yeast in a functional form and capable of interacting with the drug and yeast TOR kinase . Even though the wild type SmFKBP12 appeared to restore a large part of RAPA sensitivity, a mutation of Asp(89)-Lys(90) to Pro(89)-Gly(90) in the schistosome protein was found to be more effective and restored drug sensitivity to the same level as the endogenous yeast protein . Despite ternary complex formation, our results suggest that additional unknown factors other than a functional drug receptor are implicated in drug resistance mechanisms.

Bioorg Med Chem, 2003 Jan 2, 11(1), 43 - 52
Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase; DeSantis G et al.; 2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate . It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes . In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated . Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose . Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose . Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor . This azidoaldehyde is not a substrate for the wild-type enzyme . Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates . An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones . In addition, to facilitate the discovery of new enzymatic reactions, the engineered E . coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1318 - 23
Adsorption of cadmium ion and gallium ion to immobilized metallothionein fusion protein; Terashima M et al.; A fusion protein made from maltose binding protein (pmal) and human metallothionein (MT) was expressed using E . coli . The purified recombinant protein (pmal-MT) was immobilized on Chitopearl resin, and characteristics of pmal-MT for metal binding were evaluated . As expected from the tertiary structure of metallothionein, the pmal-MT ligand adsorbed 12.1 cadmium molecules per one molecule of the ligand at pH 5.2 . The pmal-MT ligand also bound 26.6 gallium molecules per one molecule of the ligand at pH 6.5 . Neither cadmium ion nor gallium ion bound to a control protein bovine serum albumin (BSA) . Adsorption isotherms for both ions were correlated by Langmuir-type equations . Two types of binding sites have been elucidated on the basis of HSAB (hard and soft acid and base) theory . It was suggested that gallium ion specifically binds to amino acid residues containing oxygen and nitrogen atoms, while cadmium ion binds to specific binding sites formed by multiple cysteine residues . The pmal-MT ligand bound these metals in the concentration range of 0.2-1.0 mM, and the bound metal ions could be eluted under relatively mild conditions (pH 2.0) . The pmal-MT Chitopearl resin was stable and could be used repeatedly without loss of binding activity . Thus, this new ligand would be useful for recovery of toxic heavy metals and/or valuable metal ions from various aqueous solutions.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1176 - 82
Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein; Yilmaz JL et al.; In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine . To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E . coli and in Nicotiana tabacum . The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured . The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition) . At high choline concentrations, E . coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only . The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity . Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated . On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.

J Org Chem, 2002 Dec 13, 67(25), 8890 - 4
Rapid one-pot synthesis of riboflavin isotopomers; Romisch W et al.; Flavocoenzymes labeled with stable isotopes are important reagents for the study of flavoproteins using isotope-sensitive methods such as NMR, ENDOR, infrared, and Raman spectroscopy . We describe highly versatile one-pot methods for the preparation of riboflavin isotopomers labeled with (13)C in every desired position of the xylene moiety . The starting materials are commercially available (13)C-labeled glucose samples, which are converted into riboflavin using enzymes of the oxidative pentose phosphate pathway in combination with recombinant enzymes of the riboflavin biosynthetic pathway . The overall reaction comprises six enzyme-catalyzed reaction steps for the synthesis of the vitamin and two auxiliary enzymes for in situ recycling of cofactors . The overall yields of riboflavin based on isotope-labeled glucose are 35-50%.

J Org Chem, 2002 Dec 13, 67(25), 8847 - 54
Synthesis of helix 69 of Escherichia coli 23S rRNA containing its natural modified nucleosides, m(3)Psi and Psi; Chui HM et al.; The synthesis of 3-methylpseudouridine (m(3)Psi) phosphoramidite, 5'-O-{benzhydryloxybis(trimethylsilyloxy)silyl}-2'-O-{bis(2-acetoxyethoxy)methyl}-3-methylpseudouridine-3'-(methyl-N,N-diisopropyl)phosphoramidite, is reported . Selective pivaloyloxymethyl protection of the Psi N1 followed by methylation at N3 was used to generate the naturally occurring pseudouridine analogue . The m(3)Psi phosphoramidite was used in combination with pseudouridine (Psi) and standard base phosphoramidites to synthesize a 19-nucleotide RNA representing helix 69 of Escherichia coli 23S ribosomal RNA (rRNA) (residues 1906-1924), containing a single m(3)Psi at position 1915 and two Psi's at positions 1911 and 1917 . Our synthesis of the fully modified helix 69 RNA demonstrates the ability to make milligram quantities of RNA that can be used for further high-resolution structure studies . Site-selective introduction of the methyl group at the N3 position of pseudouridine at position 1915 causes a slight increase in the thermodynamic stability of the RNA hairpin relative to pseudouridine; RNAs containing either uridine or 3-methyluridine at position 1915 have similar stability . One-dimensional imino proton NMR and circular dichroism spectra of the modified RNAs reveal that the methyl group does not cause any substantial changes in the RNA hairpin structure.

Nucleic Acids Res . 2002 Dec 1;30(23):e135.
Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry; Mengel-Jorgensen J et al.; Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis . We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines . The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1 . Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da . The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment . The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNA(TyrII) from Escherichia coli . Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2'-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications.

Nucleic Acids Res . 2002 Dec 1;30(23):e128.
Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery; Ilegems E et al.; A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells . It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli . Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA . Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy . Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases . This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types.

Nucleic Acids Res, 2002 Dec 1, 30(23), 5074 - 86
Localisation of the human hSuv3p helicase in the mitochondrial matrix and its preferential unwinding of dsDNA; Minczuk M et al.; We characterised the human hSuv3p protein belonging to the family of NTPases/helicases . In yeast mitochondria the hSUV3 orthologue is a component of the degradosome complex and participates in mtRNA turnover and processing, while in Caenorhabditis elegans the hSUV3 orthologue is necessary for viability of early embryos . Using immunofluorescence analysis, an in vitro mitochondrial uptake assay and sub-fractionation of human mitochondria we show hSuv3p to be a soluble protein localised in the mitochondrial matrix . We expressed and purified recombinant hSuv3p protein from a bacterial expression system . The purified enzyme was capable of hydrolysing ATP with a K(m) of 41.9 micro M and the activity was only modestly stimulated by polynucleotides . hSuv3p unwound partly hybridised dsRNA and dsDNA structures with a very strong preference for the latter . The presented analysis of the hSuv3p NTPase/helicase suggests that new functions of the protein have been acquired in the course of evolution.

J Gen Virol, 2002 Dec, 83(Pt 12), 3035 - 43
Structural relationship between nucleocapsid-binding activity of the rabies virus phosphoprotein (P) and exposure of epitope 402-13 located at the C terminus; Toriumi H et al.; The structural changes of the nominal phosphoprotein (P) of rabies virus using a monoclonal antibody, mAb #402-13, was investigated . This mAb recognized a linear epitope that was mapped roughly to a C-terminal region of the P protein, ranging from aa 256 to 297 . The P gene products were detected by the mAb in immunoblot assays, the products of which were produced either in BHK-21 cells or in Escherichia coli cells . The mAb, however, detected very low levels of P gene products in immunoprecipitation assays . The mAb recognized the nucleocapsid (NC)-associated P proteins but recognized free P protein and free N-P complex produced in the infected cells much less efficiently . When the P proteins were released from the NC, however, they were no longer recognized by the mAb . Similar results were obtained from BHK-21 cells co-transfected with P and N cDNAs . Furthermore, studies with C-terminally truncated P protein mutants revealed that the NC-binding ability of the P protein was dependent on the presence of the C-terminal epitope region . From these results, it is thought that the 402-13 epitope region is concealed when the P protein is present in a free form or free N-P complex but is exposed when it is associated with the NC . The C-terminal epitope region seemed to be essential for the P protein to be associated with the NC but not for the formation of free N-P complexes with newly synthesized N protein.

J Med Microbiol, 2002 Dec, 51(12), 1071 - 9
Recombinant GroES in combination with CpG oligodeoxynucleotides protects mice against Mycobacterium avium infection; Fattorini L et al.; The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and the recombinant GroES protein was purified by affinity chromatography . The GroES preparation showed high purity by electrophoresis and immunoblotting . Immuno-electron microscopy showed that GroES was located both in the cytoplasm and on the surface of the mycobacterial cells and thus is readily available to interact with the host immune system . BALB/c mice were immunised intranasally with recombinant GroES, alone or in combination with a synthetic oligodeoxynucleotide containing unmethylated CpG motifs, and tested for protection against infection with M . avium . Neither GroES nor CpG alone provided any protection against subsequent challenge with M . avium, whereas a combination of the two significantly protected the lungs and spleen against colonisation by M . avium after intranasal challenge with a low dose of the organism . This indicates that intranasal administration of GroES and CpG oligodeoxynucleotides increases the resistance of BALB/c mice to M . avium infection.

J Biol Chem, 2003 Feb 21, 278(8), 5685 - 93 Epub 2002 Dec 03.
Selective strand annealing and selective strand exchange promoted by the N-terminal domain of hepatitis delta antigen; Huang ZS et al.; We have previously shown that the N-terminal domain of hepatitis delta virus (NdAg) has an RNA chaperone activity in vitro (Huang, Z . S., and Wu, H . N . (1998) J . Biol . Chem . 273, 26455-26461) . Here we investigate further the basis of the stimulatory effect of NdAg on RNA structural rearrangement: mainly the formation and breakage of base pairs . Duplex dissociation, strand annealing, and exchange of complementary RNA oligonucleotides; the hybridization of yeast U4 and U6 small nuclear RNAs and of hammerhead ribozymes and cognate substrates; and the cis-cleavage reaction of hepatitis delta ribozymes were used to determine directly the role of NdAg in RNA-mediated processes . The results showed that NdAg could accelerate the annealing of complementary sequences in a selective fashion and promote strand exchange for the formation of a more extended duplex . These activities would prohibit NdAg from modifying the structure of a stable RNA, but allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a more extensively matched target in cognate substrate . These and other results suggest that hepatitis delta antigen may have a biological role as an RNA chaperone, modulating the folding of viral RNA for replication and transcription.

J Biol Chem, 2003 Feb 21, 278(8), 5694 - 701 Epub 2002 Dec 03.
Salt dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA polymerases; Datta K et al.; DNA binding properties of the Type 1 DNA polymerases from Thermus aquaticus (Taq, Klentaq) and Escherichia coli (Klenow) have been examined as a function of {KCl} and {MgCl(2)} . Full-length Taq and its Klentaq "large fragment" behave similarly in all assays . The two different species of polymerases bind DNA with sub-micromolar affinities in very different salt concentration ranges . Consequently, at similar {KCl} the binding of Klenow is approximately 3 kcal/mol (150x) tighter than that of Taq/Klentaq to the same DNA . Linkage analysis reveals a net release of 2-3 ions upon DNA binding of Taq/Klentaq and 4-5 ions upon binding of Klenow . DNA binding of Taq at a higher temperature (60 degrees C) slightly decreases the ion release . Linkage analysis of binding versus {MgCl(2)} reports the ultimate release of approximately 1 Mg(2+) ion upon complex formation . However, the MgCl(2) dependence for Klenow, but not Klentaq, shows two distinct phases . In 10 mm EDTA, both polymerase species still bind DNA, but their binding affinity is significantly diminished, Klenow more than Klentaq . In summary, the two polymerase species, when binding to identical DNA, differ substantially in their sensitivity to the salt concentration range, bind with very different affinities when compared under similar conditions, release different numbers of ions upon binding, and differ in their interactions with divalent cations.

J Biol Chem, 2003 Feb 14, 278(7), 4932 - 42 Epub 2002 Dec 03.
Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica; Waditee R et al.; Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses . Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica . One of gene products (ORF1) catalyzed the methylation reactions of glycine and sarcosine with S-adenosylmethionine acting as the methyl donor . The other one (ORF2) specifically catalyzed the methylation of dimethylglycine to betaine . Both enzymes are active as monomers . Betaine, a final product, did not show the feed back inhibition for the methyltransferases even in the presence of 2 m . A reaction product, S-adenosyl homocysteine, inhibited the methylation reactions with relatively low affinities . The co-expressing of two enzymes in Escherichia coli increased the betaine level and enhanced the growth rates . Immunoblot analysis revealed that the accumulation levels of both enzymes in A . halophytica cells increased with increasing the salinity . These results indicate that A . halophytica cells synthesize betaine from glycine by a three-step methylation . The changes of amino acids Arg-169 to Lys or Glu in ORF1 and Pro-171 to Gln and/or Met-172 to Arg in ORF2 significantly decreased V(max) and increased K(m) for methyl acceptors (glycine, sarcosine, and dimethylglycine) but modestly affected K(m) for S-adenosylmethionine, indicating the importance of these amino acids for the binding of methyl acceptors . Physiological and functional properties of methyltransferases were discussed.

J Biol Chem, 2003 Feb 14, 278(7), 4654 - 9 Epub 2002 Dec 03.
Signal recognition particle (SRP)-mediated targeting and Sec-dependent translocation of an extracellular Escherichia coli protein; Sijbrandi R et al.; Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenic Escherichia coli strain via the autotransporter mechanism . Little is known about the earliest steps in autotransporter secretion, i.e . the targeting to and translocation across the inner membrane . Here, we present evidence that Hbp interacts with the signal recognition particle (SRP) and the Sec-translocon early during biogenesis . Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane . SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP . Hbp is synthesized with an unusually long signal peptide that is remarkably conserved among a subset of autotransporters . We propose that these autotransporters preferentially use the co-translational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.

J Am Chem Soc, 2002 Dec 11, 124(49), 14616 - 25
Side chain orientation from methyl 1H-1H residual dipolar couplings measured in highly deuterated proteins; Sibille N et al.; High-level deuteration is a prerequisite for the study of high molecular weight systems using liquid-state NMR . Here, we present new experiments for the measurement of proton-proton dipolar couplings in CH(2)D methyl groups of (13)C labeled, highly deuterated (70-80%) proteins . (1)H-(1)H residual dipolar couplings (RDCs) have been measured in two alignment media for 57 out of 70 possible methyl containing residues in the 167-residue flavodoxin-like domain of the E . coli sulfite reductase . These data yield information on the orientation of the methyl symmetry axis with respect to the molecular alignment frame . The alignment tensor characteristics were obtained very accurately from a set of backbone RDCs measured on the same protein sample . To demonstrate that accurate structural information is obtained from these data, the measured methyl RDCs for Valine residues are analyzed in terms of chi(1) torsion angles and stereospecific assignment of the prochiral methyl groups . On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines, assuming a single-rotamer model . Methyl RDCs are complementary to other NMR data, for example, methyl-methyl NOE, to determine side chain conformation in high molecular weight systems.

J Am Chem Soc, 2002 Dec 11, 124(49), 14586 - 90
Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs; Taki M et al.; Four-base codon strategy was applied to incorporate a fluorophore-quencher pair into specific positions on a single protein; beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap) was employed as a fluorophore and p-nitrophenylalanine (ntrPhe) as a quencher . Their positions were directed by the CGGG/CCCG and GGGC/CCCG four-base codon/anticodon pairs and two doubly mutated streptavidins, i.e., ((52)atnDap, (84)ntrPhe) and ((54)ntrPhe, (84)atnDap) mutants were synthesized through Escherichia coli in vitro protein synthesizing systems . Intramolecular photoinduced electron transfer (ET) was observed as the decrease of intensity in steady-state fluorescence spectroscopy and as the shortening of fluorescence decaytimes . The quenching data indicated that the ET rate reflects the detailed structure of the protein.

Chembiochem, 2002 Dec 2, 3(12), 1242 - 50
Stereochemical course of Escherichia coli RNase H; Krakowiak A et al.; A new enzymatic method has allowed the assignment of the stereochemistry of E . coli RNase-H-assisted hydrolysis of RNA labelled within the scissile bond with (R(p))-phosphorothioate . This method is based on a stereospecific, two-step enzymatic conversion of cytidine 5'-{(18)O}phosphorothioate into the corresponding 5'-alpha-{(18)O}thiotriphosphate, which is then further used for stereospecific transfer of cytidine 5'-{(18)O}phosphorothioate to the 3'-OH group of a short oligonucleotide with the aid of terminal deoxyribonucleotidyl transferase . Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of the resulting elongated primer revealed that RNase-H-assisted hydrolysis proceeds with inversion of configuration at the phosphorus atom . This result is discussed in the context of current knowledge of the architecture of the active site of the enzyme.

Can J Gastroenterol, 2002 Nov, 16(11), 771 - 8
Enteropathogenic and enterohemorrhagic Escherichia coli infections: emerging themes in pathogenesis and prevention; Vallance BA et al.; Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E coli (EHEC) are important causes of infectious diarrhea, particularly among pediatric populations . While EPEC is a significant health threat in the developing world, EHEC causes sporadic but deadly outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome in North America and other developed areas . The present review discusses emerging themes in the pathogenesis of EPEC and EHEC, including the discovery and characterization of novel bacterial proteins that are injected by the pathogen into host cells . Recent advances have also been made in the development of relevant animal models, while bacterial virulence factors are being investigated as potential vaccination targets for humans and animals . It is hoped that these new areas of study will not only further our knowledge of the pathogenesis of EPEC- and EHEC-induced disease but also provide opportunities for reducing infection rates and improving treatment options in the future.

Osteoarthritis Cartilage, 2002 Dec, 10(12), 961 - 7
An in-vitro screening assay for the detection of inhibitors of proinflammatory cytokine synthesis: a useful tool for the development of new antiarthritic and disease modifying drugs; Laufer S et al.; OBJECTIVE: This work targets the development of a new tool to help develop new anticytokine drugs that prevent or reduce the progression of arthritic diseases . The specific aim of our study was to establish a fast and reliable in vitro screening assay of cytokine synthesis inhibitors (TNFalpha, IL-1beta) which shows better correlation with enzyme assays than previously reported in vitro assays . The test system should be able to detect p38-MAP kinase inhibitors . MATERIAL AND METHODS: Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from human EDTA-potassium whole blood . Cells were adjusted at 1 x 10(6) cells/ml . PBMCs were stimulated with lipopolysaccharide (LPS; E . coli serotype 026:B6: 1 microg/ml) in the presence of test compound (10(-5)-10(-8)M) for 4h at 37 degrees C in a 5% CO(2)-incubator . Induced TNFalpha and IL-1beta protein were measured by ELISA . RESULTS: The following are representative examples of inhibitors which effect cytokine synthesis . Corticoid Dexamethasone inhibits IL-1beta and TNFalpha synthesis at IC(50) of 38 nM and 25 nM, respectively . ERK1/ERK2 inhibitor U0126 effects cytokine synthesis at IC(50) of 0.34 microM for IL-1beta production and 0.26 microM for TNFalpha synthesis.p38-MAP kinase inhibitor SB 203580 inhibits IL-1beta- and TNF-alpha-synthesis (IC(50)sof 0.052 microM and 0.46 microM) in the same degree as p38-MAP kinase activity (IC(50): 0.34 microM) . Same results could be shown for SB 210313, which had same efficacy on IL-1beta and TNFalpha biosynthesis (IC(50)'s: 1.88 microM and 1.01 microM) and on p38-MAP kinase (IC(50): 6.85 microM) . Also for SB 202190 this correlation in inhibition of IL-1beta and TNFalpha synthesis (IC(50)'s: 0.055 microM and 1.01 microM) and p38-MAP kinase inhibition (IC(50): 0.088 microM) could be shown . CONCLUSION: This study shows the screening assay using PBMCs stimulated with LPS for IL-1beta and TNFalpha synthesis is a reliable test system for the quantification of the effectiveness of new drugs modulating IL-1beta and TNFalpha synthesis which is mainly mediated by p38-MAP Kinase . These assay allows fast detection of IL-1beta and TNFalpha synthesis inhibitors with different modes of action, including p38-MAP kinase inhibitors . The results obtained with our in-vitro screening assay show good correlation with results from enzyme assays .

Int J Parasitol, 2002 Dec 19, 32(14), 1739 - 46
Cloning and characterisation of a highly immunoreactive 37 kDa antigen with multi-immunoglobulin domains from the swine roundworm Ascaris suum; Tsuji N et al.; Antigens from larval stages of Ascaris suum have been shown to induce protection against challenge infection with infective A . suum eggs . We previously identified several antigens that reacted strongly with serum from pigs inoculated with infective eggs containing L3 . In this study, we isolated an antigen with a molecular mass of 37 kDa and a pI of 4.8 (As37) from A . suum infective eggs using two-dimensional electrophoresis, and obtained a full-length cDNA by reverse transcription-polymerase chain reaction using primers designed based on the internal amino acid sequence of As37 . The cDNA sequence consisted of 1,540 bp coding for a protein of 321 amino acids with a complex domain organisation . Simple modular architecture research tool (SMART) analysis indicated that As37 contains three immunoglobulin domains, indicating that it is a member of immunoglobulin superfamily (IgSF) . A homology search of GenBank showed that As37 has significant similarity to Caenorhabditis elegans DIM-1 protein and has low similarity to part of the multi-repeat Ig domain from nematode twitchin and mammalian skeleton muscle titin, and to members of the IgSF at the amino acid sequence level . Localisation analysis revealed that antibodies to Escherichia coli-expressed recombinant As37 (rAs37) bound to muscle cells and the hypodermis . The antibodies identified a 37 kDa native antigen in human and dog roundworms, suggesting that there are As37 homologues in ascarid nematodes . Sera from mice, rabbits and pigs immunised with A . suum infective eggs reacted with rAs37 in immunoblot analyses . The potential use of rAs37 for protection against A . suum infection is discussed.

Int J Parasitol, 2002 Dec 19, 32(14), 1683 - 92
Identification of a second proliferating cell nuclear antigen in the human malarial pathogen Plasmodium falciparum; Li JL et al.; Proliferating cell nuclear antigen seems to exist as a single form in higher eukaryotic cells and plays multiple roles in nucleic acid metabolism . We have identified a second additional proliferating cell nuclear antigen (PfPCNA2) in Plasmodium falciparum on the basis of several lines of evidence . (1) PfPCNA2, consisting of 264 amino acid residues with a predicted molecular mass of 30.2kDa, shares only 29% identity and 53% similarity with PfPCNA1 at the amino acid level . (2) Southern blot analyses revealed that the hybridisation pattern of the Pfpcna2 gene is completely different from that of the Pfpcna1 gene . (3) Chromosomal localisation studies showed that Pfpcna2 is located on chromosome 12 while Pfpcna1 is located on chromosome 13 . Northern blot analyses revealed two different transcripts of Pfpcna2, one expressed in both asexual and sexual erythrocytic stages, while the other existed only in the sexual stage, implying that PfPCNA2 may play multiple roles in DNA metabolism in different stages of the parasite . Recombinant protein of PfPCNA2, overexpressed in Escherichia coli, has been purified to near homogeneity and shown to form an oligomer, probably a trimer, as revealed by a size-exclusion chromatography and a native gel electrophoresis, suggesting that PfPCNA2, like its higher eukaryotic counterparts, may serve as a sliding platform which is capable of interaction with diverse proteins and regulation of their activities.

Arch Biochem Biophys, 2003 Jan 1, 409(1), 235 - 41
CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime; Naur P et al.; The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime . Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications . The native construct gave the highest yield as estimated by enzymatic activity per liter of culture . Spheroplasts of E . coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E . coli to obtain enzymatic activity . This indicates that the E . coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity . Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1) . The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A . thaliana, and 96% to CYP79B5 (Accession No . AF453287) from Brassica napus . The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.

Arch Biochem Biophys, 2003 Jan 1, 409(1), 102 - 12
Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy; Niaura G et al.; Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates . In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine . About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam . Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding . Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation . This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.

Arch Biochem Biophys, 2003 Jan 1, 409(1), 92 - 101
Analysis of homotropic and heterotropic cooperativity of diazepam oxidation by CYP3A4 using site-directed mutagenesis and kinetic modeling; He YA et al.; The structural basis for the cooperativity of diazepam oxidation catalyzed by human cytochrome P450 3A4 (CYP3A4) and 40 mutants has been investigated . An ordered two-site model in which substrates bind first to a catalytic/effector site and then to the catalytic site was used to explain sigmoidal kinetics for temazepam formation but hyperbolic kinetics for nordiazepam formation . In this model diazepam binds to the enzyme-substrate complex with a greater affinity (K(S2)=140 microM) than to free enzyme (K(S1)=960 microM) . Residues 107, 119, 211, 301, 304, 309, 369, 370, and 373 play an important role in determining regioselectivity of diazepam oxidation . Interestingly, S119F and A370F displayed sigmoidal kinetics for nordiazepam formation, whereas I301F exhibited hyperbolic kinetics for both products . In the presence of increasing concentrations of testosterone, K(S1) for diazepam decreased, whereas K(S2) increased . The data suggest that three sites exist within the active pocket.

Anim Genet, 2002 Dec, 33(6), 441 - 7
Fine-mapping of the intestinal receptor locus for enterotoxigenic Escherichia coli F4ac on porcine chromosome 13; Python P et al.; The aim of this study was to refine the localization of the receptor locus for fimbriae F4ac . Small intestinal enterocyte preparations from 187 pigs were phenotyped by an in vitro adhesion test using two strains of Escherichia coli representing the variants F4ab and F4ac . The three-generation pedigree comprised eight founders, 18 F1 and 174 F2 animals, for a total of 200 pigs available for the linkage analysis . Results of the adhesion tests on 171 F2 pigs slaughtered at 8 weeks of age show that 23.5% of the pigs were adhesive for F4ab and non-adhesive for F4ac (phenotype F4abR+/F4acR-; R means receptor) . Pigs of this phenotype were characterized by a weak adhesion receptor for F4ab . No pigs were found expressing only F4acR and lacking F4abR . Receptors for F4ab and F4ac (F4abR+/F4acR+) were expressed by 54.5% of the pigs . Animals of this phenotype strongly bound both F4ab and F4ac E . coli . In the segregation study, the serum transferrin (TF) gene and 10 microsatellites on chromosome 13 were linked with F4acR (recombination fractions (theta) between 0.00 and 0.11 and lod score values (Z) between 11.4 and 40.4) . The 11-point analysis indicates the F4acR locus was located in the interval S0068-Sw1030 close to S0075 and Sw225, with recombination fractions (theta) of 0.05 between F4acR and S0068, 0.04 with Sw1030, and 0.00 with S0075 and Sw225 . The lack of pigs displaying the F4abR-/F4acR+ phenotype and the presence of two phenotypes for F4abR (a strong receptor present in phenotype F4abR+/F4acR+ and a weak receptor in phenotype F4abR+/F4acR-) led us to conclude that the receptor for F4ac binds F4ab bacteria as well, and that it is controlled by one gene localized between S0068 and Sw1030 on chromosome 13.

Biochemistry, 2002 Dec 10, 41(49), 14552 - 9
Participation of active-site carboxylates of Escherichia coli DNA polymerase I (Klenow fragment) in the formation of a prepolymerase ternary complex; Gangurde R et al.; We have investigated the roles of four active-site carboxylates in the formation of a prepolymerase ternary complex of Escherichia coli DNA polymerase I (Klenow fragment), containing the template-primer and dNTP . The analysis of nine mutant enzymes with conserved and nonconserved substitutions of Asp(705), Glu(710), Asp(882), and Glu(883) clearly shows that both catalytically essential aspartates, Asp(705) and Asp(882), are required for the formation of a stable ternary complex . Of the two glutamates, only Glu(710) is required for ternary complex formation, while Glu(883) does not participate in this process . This investigation also reveals two interesting properties of the Klenow fragment with regard to enzyme-template-primer binary and enzyme-template-primer-dNTP ternary complex formation . These are (a) the significant resistance of enzyme-template-primer-dNTP ternary complexes to the addition of high salt or template-primer challenge and (b) the ability of the Klenow fragment to form ternary complexes in the presence of noncatalytic divalent cations such as Ca(2+), Co(2+), Ni(2+), and Zn(2+).

Biochemistry, 2002 Dec 10, 41(49), 14472 - 81
Fluorescence anisotropy studies of enzyme-substrate complex formation in stearoyl-ACP desaturase; Haas JA et al.; Stearoyl-acyl carrier protein Delta(9)-desaturase (delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein . Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced delta9D . Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting delta9D was observed with K(D1) = 13 +/- 3 nM . No significant difference in the K(D1) value was observed for 4e(-) delta9D . An approximately 4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0 . In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting delta9D was estimated to have K(D2) approximately 350 +/- 40 nM . The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values . The k(off) values measured by stopped-flow anisotropy measurements for reversal of the enzyme-substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-)(1)) relative to 18:0-ACP (1 s(-)(1)) . Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values . These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme-substrate complex between substrate release and subsequent steps in catalysis.

Vet J, 1997 Mar, 153(2), 163 - 9
Evaluation of a glutamine-containing oral rehydration solution for the treatment of calf diarrhoea using an Escherichia coli model; Brooks HW et al.; A high-calorie oral rehydration solution (ORS) with glutamine (n=11) was more effective in correcting plasma, extracellular fluid and blood volume than solutions without (one WHO-type solution, n=6, and two high-glucose but glutamine-free solutions, n=7, n=12) . It was the only solution to improve plasma volume significantly within 48 h and sustain the improvement throughout treatment; similarly, it was the only solution to correct packed-cell volume within 48 h and sustain the benefit to the end of treatment . At the end of treatment, the glutamine-treated calves were the only ones to avoid a significant weight loss compared with their pre-diarrhoeic values . The crucial difference between this solution and those used with glutamine previously is that it gave significant nutritional support whereas WHO type solutions did not . It also had more favourable effects on hyponatraemia and metabolic acidosis than a standard ORS . Use of a high-calorie ORS for 4 days (rather than 2 days of 50:50 admixture with milk replacer) brought additional beneficial effects on blood glucose and body weight.

J Anim Sci, 2002 Nov, 80(11), 2895 - 903
Response of early-weaned pigs to spray-dried porcine or animal plasma-based diets supplemented with egg-yolk antibodies against enterotoxigenic Escherichia colil; Owusu-Asiedu A et al.; Two experiments involving 168 10-d-old weaned pigs were conducted to compare growth-promoting properties of dietary spray-dried animal plasma (SDAP), spray-dried porcine plasma (SDPP), and chicken egg-yolk antibodies (EYA) or egg-yolk powder (EYP, contains no specific antibodies) from d 0 to 14 postweaning . In Exp . 1, 96 pigs (3.2 +/- 0.2 kg BW) were used to test the hypothesis that the superior performance of piglets fed SDPP-based diets was partly due to the presence of specific antibodies against enterotoxigenic Escherichia coli (ETEC), which could be replaced with EYA . Four experimental diets in a completely randomized design and arranged in a 2 x 2 factorial (SDPP without or with autoclaving {AuSDPP} and without {EYP} or with supplementation of EYA) were used . Autoclaving SDPP at 121degrees C for 15 min completely destroyed anti-K88/F18 antibodies . Overall feed intake and gain:feed ratio were similar (P > 0.05) among treatments and averaged 122.7 g/d and 0.688, respectively . However, pigs fed AuSDPP+EYP diets had poorer (P < 0.001) ADG compared with those fed SDPP+EYP or SDPP+EYA from 0 to 14 d . Scours were four times higher (P < 0.05) for treatment AuSDPP+EYP compared with all other treatments . Plasma urea nitrogen concentration was higher (P < 0.05) in AuSDPP+EYP- and AuSDPP+EYA-fed pigs . Also twice the number of piglets fed AuSDPP+EYP appeared unhealthy compared with piglets on treatment AuSDPP+EYA . In Exp . 2, 72 10-d-old weaned pigs (3.5 kg BW) were used to compare the effect of EYA supplementation and oral challenge of ETEC strain F18 on performance and visceral organ weights . The experimental diets consisted of SDAP+EYP, SDAP+EYA, SDPP+EYP, and SDPP+EYA . From d 0 to 7, and the entire experimental period, dietary treatment did not influence (P > 0.05) growth rate and feed consumption . Plasma urea N concentration was higher (P < 0.05) in piglets fed the SDAP+EYP diet before and after the oral challenge . Gain:feed ratio, organ weights, villi heights, and crypt depths were not affected (P > 0.05) by dietary treatments . The results indicate that SDPP contains specific anti-ETEC antibodies, which is one of the factors responsible for its superior growth-enhancing effects . Spray-dried animal plasma, SDPP and EYA have similar growth promoting effect in early-weaned pigs.

Plant Cell Physiol, 2002 Nov, 43(11), 1276 - 84
Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta; Anderca MI et al.; We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR . The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein . The predicted 24-kDa protein was named DtNDK1 . It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides . The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity . Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus . The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity . Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.

Plant Cell Physiol, 2002 Nov, 43(11), 1259 - 65
Purification and cDNA cloning of UDP-D-glucuronate carboxy-lyase (UDP-D-xylose synthase) from pea seedlings; Kobayashi M et al.; Uridine diphospho-D-glucuronate carboxy-lyase (UDP-D-xylose synthase; EC 4.1.1.35), which catalyzes the conversion of UDP-D-glucuronate to UDP-D-xylose, was purified to apparent homogenity from pea (Pisum sativum L.) seedlings . The pH optimum for enzyme activity was around 5-6, and the activity was not affected by exogeneously supplied NAD+ and NADH . The purified enzyme had a molecular weight of 250 kDa and consisted of 42 kDa polypeptides . Based on the amino acid sequence, a probe (400 bp) was prepared with degenerate primers by a reverse transcriptase-PCR . Using this probe, a clone encoding 346 amino acid residues was screened from a pea cDNA library . The recombinant protein expressed in Escherichia coli catalyzed conversion of UDP-D-glucuronate to UDP-D-xylose, confirming that the isolated clone encoded UDP-D-glucuronate carboxy-lyase.

Protein Expr Purif, 2002 Dec, 26(3), 476 - 88
Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein; Singleton SF et al.; The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction . Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes . The RecA protein has been overproduced in E . coli and purified using a variety of purification schemes requiring multiple, time-intensive steps . The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps . In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence . Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained . The protein displays in vitro activities that are identical to those of protein isolated using classical procedures . The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.

Protein Expr Purif, 2002 Dec, 26(3), 462 - 6
Protein-protein cross-linking in the use of the eukaryotic eGST-fusion system; Papaioannou M et al.; We describe here an unusual phenomenon in the isolation of protein complexes from eukaryotic cells using expressed GST-fusion proteins . Protein complexes are involved in a large number of regulatory mechanisms . Therefore, the use of tagged fusion proteins is an important tool for isolation of such protein complexes . For this purpose, we used the nuclear factor Alien, described as a corepressor for the thyroid hormone receptor, fused to the eukaryotic eGST and expressed this fusion in human cells . After affinity purification over glutathione-Sepharose using stringent washing steps, we observed several co-purifying bands migrating at molecular weights higher than the GST-Alien fusion protein . These bands appeared specifically in the GST-Alien transfected cell preparations . Surprisingly, using both Western blotting and MALDI-analyses, we revealed that these bands are composed of the GST-Alien protein itself . We hypothesize that overexpressed factors may generate unexpected cross-linking products which can confound the analyses of such affinity-purified complexes . The cross-linking products could not be eliminated by using beta-mercaptoethanol in the gel system and by boiling in SDS-sample buffer . Also, we demonstrate that Western blotting analyses using antibodies directed against both the tag-epitope and the expressed protein of interest can rapidly, reliably, and in a cost-saving manner identify such artifacts, eliminating them from the analyses of potentially interesting interaction partners . Our findings clearly show that the overexpression and purification of proteins from eukaryotic cells may generate unusual structural features that strongly influence complex formation and the migration in SDS-PAGE.

Protein Expr Purif, 2002 Dec, 26(3), 455 - 61
Engineering S-protein fragments of bovine ribonuclease A for targeted drug delivery; Backer MV et al.; High affinity interaction between S-protein and S-peptide fragments of bovine pancreatic RNase A has been recently used for construction of molecular vehicles for targeted drug delivery . The vehicle is assembled as a complex of drug carrier conjugated S-protein with S-peptide-tagged targeting protein . To avoid random chemical crosslinking of drug carriers to S-protein, we constructed a mutant 16-124aa fragment of RNase A in which 122ala is replaced with a cysteine residue . The mutant and the corresponding wild type fragments expressed in Escherichia coli are refolded into functional conformations only in the presence of S-peptide . After the removal of S-peptide, both fragments retain the ability to bind S-peptide and S-peptide-tagged proteins . The 122cys residue in the mutant fragment is available for site-specific conjugation.

Protein Expr Purif, 2002 Dec, 26(3), 449 - 54
Localized production of human E-cadherin-derived first repeat in Escherichia coli; Makagiansar IT et al.; E-cadherin is a cell surface adhesion molecule that is expressed in both epithelial and endothelial tissues . In this study, an improved method for the simple production of the human E-cadherin-derived first repeat E-CAD1 was developed by exporting it into the periplasmic space of Escherichia coli . Localization of the recombinant protein into the periplasm allowed the isolation of E-CAD1 without cell lysis . The N-terminus of E-CAD1 is fused to a streptavidin-derived peptide to allow single-step purification using a Streptag affinity column . Optimal expression in LB medium produced 3.2 mg/L while expression in minimal medium containing 15NH(4)Cl as the sole source of nitrogen produced 4.2 mg/L purified (15)N-labeled E-CAD1 . Heteronuclear NMR spectroscopy confirmed that the purified E-CAD1 produced in this manner was correctly folded . The expression and purification protocol for unlabeled and isotopically labeled E-CAD1 permits rapid preparative production of this protein for mechanistic and structural studies.

Biochim Biophys Acta, 2002 Dec 2, 1556(2-3), 217 - 25
The NADP-reducing hydrogenase from Desulfovibrio fructosovorans: functional interaction between the C-terminal region of HndA and the N-terminal region of HndD subunits; Dermoun Z et al.; The hndABCD operon from Desulfovibrio fructosovorans encodes an uncommon heterotetrameric NADP-reducing iron hydrogenase . The presence of a {2Fe-2S} cluster likely located in the C-terminal region of the HndA subunit has already been revealed . We have cloned and expressed the truncated hndA gene in Escherichia coli to isolate the structural {2Fe-2S} module . Optical and EPR spectra are found identical to that of the native HndA subunit and the midpoint redox potential (-385 mV) is similar to that of the native protein (-395 mV) . These results clearly demonstrate that the C-terminal region of HndA is a structurally independent {2Fe2S} ferredoxin-like domain . In the same way, the N-terminal domain of the HndD subunit was overproduced in E . coli and characterized . The presence of a {2Fe-2S} cluster was evidenced by optical spectroscopy . The midpoint redox potential (-380 mV) of this domain was found very close to that of the truncated HndA subunit but the EPR properties were significantly different . The various EPR properties allowed us to observe an electron exchange between the two {2Fe-2S} ferredoxin-like domains of the HndA and HndD subunits . Moreover, domain-domain interactions, observed by far-western experiments, indicate that these subunits are direct partners in the native complex.

J Mol Biol, 2002 Dec 6, 324(4), 823 - 39
Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility, conformation and mapping of contact regions; Haussinger D et al.; Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion . The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations . Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms . Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts . Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments . The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure . In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible . This mechanism provides a rational for the calcium-induced adhesiveness . Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2 . These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations . Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2 . According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.

J Mol Biol, 2002 Dec 6, 324(4), 763 - 74
Fluorescently labelled guanine nucleotide binding proteins to analyse elementary steps of GAP-catalysed reactions; Kraemer A et al.; Downregulation of small guanine nucleotide-binding proteins (GNBPs) requires the interaction with their corresponding GTPase-activating proteins (GAPs), which increase the slow intrinsic GTPase reaction by several orders of magnitude . On the basis of the structure of H-Ras in complex with the catalytic domain of p120-GAP, we have developed a set of site-specifically labelled Ras-variants, one of which turned out to be particularly sensitive for studying the interaction with Ras-specific GAPs . This specific fluorescent reporter group and the use of manganese to increase the rate of the chemical reaction step allowed us to identify differences in the rate-limiting step of either the GAP-334 or NF1-333 catalyzed reaction . The assay was also applied to study the interaction of the Ras-related protein Rap1B with Rap1GAP, for which no detailed kinetic analysis was available . Single-turnover experiments of this reaction show that the low affinity of the complex (50 microM) is due to a slow association rate as well as a fast dissociation rate . RapGAP promotes AlFx binding to Rap1B, even though it does not contain a catalytic arginine . The rate-limiting step of the RapGAP catalysed reaction is release of inorganic phosphate, which is about five times slower than the chemical cleavage step . Our data reveal marked differences in GAP/target interactions even between closely related systems and suggest that the fluorescent reporter group method might be generally applicable to many other GNBPs and their cognate GAPs.

J Mol Biol, 2002 Dec 6, 324(4), 755 - 62
Observation of additional calcium ion in the crystal structure of the triple mutant K56,120,121M of bovine pancreatic phospholipase A2; Rajakannan V et al.; Phospholipase A(2) catalyses hydrolysis of the ester bond at the C2 position of 3-sn-phosphoglycerides . Here we report the 1.9A resolution crystal structure of the triple mutant K56,120,121M of bovine pancreatic phospholipase A(2) . The structure was solved by molecular replacement method using the orthorhombic form of the recombinant phospholipase A(2) . The final protein model contains all the 123 amino acid residues, two calcium ions, 125 water molecules and one 2-methyl-2-4-pentanediol molecule . The model has been refined to a crystallographic R-factor of 19.6% (R(free) of 25.9%) for all data between 14.2A and 1.9A . The residues 62-66, which are in a surface loop, are always disordered in the structures of bovine pancreatic phospholipase A(2) and its mutants . It is interesting to note that the residues 62-66 in the present structure is ordered and the conformation varies substantially from those in the previously published structures of this enzyme . An unexpected and interesting observation in the present structure is that, in addition to the functionally important calcium ion in the active site, one more calcium ion is found near the N terminus . Detailed structural analyses suggest that binding of the second calcium ion could be responsible for the conformational change and the ordering of the surface loop . Furthermore, the results suggest a structural reciprocity between the k(cat)(*) allosteric site and surface loop at the i-face, which represents a newly identified structural property of secreted phospholipase A(2).

J Mol Biol, 2002 Dec 6, 324(4), 649 - 65
Attenuating functions of the C terminus of lambda integrase; Tekle M et al.; The tyrosine family site-specific recombinases, in contrast to the related type I topoisomerases, which act as monomers on a single DNA molecule, rely on multi-protein complexes to synapse partner DNAs and coordinate two sequential strand exchanges involving four nicking-closing reactions . Here, we analyze three mutants of the catalytic domain of lambda integrase (Int), A241V, I353M and W350ter that are defective for normal recombination, but possess increased topoisomerase activity . The mutant enzymes can carry out individual DNA strand exchanges using truncated substrates or Holliday junctions, and they show more DNA-cleavage activity than wild-type Int on isolated att sites . Structural modeling predicts that the substituted residues may destabilize interactions between the C-terminal beta-strand (beta7) of Int and the core of the protein . The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on beta6 and the catalyst K235 located on the flexible beta2-beta3 loop, relative to their positions in a crystal structure of the inactive conformation . We propose that the anchoring of beta7 against the protein core restrains the movement of Tyr342 and/or Lys235, causing an attenuation of cleavage activity in most contexts . Within a bona fide recombination complex, the release of strand beta7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex . The loss of beta7 packing by misalignment or truncation in the mutant proteins described here causes a loss of regulated activity, thereby favoring DNA cleavage activity in monomeric complexes and forfeiting the coordination of strand-exchange necessary for efficient recombination.

J Mol Biol, 2002 Dec 6, 324(4), 637 - 47
Structural properties of polyubiquitin chains in solution; Varadan R et al.; Because polyubiquitin chain structure modulates Ub-mediated signaling, knowledge of the physiological conformations of chain signals should provide insights into specific recognition . Here, we characterized the solution conformations of K48-linked Ub(2) and Ub(4) using a combination of NMR techniques, including chemical shift mapping of the interdomain interface, domain orientation measurements on the basis of 15N relaxation and residual dipolar couplings, and the solvent accessibility studies . Our data indicate a switch in the conformation of Ub(2), from open to closed, with increasing pH . The closed conformation features a well-defined interface that is related to, but distinguishable from, that observed in the Ub(2) crystal structure . This interface is dynamic in solution, such that important hydrophobic residues (L8, I44, V70) that are sequestered at the interface in the closed conformation may be accessible for direct interactions with recognition factors . Our results suggest that the distal two units of Ub(4), which is the minimum signal for efficient proteasomal degradation, may adopt the closed Ub(2) conformation.

J Mol Biol, 2002 Dec 6, 324(4), 599 - 610
Regulation of the Escherichia coli allantoin regulon: coordinated function of the repressor AllR and the activator AllS; Rintoul MR et al.; The allantoin regulon of Escherichia coli, formed by three operons expressed from promoters allA(P), gcl(P) and allD(P), is involved in the anaerobic utilization of allantoin as nitrogen source . The expression of these operons is under the control of the repressor AllR . The hyperinduction of one of these promoters (allD(P)) by allantoin in an AllR defective mutant suggested the action of another regulator, presumably of activator type . In this work we have identified ybbS (proposed gene name allS), divergently transcribed from allA, as the gene encoding this activator . Analysis of the expression of the three structural operons in DeltaallS mutant showed that the expression from allD(P) was abolished, suggesting that AllS is essential for the expression of the corresponding operon . In a wild-type strain expression of allS takes place mainly anaerobically and is hyperinduced when the nitrogen source limits growth . However, expression of allS is independent of regulators of the Ntr response, NtrC or Nac . Band shift experiments showed that AllR binds to DNA containing the allS-allA intergenic region and the gcl(P) promoter and its binding is abolished by glyoxylate . Both DNA fragments contain a highly conserved inverted repeat, which after site-directed mutagenesis, has been proven to be the AllR-binding site . This site displays similarity with the IclR family recognized consensus . Interaction of AllR with the single operator present in the allS-allA intergenic region prevented binding of RNA polymerase to either of the two divergent promoters . The regulator AllS interacts only with allD(P) even in the absence of allantoin . Analysis of this promoter allowed us to identify an inverted repeat as a motif for AllS binding . We propose a model for the coordinate control of the allantoin regulon by AllR and AllS.

J Mol Biol, 2002 Dec 6, 324(4), 573 - 6
Genetic perturbations of RNA reveal structure-based recognition in protein-RNA interaction; Choi H et al.; Protein-RNA recognition is an essential foundation of cellular processes, yet much remains unknown about these important interactions . The recognition between aminoacyl-tRNA synthetases and their cognate tRNA substrates is highly specific and essential for cell viability, due to the necessity for accurate translation of the genetic code into protein sequences . We selected an active tRNA that is highly mutated in the recognition nucleotides of the acceptor stem region in the alanine system . The functional properties of this mutant and its secondary derivatives demonstrate that recognition cannot be reduced to isolated structural elements, but rather the amino acid acceptor stem is being recognized as a unit.

Plasmid, 2002 Nov, 48(3), 186 - 92
Conjugative DNA synthesis: R1162 and the question of rolling-circle replication; Parker C et al.; Strand-replacement synthesis during conjugative mating has been characterized by introducing into donor cells R1162 plasmid DNA containing a base-pair mismatch . Conjugative synthesis in donors occurs in the absence of vegetative plasmid replication, but with a lag between rounds of transfer, and with most strands being initiated at the normal site within the replicative origin . These characteristics argue against the idea that multiple plasmid copies are generated for successive rounds of transfer by rolling-circle replication . However, the R1162 relaxase protein can process molecules containing multiple transfer origins in the manner expected for the conversion of single-strand multimers, generated by rolling-circle replication, to unit-length molecules . This capability appears to be the result of a secondary cleavage reaction carried out by the protein . The possibility is raised that the processing of molecules with more than one origin of transfer might be a repair mechanism directed against adventitious DNA synthesis during transfer.

Lett Appl Microbiol, 2002, 35(6), 533 - 7
The effect of starvation stress on the porin protein expression of Escherichia coli in lake water; Ozkanca R et al.; AIMS: The aim was to identify changes in outer membrane proteins (omps), OmpA, OmpC and OmpF, in Escherichia coli under starvation conditions in lake water microcosms studied at different temperatures . METHODS AND RESULTS: Escherichia coli was incubated in lake water microcosms at a variety of temperatures and the omps studied using quantitative densitometric analysis of protein bands of sodium dodecyl sulphate gels of omp preparations . The amount of OmpF increased over the incubation period relative to that of OmpC whereas the relative abundance of OmpA declined, most notably at 25 and 37 degrees C . This change was linked to changes in peak cell volume as determined by cell measurements . CONCLUSIONS: Major changes to the omps of E . coli accompany the adaptation of the organism to starvation conditions in lake water microcosms . SIGNIFICANCE AND IMPACT OF THE STUDY: Prolonged starvation affects the relative amounts of outer membrane porins . This study furthers the understanding of the role played by changes in the omp composition in E . coli during survival in lake water environments.

Environ Microbiol, 2002 Nov, 4(11), 713 - 20
Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries; Schramm A et al.; A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH) . Several different cloning approaches and treatments to generate target-rRNA in the clones were compared . Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase . Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli . Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences . Melting behaviour and calculated T(d) values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions . The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest . In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes . In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries.

Dev Genes Evol, 2002 Dec, 212(11), 513 - 9 Epub 2002 Sep 18.
Cloning and characterisation of PKB and PRK homologs from Hydra and the evolution of the protein kinase family; Herold M et al.; Two new serine/threonine protein kinases have been cloned from Hydra cDNA . The first of these kinases belongs to the PKB/Akt family . It is expressed ubiquitously in Hydra at a relatively low level but is upregulated during head regeneration . The second kinase is a member of the PRK/PKN family . It is ubiquitously expressed in Hydra tissue, albeit at a higher level than PKB . Construction of a phylogenetic tree including the Hydra PRK and PKB kinases and two PKC homologs previously cloned by Hassel and comparing them with members of the PKC, PKB and PRK families from porifera, Dictyostelium,yeast, Drosophila, Caenorhabditis and humans provide support for a simple model for the evolution of these kinase families . An ancestral precursor which contained a pleckstrin homology domain in its N-terminus and a C-terminal kinase domain gave rise to PKB in Dictyostelium . From this ancestor the PKB/PRK and PKC families evolved . The pleckstrin homology domain was lost in the PKC and PRK families and kept in the PKB family . PKB homologs have now been found in a variety of multicellular animals with Hydra being the phylogenetically earliest representative . Members of the PRK/PKC family, on the other hand, are also present in fungi . The precursor for these kinases must have contained N-terminal regulatory domains that were retained in fungal PRKs but subsequently partitioned between kinases of the PKC and PRK groups in metazoans.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 226 - 32 Epub 2002 Oct 16.
Nitric oxide and peroxynitrite promote complete disruption of the {4Fe-4S} cluster of recombinant human iron regulatory protein 1; Soum E et al.; Iron regulatory protein 1 (IRP1) is a metalloprotein which regulates several proteins involved in mammalian iron homeostasis at a post-transcriptional level, by binding to specific mRNA sequences termed iron responsive elements (IREs) . IRP1 exhibits two mutually exclusive activities, either aconitase or mRNA-binding protein, depending on the intactness of a versatile {4Fe-4S} cluster . Here we asked whether NO and peroxynitrite act directly on IRP1 and how they modify its functions . Recombinant human IRP1 was purified from Escherichia coli as a {4Fe-4S} cluster-containing protein, and exposed to 3-morpholinosydnonimine (SIN-1), which was used either as a peroxynitrite donor or as an NO donor when added with an excess of superoxide dismutase (SOD) . We showed that, in both settings, IRP1 lost aconitase activity and iron concomitantly . Iron release reached 3.5-4 iron atoms per IRP1 molecule, proving that the Fe-S cluster was completely disrupted . An increase in IRP1 IRE-binding was observed upon the sequential addition of SIN-1/SOD and low concentrations of 2-mercaptoethanol, whereas SIN-1 alone induced a decrease in binding capacity which was not reversed by 2-mercaptoethanol, even at high concentrations . Moreover, nitrotyrosine adducts were detected on SIN-1-treated IRP1 by Western blot analysis.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 185 - 94 Epub 2002 Sep 27.
Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli; Wernimont AK et al.; PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli . Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences . Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known . Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins . The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded beta barrel with the methionines unexpectedly housed on a solvent-exposed loop . Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC . In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit . In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface . These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA . Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein . Dimerization is significantly enhanced upon binding Cu(I) with a measured delta(deltaG degrees )<or=-8.0 kJ/mole, suggesting that copper might bind at the dimer interface.

FEBS Lett, 2002 Dec 4, 532(1-2), 207 - 10
N-terminal acetyl group is essential for the inhibitory function of carboxypeptidase Y inhibitor (I(C)); Mima J et al.; Carboxypeptidase Y (CPY) inhibitor, I(C), a yeast cytoplasmic inhibitor in which the N-terminal amino acid is acetylated, was expressed in Escherichia coli and produced as an unacetylated form of I(C) (unaI(C)) . Circular dichroism and fluorescence measurements showed that unaI(C) and I(C) were structurally identical and produce identical complexes with CPY . However, the K(i) values for unaI(C) for anilidase and peptidase activity of CPY were much larger, by 700- and 60-fold, respectively, than those of I(C) . The reactivities of phenylmethylsulfonyl fluoride and p-chloromercuribenzoic acid toward the CPY-unaI(C) complex were considerably higher than those toward the CPY-I(C) complex . Thus, the N-terminal acetyl group of I(C) is essential for achieving a tight interaction with CPY and for its complete inactivation.

Mutat Res, 2002 Dec 29, 510(1-2), 81 - 90
Efficiency, specificity and DNA polymerase-dependence of translesion replication across the oxidative DNA lesion 8-oxoguanine in human cells; Avkin S et al.; The oxidation product of guanine, 8-oxoguanine, is a major lesion formed in DNA by intracellular metabolism, ionizing radiation, and tobacco smoke . Using a recently developed method for the quantitative analysis of translesion replication, we have studied the bypass of 8-oxoguanine in vivo by transfecting human cells with a gapped plasmid carrying a site-specific 8-oxoguanine in the ssDNA region . The efficiency of bypass in the human large-cell lung carcinoma cell line H1299 was 80%, and it was similar when assayed in the presence of aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon . A similar extent of bypass was observed also in XP-V cells, defective in pol eta, both in the absence and presence of aphidicolin . DNA sequence analysis indicated that the major nucleotide inserted opposite the 8-oxoguanine was the correct nucleotide C, both in H1299 cells (81%) and in XP-V cells (77%) . The major mutagenic event was the insertion of an A, both in H1299 and XP-V cells, and it occurred at a frequency of 16-17%, significantly higher than previously reported . Interestingly, the misinsertion frequency of A opposite 8-oxoguanine was decreased in XP-V cells in the presence of aphidicolin, and misinsertion of G was observed . This modulation of the mutagenic specificity at 8-oxoguanine is consistent with the notion that while not essential for the bypass reaction, pol eta and pol delta, when present, are involved in bypass of 8-oxoguanine in vivo.

Mutat Res, 2002 Dec 29, 510(1-2), 45 - 54
The proofreading 3'-->5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis; Khare V et al.; The 3'-->5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors . A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair . The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion . The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase gamma (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts . However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions . DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'-->5' exonuclease and polymerase activities . Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity . Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions . Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity.

Insect Biochem Mol Biol, 2003 Jan, 33(1), 73 - 80
Complete sequence, expression and evolution of two members of the hexamerin protein family during the larval development of the rice moth, Corcyra cephalonica; Nagamanju P et al.; Three distinct types of storage hexamerins are expressed in the "last-instar" larvae of the rice moth, Corcyra cephalonica . A cDNA expression library was constructed from fat body-RNA and screened with a polyclonal antibody raised against purified hexamerin (SP2) of Corcyra cephalonica . Two slightly different "full-length" hexamerin cDNA clones (Hex2a and Hex2b) were isolated and sequenced . Both include open reading frames of 2109 bp which are translated into polypeptides of 703 amino acids with 92.5% identity . Signal peptides of 19 amino acids are present at the N-termini . The 684 amino acids native proteins have a high content of aryl groups (17.6%) . According to both the criteria for amino acid composition and the phylogenetic analysis, Hex2a and Hex2b belong to the lepidopteran arylphorins . Northern blot studies revealed that the Hex2 genes are species- and tissue-specifically expressed in fat body cells of "last-instar" (= 5th) larvae.

Biochem Biophys Res Commun, 2002 Dec 13, 299(4), 621 - 7
Characterization of a novel enoyl-acyl carrier protein reductase of diazaborine-resistant Rhodobacter sphaeroides mutant; Lee IH et al.; Rhodobacter sphaeroides contains two enoyl-acyl carrier protein (ACP) reductases, FabI(1) and FabI(2) . However, FabI(1) displays most of the cellular enzyme activity . The spontaneous diazaborine-resistant mutation was mapped as substitution of glutamine for proline 155 (P155Q) of FabI(1) . The mutation of FabI(1){P155Q} increased the specificity constants (k(cat)/K(m)) for crotonyl-ACP and NADH by more than 2-fold, while the site-directed mutation G95S (FabI(1){G95S}), corresponding to the well-known G93 mutation of Escherichia coli FabI, rather decreased the values . Inhibition kinetics of the enzymes revealed that triclosan binds to the enzyme in the presence of NAD(+), while the diazaborine appears to interact with NADH and NAD(+) in the enzyme active site . The apparent inhibition constant K(i)(') of triclosan for FabI(1){P155Q} and FabI(1){G95S} at saturating NAD(+) were approximately 80- and 3-fold higher than that for the wild-type enzyme, respectively, implying that the inhibition was remarkably impaired by the P155Q mutation . The similar levels of K(i)(') of diazaborine for the mutant enzymes were also observed with respect to NAD(+) . Thus, the novel mutation P155Q appears to disturb the binding of inhibitors to the enzyme without affecting the catalytic efficiency.

Biochem Biophys Res Commun, 2002 Dec 13, 299(4), 581 - 6
Cloning and expression of the human N-acetylglutamate synthase gene; Caldovic L et al.; N-acetylglutamate synthase (NAGS, E.C . 2.3.1.1) is a mitochondrial enzyme catalyzing the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthase I (CPSI), the first enzyme of the urea cycle . Patients with NAGS deficiency develop hyperammonemia because CPSI is inactive without NAG . The human NAGS cDNA was isolated from a liver library based on its similarity to mouse NAGS . The deduced amino acid sequence contains an N-terminal putative mitochondrial targeting signal of 49 amino acids (63% identity with mouse NAGS) followed by a "variable domain" of 45 amino acids (35% identity) and a "conserved domain" of 440 amino acids (92% identity) . A cDNA sequence containing the "conserved domain" complements an NAGS-deficient Escherichia coli strain and the recombinant protein has arginine-responsive NAGS catalytic activity . The NAGS gene is expressed in the liver and small intestine; the intestinal transcript is smaller in size than liver transcript.

Biochem Biophys Res Commun, 2002 Dec 13, 299(4), 517 - 24
Correlation between nitric oxide and cyclooxygenase-2 pathways in head and neck squamous cell carcinomas; Gallo O et al.; We investigated the interactions between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) pathways in head and neck squamous cell carcinomas (HNSCCs) and in two carcinoma cell lines . HNSCCs showed an up-regulation of both pathways which were strongly correlated with each other (p=0.02) and with tumor vascularization (p=0.0001 and p=0.008, respectively) . In carcinoma cells, Escherichia coli lipopolysaccharide (LPS) and EGF treatment up-regulated both pathways . NOS inhibitor N(G)-monomethyl-L-arginine methyl ester (L-NAME) inhibited this up-regulation . LPS or EGF induced iNOS expression that was not altered by NOS or COX-2 inhibitors . Conversely, LPS or EGF promoted COX-2 expression that was decreased by L-NAME . The NO donor S-nitroso-acetyl-penicillamine (SNAP) up-regulated COX-2 pathway and this effect was reduced by the guanylate cyclase inhibitor methylene blue . Thus, in squamous carcinoma cells, NO increases the activity of COX-2 pathway and this effect is probably mediated by endocellular cGMP level, with potential implications on tumor growth, angiogenesis, and therapy.

Vet Immunol Immunopathol, 2002 Dec, 90(3-4), 169 - 77
Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor; Yamamoto A et al.; Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E . coli . The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form . The activity of the recombinant feline G-CSF was 3 x 10(6)U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation . When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged . Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes . No severe side effects or antibody production was observed in cats after administration of G-CSF .

RNA, 2002 Nov, 8(11), 1461 - 70
Evolutionary dynamics and population control during in vitro selection and amplification with multiple targets; Shi H et al.; Iterative cycles of in vitro selection and amplification allow rare functional nucleic acid molecules, aptamers, to be isolated from large sequence pools . Here we present an analysis of the progression of a selection experiment that simultaneously yielded two families of RNA aptamers against two disparate targets: the intended target protein (B52/SRp55) and the partitioning matrix . We tracked the sequence abundance and binding activity to reveal the enrichment of the aptamers through successive generations of selected pools . The two aptamer families showed distinct trajectories of evolution, as did members within a single family . We also developed a method to control the relative abundance of an aptamer family in selected pools . This method, involving specific ribonuclease digestion, can be used to reduce the background selection for aptamers that bind the matrix . Additionally, it can be used to isolate a full spectrum of aptamers in a sequential and exhaustive manner for all the different targets in a mixture.

RNA, 2002 Nov, 8(11), 1416 - 27
The C-terminal amino acid sequence of nascent peptide is a major determinant of SsrA tagging at all three stop codons; Sunohara T et al.; Recent studies on endogenous SsrA-tagged proteins have revealed that the tagging could occur at a position corresponding to the normal termination codon . During the study of SsrA-mediated Lacl tagging (Abo et al., EMBO J, 2000 19:3762-3769), we found that a variant Lacl (Lacl deltaC1) lacking the last C-terminal amino acid residue is efficiently tagged in a stop codon-dependent manner . SsrA tagging of Lacl deltaC1 occurred efficiently without Lacl binding to the lac operators at any one of three stop codons . The C-terminal (R)LESG peptide of Lacl deltaC1 was shown to trigger the SsrA tagging of an unrelated protein (CRP) when fused to its C terminus . Mass spectrometry analysis of the purified fusion proteins revealed that SsrA tagging occurs at a position corresponding to the termination codon . The alteration of the amino acid sequence but not the nucleotide sequence of the C-terminal portion eliminated the tagging . We also showed that the tagging-provoking sequences cause an efficient translational readthrough at UGA but not UAA codons . In addition, we found that C-terminal dipeptides known to induce an efficient translation readthrough could cause an efficient tagging at stop codons . We conclude that the amino acid sequence of nascent polypeptide prior to stop codons is a major determinant for the SsrA tagging at all three stop codons.

RNA, 2002 Nov, 8(11), 1393 - 400
Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides; Ren YG et al.; Aminoglycosides are known to bind and perturb the function of catalytic RNA . Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes . The inhibition was pH dependent and released in a competitive manner by Mg2+ . Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor . Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes . Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions . The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.

J Biol Chem, 2003 Feb 21, 278(8), 6543 - 51 Epub 2002 Nov 27.
Crystal structure of the protease domain of a heat-shock protein HtrA from Thermotoga maritima; Kim DY et al.; HtrA (high temperature requirement A), a periplasmic heat-shock protein, functions as a molecular chaperone at low temperatures, and its proteolytic activity is turned on at elevated temperatures . To investigate the mechanism of functional switch to protease, we determined the crystal structure of the NH(2)-terminal protease domain (PD) of HtrA from Thermotoga maritima, which was shown to retain both proteolytic and chaperone-like activities . Three subunits of HtrA PD compose a trimer, and multimerization architecture is similar to that found in the crystal structures of intact HtrA hexamer from Escherichia coli and human HtrA2 trimer . HtrA PD shares the same fold with chymotrypsin-like serine proteases, but it contains an additional lid that blocks access the of substrates to the active site . A corresponding lid found in E . coli HtrA is a long loop that also blocks the active site of another subunit . These results suggest that the activation of the proteolytic function of HtrA at elevated temperatures might occur by a conformational change, which includes the opening of the helical lid to expose the active site and subsequent rearrangement of a catalytic triad and an oxyanion hole.

J Biol Chem, 2003 Feb 14, 278(7), 5271 - 6 Epub 2002 Nov 27.
Heterologous expression and characterization of mouse spermine oxidase; Cervelli M et al.; Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells . Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase . This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems . The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1 . The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1 . To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells . The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines . The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0 . Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

J Biol Chem, 2003 Apr 25, 278(17), 15095 - 104 Epub 2002 Nov 27.
Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting . Implications for macromolecular mimicry and structural changes in RF2; Scarlett DJ et al.; The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA . For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits . In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination . In the presence of the cognate UGA stop codon, residues flanking the universally conserved (250)GGQ(252) motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers . In contrast, residues that flank the putative "peptide anticodon" of RF2, (205)SPF(207), were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center . Within the recently solved crystal structure of E . coli RF2, the GGQ and SPF motifs are separated by 23 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A apart . Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.

Vet Microbiol, 2003 Feb 2, 91(2-3), 265 - 83
Characterization of the functional domain of major surface protein 1a involved in adhesion of the rickettsia Anaplasma marginale to host cells; de la Fuente J et al.; The major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been shown to mediate adhesion, infection and transmission of the organism, as well as to contribute to protective immunity in cattle . MSP1a contains a variable number of tandemly repeated peptides in the amino-terminal region, while the remainder of the protein is highly conserved among isolates . The number of repeats varies among geographic isolates of A . marginale but is constant within an isolate and has been used as a stable genetic marker of isolate identity . Because the sequence of the tandem repeats is the most variable part of the protein among isolates, this region of the protein is most likely to be involved in adhesion to host cells, a prerequisite to infection . The purpose of this study was to characterize the organization and function of the MSP1a tandem repeats of A . marginale in adhesion to host cells . We demonstrated by use of recombinant mutant proteins that the tandemly repeated region of MSP1a was necessary and sufficient to mediate adhesion of MSP1a to tick cells and bovine erythrocytes . Synthetic peptides representing the predominant sequences of individual repeats were tested for their adhesive capacity for tick cell extract (TCE) . Peptides containing acidic amino acids D or E at position 20 bound to TCE, while peptides with a G as the 20th amino acid were not adhesive to TCE . Antibodies produced in rabbits against a synthetic repeat peptide neutralized A . marginale infection of cultured tick cells, and the neutralization observed was similar to that effected by antibodies produced against the whole MSP1a recombinant protein . Analysis of tandemly repeated MSP1a peptides of several geographic isolates of A . marginale revealed a complex relationship between the msp1alpha genotype and the tick-transmissible phenotype of the isolate and suggested that both the sequence and conformation of the repeated peptides influenced the adhesive properties of MSP1a . These studies demonstrated that the tandemly repeated region of the protein mediates the adhesive function of MSP1a.

Vet Microbiol, 2003 Feb 2, 91(2-3), 205 - 13
Effect of amino acid substitutions in the epitope regions of pyolysin from Arcanobacterium pyogenes; Imaizumi K et al.; Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY, cholesterol-dependent cytolysin) family of bacterial toxins . Recently, we demonstrated that the epitopes of monoclonal antibodies (mAbs) S, H, C, and G lie in the regions of amino acids regions 55-73, 123-166, 482-506, and 482-506 of PLO, respectively, by the reaction of mAbs with truncated PLOs . In this study, we substituted the amino acids in these epitope regions of PLO by site-directed mutagenesis and examined the effect of these amino acid substitutions . Mutants I70S/R71A/L73S, Y131S/P132S, and L163S/P164S for mAbs H or S completely lost the hemolytic activity of the proteins, but these mutants still bound to erythrocyte membranes . Mutants L495S/W497S and W500S/W501S for mAbs C and G also completely lost their hemolytic activity, but still bound to erythrocyte membranes . In the undecapeptide region of PLO, the cysteine residue required for thiol activation is replaced with alanine . Therefore, we substituted Ala-492 of the undecapeptide region for Cys . The hemolytic activity of this mutant A492C decreased by adding hydrogen peroxide or storing at 4 degrees C, and the decreased hemolytic activity was restored by adding L-cysteine.

Biochimie, 2002 Sep, 84(9), 859 - 68
Photoinduced cleavage by a rhodium complex at G.U mismatches and exposed guanines in large and small RNAs; Chow CS et al.; Photoinduced cleavage reactions by the rhodium complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) {Rh(DIP)(3)(3+)} with three RNA hairpins, r(GGGGU UCGCUC CACCA) (16 nucleotide, tetraloop(Ala2)), r(GGGGCUAUAGCUCUAGCUC CACCA) (24 nucleotide, microhelix(Ala)), and r(GGCGGUUAGAUAUCGCC) (17 nucleotide, 790 loop), and full-length (1542 nucleotide) 16S rRNA from Escherichia coli were investigated . The cleavage reactions were monitored by gel electrophoresis and the sites of cleavage by Rh(DIP)(3)(3+) were determined by comparisons with chemical or enzymatic sequencing reactions . In general, RNA backbone scission by the metal complex was induced at G.U mismatches and at exposed G residues . The cleavage activity was observed on the three small RNA hairpins as well as on the isolated 1542-nucleotide ribosomal RNA.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Dec 25, 782(1-2), 317 - 29
Low-mass proteome analysis based on liquid chromatography fractionation, nanoliter protein concentration/digestion, and microspot matrix-assisted laser desorption ionization mass spectrometry; Keller BO et al.; HPLC fractionation combined with mass spectrometry can become a powerful tool for analyzing the proteome in the mass range below 15 kDa where efficient protein separation by gel electrophoresis can be difficult . For sensitive and high-resolution separation of the low-mass proteome, the use of analytical rather than preparative HPLC columns is preferred . However, individual fractions collected by a conventional HPLC separation usually contain a small amount of proteins whose concentrations may not be sufficiently high for subsequent enzyme digestion and protein identification by mass spectrometry . In this work, we present a high sensitivity nanoliter sample handling technique to analyze proteins fractionated by HPLC . In this technique, an individual HPLC fraction in hundreds of microliter volume is pre-concentrated to several microliters . About 700 pl of the pre-concentrated fraction is then drawn into a 20-microm I.D . capillary and dried in a small region near the capillary's entrance . This process can be repeated many times to concentrate a sufficient amount of protein to the small region of the capillary . After protein concentration, protein digestion is achieved by drawing 1 nl of chemical or enzymatic reagent into the capillary and placing it in the same region where the dried protein sits . The resulting peptides are then deposited onto a microspot in a MALDI probe for mass analysis . The performance of this technique is demonstrated with the use of a standard protein solution . This technique is applied to the identification of low-mass proteins separated by HPLC from a complex mixture of an E . coli extract.

Virus Res, 2002 Dec, 90(1-2), 263 - 8
Recombinant Vpr (rVpr) causes augmentation of HIV-1 p24 Ag level in U1 cells through its ability to induce the secretion of TNF; Nakamura T et al.; We have found that an HIV-1 accessory gene product Vpr enhanced HIV-1 reproduction in U1 cells chiefly by the induction of TNF, a proinflammatory cytokine, which was also known to be an activator of HIV-1 reproduction . We have generated the functional HIV-1 accessory gene product Vpr in bacterial cells . Vpr was generated in an Escherichia coli system (rVpr), purified with antibodies (Ab) to the 16 C-terminal amino acids of Vpr . The purified rVpr of 15 kDa was examined for its ability to upregulate HIV-1 reproduction in U1 cells, which is a reported function of the authentic Vpr . rVpr upregulated HIV-1 reproduction in U1 cells in a dose-dependent manner and induced the secretion of TNF . The upregulation of HIV-1 by rVpr was completely inhibited not only by anti-Vpr antibodies but also by anti-TNF antibody . These findings suggested that Vpr caused an HIV-1 reproduction in U1 cells through the induction of TNF.

Virus Res, 2002 Dec, 90(1-2), 171 - 85
Mapping of B-cell epitopic sites and delineation of functional domains on the hemagglutinin-neuraminidase protein of peste des petits ruminants virus; Renukaradhya GJ et al.; A recombinant baculovirus expressing membrane bound form of hemagglutinin-neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein . Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells . The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263-368 and 538-609 and hence the epitopes identified are conformation-dependent . The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera . Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity . Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%) . Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties . All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay.

Virus Res, 2002 Dec, 90(1-2), 91 - 9
Expression of foot and mouth disease virus non-structural polypeptide 3ABC induces histone H3 cleavage in BHK21 cells; Capozzo AV et al.; Auto-processing of the non-structural polypeptide 3ABC of foot and mouth disease virus (FMDV) expressed in Escherichia coli-BL21-DE3 was prevented by mutating either four glutamic acid residues at the 3A/3B1, 3B1/2, 3B2/3 and 3B3/3C junctions (3ABCtet) or a single cysteine residue at position 383 within the 3C domain (3ABCm) . Independent expression of 3ABC and 3ABCtet genes induced expression of chaperone DnaK and degradation of ribosomal S1 protein in E . coli . They also induced cleavage of nucleosomal histone H3 when transiently expressed in BHK21 cells . 3ABCtet, 3ABCm, 3AB and 3A proteins concentrated in the perinuclear region suggesting that peptide sequences within the 3A domain specify intracellular targeting of 3ABC in BHK-21 cells . We propose that 3ABC molecules localized in the nuclear periphery are a source of protease 3C activity and are responsible for histone H3 processing during FMDV infections.

Curr Opin Microbiol, 2002 Dec, 5(6), 591 - 5
Stationary phase gene regulation: what makes an Escherichia coli promoter sigmaS-selective?
Hengge-Aronis R.
The general stress sigma factor sigma(S) and the vegetative sigma(70) are highly related and recognise the same core promoter elements . Nevertheless, they clearly control different sets of genes in vivo . Recent studies have demonstrated that Esigma(S) selectivity is based on modular combinations of several sequence and structural features of a promoter, to which also trans-acting factors can strongly contribute . These results throw novel light on the details of transcription initiation, as well as on the co-evolution of sigma factors and their cognate promoter sequences.

Biochimie, 2002 Aug, 84(8), 731 - 43
Comparative anatomy of a regulatory ribosomal protein; Worbs M et al.; Ribosomal protein L4 is a crucial folding mediator and an important architectural component of the large ribosomal subunit . Furthermore, Escherichia coli L4 produced in excess of its rRNA binding sites downregulates the transcription and translation of its own S10 operon, encoding 11 ribosomal proteins . Genetic experiments and the crystal structure of Thermotoga maritima L4 had implicated separable regions on L4 in ribosome association and expression control while RNA competition experiments and the regulatory capacity of heterologous L4 had suggested an overlap of the protein sequences involved in the two functions . We report herein that contrary to other foreign bacterial L4 proteins, L4 from T . maritima only weakly controlled expression of the S10 operon in E . coli . Also, wildtype T . maritima L4 was more weakly associated with E . coli ribosomes than with the E . coli analog . Rational mutageneses were performed to try to increase the regulatory competence of T . maritima L4 . The ribosome incorporation of the mutant proteins was also investigated . Two different deletions removing T . maritima-specific sequences had little effects on regulation although one did improve ribosome association . Interestingly, a set of multiple mutations, which rendered the region around helices alpha4 and alpha5 in T . maritima L4 more E . coli-like, had no influence on the incorporation of the protein into the large ribosomal subunit but considerably improved its regulatory potential . Therefore, the area around helices alpha4 and alpha5, which is critical for the initial folding steps of the large subunit, is also a central element of autogenous control, presumably by contacting the S10 mRNA leader . Ribosome association is compounded at later stages of assembly by additional rRNA contacts through L4 areas which do not participate in regulation . Similarly, sequences outside the alpha4/alpha5 region aid expression control.

Biochimie, 2002 Aug, 84(8), 723 - 9
Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA; Bordeau V et al.; tmRNA (10Sa RNA, ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis . Ribosomal protein S1 is required for tmRNA binding to isolated and poly U-programmed ribosomes . Mobility assays on native gels indicate that the binding curves of both recombinant and purified proteins S1 from E . coli is biphasic with apparent binding constants of approximately 90 and approximately 300 nM, respectively, suggesting that more than one protein interacts with tmRNA . Structural probing of native tmRNA in the presence and absence of the purified protein suggest that when S1 binds, tmRNA undergoes a significant conformational change . In the presence of the protein, nucleotides from tmRNA with enhanced (H2, H3, PK1, PK2, PK4, in and around the first triplet to be translated), or decreased (H5 and PK2), reactivity towards a probe specific for RNA single-strands are scattered throughout the molecule, with the exception of the tRNA-like domain that may be dispensable for the interaction . Converging experimental evidence suggests that ribosomal protein S1 binds to pseudoknot PK2 . Previous structural studies of tmRNA in solution have revealed several discrepancies between the probing data and the phylogeny, and most of these are reconciled when analyzing tmRNA structure in complex with the protein(s) . Ribosomal protein(s) S1 is proposed to set tmRNA in the mRNA mode, relieving strains that may develop when translating a looped mRNA.

Biochimie, 2002 Aug, 84(8), 705 - 11
Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain; Choi H et al.; The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis . This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules . We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation . To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain . Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated . The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis . Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.

Biochimie, 2002 Aug, 84(8), 693 - 703
The residue immediately upstream of the RNase P cleavage site is a positive determinant; Brannvall M et al.; We have studied the importance of the residue at the position immediately upstream of the RNase P RNA cleavage site using model substrates that mimic the structure at and near the cleavage site of the tRNA(His) precursor . The various model substrates were studied with respect to cleavage site recognition as well as the kinetics of cleavage using M1 RNA, the catalytic subunit of Escherichia coli RNase P . Our studies showed that the identity of the residue immediately upstream of the cleavage site critically influences both these aspects . Among the ones tested, U is the preferred nucleotide at this position . Hence, these findings rationalize why most bacterial tRNA(His) genes/transcripts harbor a U immediately upstream of the RNase P cleavage site and extend our understanding of the cleavage site recognition process in general and the unusual cleavage of the tRNA(His) precursor in particular . Based on our as well as the data of others, we suggest that the nucleotide immediately upstream of the cleavage site is a positive determinant for cleavage by RNase P in general and the expression of tRNA genes is influenced by structural elements localized outside the promoter region i.e . in the leader and spacer regions of tRNA transcripts.

Swiss Med Wkly, 2002 Aug 10, 132(31-32), 433 - 42
RecQ helicases and genome stability: lessons from model organisms and human disease; Bjergbaek L et al.; Maintaining the integrity of genetic information is fundamental for the life of a cell and the survival of a species . Cells can encounter DNA damage as a consequence of normal cellular metabolism or as a result of exposure to chemical or physical agents . Eukaryotic cells have developed a network of responses in order to deal with DNA damage thereby preserving the integrity of their genetic information . In the presence of extensive genetic insult, a surveillance mechanism or "checkpoint" is activated . The activation of this signal transduction pathway leads to an arrest of cell cycle progression to prevent replication and segregation of damaged DNA molecules and to induce transcription of several repair genes . Existing repair mechanisms are also mobilised, in a coordinated effort to restore the original DNA structure . Genes involved in either cell cycle checkpoints, DNA repair or genes that maintain the fidelity of chromosome segregation are often termed "antimutators" or "caretaker" genes, because they control the stability of the genome and prevent accumulation of mutations in so-called "gatekeeper" genes . This latter group of genes directly regulate the growth of tumours either by inhibiting growth or promoting death . A fundamental requirement for many DNA metabolism processes is the separation of the complementary strands of the DNA duplex . This is promoted by DNA helicases, which unwind nucleic-acid duplexes in an ATP-dependent manner to provide access to the template for proteins of the replication, recombination, repair and transcription machineries . Multiple DNA helicase families have been identified, all containing seven hallmark helicase motifs; members within each helicase family also share sequence homologies beyond and between these motifs . One example is the RecQ helicase family, named after the RecQ protein of Escherichia coli, which was identified during a search for mutants sensitive to thymine starvation . Five members of the RecQ family have been identified in the human genome, and mutations in three of the genes are responsible for genetic diseases that are characterised by genomic instability and a high incidence of cancer . Because mutants in RecQ family genes in other species also have unstable chromosomes, it was proposed that members of the RecQ helicase family play a central role in the maintenance of genomic stability and thereby the prevention of tumorigenesis.

Cell Tissue Res, 2002 Dec, 310(3), 321 - 30 Epub 2002 Oct 26.
Biodefense function of omental milky spots through cell adhesion molecules and leukocyte proliferation; Cui L et al.; To evaluate the immunological functions of the greater omentum in the peritoneal cavity, the localization of cell adhesion molecules (CAMs) on mesothelial cells and leukocytes in the omental milky spots were studied in normal and lipopolysaccharide (LPS)-stimulated mice by means of immunoelectron microscopy . The milky spots featured numerous leukocytes among the dome-shaped mesothelial cells, even in the normal stable state . Leukocyte integrins LFA-1, Mac-1, and VLA-4 were preferentially localized to microvilli and ruffles of macrophages and lymphocytes . The mesothelial cells of the milky spots showed higher ICAM-1 levels than did those of other omental regions, and fibronectin was detected in the stomata . The number of leukocytes markedly increased following an increase in proliferating cell nuclear antigen (PCNA)-positive cells in the milky spots after LPS stimulation . The mesothelial cells contained VCAM-1 newly restricted to the microvilli and increasing amounts of ICAM-1 . These results show that the omental milky spots are active sites for leukocyte migration and peritoneal leukocyte supply because of the presence of adhesion molecules and active cell proliferation . Proliferative active leukocytes and those that have migrated from vessels pass through the stomata via an interaction of VLA-4 and fibronectin, adhere to the microvilli of the activated mesothelial cell surface as the result of an interaction between ICAM-1/VCAM-1 and integrins, and exude into the peritoneal cavity . Much of the exudation and adhesion of leukocytes seen in the milky spots of LPS-stimulated mice may be attributable to an increase in cell proliferation and in the amounts of ICAM-1 and VCAM-1.

Proc Natl Acad Sci U S A, 2002 Dec 10, 99(25), 15898 - 903 Epub 2002 Nov 27.
Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses; Garg AK et al.; Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates . With the notable exception of the desiccation-tolerant "resurrection plants," trehalose is not thought to accumulate to detectable levels in most plants . We report here the regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice . The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation . The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters . Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions . Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3-10 times that of the nontransgenic controls . The observation that peak trehalose levels remain well below 1 mgg fresh weight indicates that the primary effect of trehalose is not as a compatible solute . Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism . These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through tissue-specific or stress-dependent overproduction of trehalose.

J Cell Sci, 2003 Jan 1, 116(Pt 1), 187 - 96
Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium; Furukawa R et al.; The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes . To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein . Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium . A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa deltaEF2) was prepared using site-directed mutagenesis and expressed in E . coli . Equilibrium dialysis using {(45)Ca}CaCl(2) revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 microM . This calcium binding is absent in the 34 kDa deltaEF2 protein . The actin-binding activity of the 34 kDa deltaEF2 protein was equivalent to wildtype but calcium insensitive in vitro . The wild-type and 34 kDa deltaEF2 proteins were expressed in 34-kDa-null and 34 kDa/alpha-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo . The 34 kDa deltaEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains . Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa deltaEF2 . Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.

Blood, 2003 Apr 1, 101(7), 2652 - 60 Epub 2002 Nov 27.
Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells; Fiuza C et al.; Systemic inflammation because of sepsis results in endothelial cell activation and microvascular injury . High-mobility group protein-1 (HMGB1), a novel inflammatory molecule, is a late mediator of endotoxin shock and is present in the blood of septic patients . The receptor for advanced glycation end products (RAGE) is expressed on endothelium and is a receptor for HMGB1 . Here we examine the effects of HMGB1 on human endothelial cell function . Recombinant human HMGB1 (rhHMGB1) was cloned and expressed in Escherichia coli and incubated with human microvascular endothelium . rhHMGB1 caused a dose- and time-dependent increase in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and RAGE . rhHMGB1 induced the secretion of tumor necrosis factor-alpha (TNFalpha), interleukin 8 (IL-8), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), and tissue plasminogen activator (tPA) (P <.01) . rhHMGB1 stimulation resulted in transient phosphorylation of mitogen-activated protein (MAP) kinases, extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK), and p38, and in nuclear translocation of transcription factors NF-kappaB and Sp1 . These effects are partially mediated by TNFalpha autocrine stimulation, as anti-TNFalpha antibodies significantly decrease chemokine and adhesion molecule responses (P </=.002) . Thus, rhHMGB1 elicits proinflammatory responses on endothelial cells and may contribute to alterations in endothelial cell function in human inflammation.

J Chromatogr A, 2002 Nov 22, 977(2), 163 - 72
Acoustic field assisted demixing of aqueous two-phase systems; Nagaraj N et al.; Acoustic field assisted demixing was employed to decrease the demixing time in aqueous two-phase systems (polyethylene glycol-maltodextrin and polyethylene glycol-potassium phosphate) . Application of acoustic field has decreased the demixing time in polyethylene glycol-maltodextrin by around twofold and up to about 3.2-fold in polyethylene glycol-potassium phosphate systems . Ultrasonication has induced mild circulation currents in the phase dispersion, which has enhanced the rate of droplet coalescence, eventually resulting in decreased demixing time . In the polyethylene glycol-maltodextrin system, phase demixing was found to depend greatly on which of the phases iscontinuous and viscosity of the continuous phase was observed to have a strong influence on the movement of the droplets and hence controlling the phase demixing rate . In case of the polyethylene glycol-potassium phosphate system, droplet coalescence was found to play a critical role in phase demixing . Addition of NaCl increased the demixing time and presence of Escherichia coli cells did not seem to have any influence on phase demixing.

Eukaryot Cell, 2002 Feb, 1(1), 137 - 51
A new, expressed multigene family containing a hot spot for insertion of retroelements is associated with polymorphic subtelomeric regions of Trypanosoma brucei; Bringaud F et al.; We describe a novel gene family that forms clusters in subtelomeric regions of Trypanosoma brucei chromosomes and partially accounts for the observed clustering of retrotransposons . The ingi and ribosomal inserted mobile element (RIME) non-LTR retrotransposons share 250 bp at both extremities and are the most abundant putatively mobile elements, with about 500 copies per haploid genome . From cDNA clones and subsequently in the T . brucei genomic DNA databases, we identified 52 homologous gene and pseudogene sequences, 16 of which contain a RIME and/or ingi retrotransposon inserted at exactly the same relative position . Here these genes are called the RHS family, for retrotransposon hot spot . Comparison of the protein sequences encoded by RHS genes (21 copies) and pseudogenes (24 copies) revealed a conserved central region containing an ATP/GTP-binding motif and the RIME/ingi insertion site . The RHS proteins share between 13 and 96% identity, and six subfamilies, RHS1 to RHS6, can be defined on the basis of their divergent C-terminal domains . Immunofluorescence and Western blot analyses using RHS subfamily-specific immune sera show that RHS proteins are constitutively expressed and occur mainly in the nucleus . Analysis of Genome Survey Sequence databases indicated that the Trypanosoma brucei diploid genome contains about 280 RHS (pseudo)genes . Among the 52 identified RHS (pseudo)genes, 48 copies are in three RHS clusters located in subtelomeric regions of chromosomes Ia and II and adjacent to the active bloodstream form expression site in T . brucei strain TREU927/4 GUTat10.1 . RHS genes comprise the remaining sequence of the size-polymorphic "repetitive region" described for T . brucei chromosome I, and a homologous gene family is present in the Trypanosoma cruzi genome.

Prep Biochem Biotechnol, 2002 Nov, 32(4), 393 - 403
The overexpressed hexahistidine-tagged human hexokinase type III is inhibited by D-glucose; Palma F et al.; Inhibition by its product, glucose, is a kinetic property of hexokinase type III . In this paper, we report the overexpression in Escherichia coli of human hexokinase type III . The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end . This modification confers to the product the ability to bind the Ni2+ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups . The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent . Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg . Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose . The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.

Res Microbiol, 2002 Nov, 153(9), 557 - 62
Role of the RNA polymerase sigma subunit in transcription initiation; Borukhov S et al.; In bacteria, sigma subunits direct the catalytically competent RNA polymerase core enzyme to promoters . Recent advances in our understanding of bacterial RNA polymerase reveal that sigma subunits are intimately involved in all aspects of transcription initiation including promoter location, promoter melting, initiation of RNA synthesis, abortive initiation and promoter escape.

Can J Microbiol, 2002 Sep, 48(9), 801 - 9
Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase; Cottrill MA et al.; When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity . Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10 . Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C . while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C . The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively . High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5 . D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively . These data support the contention that Agp is a 3-phosphatase.

Poult Sci, 2002 Nov, 81(11), 1671 - 80
Effect of addition of a detoxifying agent to laying hen diets containing uncontaminated or Fusarium toxin-contaminated maize on performance of hens and on carryover of zearalenone; Danicke S et al.; 16-wk experiment with laying hens was carried out to examine the effects of feeding of mycotoxin-contaminated maize (CM) on performance, nutrient digestibility, weight of organs, serum chemical parameters, and antibody titers to Newcastle disease virus (NDV) in serum . Also tested were fimbrien antigen K88 in egg yolk and zearalenone (ZON) residues in eggs and tissues . The Fusarium-toxin-contaminated maize contained 17,630 microg deoxynivalenol and 1,580 microg ZON/kg . Moreover, Mycofix Plus (MP), a so-called detoxifying agent, was added to both the uncontaminated control (UCM) and to the CM diet (70% dietary maize inclusion) . Each of the four resulting diets (UCM, UCM-MP, CM, CM-MP) was tested on 25 laying hybrids (Lohmann Brown) . Feeding of the CM diets significantly depressed feed intake compared to the control groups by approximately 5% . This was mainly due to the effects observed at the beginning of the experiment . Daily egg mass production/hen was 56.6, 58.4, 53.9, and 55.2 g in groups UCM, UCM-MP, CM and CM-MP, respectively . Nutrient digestibility and metabolizability of gross energy were slightly depressed by feeding the CM diets and improved by MP addition . Feeding of the CM diets resulted in a significant decrease in serum titers to NDV and to an increase in yolk titers to antigen K88 . No residues of ZON or of its metabolites were found in yolk, albumen, abdominal fat, breast meat, follicles greater than 1 cm in diameter, ovaries including follicles smaller than 1 cm in diameter, magnum, and serum . ZON and alpha-zearalenol (alpha-ZOL) were detected in livers of hens fed the CM diets at mean concentrations of 2.1 and 3.7 microg/kg, respectively . It was concluded that feeding maize which was highly contaminated with Fusarium mycotoxins adversely influenced performance of hens and modulated immune response . At the given level of zearalenone and at the indicated detection limits, no residues of ZON and its metabolites were found in eggs . The effects of the tested detoxifying agent were quite mycotoxin-independent.

Curr Drug Targets Infect Disord, 2001 Nov, 1(3), 263 - 71
Novel adjuvant systems; McCluskie MJ et al.; Vaccination remains the single most valuable tool in the prevention of infectious disease . Nevertheless, there exists a need to improve the performance of existing vaccines such that fewer boosts are needed or to develop novel vaccines . For the development of effective vaccines for humans, a great need exists for safe and effective adjuvants . A number of novel adjuvants have been reported in recent years including: i) bacterial toxins such as cholera toxin, CT, and the Escherichia coli heat-labile enterotoxin, LT; ii) less toxic derivatives of CT and LT; iii) endogenous human immunomodulators, such as IL-2, IL-12, GM-CSF; iv) hormones; v) lipopeptides; vi) saponins, such as QS-21; vii) synthetic oligonucleotides containing CpG motifs (CpG ODN); viii) lipid 'A derivatives, such as monophosphoryl lipid A, MPL, and ix) muramyl dipeptide (MDP) derivatives . Herein, we will review recent findings using these novel adjuvant systems.

Ann Hematol, 2002 Nov, 81(11), 622 - 6 Epub 2002 Nov 09.
Regeneration of splenic autotransplants; Marques RG et al.; Splenic autotransplantation seems to be the only alternative for preservation of splenic tissue after total splenectomy . This work was carried out to analyze the morphologic regeneration of autotransplanted splenic tissue in Wistar rats and to determine the bacterial phagocytic function of their macrophages . We utilized an experimental model including young and adult rats, of both sexes, submitted to total splenectomy combined with autotransplantation in the greater omentum of slices of the whole mass of spleen . Sixteen weeks later animals were intravenously inoculated with a suspension of Escherichia coli AB1157 . There was regeneration of autotransplanted splenic tissue in all animals . A similar morphological aspect among all animals was observed, with splenic tissue showing red and white pulps with a moderate architectural disarrangement . Macrophages containing bacterial aggregates were observed, as well as macrophages with hemosiderin pigments inside the cytoplasm . Blood vessels showed preserved walls, with no signs of vasculitis or thrombosis . The present results suggest that splenic autotransplants in the greater omentum of the rat acquire the macro- and microscopic architecture of a normal spleen, with reduced dimensions, and preserve bacterial phagocyte function.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2209 - 12 Epub 2002 Nov 23.
Structure of Escherichia coli pyruvate formate-lyase with pyruvate; Lehtio L et al.; The structure of inactive pyruvate formate-lyase in complex with a natural substrate, pyruvate, was solved at 2.7 A resolution . Both active sites of the homodimeric enzyme are occupied by pyruvate; additional binding sites were not found . Pyruvate was found in a cleft close to the active-site cysteines 418 and 419, with the carboxyl group in contact with arginines 176 and 435 and the methyl group within van der Waals distance of Phe327 . It is believed that the binding site of pyruvate is not the position of pyruvate as the reaction initiates, as conformational changes occur during activation of the enzyme.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2191 - 3 Epub 2002 Nov 23.
Expression, purification, crystallization and preliminary characterization of an HHED aldolase homologue from Escherichia coli K12; Wright A et al.; An ORF designated b2245 (yfaU) in the Escherichia coli K12 genome sequence, identified as an HHED aldolase homologue, was cloned into the high-expression plasmid pT7-7 and overexpressed in E . coli B835(DE3) . The enzyme was purified in three steps to 95% purity prior to crystallization . Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions . Crystals suitable for structural studies were grown from solutions containing 0.4 M ammonium dihydrogen phosphate and grew to a maximum dimension of approximately 0.5 mm . Diffraction data to 1.7 A were collected using an in-house Cu Kalpha radiation source at 100 K . The crystals belong to space group C222(1), with unit-cell parameters a = 105.1, b = 136.6, c = 123.1 A . A 90% complete data set was collected to 1.78 A from a single native crystal using in-house facilities.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2170 - 2 Epub 2002 Nov 23.
Expression, crystallization and preliminary X-ray studies of the recombinant PTB domain of human dok-5 protein; Shi N et al.; The human dok-5 PTB domain fusion protein has been overexpressed in Escherichia coli and crystallized in a form suitable for X-ray crystallographic study . Crystals were obtained by the vapour-diffusion method . The crystal has unit-cell parameters a = b = 75.9, c = 108.0 A, alpha = beta = 90, gamma = 120 degrees and belongs to space group P3(2)21 . Diffraction data were collected to 2.8 A resolution in-house . Furthermore, a selenomethionine (SeMet) derivative of dok-5 PTB domain fusion protein was overexpressed using the same expression system and was purified in a reductive environment . The derivative crystals were obtained under similar conditions . Subsequently, three different wavelength data sets were collected to 2.3 A resolution from the derivative crystal at the Advanced Photon Source, Argonne National Laboratory.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2157 - 8 Epub 2002 Nov 23.
Crystallization and preliminary X-ray diffraction studies of NusG, a protein shared by the transcription and translation machines; Andrykovitch M et al.; N-utilization factor G (NusG) from Aquifex aeolicus (Aa) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion technique . The drops consisted of 2.5 microl protein solution (approximately 30 mg ml(-1) in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM EDTA and 10 mM DTT) and 2.5 microl reservoir solution (0.085 M Na HEPES pH 7.5, 15% glycerol, 11% 2-propanol and 20% PEG 4000) derived from condition number 41 of the Hampton Cryo Screen . The crystals grew at 291 +/- 1 K and reached dimensions of 0.2 x 0.1 x 0.05 mm in 5-7 d . The crystals, which diffracted to 2.45 A resolution, belonged to space group C222(1), with unit-cell parameters a = 65.95, b = 124.58, c = 83.60 A . One AaNusG molecule is present in the asymmetric unit, corresponding to a solvent content of 59.80% (Matthews coefficient = 3.06 A(3) Da(-1)) . Crystal structure determination is in progress.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2141 - 4 Epub 2002 Nov 23.
Crystallization and preliminary X-ray analysis of the tumor metastasis factor p37; Reutzel R et al.; P37, an outer-membrane bacterial protein from Mycoplasma hyorhinis, is a molecule whose presence on the surface of many tumor cells correlates highly with increased neoplastic invasivity and metastasis . P37 was overexpressed in Escherichia coli, purified by affinity chromatography and crystallized . Useful single crystals for X-ray diffraction structural studies have been grown by oil-immersion methods from a solution of 40% PEG 4000, 0.1 M ammonium bromide in a 0.1 M citrate buffer at pH 4.0 . X-ray diffraction data were collected at the F2 beamline at CHESS with a crystal-to-CCD detector distance of 150 mm, collecting 1 degrees oscillation slices with an exposure time of 30 s per frame . A 212 degrees sweep of data (99.8% completeness) were collected from a single crystal under cryoconditions, with a maximal useful diffraction pattern to 1.8 A resolution . The crystals are shown to be monoclinic and have been assigned to space group P2(1), with unit-cell parameters a = 50.02, b = 67.26, c = 59.89 A, beta = 108.29 degrees and a scaling R(sym) of 0.076 for 34,882 unique reflections . Packing considerations indicate that there is one molecule per asymmetric unit . It is expected that in the near future the structure of p37 will be obtained using phases from traditional heavy-atom isomorphous replacement and/or halide-soak methods . Elucidation of the structure of p37 may be paramount to producing new antibody-based anticancer therapeutic agents.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2131 - 4 Epub 2002 Nov 23.
The production, purification and crystallization of a soluble heterodimeric form of a highly selected T-cell receptor in its unliganded and liganded state; Clements CS et al.; T-cell antigen receptors (TcRs) are heterodimeric cell-surface receptors that play a pivotal role in the cellular immune response . The TcR interacts specifically with a peptide-laden major histocompatability complex (pMHC) . A human TcR has been characterized that interacts with an immunodominant epitope, FLRGRAYGL, from the Epstein-Barr virus, a ubiquitous human pathogen, in complex with HLA-B8 . Despite the vast TcR repertoire, this TcR is found in up to 10% of the total T-cell population in seropositive HLA-B8+ individuals . In this report, this highly selected TcR is characterized by expressing in Escherichia coli, refolding, purifying and crystallizing the receptor . In addition, the HLA-B8-FLRGRAYGL complex has been expressed in E . coli, refolded and shown to be functionally active . Using native gel electrophoresis, the refolded TcR is shown to be capable of binding specifically to the refolded HLA-B8-FLRGRAYGL and this TcR has been crystallized in complex with the pMHC . The crystals of the unliganded and liganded TcR diffract to 1.5 and 2.5 A, respectively.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2116 - 21 Epub 2002 Nov 23.
Autotracing of Escherichia coli acetate CoA-transferase alpha-subunit structure using 3.4 A MAD and 1.9 A native data; Korolev S et al.; The automation of protein structure determination is an essential component for high-throughput structural analysis in protein X-ray crystallography and is a key element in structural genomics . This highly challenging undertaking relies at present on the availability of high-quality native and derivatized protein crystals diffracting to high or moderate resolution, respectively . Obtaining such crystals often requires significant effort . The present study demonstrates that phases obtained at low resolution (>3.0 A) from crystals of SeMet-labeled protein can be successfully used for automated structure determination . The crystal structure of acetate CoA-transferase alpha-subunit was solved using 3.4 A multi-wavelength anomalous dispersion data collected from a crystal containing SeMet-substituted protein and 1.9 A data collected from a native protein crystal.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2109 - 15 Epub 2002 Nov 23.
A medium-throughput crystallization approach; Sulzenbacher G et al.; The first results of a medium-scale structural genomics program clearly demonstrate the value of using a medium-throughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions . The structural genomics program was based on cloning in the Gateway vectors pDEST17, introducing a long 21-residue tail at the N-terminus . So far, this tail has not appeared to hamper crystallization . In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and five to structures . Furthermore, the results using a robot dispensing 50-200 nl drops indicate that smaller protein samples can be used for crystallization . These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2102 - 8 Epub 2002 Nov 23.
Parallel cloning, expression, purification and crystallization of human proteins for structural genomics; Ding HT et al.; 54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization . Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e . more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes . Four proteins containing transmembrane regions were selected for comparative tests . To date, 44 expression clones have been constructed with the Gateway cloning system (Invitrogen, The Netherlands) . Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity . Crystallization conditions were screened for the purified proteins in 96-well plates under oil . After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes . A 2.12 A data set was collected for protein NCC27 . The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2096 - 101 Epub 2002 Nov 23.
S-SAD, Se-SAD and S/Se-SIRAS using Cu Kalpha radiation: why wait for synchrotron time?
Lemke CT, Smith GD, Howell PL.
The structure of Escherichia coli argininosuccinate synthetase (EAS) has been determined using S-SAD, Se-SAD and S/Se-SIRAS data measured with Cu Kalpha radiation . EAS contains 16 methionines and three cysteines in 455 amino acids . At a wavelength of 1.54 A (Cu Kalpha), the native (S-Met) and derivative (Se-Met) proteins yield anomalous signals of approximately 0.86 and 1.6%, respectively . Highly redundant data were measured to 2.0 A from native and derivative EAS crystals . All three structure determinations were carried out in a highly automated manner using SnB and SOLVE/RESOLVE . Despite the minute Bijvoet differences at 1.54 A, the signal was sufficient to determine the heavy-atom substructure and produce high-quality electron-density maps in all three cases . These maps were readily interpretable by the RESOLVE automated building algorithm, which modeled greater than 75% of all three structures . The success of these methods has profound implications for crystallographers experiencing difficulty with heavy-atom incorporation or with limited access to a synchrotron source.

J Clin Microbiol, 2002 Dec, 40(12), 4486 - 92
Genetic diversity of intimin genes of attaching and effacing Escherichia coli strains; Zhang WL et al.; In this study, we determined the sequences of four intimin variant genes detected in attaching and effacing Escherichia coli isolates of human origin . Three of them were novel and were designated eae-eta (eta), eae-iota (iota), and eae-kappa (kappa) . The fourth was identical to the recently described eae-zeta (zeta), isolated from a bovine E . coli O84:NM isolate . We compared these sequences with those of published intimin-alpha, intimin-beta, intimin-gamma1, intimin-gamma2, intimin- epsilon, and intimin-theta alleles . Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family in E . coli . The genetic diversity was more prominent in the 3' region (starting at bp 2,112), which encodes the binding domain of intimin . Phylogenetic analyses revealed four groups of closely related intimin genes: alpha and zeta; beta and kappa; gamma1 and gamma2/theta; and epsilon and eta . Calculation of homoplasy ratios of sequences of the 5' region of eae (positions 1 to 2,111) revealed evidence for intragenic recombination . Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history of eae . In conclusion, we recommend an eae nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E . coli eae.

J Clin Microbiol, 2002 Dec, 40(12), 4445 - 9
Differences in virulence factors among clinical isolates of Escherichia coli causing cystitis and pyelonephritis in women and prostatitis in men; Ruiz J et al.; Differences in the presence of nine urovirulence factors among clinical isolates of Escherichia coli causing cystitis and pyelonephritis in women and prostatitis in men have been studied . Hemolysin and necrotizing factor type 1 occur significantly more frequently among isolates causing prostatitis than among those causing cystitis (P < 0.0001) or pyelonephritis (P < 0.005) . Moreover, the papGIII gene occurred more frequently in E . coli isolates associated with prostatitis (27%) than in those associated with pyelonephritis (9%) (P < 0.05) . Genes encoding aerobactin and PapC occurred significantly less frequently in isolates causing cystitis than in those causing prostatitis (P < 0.01 and P < 0.0001, respectively) and pyelonephritis (P < 0.01 and P < 0.0001, respectively) . No differences in the presence of Sat or type 1 fimbriae were found . Finally, AAFII and Bfp fimbriae are no longer considered uropathogenic virulence factors since they were not found in any of the strains analyzed . Overall, the results showed that clinical isolates producing prostatitis need greater virulence than isolates producing pyelonephritis in women or, in particular, cystitis in women (P < 0.05) . Overall, the results suggest that clinical isolates producing prostatitis are more virulent that those producing pyelonephritis or cystitis in women.

Genetics, 2002 Nov, 162(3), 1055 - 62
Enrichment and elimination of mutY mutators in Escherichia coli populations; Notley-McRobb L et al.; The kinetics of mutator sweeps was followed in two independent populations of Escherichia coli grown for up to 350 generations in glucose-limited continuous culture . A rapid elevation of mutation rates was observed in both populations within 120-150 generations, as was apparent from major increases in the proportion of the populations with unselected mutations in fhuA . The increase in mutation rates was due to sweeps by mutY mutators . In both cultures, the enrichment of mutators resulted from hitchhiking with identified beneficial mutations increasing fitness under glucose limitation; mutY hitchhiked with mgl mutations in one culture and ptsG in the other . In both cases, mutators were enriched to constitute close to 100% of the population before a periodic selection event reduced the frequency of unselected mutations and mutators in the cultures . The high proportion of mutators persisted for 150 generations in one population but began to be eliminated within 50 generations in the other . The persistence of mutator, as well as experimental data showing that mutY bacteria were as fit as near-isogenic mutY(+) bacteria in competition experiments, suggest that mutator load by deleterious mutations did not explain the rapidly diminishing proportion of mutators in the populations . The nonmutators sweeping out mutators were also unlikely to have arisen by reversion or antimutator mutations; the mutY mutations were major deletions in each case and the bacteria sweeping out mutators contained intact mutY . By following mgl allele frequencies in one population, we discovered that mutators were outcompeted by bacteria that had rare mgl mutations previously as well as additional beneficial mutation(s) . The pattern of appearance of mutY, but not its elimination, conforms to current models of mutator sweeps in bacterial populations . A mutator with a narrow mutational spectrum like mutY may be lost if the requirement for beneficial mutations is for changes other than GC --> TA transversions . Alternatively, epistatic interactions between mutator mutation and beneficial mutations need to be postulated to explain mutator elimination.

Genetics, 2002 Nov, 162(3), 1031 - 43
A novel class of secA alleles that exert a signal-sequence-dependent effect on protein export in Escherichia coli; Khatib K et al.; The murine plasminogen activator inhibitor 2 (PAI2) signal sequence inefficiently promotes the export of E . coli alkaline phosphatase (AP) . High-level expression of PAI2::AP chimeric proteins from the arabinose P(BAD) promoter is toxic and confers an Ara(S) phenotype . Most Ara(R) suppressors map to secA, as determined by sequencing 21 independent alleles . Mutations occur throughout the gene, including both nucleotide binding domains (NBDI and NBDII) and the putative signal sequence binding domain (SSBD) . Using malE and phoA signal sequence mutants, we showed that the vast majority of these secA suppressors exhibit weak Sec phenotypes . Eight of these secA mutations were further characterized in detail . Phenotypically, these eight suppressors can be divided into three groups, each localized to one domain of SecA . Most mutations allow near-normal levels of wild-type preprotein export, but they enhance the secretion defect conferred by signal sequence mutations . Interestingly, one group exerts a selective effect on the export of PAI2::AP when compared to that of AP . In conclusion, this novel class of secA mutations, selected as suppressors of a toxic signal sequence, differs from the classical secA (prlD) mutations, selected as suppressors of defective signal sequences, although both types of mutations affect signal sequence recognition.

J Biol Chem, 2003 Feb 14, 278(7), 4963 - 71 Epub 2002 Nov 25.
Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase . New insights into the phosphoryl transfer mechanism; Haouz A et al.; The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported . The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring . The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion . The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A . It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99) . Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group . The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures . This alternative binding pocket opens a way for the design of a new family of specific inhibitors.

J Biol Chem, 2003 Feb 21, 278(8), 5646 - 51 Epub 2002 Nov 25.
Phosphorylation of supernatant protein factor enhances its ability to stimulate microsomal squalene monooxygenase; Singh DK et al.; Supernatant protein factor is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway . The mechanism of stimulation is poorly understood, although supernatant protein factor belongs to a family of lipid-binding proteins that includes Sec14p and alpha-tocopherol transfer protein . Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a relatively weak ability to activate microsomal squalene monooxygenase, we investigated the possibility that cofactors or post-translational modifications were necessary for full activity . Addition of ATP to rat liver cytosol increased supernatant protein factor activity by more than 2-fold and could be prevented by the addition of inhibitors of protein kinases A and C . Incubation of purified recombinant supernatant protein factor with ATP and protein kinases A or C delta similarly increased activity by more than 2-fold . Addition of protein phosphatase 1 gamma, a serine/threonine phosphatase, to rat liver cytosol reduced activity by 50%, suggesting that supernatant protein factor is partially phosphorylated in vivo . To determine whether dietary cholesterol influenced the phosphorylation state, cytosols were prepared from livers of rats fed a high fat diet . Although supernatant protein factor activity was reduced by more than one-half, it could not be restored by the addition of ATP or protein kinase C delta with ATP, suggesting that dietary cholesterol reduced the expression of this protein . Supernatant protein factor thus appears to be regulated both post-translationally through phosphorylation and at the level of expression . Phosphorylation may provide a means for the rapid short term modulation of cholesterol synthesis.

Mol Cell, 2002 Nov, 10(5), 1247 - 53
RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators; Harris RS et al.; APOBEC1 is the catalytic component of an RNA editing complex but shows homology to activation-induced cytidine deaminase (AID), a protein whose function is to potentiate diversification of immunoglobulin gene DNA . Here, we show that APOBEC1 and its homologs APOBEC3C and APOBEC3G exhibit potent DNA mutator activity in an E . coli assay . Indeed, like AID, these proteins appear to trigger DNA mutation through dC deamination . However, each protein exhibits a distinct local target sequence specificity . The results reveal the existence of a family of potential active dC/dG mutators, with possible implications for cancer.

Mol Cell, 2002 Nov, 10(5), 1151 - 62
Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination; Komissarova N et al.; Passage of E . coli RNA polymerase through an intrinsic transcription terminator, which encodes an RNA hairpin followed by a stretch of uridine residues, results in quick dissociation of the elongation complex . We show that folding of the hairpin disrupts the three upstream base pairs of the 8 bp RNA:DNA hybrid, a major stability determinant in the complex . Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex . During termination, the hairpin does not directly compete for base pairing with the 8 bp hybrid . Thus, melting of the hybrid seems to result from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem . Our results suggest that a similar mechanism disrupts elongation complexes of yeast RNA polymerase II in vitro.

Mol Cell, 2002 Nov, 10(5), 1007 - 17
Topography for independent binding of alpha-helical and PPII-helical ligands to a peroxisomal SH3 domain; Douangamath A et al.; While the function of most small signaling domains is confined to binary ligand interactions, the peroxisomal Pex13p SH3 domain has the unique capacity of binding to two different ligands, Pex5p and Pex14p . We have used this domain as a model to decipher its structurally independent ligand binding sites . By the combined use of X-ray crystallography, NMR spectroscopy, and circular dichroism, we show that the two ligands bind in unrelated conformations to patches located at opposite surfaces of this SH3 domain . Mutations in the Pex13p SH3 domain that abolish interactions within the Pex13p-Pex5p interface specifically impair PTS1-dependent protein import into yeast peroxisomes.

Mol Microbiol, 2002 Dec, 46(5), 1415 - 27
Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be site; Collis CM et al.; Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron-encoded IntI integrases . IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved . Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays . IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette-associated 59-be site and the cognate attI1 or attI3 site . Integrative recombination events involving a 59-be and a non-cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely . In cassette excision assays, the non-cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3 . The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.

Mol Microbiol, 2002 Dec, 46(5), 1399 - 413
MglA, a small GTPase, interacts with a tyrosine kinase to control type IV pili-mediated motility and development of Myxococcus xanthus; Thomasson B et al.; The mglA gene encodes a 22 kDa GTPase that is critical for single-cell (A) gliding, type IV pili-mediated (S) gliding and development of Myxococcus xanthus . To identify components that interact with MglA to control these processes, second-site mutations that restore movement to non-motile mglA mutants were sought . An allele-specific extragenic suppressor of mglA8, named mas815 (mglA8 suppressor 15), was obtained . mas815 does not bypass the requirement for MglA, yet it restores type IV pili-mediated motility and starvation-induced development . Single-cell (A) motility is not restored . The suppressing mutation maps to the 3' end of a gene, masK, in an operon immediately upstream of the mglBA operon . masK encodes a protein of the STY kinase family . When the masK gene was used as bait against a library carrying M . xanthus DNA in the yeast two-hybrid system, eight positive, independent clones containing fusions of mglA to GAL4 were obtained, thus confirming the interaction between MglA and MasK . MasK, expressed in Escherichia coli, was shown to phosphorylate at a tyrosine residue(s) . The gain-of-function in the masK815 mutant was correlated with increased production of extracellular fibrils, which are required for adhesion, cell-cell contact and sensing phosphatidylethanolamine chemoattractants . These data suggest that the interaction between MasK and MglA is an essential part of a signal transduction pathway controlling motility and development.

Mol Microbiol, 2002 Dec, 46(5), 1391 - 7
In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperatures; Gur E et al.; The formation of protein aggregates is associated with unfolding and denaturation of proteins . Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress . Here, we show that the aggregation of one single E . coli cytoplasmic protein limits growth at elevated temperatures in minimal media . Homoserine trans-succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temperatures higher than 44 degrees C in vitro . Above this temperature, we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the soluble fraction . Moreover, reducing the in vivo level of HTS aggregation enables growth at non-permissive temperatures . This is the first demonstration of the physiological role of aggregation of a specific protein in the growth of wild-type bacteria.

Mol Microbiol, 2002 Dec, 46(5), 1283 - 94
Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator; Yoshida T et al.; EnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins . Here, we re-examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR-P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo . Using an identical method, the Kd value for the interaction of the fluorescein-labelled OmpR (Fl-OmpR) with EnvZc was determined to be 1.96 +/- 0.28 micro M . We demonstrated that OmpR-P as well as OmpR inhibited the interaction of Fl-OmpR with EnvZc . Their 50% inhibitory concentrations were 1.09 +/- 0.25 micro M and 0.89 +/- 0.14 micro M, respectively, under the conditions used . The interaction between His-10-OmpR and EnvZc was also inhibited almost equally with OmpR-P and OmpR . Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR-P (phosphatase), indicating that EnvZc is able to interact with Fl-OmpR or Fl-OmpR-P as well as with OmpR or OmpR-P as a substrate . We demonstrated that OmpR-P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase . These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR-P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.

Mol Microbiol, 2002 Dec, 46(5), 1273 - 82
Formation of the stoichiometric complex of EnvZ, a histidine kinase, with its response regulator, OmpR; Yoshida T et al.; EnvZ, a histidine kinase, and its cognate response regulator OmpR of Escherichia coli are responsible for adaptation to external osmotic changes by regulating the levels of the outer membrane porin proteins, OmpF and OmpC . The osmosensor, EnvZ, has dual enzymatic functions with OmpR kinase and OmpR-P phosphatase . Here, we demonstrate that the cytoplasmic kinase domain of EnvZ (EnvZc) and OmpR are able to form a 1:1 complex detected by native PAGE . This indicates that two OmpR molecules can bind to one EnvZc dimer . As this 1:1 EnvZc/OmpR complex is formed even in the presence of a large excess of EnvZc, OmpR binding to EnvZc is co-operative . The complex formation is also observed between EnvZc and phosphorylated OmpR for the phosphatase reaction . OmpR-P bound to EnvZc was readily released upon the addition of OmpR, indicating that OmpR and OmpR-P can compete for the binding to EnvZ . On the basis of these results, a model is discussed to explain how cellular OmpR-P concentrations are regulated in response to medium osmolarity.

Mol Microbiol, 2002 Dec, 46(5), 1247 - 57
Governor of the glnAp2 promoter of Escherichia coli; Atkinson MR et al.; Low-affinity sites for the activator NRI-P (NtrC-P) that map between the enhancer and the glnAp2 promoter were responsible for limiting promoter activity at high concentrations of NRI approximately P in intact cells and in an in vitro transcription system consisting of purified bacterial components . That is, the low-affinity sites constitute a 'governor', limiting the maximum promoter activity . As the governor sites are themselves far from the promoter, they apparently act either by preventing the formation of the activation DNA loop that brings the enhancer-bound activator and the promoter-bound polymerase into proximity or by preventing a productive interaction between the enhancer-bound activator and polymerase . The combination of potent enhancer and governor sites at the glnAp2 promoter provides for efficient activation of the promoter when the activator concentration is low, while limiting the maximum level of promoter activity when the activator concentration is high.

Eur J Neurosci, 2002 Nov, 16(10), 1949 - 58
Prominent expression of Narp in central vestibular pathways: selective effect of labyrinth ablation; Reti IM et al.; Recent in vitro studies demonstrated that Narp, a secreted immediate early gene (IEG) product, induces AMPA receptor clustering . Accordingly, Narp has been implicated in mediating activity-dependent changes in synaptic efficacy . To help define the role of Narp in vivo, we conducted immunohistochemical studies of Narp in rat brain . Unexpectedly, we found robust Narp expression in several discrete areas linked to the vestibular system: the anterodorsal nucleus (ADN) of the thalamus, which relays head orientation information to the cortex, the lateral vestibulospinal (Deiters') nucleus and Purkinje cells in the flocculonodular lobe of the cerebellum . Although strong Narp expression in Deiters' nucleus and the cerebellum was present consistently, Narp expression in the ADN displayed a high degree of variability among animals . To check if this variability in ADN Narp expression reflects its dependence on fluctuating levels of vestibular input, we monitored Narp immunostaining following bilateral labyrinth ablation . This procedure significantly suppressed Narp immunostaining in the ADN, indicating that it is stimulated by naturally occurring vestibular input . In contrast, labyrinth ablation did not affect Narp staining in Deiters' nucleus or the flocculonodular lobe of the cerebellum, presumably because these areas are driven by inputs from multiple systems . As previous studies implicate Narp in synaptic plasticity, these findings suggest that this IEG may mediate ongoing adjustments in synaptic strength or connectivity in several pathways linked to the vestibular system.

J Appl Microbiol, 2002, 93(6), 1026 - 33
Serotypes and virulence factors of Shiga toxin-producing Escherichia coli isolated from healthy Norwegian sheep; Urdahl AM et al.; AIMS: To characterize a number of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep and to discuss the potential of these isolates as human pathogens . METHODS AND RESULTS: Twelve different O-groups and seven different H-types were identified by standard serotyping methods . The most common serotypes were O5:NM, O6:H10, O91:NM and O128:NM . Polymerase chain reaction (PCR) was used for the detection of virulence factor genes . Of 102 isolates, 86.3% carried stx1 and 83% of these were also positive in the stx1OX3-specific PCR . stx2 was carried by 55.9% of the isolates and 77.2% of these were also positive in the stx2d-specific PCR . The Vero cell assay showed high toxin production in 70.6% of the isolates . None of the isolates carried eae . CONCLUSIONS: The study supports the animal-host relationship suggested in other studies with STEC serogroups O5, O91 and O128 strongly associated with sheep . Most sheep STEC carry stx1OX3 (except O91) and the dominating stx2 variant is stx2d . One stx profile clearly dominates within a serotype . SIGNIFICANCE AND IMPACT OF THE STUDY: In spite of the predominance of certain sheep-associated STEC, sheep cannot be excluded as carriers of human pathogenic STEC.

Biotechnol Appl Biochem, 2002 Dec, 36(Pt 3), 213 - 8
The calcium-binding protein of Entamoeba histolytica as a fusion partner for expression of peptides in Escherichia coli; Reddi H et al.; We describe the construction of an Escherichia coli expression vector, CBP that allows the C-terminal fusion of heterologous proteins to the calcium-binding protein (CaBP) of the parasitic protozoan Entamoeba histolytica . The intrinsic nature of this protein to remain soluble on heat treatment has been exploited in its use as a novel fusion partner . The presence of a histidine tag and an enterokinase recognition site, aid in the affinity purification and proteolytic cleavage of the fusion protein . The efficacy of the vector was tested using the preS1 region of the envelope protein of the hepatitis B virus . The CaBP-preS1 fusion protein partitioned in the soluble fraction on heat treatment and this facilitated its rapid purification.

J Agric Food Chem, 2002 Dec 4, 50(25), 7264 - 70
Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability; Lin MW et al.; A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach . Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues . The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species . Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study . To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3) . Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow . The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg . The activity of the dimer was 65% higher than that of the monomer . The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min . Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS . The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure . Imidazole caused the dimer to dissociate into monomers . These studies suggested subunit interaction might be important for enzyme stability.

J Agric Food Chem, 2002 Dec 4, 50(25), 7258 - 63
A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid; Chen KC et al.; A cDNA encoding a small cysteine-rich protein designated VrCRP was isolated from a bruchid-resistant mungbean . VrCRP encodes a protein of 73 amino acids containing a 27 amino acid signal peptide and 8 cysteines . On the basis of the amino acid sequence similarity and conserved residues, it is suggested that VrCRP is a member of the plant defensin family . VrCRP protein was obtained by overexpression of VrCRP with a truncated signal peptide in an IMPACT system . Artificial seeds containing 0.2% (w/w) of the purified VrCRP-TSP were lethal to larvae of the bruchid Callosobruchus chinensis . VrCRP is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C . chinensis.

Biol Chem, 2002 Oct, 383(10), 1611 - 9
Three-state equilibrium of Escherichia coli trigger factor; Patzelt H et al.; Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli . Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2-3 fold molar excess over ribosomes and a fraction of it is not ribosome-associated after cell lysis . Here we show that TF follows a three-state equilibrium . Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 microM . Dimerization is mediated by the N-terminal ribosome binding domain and the C-terminal domain of TF, whereas the central peptidyl prolyl isomerase (PPlase) and substrate binding domain does not contribute to dimerization . Crosslinking experiments showed that TF is monomeric in its ribosome-associated state . Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TF-ribosome complex of approximately 1.2 microM . From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF . Uncomplexed TF, however, is in a monomer-dimer equilibrium with approximately two thirds of TF existing in a dimeric state.

Ai Zheng, 2002 Jun, 21(6), 636 - 9
{Preparation of phage-displayed single chain variable fragments of monoclonal antibody MC5 recognizing colorectal carcinoma}; He FT et al.; BACKGROUND & OBJECTIVE: MC5 is a murine monoclonal antibody with a good specificity to human colorectal carcinoma and smaller murine antibody can significantly decrease the possibility of developing human antimouse antibody response in vivo study . The aim of this study was to prepare single chain variable fragments (ScFv) of MC5 . METHODS: mRNA was isolated from the hybridoma cell line producing MC5, and the DNAs encoding variable domains of heavy and light chains(VH and VL DNAs) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA . The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E . coli TG1 . The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFvs . After two rounds of panning with cell line SW480 highly expressing MC5-binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by ELISA, and the affinity of the positive phage clones was assayed by competition ELISA . RESULTS: The VH, VL, and ScFv DNAs were about 340 bp, 320 bp, and 750 bp, respectively . Ten phage clones displayed ScFv of MC5 were selected from 25 enriched phage clones, and 3 of the 10 phage clones had higher affinity of binding to the antigen . CONCLUSION: The phage-displayed ScFv fragments of monoclonal antibody MC5 are successfully produced by phage display technique, which may provide a way for broadening the application range of the antibody.

Ai Zheng, 2002 Mar, 21(3), 245 - 8
{High expression of human angiogenesis inhibitor HIAF-1 in insect cells and biological activity}; Guo WZ et al.; BACKGROUND & OBJECTIVE: Human inhibiting angiogenesis factor-1(HIAF-1) expressed in E coli . shown the activity of inhibiting the angiogenesis of tumors . This study was designed to investigate the expression of HIAF-1 in insect cells and to elucidate its biological activity . METHODS: Recombinant baculovirus expression vector pAcuW51-HIAF-1 was constructed by recombinant DNA technique and cotransfected with linearized baculovirus DNA into sf9 cells to produce recombinant virus . The expression of recombinant HIAF-1 in insect cells infected by recombinant virus was detected by SDS-PAGE and Western blot . The recombinant protein was purified by affinity chromatography . In vitro endothelial proliferation inhibiting activity of recombinant HIAF-1 was examined by MTT method, its antitumor activity in vivo was studied in human esophageal cancer transplanted in nude mice . RESULTS: HIAF-1 was effectively expressed in insect cells as 26 KD fusing protein and its expression level was about 5-10% of insect cellular total soluble proteins . Recombinant HIAF-1 protein could inhibit endothelial cell proliferation in vitro with IC50 value of 3.1 micrograms/ml and inhibited remarkably growth of human esophageal cancer transplanted in nude mice . CONCLUSIONS: The recombinant HIAF-1 with better activity was successfully expressed in insect cells and establish a base for clinical application.

Biotechnol Bioeng, 2003 Jan 20, 81(2), 221 - 32
Coupling of chemical extraction and expanded-bed adsorption for simplified inclusion-body processing: optimization using surface plasmon resonance; Choe WS et al.; Integration of the chemical extraction of recombinant inclusion-body protein from Escherichia coli, and its recovery by metal-affinity expanded-bed adsorption (IMAC-EBA) under denaturing conditions, was investigated . The viral coat protein L1 with a hexa-histidine tag was expressed in Escherichia coli HMS174(DE3) as a model protein . Interference of released host DNA with adsorbent fluidization in the EBA step was solved by selective precipitation using spermine and low-speed centrifugation . However, the capacity and selectivity of the adsorbent for L1 remained lower than anticipated . The binding of L1 to immobilized Ni(2+) was therefore studied in detail using surface plasmon resonance (SPR) . The Tris buffer and ethylene-diamine tetraacetic acid (EDTA) used in the extraction mixture were found to interfere significantly with the L1-Ni(2+) interaction . The SPR studies suggest that L1 binding could be improved by replacing the Tris buffer with HEPES and by adding CaCl(2) to inactivate the EDTA . The modified chemical extraction conditions resulted in effective L1 extraction from cytoplasmic inclusion bodies, at high cell density (OD(600 )= 80) and without the use of reducing agent, into a medium optimized for subsequent IMAC recovery . The modified buffer conditions resulted in an improved binding capacity and a good L1 purification factor (12.7) and recovery yield (71%) . This work demonstrates that it is possible to reduce the complexity and hence the cost associated with traditional processes used to prepare purified denatured protein, ready for refolding, from cytoplasmic inclusion bodies .

Biotechnol Bioeng, 2003 Jan 20, 81(2), 158 - 66
Limiting factors in Escherichia coli fed-batch production of recombinant proteins; Sanden AM et al.; Fed-batch production of recombinant beta-galactosidase in E . coli was studied with respect to the specific growth rate at induction . The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate . Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein . Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor . The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate . At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level . Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity . The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose . The altered physiological status was also detected by the formation of acetic acid . However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production . Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin . In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited .

Am J Respir Crit Care Med, 2002 Dec 1, 166(11), 1436 - 42
Aerosolized linear polyethylenimine-nitric oxide/nucleophile adduct attenuates endotoxin-induced lung injury in sheep; Kirov MY et al.; Pulmonary hypertension and edema are mainstays of acute lung injury (ALI) . We synthesized linear polyethylenimine-nitric oxide/nucleophile adduct (DS-1), a water-soluble nitric oxide donor, and demonstrated that it is a potent relaxant of precontracted rat aortic rings without inducing desensitization . Moreover, DS-1 does not suppress the viability of human pulmonary epithelial cells in vitro . We also tested whether DS-1 counteracts ALI in endotoxemic sheep . Animals were instrumented for a chronic study . In 16 awake, spontaneously breathing sheep, Escherichia coli endotoxin (10 ng/kg/minute) was infused for 8 hours . From 2 hours of endotoxemia, sheep received either nebulized DS-1 (1 mg/kg/hour) or isotonic saline . DS-1 reduced endotoxin-induced rises in pulmonary arterial and microwedge pressures and vascular resistance index by 40-70% . In parallel, DS-1 decreased the accumulation of extravascular lung water by 60-70% and reduced the increment in right ventricle stroke work index and the falls in right ventricle ejection fraction, stroke volume, and left ventricle stroke work indices . Furthermore, DS-1 reduced venous admixture and improved arterial oxygen saturation . In four healthy animals, DS-1 alone slightly increased arterial oxygenation but had no other effects . Thus, aerosolized DS-1 attenuates endotoxin-induced ALI in sheep by reducing pulmonary hypertension and edema and improving myocardial function and gas exchange.

Appl Environ Microbiol, 2002 Dec, 68(12), 6029 - 35
Photoreactivation of Escherichia coli after low- or medium-pressure UV disinfection determined by an endonuclease sensitive site assay; Oguma K et al.; Photoreactivation of Escherichia coli after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated . An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of E . coli, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of E . coli . In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure . The CFA ratios of E . coli recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively . Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation . On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers . Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.

Appl Environ Microbiol, 2002 Dec, 68(12), 6021 - 8
Cloning and characterization of lin genes responsible for the degradation of Hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain B90; Kumari R et al.; Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes . Commercial formulations of HCH consist of a mixture of four isomers, alpha, beta, gamma, and delta . While all these isomers pose serious environmental problems, beta-HCH is more problematic due to its longer persistence in the environment . We have studied the degradation of HCH isomers by Sphingomonas paucimobilis strain B90 and characterized the lin genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers . Two nonidentical copies of the linA gene encoding HCH dehydrochlorinase, which were designated linA1 and linA2, were found in S . paucimobilis B90 . The linA1 and linA2 genes could be expressed in Escherichia coli, leading to dehydrochlorination of alpha-, gamma-, and delta-HCH but not of beta-HCH, suggesting that S . paucimobilis B90 contains another pathway for the initial steps of beta-HCH degradation . The cloning and characterization of the halidohydrolase (linB), dehydrogenase (linC and linX), and reductive dechlorinase (linD) genes from S . paucimobilis B90 revealed that they share approximately 96 to 99% identical nucleotides with the corresponding genes of S . paucimobilis UT26 . No evidence was found for the presence of a linE-like gene, coding for a ring cleavage dioxygenase, in strain B90 . The gene structures around the linA1 and linA2 genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between linA1 and linA2, which formed linA of strain UT26.

Appl Environ Microbiol, 2002 Dec, 68(12), 5965 - 72
Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164; Casadei MA et al.; The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164 . The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C . By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C . The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry . Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance . When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C) . Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.

Biochemistry, 2002 Dec 3, 41(48), 14438 - 46
Analysis of the stimulation of DNA polymerase V of Escherichia coli by processivity proteins; Maor-Shoshani A et al.; Bypass of replication-blocking lesions in Escherichia coli is carried out by DNA polymerase V (UmuC) in a reaction that requires UmuD', RecA, and single-strand DNA-binding protein (SSB) . The activity of this four-component basic bypass system is a low-fidelity and low-processivity activity . Addition of the processivity subunits of pol III, the beta subunit sliding DNA clamp, and the five-subunit gamma complex clamp loader increased the rate of translesion replication approximately 3-fold . This stimulation was specific to the lesion bypass step, with no effect on the initiation of synthesis by pol V . The beta subunit and gamma complex increased the processivity of pol V from 3 to approximately 14-18 nucleotides, providing a mechanistic basis for their stimulatory effect . Stimulation of bypass was observed over a range of RecA and SSB concentrations . ATPgammaS, which strongly inhibits translesion replication by pol V, primarily via inhibition of the initiation stage, caused the same inhibition also in the presence of the processivity proteins . The in vivo role of the processivity proteins in translesion replication was examined by assaying UV mutagenesis . This was done in a strain carrying the dnaN59 allele, encoding a temperature-sensitive beta subunit . When assayed in an excision repair-defective background, the dnaN59 mutant exhibited a level of UV mutagenesis reduced up to 3-fold compared to that of the isogenic dnaN(+) strain . This suggests that like in the in vitro system, the beta subunit stimulates lesion bypass in vivo.

Biochemistry, 2002 Dec 3, 41(48), 14383 - 90
Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases; Tomson F et al.; Infrared spectroscopy, isotopic labeling ({(15)N(delta,epsilon)}histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases . The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E . coli . Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)) . Two of these, at ca . 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E . coli enzyme . Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone . This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.

Biochemistry, 2002 Dec 3, 41(48), 14263 - 71
The biotin repressor: thermodynamic coupling of corepressor binding, protein assembly, and sequence-specific DNA binding; Streaker ED et al.; The Escherichia coli biotin repressor, an allosteric transcriptional regulator, is activated for binding to the biotin operator by the small molecule biotinyl-5'-AMP . Results of combined thermodynamic, kinetic, and structural studies of the protein have revealed that corepressor binding results in disorder to order transitions in the protein monomer that facilitate tighter dimerization . The enhanced stability of the dimer leads to stabilization of the resulting biotin repressor-biotin operator complex . It is not clear, however, that the allosteric response in the system is transmitted solely through the protein-protein interface . In this work, the allosteric mechanism has been quantitatively probed by measuring the biotin operator binding and dimerization properties of three biotin repressor species: the apo or unliganded form, the biotin-bound form, and the holo or bio-5'-AMP-bound form . Comparisons of the pairwise differences in the bioO binding and dimerization energetics for the apo and holo species reveal that the enhanced DNA binding energetics resulting from adenylate binding track closely with the enhanced assembly energetics . However, when the results for repressor pairs that include the biotin-bound species are compared, no such equivalence is observed.

Biochemistry, 2002 Dec 3, 41(48), 14255 - 62
Mutagenic events in Escherichia coli and mammalian cells generated in response to acetylaminofluorene-derived DNA adducts positioned in the Nar I restriction enzyme site; Tan X et al.; Comparative mutagenesis studies of N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) adducts positioned in the Nar I restriction enzyme site were performed using Escherichia coli (E . coli) and simian kidney (COS-7) cells . Oligodeoxynucleotides ((5)(')TCCTCG(1)G(2)CG(3)CCTCTC) containing a recognition sequence for the Nar I restriction enzyme were modified site-specifically with dG-AAF or dG-AF . Modified and unmodified oligomers inserted into single-stranded phagemid shuttle vectors were used to transform E . coli or to transfect COS-7 cells . Following replication in host cells, progeny plasmids were recovered and analyzed for mutations . In SOS-induced E . coli, dG-AAF primarily induced one- and two-base deletions . The mutational frequency varied, depending on the position modified in the Nar I site; 91% two-base deletions were observed at G(3), while 8.4% and 2.8% deletions were detected at G(2) and G(1), respectively . In contrast, dG-AF at any position in the Nar I site failed to produce deletions, generating primarily G --> T transversions (mutational frequency, 7.6-8.4%) . In COS-7 cells, both dG-AAF and dG-AF primarily induced G --> T transversions . Mutation frequencies for dG-AAF were 9.4-24%, the highest values being at G(1) and G(3) . Mutation frequencies for dG-AF were 9.3-21%, the higher value at G(2) . We conclude from this study that the mutation potential of dG-AAF and dG-AF depends on the structure of the adduct, the sequence context of the lesion, and the host cell used for the experiment.

Biochemistry, 2002 Dec 3, 41(48), 14216 - 24
Folding and assembly of lambda Cro repressor dimers are kinetically limited by proline isomerization; Satumba WJ et al.; Cro binds to operator sites in lambda DNA as a dimer . Dimerization of this small repressor protein is weak, however, and proline residues in the dimer interface suggest that folding and assembly of active repressors may be complex . Cro and selected variants have been studied by circular dichroism and fluorescence . Fluorescent probes include a unique tryptophan residue in the dimer interface and extrinsic resonance energy transfer probes that monitor dimerization . Both folding and unfolding are characterized by two distinct kinetic phases . Fast processes that are complete within the 5-10 ms dead time of stopped flow experiments account for the majority of the change in the CD signal and abrupt changes in both tryptophan fluorescence and energy transfer . The slow phases show all the hallmarks of proline isomerization . The rates of the slow phases are between 0.005 and 0.02 s(-1), are relatively independent of protein and denaturant concentration, display activation energies of 20 kcal/mol, and are accelerated by the peptidyl-prolyl isomerase SlyD . Although CD measurements indicate that more than 70% of the secondary structure is regained in the refolding burst phase, intermolecular fluorescence resonance energy transfer experiments indicate that less than 25% of these subunits are assembled into dimers . Full folding and dimerization requires isomerization of the non-native prolyl isomers over hundreds of seconds.

Biochemistry, 2002 Dec 3, 41(48), 14206 - 15
Crystal structures of human GAR Tfase at low and high pH and with substrate beta-GAR; Zhang Y et al.; Glycinamide ribonucleotide transformylase (GAR Tfase) is a key folate-dependent enzyme in the de novo purine biosynthesis pathway and, as such, has been the target for antitumor drug design . Here, we describe the crystal structures of the human GAR Tfase (purN) component of the human trifunctional protein (purD-purM-purN) at various pH values and in complex with its substrate . Human GAR Tfase exhibits pH-dependent enzyme activity with its maximum around pH 7.5-8 . Comparison of unliganded human GAR Tfase structures at pH 4.2 and pH 8.5 reveals conformational differences in the substrate binding loop, which at pH 4.2 occupies the binding cleft and prohibits substrate binding, while at pH 8.5 is permissive for substrate binding . The crystal structure of GAR Tfase with its natural substrate, beta-glycinamide ribonucleotide (beta-GAR), at pH 8.5 confirms this conformational isomerism . Surprisingly, several important structural differences are found between human GAR Tfase and previously reported E . coli GAR Tfase structures, which have been used as the primary template for drug design studies . While the E . coli structure gave valuable insights into the active site and formyl transfer mechanism, differences in structure and inhibition between the bacterial and mammalian enzymes suggest that the human GAR Tfase structure is now the appropriate template for the design of anti-cancer agents.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2287 - 91
Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor, cystatin, from Job's tears (Coix lacryma-jobi L . var . Ma-yuen Stapf); Yoza K et al.; A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears . A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library . The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues . The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn . The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found . A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice . The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein . The purified recombinant protein inhibited papain.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2194 - 200
Gene cloning and polymerase chain reaction with proliferating cell nuclear antigen from Thermococcus kodakaraensis KOD1; Kitabayashi M et al.; The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1 . The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus . Tk-PCNA was overexpressed in Escherichia coli and purified . This protein stimulated the primer extension abilities of the DNA polymerase from T . kodakaraensis KOD1 'KOD DNA polymerase' . The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA . The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase . This is the first report in which a replication-related factor worked on PCR.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2134 - 45
Molecular cloning of mouse collectin liver 1; Kawai T et al.; Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense . We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1) . In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1 . Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos . In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age . In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac . The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q . CL-L1 was a highly conserved protein in mammals, birds, and fish.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2112 - 24
Novel trimeric adenylate kinase from an extremely thermoacidophilic archaeon, Sulfolobus solfataricus: molecular cloning, nucleotide sequencing, expression in Escherichia coli, and characterization of the recombinant enzyme; Okajima T et al.; A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus . The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325 . Although the S . solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S . solfataricus enzyme . The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S . acidocaldarius . Furthermore, gel filtration and sedimentation analyses showed that the S . solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.

Biosci Biotechnol Biochem, 2002 Oct, 66(10), 2083 - 9
Two genes encoding fruit body lectins of Pleurotus cornucopiae: sequence similarity with the lectin of a nematode-trapping fungus; Iijima N et al.; Our previous studies on the fruit body lectin of Pleurotus cornucopiae revealed the existence of three isolectins, composed of two homodimers and one heterodimer of 16- and 15-kDa subunits . In this study, two genes encoding the lectins were cloned and characterized . Both genes encoded 144 amino acids and only 5 amino acids were different within the coding region, but the nucleotide sequences of the 5'-upstream and 3'-downstream regions differed extensively . Southern hybridization with gene-specific probes showed that one gene encoded the 16-kDa and the other encoded the 15-kDa subunit . Functional lectins were synthesized in Escherichia coli under the direction of these genes . On SDS-PAGE, the recombinant lectins showed the same banding patterns as the native lectins . In amino acid sequence, these lectins showed extensive similarity with the lectin from a nematode-trapping ascomycete fungus, Arthrobotrys oligospora, suggesting that the lectins might also function in capturing nematodes.

Genetika, 2002 Oct, 38(10), 1422 - 7
{Mutations in beta'-subunit of the Escherichia coli RNA-polymerase influence interaction with downstream duplex DNA in the elongation complex}; Kul'bachinskii AV et al.; RNA polymerase (RNAP) exhibits absolute processivity being capable of synthesizing RNA 10(3)-10(5) nucleotides in length without breaking contact with the DNA template . Stability of the elongation complex is thought to depend, in particular, on the RNAP-DNA interactions downstream along the run of transcription . We studied the effects of several deletions and insertions in the RNAP beta'-subunit N-terminal region, which presumably interacts with the downstream duplex DNA in the elongation complex . Most of the mutations obtained led to gross defects in RNAP assembly and disturbed catalytic activity of the enzyme . The mutations reduced stability of both promoter and elongation complexes, probably because they altered the contacts between RNAP and the downstream duplex DNA.

Biotechniques, 2002 Nov, 33(5), 1050, 1052 - 4
Negative purification method for the selection of specific antibodies from polyclonal antisera; Benet C et al.; We developed a protocol to remove non-specific antibodies from polyclonal antisera by adsorption on non-target antigens immobilized on nitrocellulose membranes . This "negative" purification method is simple and provides better immunoreagents than the blocking of nonspecific antibodies in solution or the enrichment of specific antibodies on nitrocellulose membranes . For routine applications, this method is quicker and cheaper than the purification protocols based on selective precipitations and affinity chromatography.

J Vet Med B Infect Dis Vet Public Health, 2002 Oct, 49(8), 409 - 10
Entropion, corneal ulcer and corneal haemorrhages in a one-humped camel (Camelus dromedarius); Yeruh I et al.; An unusual case of entropion, corneal ulcer and corneal haemorrhages in a one-humped camel (Camelus dromedaries) is described . The most prominent clinical findings were entropion of both eyelids, severe blephrospasm, epiphora, conjunctivitis, conjunctival oedema, mucopurulent conjunctival discharges, hyperaemia, lacrimation and photophobia . Corneal ulcers and corneal haemorrhages were also observed.

Kansenshogaku Zasshi, 2002 Oct, 76(10), 893 - 7
{Fatal septic shock caused by transrectal needle biopsy of the prostate; a case report}; Hasegawa T et al.; A 46-year-old man refer to us because of hemospermia . The prostatic gland was normal in size and consistency at rectal examination . Serum prostate specific antigen was 7.04 ng/ml . Magnetic resonance imaging showed an area of low signal intensity on T2-weighted images in the left peripheral gland, possibly indicative of carcinoma . Transrectal prostate biopsy was performed after intravenous administration of piperacillin . He developed chills and fever (39 degrees C) the next morning following biopsy . He was taken unconscious into the hospital where a diagnosis of septic shock caused by Escherichia coli was made . Five days later, he died . His general condition deteriorated notwithstanding intensive treatment . Postmortem blood cultures were positive for a piperacillin resistant Escherichia coli . Histological examination of the biopsies showed a benign prostatic hyperplasia . Autopsy showed diffuse tissue damage in the heart, lung, liver and kidneys . The prostate had numerous microabscesses . Currently, transrectal prostate biopsy is considered a generally reliable procedure to detect adenocarcinoma of the prostate . Our case seems to the sixth case report of fatal complications.

Kansenshogaku Zasshi, 2002 Oct, 76(10), 842 - 8
{PulseNet Japan: surveillance system for the early detection of diffuse outbreak based on the molecular epidemiological method}; Watanabe H et al.; As the foods are stocked below freezing and widely distributed, a kind of food-borne outbreak which occurs in separate regions or in different time, so called "diffuse outbreak", has been found at the present day . Unless the outbreak is early recognized, the number of victims would increase . Some methods have been developed to analyze the relatedness of bacteria isolated from the patients of enteric infections . PFGE, pulsed-field gel electrophoresis, is one of the methods and powerful to discriminate the difference in nucleotide sequences among bacterial genomes . Availability of PFGE analysis is appreciated to examine the linkage of each incident of food-borne infections in epidemiological investigation . A PFGE network, PulseNet Japan, is now under construction among National Institute of Infectious Diseases, local Health Institutes and Ministry of Health, Labour and Welfare.

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