Infect Immun, 2004 Nov, 72(11), 6722 - 8 Comparative analysis of locus of enterocyte effacement pathogenicity islands of atypical enteropathogenic Escherichia coli; Gartner JF et al.; The pathogenicity of enteropathogenic Escherichia coli (EPEC) is linked to the locus of enterocyte effacement, or LEE, encoding a type III secretion system (T3SS) that directly transfers bacterial effector proteins into eukaryotic cells . Atypical diffusely adhering EPEC (DA-EPEC) strains that harbor homologues of the LEE but lack the EPEC adherence factor plasmid have been increasingly associated with outbreaks of diarrhea . In this study, we have completely sequenced and functionally characterized LEE pathogenicity islands derived from the clinical DA-EPEC isolates 3431 (O8:H-) and 0181 (O119:H9:K61) . LEE3431 and LEE0181 exhibit genetic organization analogous to that of the prototype LEE(E2348/69) . Genes constituting the T3SS apparatus are highly conserved . However, LEE-encoded effector proteins exhibit major differences . Transfer and functional expression of LEE0181 in an E . coli XL1 blue MR background demonstrated that LEE0181 contains all the information for signal transduction and pedestal formation. Infect Immun, 2004 Nov, 72(11), 6554 - 60 Association of iss and iucA, but not tsh, with plasmid-mediated virulence of avian pathogenic Escherichia coli; Tivendale KA et al.; Avian pathogenic Escherichia coli (APEC) is an economically important respiratory pathogen of chickens worldwide . Factors previously associated with the virulence of APEC include adhesins, iron-scavenging mechanisms, the production of colicin V (ColV), serum resistance, and temperature-sensitive hemagglutination, but virulence has generally been assessed by parenteral inoculation, which does not replicate the normal respiratory route of infection . A large plasmid, pVM01, is essential for virulence in APEC strain E3 in chickens after aerosol exposure . Here we establish the size of pVM01 to be approximately 160 kb and show that the putative virulence genes iss (increased serum survival) and tsh (temperature-sensitive hemagglutinin) and the aerobactin operon are on the plasmid . These genes were not clustered on pVM01 but, rather, were each located in quite distinct regions . Examination of APEC strains with defined levels of respiratory pathogenicity after aerosol exposure showed that both the aerobactin operon and iss were associated with high levels of virulence in APEC but that the possession of either gene was sufficient for intermediate levels of virulence . In contrast, the presence of tsh was not necessary for high levels of virulence . Thus, both the aerobactin operon and iss are associated with virulence in APEC after exposure by the natural route of infection . The similarities between APEC and extraintestinal E . coli infection in other species suggests that they may be useful models for definition of the role of these virulence genes and of other novel virulence genes that may be located on their virulence plasmids. Infect Immun, 2004 Nov, 72(11), 6519 - 27 Phase I testing of a malaria vaccine composed of hepatitis B virus core particles expressing Plasmodium falciparum circumsporozoite epitopes; Nardin EH et al.; We report the first phase I trial to assess the safety and immunogenicity of a malaria vaccine candidate, ICC-1132 (Malarivax), composed of a modified hepatitis B virus core protein (HBc) containing minimal epitopes of the Plasmodium falciparum circumsporozoite (CS) protein . When expressed in Escherichia coli, the recombinant ICC-1132 protein forms virus-like particles that were found to be highly immunogenic in preclinical studies of mice and monkeys . Twenty healthy adult volunteers received a 20- or a 50-microg dose of alum-adsorbed ICC-1132 administered intramuscularly at 0, 2, and 6 months . The majority of volunteers in the group receiving the 50-microg dose developed antibodies to CS repeats as well as to HBc . Malaria-specific T cells that secreted gamma interferon were also detected after a single immunization with ICC-1132-alum . These studies support ICC-1132 as a promising malaria vaccine candidate for further clinical testing using more-potent adjuvant formulations and confirm the potential of modified HBc virus-like particles as a delivery platform for vaccines against other human pathogens. Infect Immun, 2004 Nov, 72(11), 6351 - 8 The Type II heat-labile enterotoxins LT-IIa and LT-IIb and their respective B pentamers differentially induce and regulate cytokine production in human monocytic cells; Hajishengallis G et al.; The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties . However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT) . We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells . We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1beta (IL-1beta) and IL-8 . However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae . Induction of proinflammatory (tumor necrosis factor alpha {TNF-alpha}) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1beta) was upregulated by the enterotoxins . These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity . Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor kappaB (NF-kappaB) activation . In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties . In this regard, the ability of LT-IIbB to activate NF-kappaB and induce TNF-alpha and IL-8 was antagonized by the LT-IIb holotoxin . These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers . Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen. FEMS Microbiol Lett, 2004 Nov 1, 240(1), 41 - 7 Antibodies produced against a fragment of filamentous haemagglutinin (FHA) of Bordetella pertussis are able to inhibit hemagglutination induced by the whole adhesin; Colombi D et al.; Filamentous hemagglutinin adhesin (FHA) is important for the adherence of Bordetella pertussis to the host ciliary epithelial cells of the respiratory tract . Several binding domains have been characterized in the FHA molecule . For example, an putative heparin-binding domain of FHA was previously located in the FHA(442-863) region . In this work, the HEP fragment, corresponding to FHA(430-873) was amplified by PCR and subcloned in an Escherichia coli expression plasmid . Purified recombinant HEP was used to produce polyclonal antibodies in mice that were able to recognize HEP and FHA in ELISA and in Western-blot assays . Although recombinant HEP displayed low ability to bind heparin and no hemagglutination activity, the anti-HEP antibodies were able to inhibit FHA mediated hemagglutination activity in goose erythrocytes . These results indicate that other amino acid residues that are not present in the FHA(430-873) fragment may be necessary for heparin binding . Further studies to address the immunogenic response against HEP are also required. Res Vet Sci, 2005 Feb, 78(1), 77 - 83 Isopathic and pluralist homeopathic treatment of commercial broilers with experimentally induced colibacillosis; Velkers FC et al.; This study sought to determine the efficacy of isopathic and pluralist homeopathic treatment of colibacillosis in broiler chickens and thereby contribute to the evaluation of homeopathy in general . In each of two experiments three groups of broilers, infected intratracheally at 8 days of age with E . coli (O78:K80), were treated with different combinations of homeopathic remedies . Control groups and an infected, doxycyline-treated group were included . Experiments differed only in the dose of E . coli . Efficacy of treatment was evaluated based on the parameters mortality, body weight gain and colibacillosis lesions . In both experiments doxycyline prevented mortality and reduced E . coli lesions and stunting . None of the homeopathically treated groups differed significantly with respect to any of the parameters from the non-medicated, infected control group . It is concluded that the results of this study do not justify use of these homeopathic remedies for treatment of colibacillosis in broilers . Furthermore, no significant effects of this homeopathic treatment were established. Plant J, 2004 Nov, 40(4), 453 - 61 Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase; Basset GJ et al.; In plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated . In Escherichia coli, this step is catalyzed by the PabC protein, a beta-lyase that converts 4-amino-4-deoxychorismate (ADC)--the reaction product of the PabA and PabB enzymes--to PABA and pyruvate . So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E . coli PabA and PabB and is located in plastids . ADC synthase has no lyase activity, implying that plants have a separate ADC lyase . No such lyase is known in any eukaryote . Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides . These cDNAs were shown to encode functional enzymes by complementation of an E . coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA . Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates . The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments . These data indicate that ADC lyase, like ADC synthase, is present in plastids . As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated. Biochem J, 2004 Nov 1, 383(Pt . 3), 537 - 42 Maturation of the unusual single-cysteine (XXXCH) mitochondrial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway; Allen JW et al.; The c-type cytochromes are characterized by the covalent attachment of haem to the polypeptide via thioether bonds formed from haem vinyl groups and, normally, the thiols of two cysteines in a CXXCH motif . Intriguingly, the mitochondrial cytochromes c and c1 from two euglenids and the Trypanosomatidae contain only a single cysteine within the haem-binding motif (XXXCH) . There are three known distinct pathways by which c-type cytochromes are matured post-translationally in different organisms . The absence of genes encoding any of these c-type cytochrome biogenesis machineries is established here by analysis of six trypanosomatid genomes, and correlates with the presence of single-cysteine cytochromes c and c1 . In contrast, we have identified a comprehensive catalogue of proteins required for a typical mitochondrial oxidative phosphorylation apparatus . Neither spontaneous nor catalysed maturation of the single-cysteine Trypanosoma brucei cytochrome c occurred in Escherichia coli . However, a CXXCH variant was matured by the E . coli cytochrome c maturation machinery, confirming the proposed requirement of the latter for two cysteines in the haem-binding motif and indicating that T . brucei cytochrome c can accommodate a second cysteine in a CXXCH motif . The single-cysteine haem attachment conserved in cytochromes c and c1 of the trypanosomatids is suggested to be related to their cytochrome c maturation machinery, and the environment in the mitochondrial intermembrane space . Our genomic and biochemical studies provide very persuasive evidence that the trypanosomatid mitochondrial cytochromes c are matured by a novel biogenesis system. Arzneimittelforschung, 2004, 54(9), 545 - 50 Immunostimulatory effect of coumarin derivatives before and after infection of mice with the parasite Schistosoma mansoni; Maghraby A et al.; Coumarins (pyranobenzopyran derivatives) (coumarin: CAS 91-64-5), organic compounds of known chemotherapeutic importance against bacteria, infectious diseases and tumors, were tested for their immunomodulatory effects . The compounds used in the present study were 1H,9H-3-amino-7-methyl-9-oxo-1-phenylpyran(2,3-h)(1)benzopyran-2-carbonitrile; 1H,9H-2-carboxamido-7-methyl-3,9-dioxo-1-phenylpyran(2,3-h)(1)benzopyran; 4H,8H-2-amino-7-bromo-6-methyl-10-nitro-8-oxo-4-(p-nitrophenyl)-pyran(3,2-g)(1)benzopyran-3-carbonitrile; 4H-7-bromo-3-carboxamido-2,8-dioxo-6-methyl-10-nitro-4-(p-nitrophenyl)2,8-dihydropyran(3,2g)(1) benzopyran . Mice were injected subcutaneously (0.75 mg/mouse) with each of the compounds for three successive days, then each animal was exposed to 100 Schistosoma mansoni cercariae . The effect on the humoral immune response was detected by measuring immunoglbulin G (IgG) levels using enzyme-linked immunosorbent assay (ELISA) against E . coli lysate, soluble worm antigen preparation (SWAP) and cancer bladder tissue homogenates . The reactivity of IgG to E . coli lysate was measured in sera of treated mice before and after infection . However, for SWAP and cancer tissue homogenates the IgG was measured only after infection . Mice given compounds 2, 3 and 4 showed a concentration dependent increase in IgG level against E . coli lysate as compared to untreated uninfected mice . Animals given compounds 3 and 4 followed by S . mansoni infection showed a significant increase (p< 0.05) in IgG level against the same antigen . Moreover, compounds 1, 2 and 4 significantly (p < 0.05) stimulated IgG production when measured against SWAP . Compounds 2, 3 and 4 induced a significant (p < 0.05) IgG response to cancer bladder tissue homogenates as compared to infected control mice . At the cellular level, treatment with compounds 1, 2, 3 and 4 caused a significant increase (p < 0.05) in the mean percentage of CD4+-T cells as compared with normal control, whereas, compounds 1, 2 and 3 stimulated a significant increase (p < 0.05) in the mean percentage of CD8+-T cells . Six weeks post-infection compounds 3 and 4 induced a significant stimulation (p< 0.05) in the mean number of both CD4+ and CD8+-T cells . The study showed that the compounds used have an immunomodulatory effect at both humoral and cellular levels. J Enzyme Inhib Med Chem, 2004 Jun, 19(3), 249 - 56 Plasmodium falciparum carbonic anhydrase is a possible target for malaria chemotherapy; Reungprapavut S et al.; Plasmodiumfalciparum is responsible for the majority of life-threatening cases of human malaria . The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets . Carbonic anhydrase (CA) is present at high levels in human red cells and in P . falciparum . Existence of at least three isozymes of the alpha < class was demonstrated in P . falciparum and a rodent malarial parasite Plasmodium berghei . The major isozyme CA1 was purified and partially characterized from P . falciparum (PfCA1) . A search of the malarial genome database yielded an open reading frame similar to the alpha-CAs from various organisms, including human . The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA) . The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively . The highly conserved active site residues were also found among organisms having alpha-CAs . The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli . The purified recombinant PfCA1 enzyme was catalytically active . It was sensitive to acetazolamide and sulfanilamide inhibition . Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P . falciparum in vitro culture . Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P . falciparum . Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P . falciparum carbonic anhydrase. J Plant Physiol, 2004 Sep, 161(9), 1053 - 60 The pyridoxal kinase gene TaPdxK from wheat complements vitamin B6 synthesis-defective Escherichia coli; Wang H et al.; Pyridoxal kinase (EC 2.7.1.35) is a key enzyme in the conversion of vitamin B6 to pyridoxal 5'-phosphate (PLP) . PLP is the crucial cofactor required by numerous enzymes involved in amino acids metabolism . Recently, studies with Arabidopsis salt overly sensitive 4 mutants demonstrated that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na+ and K+ homeostasis in plants . We describe here the TaPdxK gene which encodes a pyridoxal kinase, cloned from Triticum aestivum by RACE PCR method . The putative amino acid sequence of TaPdxK is 78% identical to Arabidopsis AtSOS4 . Southern analysis suggests that there are at least two copies of pyridoxal kinase genes in wheat genome . The expression of TaPdxK cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase . TaPdxK transcripts were detected in roots, shoots, spikes and anthers by RT-PCR analysis . TaPdxK expression level was not regulated by salt, ABA, and osmotic stress. Bioessays, 2004 Nov, 26(11), 1151 - 5 Exploring the multiple facets of the meiotic recombinase Dmc1; Sauvageau S et al.; Meiotic recombination in eukaryotic cells requires two homologs of E . coli RecA protein, Rad51 and Dmc1 . Until recently, the role of Dmc1 in meiotic recombination was mostly attributed to genetic studies as purified Dmc1 was found to be a much weaker recombinase than Rad51 in the test tube . Now, Sehorn and colleagues1 have reported that, like Rad51, human Dmc1 is an efficient recombinase in vitro . Dmc1 forms helical nucleoprotein filaments--the signature of classical recombinases such as Rad51 . These observations reveal a high level of similitude between the Dmc1 and the Rad51 family of recombination enzymes in higher eukaryotes. J Infect Dis, 2004 Nov 15, 190(10), 1812 - 20 Epub 2004 Sep 30. Recombinant Ascaris 16-Kilodalton protein-induced protection against Ascaris suum larval migration after intranasal vaccination in pigs; Tsuji N et al.; We recently cloned a protective antigen that is commonly expressed in Ascaris species that infect humans and pigs . We evaluated the vaccinal effects of this 16-kilodalton protein (As16) in pigs, the natural host of Ascaris suum, by intranasal immunization . Pigs that received Escherichia coli-expressed recombinant As16 (rAs16) coupled with cholera toxin (CT) had significantly elevated levels of rAs16-specific serum immunoglobulin G (IgG) and mucosal-associated IgA antibodies . rAs16 evoked a type II immune response characterized by elevated levels of interleukin-4 and -10 in the culture supernatants of peripheral blood mononuclear cells of the vaccinated pigs . An increased level of rAs16-specific serum IgG1 was also detected . Pigs vaccinated with rAs16-CT were protected from migration of A . suum larvae through the lungs, as indicated by a 58% reduction in the recovery of lung-stage third-stage larvae (L3), compared with that in nonvaccinated controls . Purified immunoglobulin from rAs16-CT-vaccinated pigs inhibited survival of infective L3 and interrupted the molting of lung-stage L3 . Immunofluorescence studies revealed that this immunoglobulin bound to the digestive tracts of L3, suggesting that it might inactivate functions of the gut tissues of Ascaris species . We conclude that rAs16 is a promising mucosal vaccine candidate for pig and human ascariasis. Drug Metab Pharmacokinet, 2004 Apr, 19(2), 120 - 9 CYP3A5 Contributes significantly to CYP3A-mediated drug oxidations in liver microsomes from Japanese subjects; Yamaori S et al.; The purpose of this study was to evaluate a contribution of polymorphic cytochrome P450 (CYP) 3A5 to the oxidation of diltiazem, midazolam and testosterone by liver microsomes from Japanese subjects . Twenty-seven liver samples were classified into three groups according to the CYP3A5 genotypes; CYP3A5(*)1/(*)1 (n=3), (*)1/(*)3 (n=12) and (*)3/(*)3 (n=12) . The results of genotyping and immunochemical quantitation of CYP3A5 protein showed a good accordance between the CYP3A5 genotype and CYP3A5 content but not CYP3A4 content in liver microsomes . The expression levels of hepatic CYP3A5 protein ranged from 20 to 60% of the sum of CYP3A4 and CYP3A5 contents in subjects with at least one wild type allele ((*)1) . The CYP3A5 contents correlated well with liver microsomal activities of diltiazem N-demethylation, midazolam 1'- and 4-hydroxylations and testosterone 6beta-hydroxylation among subjects carrying at least one (*)1 allele . In addition, the correlation coefficients of CYP3A5 contents with the rates of diltiazem N-demethylation, midazolam 1'-hydroxylation and testosterone 6beta- hydroxylation were higher than those of CYP3A4, although the value of CYP3A5 with the midazolam 4-hydroxylation rate was similar to that of CYP3A4 . Kinetic analyses revealed a biphasic diltiazem N-demethylation in liver microsomes from subjects carrying the (*)1 allele . The apparent V(max)/K(m) values for recombinant CYP3A5 indicated the greater contributions to diltiazem N-demethylation and midazolam 1'-hydroxylation as compared with CYP3A4 . These results suggest that polymorphic CYP3A5 contributes markedly to the drug oxidations, particularly diltiazem N-demethylation, midazolam 1'- hydroxylation and testosterone 6beta-hydroxylation by liver microsomes from Japanese subjects. Drug Metab Pharmacokinet, 2004 Feb, 19(1), 33 - 40 Lipopolysaccharide transport system across colonic epithelial cells in normal and infective rat; Tomita M et al.; To clarify whether lipopolysaccharide (LPS) is transported in rat intestinal epithelial cells, the transport of FITC-LPS across colonic epithelial cells in normal and LPS-exposured rats using a diffusion chamber was examined . The expression of CD14 and Toll-like receptor 4 (TLR4) was also examined . Rats were given 10 mg/kg LPS i.p . injection at 4 hr prior to the isolation of colonic epithelial tissues . The permeation rate across colonic mucosa by FITC-LPS was several times greater in the mucosal to serosal (M to S) direction than in the opposite direction in both normal and LPS-exposured rats . Increased M to S permeation by FITC-LPS was evident at 37 degrees C, but not at 4 degrees C . The permeability of FITC-LPS in both the M to S and S to M directions was inhibited by unlabeled LPS, anti-CD14 antibody or anti-TRL4 antibody in normal rat . In LPS-exposured rat, the inhibition in the M to S direction was observed by anti-TLR4 antibody, but not by unlabeled LPS and anti- CD14 antibody . In contrast, the permeability in the S to M direction was decreased only by unlabeled LPS in LPS-exposured rat . In normal rat, the expression of CD14 and TLR4 was found in the mucosal and serosal sides . In LPS-exposured rat, the expression of CD14 was not observed in the mucosal side . The electrophysiological parameters by LPS exposure remain unchanged . These findings suggest the possibility that colonic epithelial cells contain specific transport systems for LPS, one of which shows some degree of substrate specificity with the interaction of CD14 and/or that of TLR4. Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15585 - 90 Epub 2004 Oct 21. Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains; Hahn M et al.; Nonribosomal peptide synthetases (NRPSs) catalyze the formation of structurally diverse and biologically important peptides . Given their modular organization, NRPSs provide an enormous potential for biocombinatorial approaches to generate novel bioactive compounds . Crucial for the exploitation of this potential is a profound knowledge of the intermolecular communication between partner NRPSs . The overall goal of this study was to understand the basis of protein-protein communication that facilitates the selective interaction in these multienzyme complexes . On this account, we studied the relevance of short regions at the termini of the NRPSs tyrocidine (Tyc) synthetases TycA, TycB, and TycC, constituting the Tyc biosynthetic template . In vitro and in vivo investigations of C-terminal deletion mutants of the initiation module TycA provided evidence for the existence and impact of short communication-mediating (COM) domains . Their decisive role in protein-protein recognition was subsequently proven by means of COM domain-swapping experiments . Substitution of the terminal COM domains between the donor modules TycA and TycB3, as well as between the acceptor modules TycB1 and TycC1, clearly demonstrated that matching pairs of COM domains are both necessary and sufficient for the establishment of communication between partner NRPSs in trans . These results corroborated the generality of COM domains, which were subsequently exploited to induce crosstalk, even between NRPSs derived from different biosynthetic systems . In conclusion, COM domains represent interesting tools for biocombinatorial approaches, which, for example, could be used for the generation of innovative natural product derivatives. Antiviral Res, 2004 Nov, 64(2), 85 - 92 Evaluation of a Western Equine Encephalitis recombinant E1 protein for protective immunity and diagnostics; Das D et al.; The E1 and E2 glycoproteins of Western Equine Encephalitis (WEE) are candidate antigens for WEE subunit vaccine development . We have cloned the E1 gene of WEE virus and expressed it in Escherichia coli as inclusion bodies . The inclusion bodies were successfully solubilised, refolded and the immunogenicity of this unglycosylated protein was assessed in mice . Immunization of mice with recombinant E1 protein generated both humoral and cell-mediated immune responses, indicating the recombinant E1 protein is immunogenic . Challenge of E1-immunized mice with live WEE virus demonstrated little or no protection from this E . coli-derived non-glycosylated subunit. J Theor Biol, 2005 Jan 7, 232(1), 55 - 69 DEMSIM: a discrete event based mechanistic simulation platform for gene expression and regulation dynamics; Dasika MS et al.; In this paper, a discrete event based mechanistic simulation platform DEMSIM is developed for testing and validating putative regulatory interactions . The proposed framework models the main processes in gene expression, which are transcription, translation and decay processes, as stand-alone modules while superimposing the regulatory circuitry to obtain an accurate time evolution of the system . The stochasticity inherent to gene expression and regulation processes is captured using Monte Carlo based sampling . The proposed framework is applied to the extensively studied lac operon system, the SOS response system and the araBAD operon system of Escherichia coli . The results for the lac gene system demonstrate the simulation framework's ability to capture the dynamics of gene regulation, whereas the results for the SOS response system indicate that the framework is able to make accurate predictions about system behavior in response to perturbations . Finally, simulation studies for the araBAD system suggest that the developed framework is able to distinguish between different plausible regulatory mechanisms postulated to explain observed gene expression profiles . Overall, the obtained results highlight the effectiveness of DEMSIM at describing the underlying biological processes involved in gene regulation for querying alternative regulatory hypotheses. FEBS Lett, 2004 Oct 22, 576(3), 442 - 4 The iron-sulfur cluster in the L-serine dehydratase TdcG from Escherichia coli is required for enzyme activity; Burman JD et al.; The anaerobically inducible L-serine dehydratase, TdcG, from Escherichia coli was characterized . Based on UV-visible spectroscopy, iron and labile sulfide analyses, the homodimeric enzyme is proposed to have two oxygen-labile {4Fe-4S}2+ clusters . Anaerobically isolated dimeric TdcG had a kcat of 544 s(-1) and an apparent KM for L-serine of 4.8 mM . L-threonine did not act as a substrate for the enzyme . Exposure of the active enzyme to air resulted in disappearance of the broad absorption band at 400-420 nm, indicating a loss of the {4Fe-4S}2+ cluster . A concomitant loss of dehydratase activity was demonstrated, indicating that integrity of the {4Fe-4S}2+ cluster is essential for enzyme activity. FEBS Lett, 2004 Oct 22, 576(3), 417 - 22 Membrane-permeabilizing motif in Semliki forest virus E1 glycoprotein; Nieva JL et al.; Cell infection by alphaviruses is accompanied by membrane permeability changes . New predictive approaches, including the computation of interfacial affinity and corresponding hydrophobic moments, suggest a segmented amphipathic-at-interface domain in the stem region of Semliki Forest virus fusion protein E1 . Expression of E1 sequences in Escherichia coli cells confirmed that the membrane proximal plus transmembrane (TM) domain unit permeabilizes cells as efficiently as the 6K viroporin . Both our predictive and experimental data support the involvement of the E1 stem-TM region in membrane insertion and permeabilization . We propose to combine Wimley-White hydrophobicity analysis with expression-coupled permeability assays in order to identify viral products implied in breaching cell membrane barriers during infection. FEBS Lett, 2004 Oct 22, 576(3), 387 - 90 Expression of a cascading genetic network within liposomes; Ishikawa K et al.; Liposomes have long been used as possible compartments for artificial cells, and it has been shown that liposomes can sustain various types of biochemical reactions . To elevate the degree of molecular complexity of the system in liposomes, we have constructed a two-stage genetic network encapsulated in liposomes . This two-stage genetic network was constructed with the plasmid pTH, in which the protein product of the first stage (T7 RNA polymerase) is required to drive the protein synthesis of the second stage (GFP) . We show that the two-stage genetic network constructed in a cell-free expression system is functional within liposomes. FEBS Lett, 2004 Oct 22, 576(3), 325 - 30 Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay; Kao RY et al.; Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV Mpro) has been proposed as a prime target for anti-SARS drug development . We have cloned and overexpressed the SARS-CoV Mpro in Escherichia coli, and purified the recombinant Mpro to homogeneity . The kinetic parameters of the recombinant SARS-CoV Mpro were characterized by high performance liquid chromatography-based assay and continuous fluorescence-based assay . Two novel small molecule inhibitors of the SARS-CoV Mpro were identified by high-throughput screening using an internally quenched fluorogenic substrate . The identified inhibitors have Ki values at low microM range with comparable anti-SARS-CoV activity in cell-based assays. FEBS Lett, 2004 Oct 22, 576(3), 301 - 5 Medical implications from the crystal structure of a copper-containing amine oxidase complexed with the antidepressant drug tranylcypromine; Wilmot CM et al.; The X-ray crystal structure of the copper-containing quinoprotein amine oxidase from E . coli has been determined in complex with the antidepressant drug tranylcypromine to 2.4 A resolution . The drug is a racemic mix of two enantiomers, but only one is seen bound to the enzyme . The other enantiomer is not acting as a substrate for the enzyme as no catalytic activity was detected when the enzyme was initially exposed to the drug . The inhibition of human copper amine oxidases could be a source of side-effects in its use as an antidepressant to inhibit the flavin-containing monoamine oxidases in the brain. FEBS Lett, 2004 Oct 22, 576(3), 291 - 6 Molecular cloning in yeast by in vivo homologous recombination of the yeast putative alpha1 subunit of the voltage-gated calcium channel; Iida K et al.; Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ channel pore-forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli . Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E . coli . Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch-activated Ca2+ channel component, does not increase Ca2+ uptake activity, but co-overexpression results in a 2- to 3-fold increase . Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa . These results indicate that co-overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity. Curr Biol, 2004 Oct 26, 14(20), R895 - 7 Damage signaling: RecQ sends an SOS to you; Heyer WD; The DNA helicase RecQ is required for proper induction of the SOS response to replication stress in Escherichia coli . Unwinding of stalled replication forks by RecQ family helicases in bacteria, and possibly in eukaryotes, may provide a means of damage signaling and recovering stalled replication forks. Chin Med J (Engl), 2004 Oct, 117(10), 1464 - 70 Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells; Cao HQ et al.; BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty . Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E . coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively . We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing . To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells . METHODS: Adenoviral vectors, including Ad-EF1alpha-CD-cytomegalovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1alpha-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA + E . coli cells . Integration and expression of CD and/or TK gene were identified by PCR and Western blot . Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV . Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays . Flow cytometry analysis was used to detect cell death . Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe . Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis . RESULTS: Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed . Both single and double suicide genes could be integrated into adenoviral genome and expressed . Cytotoxic effects of Ad-EF1alpha-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1alpha-CD single gene groups . The rate of cell survival was only (9 +/- 3)% in the Ad-EF1alpha-CD-CMV-TK group, but (37 +/- 3)% in the Ad-CMV-TK and (46 +/- 4)% in the Ad-EF1alpha-CD groups (P < 0.05) . Flow cytometry analysis indicated that the killing mechanisms of the groups were different . Necrosis and apoptosis were involved in the mechanism of the double gene group . Based on the DNA stainability with Hoechst 33342, the apoptotic rates of VSMCs in the Ad-EF1alpha-CD-CMV-TK {(11.0 +/- 2.1)%} and Ad-CMV-TK {(12.0 +/- 2.2)%} groups were higher than those in Ad-CMV-LacZ {(1.2 +/- 0.11)%} and Ad-EF1alpha-CD {(5.0 +/- 1.8)%} groups (P < 0.05, respectively) . DNA smear could be observed in both Ad-CMV-TK and Ad-EF1alpha-CD-CMV-TK groups after administration of prodrugs . CONCLUSIONS: The killing effect on rat VSMCs mediated by adenoviral CD/TK double suicide genes is superior to that of single suicide gene . The killing mechanism of recombinant adenovirus coexpressing CD/TK double suicide genes is mainly through cytotoxic effect and apoptosis. J Nat Prod, 2004 Oct, 67(10), 1672 - 80 New pseudopterosin and seco-pseudopterosin diterpene glycosides from two Colombian isolates of Pseudopterogorgia elisabethae and their diverse biological activities; Rodriguez II et al.; As part of an ongoing program to explore the chemical constituents of Caribbean marine invertebrates, a family of 13 new diterpene glycosides, pseudopterosins P-Z (1-11) and seco-pseudopterosins H (12) and I (13), have been isolated from the organic extracts of two collections of the sea whip Pseudopterogorgia elisabethae procured near the Colombian Southwestern Caribbean Sea . The structures of compounds 1-13, including absolute stereochemistry, have been proposed on the basis of comprehensive spectral analyses, chemical transformations, specific rotation, and TLC chromatographic analyses . Pseudopterosin Q (2) inhibited thromboxane B2 (TXB2) (IC50 = 4.7 microM) and superoxide anion (O2-) (IC50 = 11.2 microM) generation from E . coli lipopolysaccharide (LPS) activated rat neonatal microglia in vitro . In contrast, pseudopterosins P (1), U (6), V (7), W (8), and X (9) as well as seco-pseudopterosins H (12) and I (13) demonstrated minimal effects on both TXB2 and O2- release . In addition, some of the new compounds displayed strong antituberculosis, antiviral, antimalarial, and anticancer activity. Intern Med, 2004 Sep, 43(9), 811 - 5 Post-operative constrictive pericarditis complicated with lymphocytopenia and hypoglobulinemia; Ohsawa M et al.; A 71-year-old man who had a history of open chest surgery was admitted due to anasarca and bilateral pleural effusions . Although imaging modalities could not demonstrate any pericardial abnormalities, right-sided cardiac catheterization revealed 'dip and plateau' in diastolic pressure waveform . He was admitted frequently because of the episodic right-sided congestive heart failure and hypoproteinemia due to protein-losing enteropathy . The peripheral lymphocyte count and serum gamma-globulin concentration were gradually decreased, and finally showed lymphocytopenia and hypoglobulinemia . On the last admission, the patient showed extensive cellulitis on both legs, and he developed septicemia, and finally died due to septic shock . Post-mortem examination showed that both visceral and parietal layers of the pericardium adhered tightly with mediastinal fibrosis . This case report suggested that constrictive pericarditis should be considered even if there is a lack of typical abnormal pericardial imaging findings when patients have a history of open chest surgery and recurrent right-sided congestive heart failure . In addition, we should be aware of a serious outcome due to immune compromised conditions such as lymphocytopenia and dysglobulinemia in this disorder. EMBO J, 2004 Nov 10, 23(22), 4434 - 42 Epub 2004 Oct 21. RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences; Akiyama Y et al.; Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment . However, neither its enzymatic activity nor the substrate cleavage position has been established . Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo . Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components . Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity . These results show that RseP has potential ability to cut a broad range of membrane protein sequences . Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP. J Biochem (Tokyo), 2004 Aug, 136(2), 211 - 20 Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli; Omata Y et al.; The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase . Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied . We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase . The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate . The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state . Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss . From the crystal structure of human uro'gen III synthase {Mathews et al . (2001) EMBO J . 20, 5832-5839}, this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity. RNA, 2004 Nov, 10(11), 1798 - 812 The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits; Lovgren JM et al.; The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes . A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA . To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed . A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM . The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain . A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant . A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants . Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants . However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant . Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19 . A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S . Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b. Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15573 - 8 Epub 2004 Oct 20. Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster; Kodumal SJ et al.; To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important . An approach proposed toward this end involves the synthesis of approximately 5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods {Smith, H . O., Hutchinson, C . A., III, Pfannkoch, C . & Venter, J . C . (2003) Proc . Natl . Acad . Sci . USA 100, 15440-15445} . The major current impediment to the success of this tactic is the difficulty of building the approximately 5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks . We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences . Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp "synthons" with low error frequency by automated PCR-based gene synthesis . By parallel processing, these synthons are efficiently joined into multisynthon approximately 5-kb segments by using only three endonucleases and "ligation by selection." These large segments can be subsequently assembled into very long sequences by conventional cloning . We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli. Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15760 - 5 Epub 2004 Oct 20. A biochemical oscillator explains several aspects of Myxococcus xanthus behavior during development; Igoshin OA et al.; During development, Myxococcus xanthus cells produce a series of spatial patterns by coordinating their motion through a contact-dependent signal, the C-signal . C-signaling modulates the frequency at which cells reverse their gliding direction . It does this by interacting with the Frz system (a homolog of the Escherichia coli chemosensory system) via a cascade of covalent modifications . Here we show that introducing a negative feedback into this cascade results in oscillatory behavior of the signaling circuit . The model explains several aspects of M . xanthus behavior during development, including the nonrandom distribution of reversal times, and the differences in response of the reversal frequency to both moderate and high levels of C-signaling at different developmental stages . We also propose experiments to test the model. J Leukoc Biol, 2005 Jan, 77(1), 71 - 9 Epub 2004 Oct 20. Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and -independent pathways; Yasuda K et al.; Previously, we showed that bacterial DNA and vertebrate DNA/cationic liposome complexes stimulate potent inflammatory responses in cultured mouse macrophages . In the present study, we examined whether endocytosis and subsequent acidification are associated with these responses . The endocytosis inhibitor, cytochalasin B, reduced tumor necrosis factor alpha (TNF-alpha) production by a plasmid DNA (pDNA)/cationic liposome complex . The endosomal acidification inhibitor, monensin, inhibited cytokine production by pDNA or a calf thymus DNA/liposome complex . These results suggest, similarly to CpG motif-dependent responses, that endocytosis and subsequent endosomal acidification are also required for these inflammatory responses . It is intriguing that another inhibitor of endosomal acidification, bafilomycin A, stimulated the production of TNF-alpha mRNA and its protein after removal of the pDNA/liposome complex and inhibitors, although it inhibited the release of interleukin-6 . Similar phenomena were observed in the activation of macrophages by CpG oligodeoxynucleotide, calf thymus DNA, and Escherichia coli DNA complexed with liposomes . Moreover, bafilomycin A also induced a high degree of TNF-alpha release after stimulation with naked pDNA . These results suggest that bafilomycin A increases TNF-alpha production induced by DNA at the transcriptional level via an as-yet unknown mechanism . Furthermore, we investigated the contribution of Toll-like receptor 9 (TLR9), the receptor of CpG motifs, to the cell activation by the DNA/cationic liposome complex using the macrophages from TLR9(-/-) mice . We observed a reduced inflammatory cytokine release from macrophages of TLR9(-/-) mice compared with wild-type mice . However, the cytokine production was not completely abolished, suggesting that the DNA/cationic liposome complex can induce macrophage activation via TLR9-dependent and -independent pathways. J Leukoc Biol, 2005 Jan, 77(1), 16 - 23 Epub 2004 Oct 20. Linking the "two-hit" response following injury to enhanced TLR4 reactivity; Murphy TJ et al.; Severe injury can initiate an exaggerated systemic inflammatory response and multiple organ failure (MOF) if a subsequent immune stimulus, "second hit", occurs . Using a mouse thermal injury model, we tested whether changes in innate immune cell reactivity following injury can contribute to the development of heightened inflammation and MOF . Using high-purity Escherichia coli lipopolysaccharide (LPS) to selectively stimulate Toll-like receptor 4 (TLR4), we demonstrate augmented interleukin (IL)-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by 1 day but particularly, at 7 days after injury . The in vivo significance of enhanced TLR4 responsiveness was explored by challenging sham or burn mice with LPS at 1 or 7 days after injury and determining mortality along with in vivo cytokine and chemokine levels . Mortality was high (75%) in LPS-challenged burn but not sham mice at 7 days, although not at 1 day, after injury . Death was associated with leukocyte sequestration in the lungs and livers along with increased proinflammatory cytokine and chemokine levels in these organs . Blocking TNF-alpha activity prevented this mortality, suggesting that excessive TNF-alpha production contributes to this lethal response . These findings demonstrate the potential lethality of excessive TLR4 reactivity after injury and provide an explanation for the exaggerated inflammatory response to a second hit, which can occur following severe injury. J Med Microbiol, 2004 Nov, 53(Pt 11), 1145 - 9 Distribution of espI among clinical enterohaemorrhagic and enteropathogenic Escherichia coli isolates; Mundy R et al.; Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli are important diarrhoeagenic pathogens; infection is dependent on translocation of a number of type III effector proteins . Until recently all the known effectors were encoded on the LEE pathogenicity island, which also encodes the adhesin intimin and the type III secretion apparatus . Recently, a novel non-LEE effector protein, EspI/NleA, which is required for full virulence in vivo and is encoded on a prophage, was identified . The aim of this study was to determine the distribution of espI among clinical EHEC and EPEC isolates . espI was detected in 86 % and 53 % of LEE+ EHEC and EPEC strains, respectively . Moreover, the espI gene was more commonly found in patients suffering from a more severe disease. J Med Microbiol, 2004 Nov, 53(Pt 11), 1137 - 44 Association of atypical enteropathogenic Escherichia coli (EPEC) with prolonged diarrhoea; Afset JE et al.; The aim of the present case control study was to investigate the prevalence of atypical enteropathogenic Escherichia coli (EPEC) and its possible role in causing diarrhoea among children < 5 years of age in Norway . Stool specimens received in the laboratory from children with suspected gastroenteritis (n = 251) were, in addition to routine testing, analysed for the presence of EPEC by PCR of the eae, bfpA and stx genes . Specimens from healthy children (n = 210) recruited from Maternal and Child Health Centres were analysed for EPEC only . EPEC isolates (eae+, stx-) were classified as typical (bfpA+) or atypical (bfpA-), and were tested for O : K serogroup . Information on duration of diarrhoea was recorded in a questionnaire and from referral forms . Atypical EPEC was diagnosed in 37 patients (14.7 %) compared to 21 (10.0 %) of the healthy controls {Odds ratio (OR) = 1.4, P = 0.3} . Only three isolates, all from patients, belonged to EPEC serogroups . One patient had typical EPEC . Twenty (22.5 %) of 89 patients with diarrhoea lasting > or = 14 days had atypical EPEC . The association between atypical EPEC and prolonged diarrhoea (OR = 2.1, P = 0.04) was caused by a high prevalence among female patients (40.6 %) . In conclusion, atypical EPEC was found to be slightly more prevalent in patients than controls, without any overall significant association with diarrhoea . However, a significant association was observed with diarrhoea lasting 14 days or more, a finding that may indicate a role for atypical EPEC in prolonged disease. J Gene Med, 2004 Dec, 6(12), 1304 - 19 Complexation of retroviruses with charged polymers enhances gene transfer by increasing the rate that viruses are delivered to cells; Landazuri N et al.; BACKGROUND: We have previously found that retrovirus transduction is enhanced when an anionic polymer (chondroitin sulfate C) is added to virus stocks that contain an equal weight concentration of a cationic polymer (Polybrene) . This observation was unexpected given that previous work has shown that cationic polymers enhance transduction while anionic polymers have the opposite effect . METHODS: Using model recombinant retroviruses and lentiviruses that encode for the Escherichia coli lacZ gene and quantitative assays of virus adsorption and transduction, we examined the mechanism of enhancement . RESULTS: We found that addition of oppositely charged polymers (Polybrene and chondroitin sulfate C) to virus stocks enhanced gene transfer by increasing the flux of active viruses to the cells . Virus-polymer complexes formed that did not reduce the stability of the viruses, yet were large enough to sediment, delivering the viruses to the cells more rapidly than by simple diffusion . The size of the complexes, the rate of sedimentation, and the levels of gene transfer increased with increasing concentrations of polymers . The degree to which transduction was enhanced ranged from 2- to nearly 40-fold, and varied depending on the type of cells and viruses used . Interestingly, we found that association of the viruses with the polymer complexes did not significantly hinder their ability to complete post-binding steps of transduction . CONCLUSIONS: Complexation of retroviruses with charged polymers significantly improves the efficiency of ex vivo gene transfer by increasing the number of active viruses that reach the cells . Copyright (c) 2004 John Wiley & Sons, Ltd. J Immunol, 2004 Nov 1, 173(9), 5749 - 56 Toll receptor-mediated regulation of NADPH oxidase in human dendritic cells; Vulcano M et al.; Activation of NADPH oxidase represents an essential mechanism of defense against pathogens . Dendritic cells (DC) are phagocytic cells specialized in Ag presentation rather than in bacteria killing . Human monocyte-derived DC were found to express the NADPH oxidase components and to release superoxide anions in response to phorbol esters and phagocytic agonists . The NADPH oxidase components p47phox and gp91phox were down-regulated during monocyte differentiation to DC, and maturation of DC with pathogen-derived molecules, known to activate TLRs, increased p47phox and gp91phox expression and enhanced superoxide anions release . Similar results were obtained with plasmacytoid DC following maturation with influenza virus . In contrast, activation of DC by immune stimuli (CD40 ligand) did not regulate NADPH oxidase components or respiratory burst . NADPH oxidase-derived oxygen radicals did not play any role in DC differentiation, maturation, cytokine production, and induction of T cell proliferation, as based on the normal function of DC generated from chronic granulomatous disease patients and the use of an oxygen radical scavenger . However, NADPH oxidase activation was required for DC killing of intracellular Escherichia coli . It is likely that the selective regulation of oxygen radicals production by pathogen-activated DC may function to limit pathogen dissemination during DC trafficking to secondary lymphoid tissues. Nucleic Acids Res, 2004 Oct 19, 32(18), 5609 - 20 Print 2004. Processing of bistranded abasic DNA clusters in gamma-irradiated human hematopoietic cells; Georgakilas AG et al.; Clustered DNA damages--two or more lesions on opposing strands and within one or two helical turns--are formed in cells by ionizing radiation or radiomimetic antitumor drugs . They are hypothesized to be difficult to repair, and thus are critical biological damages . Since individual abasic sites can be cytotoxic or mutagenic, abasic DNA clusters are likely to have significant cellular impact . Using a novel approach for distinguishing abasic clusters that are very closely spaced (putrescine cleavage) or less closely spaced (Nfo protein cleavage), we measured induction and processing of abasic clusters in 28SC human monocytes that were exposed to ionizing radiation . gamma-rays induced approximately 1 double-strand break: 1.3 putrescine-detected abasic clusters: 0.8 Nfo-detected abasic clusters . After irradiation, the 28SC cells rejoined double-strand breaks efficiently within 24 h . In contrast, in these cells, the levels of abasic clusters decreased very slowly over 14 days to background levels . In vitro repair experiments that used 28SC cell extracts further support the idea of slow processing of specific, closely spaced abasic clusters . Although some clusters were removed by active cellular repair, a substantial number was apparently decreased by 'splitting' during DNA replication and subsequent cell division . The existence of abasic clusters in 28SC monocytes, several days after irradiation suggests that they constitute persistent damages that could lead to mutation or cell killing. Proc Natl Acad Sci U S A, 2004 Oct 26, 101(43), 15335 - 40 Epub 2004 Oct 19. Probing the instabilities in the dynamics of helical fragments from mouse PrPC; Dima RI et al.; The first step in the formation of the protease resistant form (PrPSc) of prion proteins involves a conformational transition of the monomeric cellular form of PrPC to a more stable aggregation prone state PrPC* . A search of PDBselect and Escherichia coli and yeast genomes shows that the exact pattern of charges in helix 1 (H1) is rare . Among the 23 fragments in PDBselect with the pattern of charges that match H1, 83% are helical . Mapping of the rarely found (in E . coli and yeast genomes) hydrophobicity patterns in helix 2 (H2) to known secondary structures suggests that the PrPC-->PrPC* transition must be accompanied by alterations in conformations in second half of H2 . We probe the dynamical instability in H1 and in the combined fragments of H2 and helix 3 (H3) from mPrPC (H2+H3), with intact disulfide bond, using all atom molecular dynamics (MD) simulations totaling 680 ns . In accord with recent experiments, we found that H1 is helical, whereas the double mutant H1{D147A-R151A} is less stable, implying that H1 is stabilized by the (i,i + 4) charged residues . The stability of H1 suggests that it is unlikely to be involved in the PrPC-->PrPC* transition . MD simulations of H2+H3 shows that the second half of H2 (residues 184-194) and parts of H3 (residues 200-204 and 215-223) undergo a transition from alpha-helical conformation to a beta and/or random coil state . Simulations using two force fields (optimized potentials for liquid simulations and CHARMM) give qualitatively similar results . We use the MD results to propose tentative structures for the PrPC* state. Proc Natl Acad Sci U S A, 2004 Oct 26, 101(43), 15341 - 5 Epub 2004 Oct 19. Fourier transform ion cyclotron resonance MS reveals the presence of a water molecule in an enzyme transition-state analogue complex; Borchers CH et al.; The structures of several powerful inhibitors of hydrolytic enzymes resemble that of the altered substrate in the transition state, except that a hydrogen atom replaces one substituent (typically the leaving group) . To test the hypothesis that a water molecule might be present in the gap resulting from this replacement, we examined a transition-state analogue complex formed by Escherichia coli cytidine deaminase by Fourier transform ion cyclotron resonance MS in electrospray mode . Upon nebularization from aqueous solution under conditions (pH 5.6) where the enzyme is active, cytidine deaminase remains dimeric in the vapor phase . In the presence of inhibitor, the enzyme's exact mass can be used to infer the presence at each active site of zinc, 5-fluoro-3,4-dihydrouridine, and a single water molecule. J Biol Chem . 2004 Oct 19; {Epub ahead of print} PRMT7: A new protein arginine methyltransferase that synthesizes symmetric dimethylarginine; Lee JH et al.; The cDNA for PRMT7, a recently discovered human protein arginine methyltransferase (PRMT), was cloned and expressed in E . coli and mammalian cells . Immuno-purified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB . Amino acid analysis showed that the modifications produced were predominantly monomethylarginine (MMA) and symmetric dimethylarginine (SDMA) . Examination of PRMT7 expressed in E . coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein and SmD3 were methylated . Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine (ADMA) were produced . SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is, by itself, sufficient for symmetric dimethylation to occur . The data demonstrate that PRMT7, like PRMT5, is a type II methyltransferase capable of producing SDMA modifications in proteins. J Biol Chem, 2004 Dec 31, 279(53), 55760 - 9 Epub 2004 Oct 19. Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis; Akhtar MW et al.; Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress . It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner . This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation . In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33 . Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures . SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species . Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population . Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species . Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins . Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress . Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress. J Biol Chem, 2004 Dec 24, 279(52), 53947 - 54 Epub 2004 Oct 19. Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis; Walker KD et al.; Biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug Taxol starts with the conversion of 2S-alpha-phenylalanine to 3R-beta-phenylalanine by phenylalanine aminomutase (PAM) . A gene cloning approach was based on the assumption that PAM would resemble the well known plant enzyme phenylalanine ammonia lyase . A phenylalanine ammonia lyase-like sequence acquired from a Taxus cuspidata cDNA library was expressed functionally in Escherichia coli and confirmed as the target aminomutase that is virtually identical to the recombinant enzyme and clone from Taxus chinensis, acquired recently by a reverse genetics approach (Bristol-Myers Squibb (August 14, 2003) U . S . Patent WO 03/066871 A2) . The full-length cDNA has an open reading frame of 2094 base pairs and encodes a protein of 698 residues with a calculated molecular mass of 76,530 Da . The recombinant mutase has a pH optimum of 8.5, a k(cat) value of 0.015 s(-1), and a K(m) of 45 +/- 8 microm for 2S-alpha-phenylalanine . The stereochemical mechanism of PAM involves the removal and interchange of the pro-3S hydrogen and the amino group, which rebonds at C-3 with retention of configuration . The recombinant enzyme appears to catalyze both the forward and reverse reactions with specificity for both 2S-alpha-phenylalanine and 3S- or 3R-beta-phenylalanine substrates, respectively, whereas the related phenylpropanoids 2S-aminocyclohexanepropanoic acid, 2R-alpha-phenylalanine, and 2S-alpha-tyrosine are not converted to their beta-isomers by the mutase. J Biol Chem, 2004 Dec 24, 279(52), 54193 - 201 Epub 2004 Oct 19. SsrA tagging of Escherichia coli SecM at its translation arrest sequence; Collier J et al.; SecM is expressed from the secM-secA operon and activates the expression of secA in response to secretion defects . The 3'-end of secM encodes an "arrest sequence," which can interact with the ribosomal exit tunnel, preventing complete secM translation under secretion-defective conditions . In a cis-acting manner, ribosome stalling enhances secA translation . Pro(166) is the last residue incorporated when SecM elongation is arrested . We report that secretion deficiencies lead to SsrA tagging of SecM after Pro(166), Gly(165), and likely Arg(163) . Northern blot analysis revealed the presence of a truncated secM transcript, likely issued from a secM-secA cleavage . The level of secM transcripts was decreased either when secM translation was totally prevented or when Pro(166) was mutated . However, the accumulation of a truncated secM transcript required secM translation and was prevented when the SecM arrest sequence was inactivated by a point mutation changing Pro(166) to Ala . We suggest that ribosome pausing at the site encoding the arrest sequence is required for formation of the truncated secM mRNA . SsrA tagging affected neither the presence of the secM mRNA nor secA expression, even under translocation-defective conditions . It is therefore likely that SsrA tagging of SecM occurs only after cleavage of secM-secA mRNA within the secM open reading frame encoding the SecM arrest sequence . Accumulation of transcripts expressing arrested SecM generated growth inhibition that was alleviated by the SsrA tagging system . Therefore, SsrA tagging of SecM would rescue ribosomes to avoid excessive jamming of the translation apparatus on stop-less secM mRNA. J Biol Chem, 2004 Dec 24, 279(52), 54202 - 9 Epub 2004 Oct 19. Contributions of pseudoknots and protein SmpB to the structure and function of tmRNA in trans-translation; Wower IK et al.; Bacteria contain transfer-messenger RNA (tmRNA), a molecule that during trans-translation tags incompletely translated proteins with a small peptide to signal the proteolytic destruction of defective polypeptides . TmRNA is composed of tRNA- and mRNA-like domains connected by several pseudoknots . Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli tmRNA in trans-translation . Site-directed mutagenesis experiments showed that pseudoknot 2 and the abutting helix 5 were particularly important for the binding of ribosomal protein S1 to tmRNA . Pseudoknot 4 not only facilitated tmRNA maturation but also promoted tagging . In addition, the three pseudoknots (pk2 to pk4) were shown to play a significant role in the proper folding of the tRNA-like domain . Protein SmpB enhanced tmRNA processing, suggesting a new role for SmpB in trans-translation . Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis. Biochem Soc Trans, 2004 Nov, 32(Pt 5), 861 - 4 AKAP (A-kinase anchoring protein) domains: beads of structure-function on the necklace of G-protein signalling; Malbon CC et al.; AKAPs (A-kinase anchoring proteins) are members of a diverse family of scaffold proteins that minimally possess a characteristic binding domain for the RI/RII regulatory subunit of protein kinase A and play critical roles in establishing spatial constraints for multivalent signalling assemblies . Especially for G-protein-coupled receptors, the AKAPs provide an organizing centre about which various protein kinases and phosphatases can be assembled to create solid-state signalling devices that can signal, be modulated and trafficked within the cell . The structure of AKAP250 (also known as gravin or AKAP12), based on analyses of milligram quantities of recombinant protein expressed in Escherichia coli, suggests that the AKAP is probably an unordered scaffold, acting as a necklace on which 'jewels' of structure-function (e.g . the RII-binding domain) that provide docking sites on which signalling components can be assembled . Recent results suggest that AKAP250 provides not only a 'tool box' for assembling signalling elements, but may indeed provide a basis for spatial constraint observed for many signalling paradigms . The spatial dimension of the integration of cell signalling will probably reflect many functions performed by members of the AKAP family. Int J Med Microbiol, 2004 Sep, 294(2-3), 103 - 13 Impact of the locus of enterocyte effacement pathogenicity island on the evolution of pathogenic Escherichia coli; Jores J et al.; This review summarizes our current knowledge and models of appearance and dissemination of the locus of enterocyte effacement (LEE) within Escherichia coli phylogenetic lineages . The LEE is a pathogenicity island (PAI) required for attaching and effacing (A/E) lesion formation induced on epithelial cells of humans and animals by enteropathogenic and numerous enterohemorrhagic E . coli strains as well as other related bacteria . The LEE encodes a type III secretion system, an adhesin (intimin) responsible for the intimate attachment of the bacteria to the cell and a number of secreted proteins involved in signal transduction events . It has been shown that the LEE varies in size from 36 to 111 kb, depending on what E . coli lineages carrying that PAI . Three tRNA genes are known as LEE integration sites selC, pheU and pheV, the latter two are identical in sequence . Beneath its functional role, intimin is considered a phylogenetic marker of the LEE . Currently, 14 different intimin types have been described, designated alpha through ksi . Beta intimin-carrying LEEs moved within certain E . coli lineages from the pheU tRNA gene into the pheV tRNA gene . Moreover, as a result of the typing of multiple LEE core regions, the appearance of two different LEE cores indicates an import of the LEE within E . coli at least two times. Sci China C Life Sci, 2004 Aug, 47(4), 359 - 67 Homology modeling three-dimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis; Yan H et al.; AnxB1, a novel annexin previously isolated from Cysticercus cellulose, shows high thrombi affinity and anticoagulant activity in vivo . In order to investigate the relationship between structure and biological function, a predicted three-dimensional (3D) model of AnxB1 was generated by homology modeling . This model contains four homologous internal-domains and the Ca trace of domain I, II and IV shows high similarity . Based on the structure characterization, four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E . coli . Two of the mutants, GST-M3 and GST-M4 reserved high anticoagulant activity (p<0.01 vs . GST) . Furthermore, compared with the wild type GST-AnxB1, the immunogenicity of GST-M3 and GST-M4 was reduced significantly (p<0.01) and the molecular weight was lowered to 27 kD and 34 kD, respectively . These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect. Adv Biophys, 2004, 38, 21 - 44 Genetic and physiological regulation of non-homologous end-joining in mammalian cells; Tachibana A; Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions . To investigate the process in NHEJ, we have established an in vitro system to clarify the measurement and analysis of the efficiency and the fidelity of rejoining of DSBs, and applied the method to investigate NHEJ in human cells derived from patients suffering from cancer-prone hereditary diseases . A DSB was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to E . coli cells, and treated with nuclear extracts from human cells . The efficiency of rejoining in the nuclear extract from an A-T cell line was comparable to that from a control cell line . However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract . All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions . The deletion spectrum caused by the A-T nuclear extract was distinct from that by the control extract . These results indicate that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA . The extract from BS cells also showed the similar activity and the lower fidelity of rejoing compared to that from normal cells . From the sequencing analysis of the junction of DSBs, it is speculated that the defect in the BLM helicase might cause irregular rejoining of DSBs . Radioadaptive response is the acquirement of cellular resistance to ionizing radiation by prior exposure to low dose . We investigated the in vitro end-joining activity of DNA ends in radioadaptive cells . Both the efficiency and the fidelity of rejoining in the cells pre-exposed to low dose are increased comparing to those without pre-exposure . We also investigated the joining activity of DNA ends in p53-deficient cells . Pre-irradiation caused no apparent alteration in both the efficiency and fidelity of end-joining . These results suggest that the exposure to low dose activates a cellular function to repair DSBs efficiently, which is dependent on p53 . These results indicate that NHEJ pathway is regulated by many factors; genetic regulation by ATM and BLM, and physiological conditions such as irradiation with ionizing radiation . The observations also suggest that in some occasions p53 might play a key role in NHEJ. Adv Biophys, 2004, 38, 3 - 20 Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli; Ikeda H et al.; The frequency of illegitimate recombination has been measured by a lambda bio transducing phage assay during the induction of the E . coli lambda cI857 lysogen . Illegitimate recombination falls into two classes, short homology-independent and short homology-dependent illegitimate recombination . The former involves sequences with virtually no homology, and is mediated by DNA topoisomerases and controlled by the DNA binding protein HU . The latter is induced by UV irradiation or other DNA damaging agents and requires short regions of homology, usually contain 4 to 13 base pairs, at sites involved in recombination . It has been shown that the RecJ exonuclease promotes short homology-dependent illegitimate recombination, but that the RecQ helicase suppresses it . In addition, we have shown that the overexpression of RecE and RecT enhances the frequencies of spontaneous and UV-induced illegitimate recombination and that the RecJ, RecF, RecO, and RecR functions are required for this RecE-mediated illegitimate recombination . Moreover, we have also indicated that RecQ plays a role in the suppression of RecE-mediated illegitimate recombination, with the participation of DnaB, Fis, ExoI, and H-NS . Models have been proposed for these modes of recombination: the DNA gyrase subunit exchange model for short homology-independent illegitimate recombination and the "double-strand break and join" model for short homology-dependent illegitimate recombination . Many features of these models remain to be tested in future studies. Yi Chuan Xue Bao, 2004 Sep, 31(9), 963 - 9 {Cloning and expression of pituitary prolactin gene in Ailuropoda melanoleuca}; Zheng X et al.; The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China . It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at molecular level . In this study,the pituitary prolactin (PRL) cDNA of giant panda was amplified by RT-PCR from pituitary total RNA and then cloned, sequenced and submitted to GenBank (GenBank accession No . AY161285) . The sequence analysis revealed that the giant panda prolactin cDNA contains a 687-nucleotide open reading frame encoding the prolactin prohormone of 229 amino acid residues . The signal peptide contains 30 amino acid residues and the mature prolactin is composed of 199 amino acid residues . Then the DNA fragment amplified was subcloned into pGEX-4T-1 procaryotic expression plasmid and protein expression was induced by IPTG in Escherichia coil BL21 . SDS-PAGE analysis revealed the PRL protein is infusible . The multiple sequence alignments revealed that the homology of giant panda is 95% to cat and pig, 80% - 70% to human, cow and goat, 52% to rat and 45.9% to mouse at the amino acid level . The 64th amino acid of giant panda prolactin is hydrophilic serine instead of hydrophobic proline of cat, goat, and cow or hydrophobic alanine of human. J Gene Med, 2004 Dec, 6(12), 1343 - 57 Purine nucleoside phosphorylase and fludarabine phosphate gene-directed enzyme prodrug therapy suppresses primary tumour growth and pseudo-metastases in a mouse model of prostate cancer; Martiniello-Wilks R et al.; Gene-directed enzyme prodrug therapy based on the E . coli purine nucleoside phosphorylase (PNP) gene produces efficient tumour cell killing . PNP converts adenosine analogs into toxic metabolites that diffuse across cell membranes to kill neighbouring untransduced cells (PNP-GDEPT) . Interference with DNA, RNA and protein synthesis kills dividing and non-dividing cells, an important consideration for slow-growing prostate tumours . This study examined the impact of administering PNP-GDEPT into orthotopically grown RM1 prostate cancers (PCas) on the growth of lung pseudo-metastases of immunocompetent mice . C57BL/6 mice bearing orthotopic RM1 PCas received a single intraprostatic injection of OAdV220 (10(10) particles), a recombinant ovine atadenovirus containing the PNP gene controlled by the Rous Sarcoma virus promoter, followed by fludarabine phosphate ( approximately 600 mg/m(2)/day) administered intraperitoneally (ip) once daily for 5 days . Pseudo-metastases were induced 2 days after intraprostatic vector administration by tail-vein injection of untransduced RM1 cells . Mice given PNP-GDEPT showed a significant reduction both in prostate volume ( approximately 50%) and in lung colony counts ( approximately 60%) . Apoptosis was increased two-fold in GDEPT-treated prostates compared with controls (P < 0.01), but was absent in the lungs . Staining for proliferating cell nuclear antigen (PCNA) indicated that proliferation of both RM1 prostate tumours (P < 0.01) and lung colonies (P < 0.01) was significantly suppressed after GDEPT . Although prostate tumour immune cell infiltration did not differ significantly between treatments, immunostaining for Thy-1.2 (CD90) showed that GDEPT promoted Thy-1.2(+) cell infiltration into the prostate tumour site . This study showed that a single course of PNP-GDEPT significantly suppressed local PCa growth and reduced lung colony formation in the aggressive RM1 tumour model . Copyright (c) 2004 John Wiley & Sons, Ltd. J Biol Chem, 2004 Dec 31, 279(53), 55080 - 8 Epub 2004 Oct 18. Chaperone activity and prodan binding at the self-associating domain of erythroid spectrin; Bhattacharyya M et al.; Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25 degrees C and that of alcohol dehydrogenase at 50 degrees C . The dimeric spectrin and the two subunits, alpha-spectrin and beta-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25% . Our results also show that spectrin binds to denatured enzymes alpha-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence . The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin . The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively . Regions in alpha- and beta-spectrins have been found to have sequence homology with known chaperone proteins . More than 50% similarities in alpha-spectrin near the N terminus with human Hsp90 and in beta-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of alpha-spectrin near the N terminus and the beta-spectrin near the C terminus . There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins. Metab Eng, 2004 Oct, 6(4), 378 - 90 Optimal re-design of primary metabolism in Escherichia coli using linlog kinetics; Visser D et al.; This paper examines the validity of the linlog approach, which was recently developed in our laboratory, by comparison of two different kinetic models for the metabolic network of Escherichia coli . The first model is a complete mechanistic model; the second is an approximative model in which linlog kinetics are applied . The parameters of the linlog model (elasticities) are derived from the mechanistic model . Three different optimization cases are examined . In all cases, the objective is to calculate the enzyme levels that maximize a certain flux while keeping the total amount of enzyme constant and preventing large changes of metabolite concentrations . For an average variation of metabolite levels of 10% and individual changes of a factor 2, the predicted enzyme levels, metabolite concentrations and fluxes of both models are highly similar . This similarity holds for changes in enzyme level of a factor 4-6 and for changes in fluxes up to a factor 6 . In all three cases, the predicted optimal enzyme levels could neither have been found by intuition-based approaches, nor on basis of flux control coefficients . This demonstrates that kinetic models are essential tools in Metabolic Engineering . In this respect, the linlog approach is a valuable extension of MCA, since it allows construction of kinetic models, based on MCA parameters, that can be used for constrained optimization problems and are valid for large changes of metabolite and enzyme levels. Metab Eng, 2004 Oct, 6(4), 364 - 77 Metabolic design based on a coupled gene expression-metabolic network model of tryptophan production in Escherichia coli; Schmid JW et al.; The presumably high potential of a holistic design approach for complex biochemical reaction networks is exemplified here for the network of tryptophan biosynthesis from glucose, a system whose components have been investigated thoroughly before . A dynamic model that combines the behavior of the trp operon gene expression with the metabolic network of central carbon metabolism and tryptophan biosynthesis is investigated . This model is analyzed in terms of metabolic fluxes, metabolic control, and nonlinear optimization . We compare two models for a wild-type strain and another model for a tryptophan producer . An integrated optimization of the whole network leads to a significant increase in tryptophan production rate for all systems under study . This enhancement is well above the increase that can be achieved by an optimization of subsystems . A constant ratio of control coefficients on tryptophan synthesis rate has been identified for the models regarding or disregarding trp operon expression . Although we found some examples where flux control coefficients even contradict the trends of enzyme activity changes in an optimized profile, flux control can be used as an indication for enzymes that have to be taken into account in optimization. Metab Eng, 2004 Oct, 6(4), 294 - 9 Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli; Vadali RV et al.; Coenzyme A (CoA) and its thioester derivatives are important precursor molecules for many industrially useful compounds such as esters, PHBs, lycopene and polyketides . Previously, in our lab we could increase the intracellular levels of CoA and acetyl-Coenzyme A (acetyl-CoA) by overexpressing one of the upstream rate-controlling enzymes pantothenate kinase with a concomitant supplementation of the precursor pantothenic acid to the cell culture medium . In this study, we showed that the CoA/acetyl-CoA manipulation system could be used to increase the productivity of industrially useful compounds derived from acetyl-CoA . We chose the production of isoamyl acetate as a model system . Isoamyl acetate is an important flavor component of sake yeast and holds a great commercial value . Alcohol acetyl transferase (AAT) condenses isoamyl alcohol and acetyl-CoA to produce isoamyl acetate . The gene ATF2, coding for this AAT was cloned and expressed in Escherichia coli . This genetic engineered E . coli produces isoamyl acetate, an ester, from intracellular acetyl-CoA when isoamyl alcohol is added externally to the cell culture medium . In the current study, we showed that in a strain bearing ATF2 gene, an increase in intracellular CoA/acetyl-CoA by overexpressing panK leads to an increase in isoamyl acetate production . Additionally, the cofactor manipulation technique was combined with more traditional approach of competing pathway deletions to further increase isoamyl acetate production . The acetate production pathway competes with isoamyl acetate production for the common intracellular metabolite acetyl-CoA . Earlier we have shown that acetate pathway deletion (ackA-pta) increases isoamyl acetate production . The acetate production pathway was inactivated under elevated CoA/acetyl-CoA conditions, which lead to a further increase in isoamyl acetate production. Mutat Res, 2004 Nov 22, 556(1-2), 1 - 9 N-methyl-N'-nitro-N-nitrosoguanidine sensitivity, mutator phenotype and sequence specificity of spontaneous mutagenesis in FEN-1-deficient cells; Shi BS et al.; Intact pZ189 DNA was allowed to replicate in FL-FEN-1(-) cell line that was established in this laboratory in which the expression of FEN-1 gene was blocked by dexamethasone-inducible expression of antisense RNA to FEN-1 . E . coli MBM7070 was transfected with the replicated plasmid, and those with mutations in the supF gene were identified . The frequency of mutants that did not contain recognizable changes in the electrophoretic mobility of the plasmid DNA was scored . The frequency of such mutants was 19.1 x 10(-4) (34/17781), significantly higher than those of 2.9 x 10(-4) (4/13668) and 3.0 x 10(-4) (3/9857) in the corresponding controls, respectively . Sequence analysis of the supF genes of these mutants showed that all (37/37) the base substitutions occurred at C:G base pairs; 68% (23/37) of the base substitutions were base transversions, while 32% (12/37) were transitions . Approximately 76% (23/37) of these base substitutions occurred frequently at nine positions; two of these sites contain triple pyrimidine (T or C) repeat upstream to the mutated base; four of these sites consist of 5'-TTN1N2 and mutations occurred at N1 site sequence; another two sites have the characteristics of triple A flanked at both 5' and 3' side by TCT, with the base substitution occurring at C in the context sequence . These data suggested that these sites are the hot spot of mutagenesis in plasmid replicated in FEN-1-deficient cells . Besides the mutator phenotype of the FEN-1-deficient cell, it was also demonstrated that FEN-1-deficient cell exhibited an increased N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) sensitive phenotype. J Mol Biol, 2004 Nov 5, 343(5), 1477 - 85 Detection and structure determination of an equilibrium unfolding intermediate of Rd-apocytochrome b562: native fold with non-native hydrophobic interactions; Feng H et al.; The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process . However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance . Rd-apocytochrome b(562), a four-helix bundle, is one such protein . Here, we found another unfolding intermediate for Rd-apocytochrome b(562) . It is based on a cooperative transition of (15)N chemical shifts of amide protons as a function of urea concentrations before the global unfolding . We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding . All four helices remained intact, but a number of hydrophobic core residues repacked . This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding. J Mol Biol, 2004 Nov 5, 343(5), 1467 - 76 Probing the high energy states in proteins by proteolysis; Park C et al.; Unless the native conformation has an unstructured region, proteases cannot effectively digest a protein under native conditions . Digestion must occur from a higher energy form, when at least some part of the protein is exposed to solvent and becomes accessible by proteases . Monitoring the kinetics and denaturant dependence of proteolysis under native conditions yields insight into the mechanism of proteolysis as well as these high-energy conformations . We propose here a generalized approach to exploit proteolysis as a tool to probe high-energy states in proteins . This "native state proteolysis" experiment was carried out on Escherichia coli ribonuclease HI . Mass spectrometry and N-terminal sequencing showed that thermolysin cleaves the peptide bond between Thr92 and Ala93 in an extended loop region of the protein . By comparing the proteolysis rate of the folded protein and a peptidic substrate mimicking the sequence at the cleavage site, the energy required to reach the susceptible state (Delta G(proteolysis)) was determined . From the denaturant dependence of Delta G(proteolysis), we determined that thermolysin digests this protein through a local fluctuation, i.e . localized unfolding with minimal change in solvent assessable surface area . Proteolytic susceptibilities of proteins are discussed based on the finding of this local fluctuation mechanism for proteolysis under native conditions. J Mol Biol, 2004 Nov 5, 343(5), 1231 - 41 Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene; Edgell DR et al.; To maximize spread of their host intron or intein, many homing endonucleases recognize nucleotides that code for important and conserved amino acid residues of the target gene . Here, we examine the cleavage requirements for I-TevI, which binds a stretch of thymidylate synthase (TS) DNA that codes for functionally critical residues in the TS active site . Using an in vitro selection scheme, we identified two base-pairs in the I-TevI cleavage site region as important for cleavage efficiency . These were confirmed by comparison of I-TevI cleavage efficiencies on mutant and on wild-type substrates . We also showed that nicking of the bottom strand by I-TevI is not affected by mutation of residues surrounding the bottom-strand cleavage site, unlike other homing endonucleases . One of these two base-pairs is universally conserved in all TS sequences, and is identical with a previously identified cleavage determinant of I-BmoI, a related GIY-YIG endonuclease that binds a homologous stretch of TS-encoding DNA . The other base-pair is conserved only in a subset of TS genes that includes the I-TevI, but not the I-BmoI, target sequence . Both the I-TevI and I-BmoI cleavage site requirements correspond to functionally critical residues involved in an extensive hydrogen bond network within the TS active site . Remarkably, these cleavage requirements correlate with TS phylogeny in bacteria, suggesting that each endonuclease has individually adapted to efficiently cleave distinct TS substrates. J Mol Biol, 2004 Nov 5, 343(5), 1171 - 82 An RNA polymerase mutant deficient in DNA melting facilitates study of activation mechanism: application to an artificial activator of transcription; Sun L et al.; Transcription initiation is a major target for the regulation of gene expression in all organisms . Transcription activators can stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a subsequent promoter-melting step . Typically, kinetic assays are required to determine whether an activator exerts its effect on the initial binding of RNAP or on the promoter-melting step . Here we take advantage of a mutant Escherichia coli RNAP that is deficient in promoter melting to assess the ability of an activator to stabilize the initial binding of RNAP to the promoter . For the well-characterized activator CRP, we show that this RNAP mutant can be used to distinguish between effects on initial binding and promoter melting; these results provide an independent confirmation of the results of kinetic analysis . We then employ the melting-deficient RNAP mutant to demonstrate an effect of an artificial activator of transcription on the initial binding of RNAP . Our findings demonstrate that a melting-deficient RNAP mutant can be used to trap a normally unstable intermediate in transcription initiation, thus providing a novel tool for probing activation mechanism. J Mol Biol, 2004 Nov 5, 343(5), 1159 - 69 Oligomeric assemblies of the Escherichia coli MalT transcriptional activator revealed by cryo-electron microscopy and image processing; Larquet E et al.; MalT, the dedicated transcriptional activator of the maltose regulon in Escherichia coli, is the prototype for a family of large (approximately 100 kDa) transcriptional activators . MalT self-association plays a key role in recognition of the target promoters, which contain several MalT sites that are cooperatively bound by the activator . The unliganded form of MalT is monomeric . The protein self-associates only in the presence of both ATP (or AMP-PNP, a non-hydrolysable analog of ATP) and maltotriose, the inducer . Here, we report cryo-electron microscopy analyses of MalT multimeric forms . We show that, in the presence of maltotriose and AMP-PNP, MalT associates into novel, polydisperse, curved homopolymers . The building block, corresponding to a MalT monomer, comprises an outer globular domain connected by a peduncle to an inner domain that mediates self-association . Image analyses highlight the significant conformational flexibility of these polymeric forms . In the presence of a DNA fragment containing a MalT-controlled promoter, malPp500, MalT forms homopolymers with a much smaller radius of curvature and a different conformation . We propose that MalT binding to the target promoters involves the assembly of a MalT homo-oligomer that is governed by the array of MalT sites present. Int J Parasitol, 2004 Oct, 34(11), 1245 - 54 Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii; Brumlik MJ et al.; Toxoplasma gondii is an Apicomplexan parasite causing significant morbidity and mortality in immunocompromised hosts . Mitogen activated protein kinases regulate diverse biologic processes including proliferation, differentiation, survival and stress responses . We searched a new T . gondii genomic database to identify a 1.6 kilobase pair (kbp) coding region with features suggesting a mitogen activated protein kinase . This gene is predicted to encode a 58kDa protein with a threonine, aspartic acid, tyrosine (TDY) activation loop, similar to parasite and plant mitogen activated protein kinases, but distinct from mammalian mitogen activated protein kinases (with threonine, glycine, tyrosine (TGY) motifs) . The predicted protein shares 45% amino acid identity with human stress-activated p38alpha mitogen activated protein kinase . Expression of the cloned gene in Escherichia coli produced a protein with an apparent molecular weight of 63kDa and which exhibited kinase activity . Following osmotic stress, the abundance of the mRNA encoding this T . gondii mitogen activated protein kinase, which we name TgMAPK-1, increased in tachyzoites . Its expression rescued hog1-deficient yeast grown under osmotic stress . These data confirm that the gene product is a stress-response mitogen activated protein kinase . Upon conversion of T . gondii tachyzoites to the latent bradyzoite form in vitro, tgMAPK-1 transcript accumulation increased, suggesting a role in parasite proliferation or stage differentiation . We previously demonstrated that pyridinylimidazole p38 mitogen activated protein kinase inhibitors block T . gondii replication . These inhibitors also blocked TgMAPK-1 autophosphorylation, suggesting that TgMAPK-1, or other parasite mitogen activated protein kinases are novel drug development targets. Oral Microbiol Immunol, 2004 Dec, 19(6), 403 - 7 Resistance to human beta-defensins is common among oral treponemes; Brissette CA et al.; BACKGROUND/AIMS: Oral treponemes are implicated in the pathogenesis of periodontal disease . We have previously shown that Treponema denticola ATCC type strains and strain GM-1 are resistant to killing by human beta-defensins (hbetaD)-1 and -2 . We hypothesize that resistance to beta-defensins is a common feature of oral treponemes, which allows colonization and persistence in the oral cavity . In this study, we tested additional isolates of T . denticola, as well as six other species of treponemes, for resistance to hbetaD-1, -2 and -3 . We also examined the four ATCC strains of T . denticola and strain GM-1 for resistance to hbetaD-3 . METHODS: Resistance was determined by motility and Alamar Blue assays for metabolic activity . RESULTS: All T . denticola strains tested were resistant to hbetaD-1, -2 and -3, with the exception of strain Ambigua, which was sensitive to hbetaD-2 and -3 . All other treponemes except Treponema vincentii were resistant to hbetaD-1 . Treponema pectinovorum was sensitive to hbetaD-2, while T . vincentii, T . pectinovorum and Treponema maltophilum were sensitive to hbetaD-3 . Escherichia coli was used as a control organism and was killed by all three defensins . CONCLUSION: Resistance to the constitutively expressed hbetaD-1 may assist treponemes in initial colonization of epithelial surfaces, while resistance to the inducible hbetaD-2 and -3 would allow some treponemes to survive in active periodontal lesions. Mol Microbiol, 2004 Nov, 54(3), 769 - 82 The role of H-NS in silencing F transfer gene expression during entry into stationary phase; Will WR et al.; The conjugative ability of the F plasmid of Escherichia coli is highly growth phase dependent, with plasmid transfer efficiency dropping rapidly as donor cells progress through the growth cycle towards stationary phase . Transfer is dependent on the expression of the plasmid transfer (tra) genes, which are controlled by three plasmid-encoded regulatory proteins: TraJ, TraY and TraM . Here, we show that the nucleoid-associated host protein, H-NS, acts to repress the expression of traM and traJ as cells enter stationary phase, thereby decreasing mating ability to barely detectable levels . Sequence analysis identified regions of predicted intrinsic curvature, to which H-NS preferentially binds, at the promoters of both traM and traJ . H-NS binding at these regions was then confirmed by electrophoretic mobility shift and DNase I protection footprinting assays . Immunoblot assays displayed a significant increase in TraJ and TraM levels in an hns mutant strain . These findings were further supported by Northern and primer extension analyses which showed that whereas both genes were only expressed in early exponential phase in wild-type cells, hns mutant cells exhibited drastic derepression throughout the growth cycle . Transcriptional fusion studies of the individual promoters demonstrated that H-NS-mediated repression was observed when the promoters of both traM and traJ were present in cis to each other . This suggests that H-NS may bind to an extended region of the F plasmid, acting as a regional silencer of promoters for traJ and traM. Mol Microbiol, 2004 Nov, 54(3), 692 - 701 Axiom of determining transcription start points by RNA polymerase in Escherichia coli; Lewis DE et al.; To investigate the determining factors in the selection of the transcription start points (tsp) by RNA polymerase of Escherichia coli, we systematically deleted or substituted single base pairs (bps) at 25 putative critical positions in the two extended -10 promoters, P1 and P2, of the gal operon . These changes extend downstream from -24 to +1 of the P1 promoter . In vitro transcription assays using supercoiled DNA templates revealed a preference for a purine in the non-template strand for tsp in both promoters . The optimal tsp is the 11th bp counting downstream from the -10 position . A single bp deletion anywhere from -10 to +1 switched the tsp to the next available purine 2-3 bp downstream on the non-template strand whereas deleting a single bp at position from -24 to -11 did not affect the tsp . The nature of the 10 bp sequence of the -10 to -1 region, while affecting promoter strength, did not influence tsp . The cAMP-CRP complex, which stimulates P1 and represses P2, did not affect the tsp selection process . The rules of tsp selection by RNA polymerase containing sigma70 in gal and pyr promoters discussed here may be applicable to others. Biochemistry, 2004 Oct 26, 43(42), 13579 - 89 Recombinant EWS-FLI1 oncoprotein activates transcription; Uren A et al.; The Ewing's sarcoma family of tumors (ESFT) contains a characteristic translocation the chimeric transcript of which is translated to become the EWS-FLI1 fusion protein . EWS-FLI1 regulates transcription and posttranscriptional splicing . Elimination of EWS-FLI1 protein from ESFT cells induces apoptosis and reduces xenograft tumor growth . Therefore the production of a biologically active recombinant EWS-FLI1 could lead to discoveries that would enhance our mechanistic understanding of ESFT . We have cloned, expressed, and purified a biologically active recombinant EWS-FLI1 in Escherichia coli using affinity column chromatography . A refolding procedure was required to render the recombinant EWS-FLI1 soluble in relatively native conditions . The structural alterations induced by the refolding procedure were monitored by SDS-gel electrophoresis, circular dichroism, and steady-state fluorescence spectroscopy . Recombinant EWS-FLI1 under native conditions approaches a largely unfolded conformation . Recombinant EWS-FLI1 protein under native conditions specifically binds to DNA and transcribes RNA . Our biologically active recombinant EWS-FLI1 oncoprotein will be useful to identify functional molecular partners and inhibitors. Biochemistry, 2004 Oct 26, 43(42), 13510 - 24 Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase; Iwig DF et al.; Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids . The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products . This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet . Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme . In the preceding paper {Iwig, D . F . and Booker, S . J . (2004) Biochemistry 43, we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved . We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet . When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet . Use of S-adenosyl-L-{methyl-d(3)}methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state. Biochemistry, 2004 Oct 26, 43(42), 13496 - 509 Insight into the polar reactivity of the onium chalcogen analogues of S-adenosyl-L-methionine; Iwig DF et al.; S-Adenosyl-L-methionine (AdoMet) is one of Nature's most diverse metabolites, used not only in a large number of biological reactions but amenable to several different modes of reactivity . The types of transformations in which it is involved include decarboxylation, electrophilic addition to any of the three carbons bonded to the central sulfur atom, proton removal at carbons adjacent to the sulfonium, and reductive cleavage to generate 5'-deoxyadenosyl 5'-radical intermediates . At physiological pH and temperature, AdoMet is subject to three spontaneous degradation pathways, the first of which is racemization of the chiral sulfonium group, which takes place in a pH-independent manner . The two remaining pathways are pH-dependent and include (1) intramolecular attack of the alpha-carboxylate group onto the gamma-carbon, affording L-homoserine lactone (HSL) and 5'-methylthioadenosine (MTA), and (2) deprotonation at C-5', initiating a cascade that results in formation of adenine and S-ribosylmethionine . Herein, we describe pH-dependent stability studies of AdoMet and its selenium and tellurium analogues, Se-adenosyl-L-selenomethionine and Te-adenosyl-L-telluromethionine (SeAdoMet and TeAdoMet, respectively), at 37 degrees C and constant ionic strength, which we use as a probe of their relative intrinsic reactivities . We find that with AdoMet intramolecular nucleophilic attack to afford HSL and MTA exhibits a pH-rate profile having two titratable groups with apparent pK(a) values of 1.2 +/- 0.4 and 8.2 +/- 0.05 and displaying first-order rate constants of <0.7 x 10(-6) s(-1) at pH values less than 0.5, approximately 3 x 10(-6) s(-1) at pH values between 2 and 7, and approximately 15 x 10(-6) s(-1) at pH values greater than 9 . Degradation via deprotonation at C-5' follows a pH-rate profile having one titratable group with an apparent pK(a) value of approximately 11.5 . The selenium analogue decays significantly faster via intramolecular nucleophilic attack, also exhibiting a pH-rate profile with two titratable groups with pK(a) values of approximately 0.86 and 8.0 +/- 0.1 with first-order rate constants of <7 x 10(-6) s(-1) at pH values less than 0.9, approximately 32 x 10(-6) s(-1) at pH values between 2 and 7, and approximately 170 x 10(-6) s(-1) at pH values greater than 9 . Degradation via deprotonation at C-5' proceeds with one titratable group displaying an apparent pK(a) value of approximately 14.1 . Unexpectedly, TeAdoMet did not decay at an observable rate via either of these two pathways . Last, enzymatically synthesized AdoMet was found to racemize at rates that were consistent with earlier studies (Hoffman, J . L . (1986) Biochemistry 25, 4444-4449); however, SeAdoMet and TeAdoMet did not racemize at detectable rates . In the accompanying paper, we use the information obtained in these model studies to probe the mechanism of cyclopropane fatty acid synthase via use of the onium chalcogens of AdoMet as methyl donors. Biochemistry, 2004 Oct 26, 43(42), 13397 - 403 Probing the configurations of formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA using endonuclease IV; Patro JN et al.; The formamidopyrimidines Fapy.dA and Fapy.dG are produced in DNA as a result of oxidative stress . These lesions readily epimerize in water, an unusual property for nucleosides . The equilibrium mixture slightly favors the beta-anomer, but the configurational status in DNA is unknown . The ability of endonuclease IV (Endo IV) to efficiently incise alpha-deoxyadenosine was used as a tool to determine the configuration of Fapy.dA and Fapy.dG in DNA . Endo IV incision of the C-nucleoside analogues of Fapy.dA was used to establish selectivity for the alpha-anomer . Incision of alpha-C-Fapy.dA follows Michaelis-Menten kinetics (K(m) = 144.0 +/- 7.5 nM, k(cat) = 0.58 +/- 0.21 min(-1)), but the beta-isomer is a poor substrate . Fapy.dA incision is considerably slower than that of alpha-C-Fapy.dA, and does not proceed to completion . Endo IV incision of Fapy.dA proceeds further upon rehybridization, suggesting that the lesion reequilibrates and that the enzyme preferentially cleaves duplex DNA containing alpha-Fapy.dA . The extent of Fapy.dA incision suggests that the lesion exists predominantly ( approximately 90%) as the beta-anomer in DNA . Endo IV incises Fapy.dG to less than 5% under comparable reaction conditions, suggesting that the lesion exists almost exclusively as its beta-anomer in DNA. Biochemistry, 2004 Oct 26, 43(42), 13390 - 6 Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB); Mendel S et al.; The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products . Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction . Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor . His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine . Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues . Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid . Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis . A catalytic mechanism involving His-179 and His-115 as acid-base catalytic resi