Protein Sci, 2003 Sep, 12(9), 2104 - 8 Backbone and side-chain heteronuclear resonance assignments and hyperfine NMR shifts in horse cytochrome c; Liu W et al.; The {H26N, H33N} mutant of horse heart cytochrome c was expressed in E . coli during growth on isotopically enriched minimal media . Complete resonance assignments of both the diamagnetic reduced (spin zero) and paramagnetic oxidized (spin (1/2)) states of the protein were obtained using standard triple resonance and total correlation spectroscopy using the previously determined (1)H chemical shifts of the wild-type protein as a guide . The correspondence of chemical shifts between the wild type and the mutant protein is excellent, indicating that they have nearly identical structures . The expanded library of chemical shifts for both redox states in both proteins allowed the refinement of the electron spin g-tensor of the oxidized states . The g-tensors of the oxidized states of the wild-type and {H26N, H33N} mutant proteins are closely similar, indicating that the subtle details of the ligand fields are nearly identical . The refined g-tensors were then used to probe for redox-dependent structure change in the two proteins. Protein Sci, 2003 Sep, 12(9), 2073 - 80 Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins; Maxwell KL et al.; Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies . Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E . coli . Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized . For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology . Our results show that a denaturation/refolding protocol is appropriate for many small proteins (<or=18 kD) that are normally soluble in vivo . In addition, refolding the purified proteins by using dialysis against a single buffer allowed us to obtain soluble protein samples of 58% of small proteins that were found in the insoluble fraction in vivo, and 10% of the initial number of proteins provided good heteronuclear single quantum coherence (HSQC) NMR spectra . We conclude that a denaturation/refolding protocol is an efficient way to generate structural samples for high-throughput studies of small proteins. Protein Sci, 2003 Sep, 12(9), 2047 - 56 Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli; Dimitrova MN et al.; The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the homodimer . Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation . Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect . A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed . That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively . Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold . With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1) . The binding of PEP to EI(H189A) is synergistic with that of Mg(2+) . Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I. Protein Sci, 2003 Sep, 12(9), 1991 - 2000 Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein; Broos J et al.; Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared . Until now, the approach has dealt primarily with soluble proteins . In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EII(mtl)) . EII(mtl) overexpression was under control of the lambdaP(R) promoter, and the E . coli Trp auxotroph M5219 was used as host . This strain constitutively expresses the heat labile repressor protein of the lambdaP(R) promoter . Together with the presence of the repressor gene on the EII(mtl) plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation . A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins . The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum . The data show that the analog content of EII(mtl) samples is very high (>95%) . In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs. Protein Sci, 2003 Sep, 12(9), 1980 - 90 Dissecting interdomain communication within cAPK regulatory subunit type IIbeta using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS); Zawadzki KM et al.; cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways . The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively) . Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding . Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation . To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIbeta subunit was probed by electrospray mass spectrometry . Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation . Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization . Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain . This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain. Nucleic Acids Res . 2003 Sep 1;31(17):e101. Improved generation of recombinant baculovirus genomes in Escherichia coli; Airenne KJ et al.; An improved method for the generation of recombinant baculoviruses by Tn7-mediated transposition is described . The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculovirus bacmids in Escherichia coli with a mutated SacB gene . Recombinant bacmids can be generated at a frequency of approximately 10(7)/microg of donor vector with a negligible background . This easy-to-use and efficient pBVboost system provides the basis for a high-throughput generation of recombinant baculoviruses as well as a more convenient way to produce single viruses . The introduced selection scheme is also useful for the construction of other vectors by transposition in E.coli. Nucleic Acids Res, 2003 Sep 1, 31(17), 5195 - 201 Comparative analysis of the base biases at the gene terminal portions in seven eukaryote genomes; Niimura Y et al.; Adenine nucleotides have been found to appear preferentially in the regions after the initiation codons or before the termination codons of bacterial genes . Our previous experiments showed that AAA and AAT, the two most frequent second codons in Escherichia coli, significantly enhance translation efficiency . To determine whether such a characteristic feature of base frequencies exists in eukaryote genes, we performed a comparative analysis of the base biases at the gene terminal portions using the proteomes of seven eukaryotes . Here we show that the base appearance at the codon third positions of gene terminal regions is highly biased in eukaryote genomes, although the codon third positions are almost free from amino acid preference . The bias changes depending on its position in a gene, and is characteristic of each species . We also found that bias is most outstanding at the second codon, the codon after the initiation codon . NCN is preferred in every genome; in particular, GCG is strongly favored in human and plant genes . The presence of the bias implies that the base sequences at the second codon affect translation efficiency in eukaryotes as well as bacteria. Nucleic Acids Res, 2003 Sep 1, 31(17), 5140 - 8 Activation of site-specific DNA integration in human cells by a single chain integration host factor; Corona T et al.; The heterodimeric integration host factor (IHF) is a site-specific DNA-binding and DNA-bending protein from Escherichia coli . It plays essential roles in a variety of DNA transactions including recombination, transcription and DNA replication . IHF's ability for concerted binding and bending of DNA is key to its biological function . Here we report the design, characterization and application of a single polypeptide chain IHF, termed scIHF2 . In a novel approach for protein engineering, we inserted almost the entire alpha-subunit of IHF into the beta-subunit . DNA binding and DNA bending assays revealed that purified wild-type IHF and scIHF2 behave very similarly . Further, scIHF2 is required for site-specific integrative recombination by phage lambda integrase and for pSC101 replication in a DeltaIHF E.coli host . It also triggers site-specific integrative and excisive recombination in vitro to the same extent as the wild-type protein . We also demonstrate that scIHF2 is stably expressed in HeLa cells, that it is localized primarily in the cell nucleus and that it triggers integrative recombination in mammalian cells by wild-type integrase . Hence, scIHF2 may be used as a novel regulatory cofactor for recombination or other DNA transactions in mammalian cells that require or benefit from sequence-specific high precision DNA bending. J Biol Chem, 2003 Nov 14, 278(46), 45555 - 62 Epub 2003 Aug 20. Export of a heterologous cytochrome P450 (CYP105D1) in Escherichia coli is associated with periplasmic accumulation of uroporphyrin; Akhtar MK et al.; This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid . A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E . coli . Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105D1 . Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm . Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b5, did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production . This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis . Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors . Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin . This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E . coli . The findings have significant implications in the ontogenesis of human uroporphyria-related diseases. J Biol Chem, 2003 Nov 14, 278(46), 45661 - 71 Epub 2003 Aug 20. Identification, characterization, and inhibition of Plasmodium falciparum beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ); Sharma SK et al.; The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials . In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention . Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria . In continuation of these efforts we report cloning and overexpression of P . falciparum beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein . The enzyme catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle . It has a Km of 199 microM and kcat/Km of 80.4 m-1 s-1 for the substrate analog beta-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (Km = 86 microM, kcat/Km = 220 m-1 s-1) . More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme . Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion . Indeed these inhibitors compromised the growth of P . falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ . We modeled the structure of PfFabZ using Escherichia coli beta-hydroxydecanoyl thioester dehydratase (EcFabA) as a template . We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ . The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria. J Biol Chem, 2003 Nov 7, 278(45), 43939 - 50 Epub 2003 Aug 20. Transforming growth factor-beta1 decreases expression of the epithelial sodium channel alphaENaC and alveolar epithelial vectorial sodium and fluid transport via an ERK1/2-dependent mechanism; Frank J et al.; Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage . We have previously reported that transforming growth factor-beta1 (TGF-beta1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability . In the present study, we hypothesized that TGF-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium . Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells . TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells . TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells . The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2 . Consistent with the in vitro results, TGF-beta1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability . These data indicate that increased TGF-beta1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance . This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity. J Biol Chem, 2003 Oct 31, 278(44), 42795 - 801 Epub 2003 Aug 20. The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction; Brooks DR et al.; Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14) . Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology . The functional roles of the cytosolic PSA are less clear . The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic . We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein . Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head . Neuronal expression was also observed in the tail of adult males . Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates . Favored substrates had positively charged or small neutral amino acids in the N-terminal position . Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin . However, the enzyme was approximately 100-fold less sensitive toward puromycin (IC50, 135 mum) than other PSA homologues . Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+ . Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality . Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period . The overall brood size was, however, unaffected . We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode. J Biol Chem, 2003 Oct 31, 278(44), 43384 - 93 Epub 2003 Aug 19. Role of the M-loop and reactive center loop domains in the folding and bridging of nucleosome arrays by MENT; Springhetti EM et al.; MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells . Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function . To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences . Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines") . Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA . Reconstitution of wild-type MENT with nucleosome arrays caused their tight folding and self-association . M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association . Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA . This oligomerization was severely reduced in the RCL mutant . We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers . Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, whereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL. J Biol Chem, 2003 Oct 31, 278(44), 43340 - 5 Epub 2003 Aug 20. Physical interactions of the peroxisomal targeting signal 1 receptor pex5p, studied by fluorescence correlation spectroscopy; Wang D et al.; We have studied Hansenula polymorpha Pex5p and Pex8p using fluorescence correlation spectroscopy (FCS) . Pex5p is the Peroxisomal Targeting Signal 1 (PTS1) receptor and Pex8p is an intraperoxisomal protein . Both proteins are essential for PTS1 protein import and have been shown to physically interact . We used FCS to analyze the molecular role of this interaction . FCS is a very sensitive technique that allows analysis of dynamic processes of fluorescently marked molecules at equilibrium in a very tiny volume . We used this technique to determine the oligomeric state of both peroxins and to analyze binding of Pex5p to PTS1 peptides and Pex8p . HpPex5p and HpPex8p were overproduced in Escherichia coli, purified by affinity chromatography, and, when required, labeled with the fluorescent dye Alexa Fluor 488 . FCS measurements revealed that the oligomeric state of HpPex5p varied, ranging from monomers at slightly acidic pH to tetramers at neutral pH . HpPex8p formed monomers at all pH values tested . Using fluorescein-labeled PTS1 peptide and unlabeled HpPex5p, we established that PTS1 peptide only bound to tetrameric HpPex5p . Upon addition of HpPex8p, a heterodimeric complex was formed consisting of one HpPex8p and one HpPex5p molecule . This process was paralleled by dissociation of PTS1 peptide from HpPex5p, indicating that Pex8p may play an important role in cargo release from the PTS1 receptor . Our data show that FCS is a powerful technique to explore dynamic physical interactions that occur between peroxins during peroxisomal matrix protein import. Zhonghua Yi Xue Za Zhi, 2003 Aug 10, 83(15), 1292 - 5 {Construction and expression of anti-CD3/anti-prostate-cancer bispecific single-chain antibody}; Wang D et al.; OBJECTIVE: To construct a recombinant vector that expresses anti-CD 3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and study its biological activities and clinical significance . METHODS: An anti-CD 3/anti-prostate-cancer BsAb was constructed by PCR and molecular biological technique of DNA cloning . The fusion gene, confirmed by sequencing, was subcloned into the pSectag2-B plasmid from the pUC18 vector by digestion with EcoR I and Hind III restriction endonucleases, whose sites exist in both the vectors . Then the recombinant plasmid was transfected into HeLa cells . The expression products in the supernatant were analyzed by both SDS-PAGE and Western blot technique, then were purified with Ni(2+)-NTA superflow affinity chromatography . Its biological activities were examined by flow cytometry (FCM) . RESULTS: A fragment of 1.5 kb was inserted into the pUC18 vector, which was sequenced and verified to be identical with that designed . The expression of anti-CD 3 x anti-prostate-cancer BsAb yielded a soluble protein with an apparent molecular mass of 61 KD . The purification rate of the expressed BsAb was up to 90.537% and the yield of purified BsAb from this procedure was 0.09 mg/ml . The positive binding rates of BsAb to PBMC and to PC-3 cell were 54.1% and 53.7% respectively . CONCLUSION: The anti-CD 3 x anti-prostate-cancer BsAb thus constructed exhibits beneficial biological activities and may play an important role in the treatment of prostate cancer. Ann Trop Med Parasitol, 2003 Jul, 97(5), 481 - 7 Reactivity of sera from cases of Plasmodium vivax malaria towards three recombinant antigens based on the surface proteins of the parasite; Suh IB et al.; Sera collected in South Korea, from 61 cases of Plasmodium vivax malaria and, as controls, 40 healthy volunteers, were tested in ELISA for IgG or IgM reacting with any of three recombinant P . vivax proteins . The antigens used, representing the parasite's major merozoite surface protein (MSP), circumsporozoite surface protein (CSP) and Duffy-binding protein (DBP), had all been expressed in an Escherichia coli system and purified . The ELISA results were recorded as optical densities (OD) . The highest ratio observed between the mean OD for a malaria serum and that for a control serum was that for IgG against MSP, although CSP gave a higher ratio than MSP or DBP in the IgM ELISA . In the ELISA for IgG, the OD for MSP were found to be correlated with those for DBP (r = 0.53; P < 0.5) but the OD for CSP were not correlated with those for MSP or DBP . As the most intense reactions observed were those between the IgG from the malaria sera and the recombinant MSP, the latter antigen may be useful in diagnostic tests and as a component of any vaccine used to protect against P . vivax malaria. Environ Mol Mutagen, 2003, 42(2), 111 - 21 Frameshift mutations induced by three classes of acridines in the lacZ reversion assay in Escherichia coli: potency of responses and relationship to slipped mispairing models; Hoffmann GR et al.; The frameshift mutagenicity of 9-aminoacridine (9AA) was compared with that of quinacrine, the acridine mustards ICR-191 and quinacrine mustard (QM), and the nitroacridine Entozon in the lacZ reversion assay in Escherichia coli . As intercalating agents, 9AA and quinacrine cause mutations through noncovalent associations with DNA . Mustards and nitroacridines form covalent adducts in DNA and give rise to different spectra of mutations . Quinacrine and 9AA most effectively induced -1 frameshifts in a run of guanine residues, with 9AA being the more potent mutagen . They also induced +G frameshifts . The acridine mustard ICR-191 was a stronger mutagen than 9AA, owing largely to its potent induction of +G frameshifts . QM induced +G frameshifts more strongly than did its nonreactive counterpart quinacrine . The nitroacridine Entozon differed from the other acridines in being a potent inducer of -2 frameshifts, but it was less effective in inducing +/-1 frameshifts . Quinacrine, although a simple intercalator, induced all five kinds of frameshift mutations detected in the assay, as did the acridine mustards . Although +A and -A frameshifts were induced, adenine runs were less susceptible to acridine mutagenesis than guanine runs . The patterns of frameshift mutagenicity in the lacZ assay are similar to those in an assay based on the reversion of mutations in the tetracycline-resistance gene of the plasmid pBR322 . The similarity suggests that the responses reflect the inherent bacterial mutagenicity of the compounds in the local sequence context and are not highly dependent on the broader sequence context . The results are interpreted with respect to slipped mispairing models of frameshift mutagenesis . Curr Genet, 2003 Nov, 44(3), 115 - 23 Epub 2003 Aug 19. PCR-based methods facilitate targeted gene manipulations and cloning procedures; Wendland J; Genome sequencing of a large number of organisms has provided a wealth of previously uncharacterized genes . Rapid functional analysis of these genes relies on efficient methods for targeted gene disruption . Gene replacement requires homologous recombination at the target locus . The efficiency of homologous recombination largely depends on the size of the flanking homology regions provided with the disruption cassette . Therefore, the ratio of targeted versus random integration into the genome governs the choice of tools applicable in any organism . PCR-based methods for gene disruption were first reported in Saccharomyces cerevisiae . Over the past years, additional tools have been developed for epitope- or green fluorescent protein-tagging of genes and for promoter exchanges . The attractiveness of these tools led to the generation of PCR modules for use in a wide variety of bacterial and fungal species . The high capacity of in vivo recombination of Sac . cerevisiae and Escherichia coli may also be used for heterologous DNA manipulations . This facilitates the generation of disruption cassettes for organisms that cannot be transformed with very short flanks of target homology regions . Furthermore, laborious cloning procedures, e.g . the generation of point mutations or the deletion of internal domains of genes, can be simplified by using these organisms as workhorses which will advance the general genetic toolkit. Bioorg Med Chem, 2003 Sep 1, 11(18), 3889 - 99 5'-(2-Nitrophenylalkanoyl)-2'-deoxy-5-fluorouridines as potential prodrugs of FUDR for reductive activation; Liu B et al.; Four 5'-(2-nitrophenylalkanoyl)-2'-deoxy-5-fluorouridines (1a-d) were designed and synthesized as potential prodrugs of FUDR for reductive activation . Two methyl groups were introduced alpha to the ester carbonyl to increase both the rate of cyclization activation and the stability of the conjugates towards serum esterases . Chemical reduction of the nitro group into an amino leads to cyclization and release of the active FUDR . Kinetic analysis of the cyclization activation process indicates that the two methyl groups alpha to the ester carbonyl restrict the rotational freedom of ground state molecule and promote the cyclization reaction . However, the two methyl groups also were found to render the conjugates as poor substrates of E . coli B nitroreductase . Conjugate 1c, without the two methyl groups, was reduced by E . coli B nitroreductase (t(1/2)=8 h) to give two products, a N-hydroxyl lactam and the drug FUDR, suggesting that the enzymatic reduction and subsequent cyclization activation proceeded through the hydroxylamine intermediate . These results indicate that cyclization activation will occur once the nitro group is reduced either to an amino or to a hydroxylamino group . The fact that the amino intermediates cyclized easily to release the incorporated drug FUDR suggests the feasibility of using peptide-linked acyl 2-aminophenylalkanoic acid esters as potential prodrugs for proteolytic activation. Anal Biochem, 2003 Sep 15, 320(2), 259 - 65 A specific ceramide kinase assay to measure cellular levels of ceramide; Bektas M et al.; Human ceramide kinase was recently cloned and characterized . Recombinant ceramide kinase is highly active and ceramide is the only lipid that it phosphorylates, indicating that it should be useful for the measurement of ceramide levels in biological samples by conversion to ceramide-1-phosphate, in a manner analogous to that of the widely used Escherichia coli diacylglycerol kinase method . Using recombinant ceramide kinase, we have now developed a rapid and specific enzymatic method to quantify mass levels of long-chain ceramides in cellular lipid extracts . This new ceramide kinase assay is more specific than the commonly used diacylglycerol kinase method because the ubiquitous lipid diacylglycerol, the preferred substrate for diacyglycerol kinase which is usually present at higher concentrations than ceramide and can interfere with ceramide phosphorylation, is completely inactive with ceramide kinase . Moreover, this high specificity eliminates the need for analysis of the lipid product by thin-layer chromatography since ceramide-1-phosphate is the only radiolabeled lipid in organic solvent extracts of ceramide kinase reactions. Anal Biochem, 2003 Sep 15, 320(2), 239 - 51 Inactivation and destruction by KMnO4 of Escherichia coli RNA polymerase open transcription complex: recommendations for footprinting experiments; Lozinski T et al.; Potassium permanganate oxidation of pyrimidine residues in single-stranded DNA is commonly used in footprinting studies on formation of open transcription complex (RPo) by RNA polymerases (RNAP) at cognate promoters . Our own experience and literature search led us to conclude that KMnO4 doses often used in such studies might cause multiple-hit oxidation of promoter DNA and oxidative damage to RNAP in RPo and lead to false interpretation of footprints . We have therefore studied as a function of KMnO4 dose (i) transcription activity of RPo formed by Escherichia coli RNAP at a model cognate promoter Pa and (ii) RPo's structural integrity, by gel electrophoresis and footprinting assays . Kinetics of formation of this complex and melting of DNA in the transcription bubble region were thoroughly characterized by us previously . Here we show that (i) RPo becomes completely inactivated at oxidant doses much lower than those needed to cause a detectable footprint of the melted DNA region, (ii) footprinting patterns of the melted promoter region remain practically unaffected by RNAP oxidation within a range of low oxidant doses causing single-hit oxidation of DNA, and (iii) at higher oxidant doses, corresponding to multiple-hit DNA oxidation, the gross structure of RPo changes progressively until its complete collapse and dissociation into constituent components, so that only approximate interpretation of the footprinting data for the melted DNA region is possible . A protocol for accurate RPo footprinting with low single-hit KMnO4 doses and interpretation of the footprinting data in terms of kinetics of oxidation of pyrimidine residues in promoter DNA is recommended. Anal Biochem, 2003 Sep 15, 320(2), 170 - 8 Nicotinamide adenine dinucleotide phosphate-regenerating system coupled to a glutathione-reductase microtiter method for determination of total glutathione concentrations in adherent growing cancer cell lines; Neumann C et al.; Improvements in the traditional glutathione (GSH)-reductase recycling method for determining total glutathione levels in adherent growing cells have been achieved by eliminating the direct use of expensive nicotinamide adenine dinucleotide phosphate (NADPH) and normalizing the levels of GSH to moles/liter instead of the more usual but more error-prone method of normalizing with cellular protein . A glucose-6-phosphate-dehydrogenase auxiliary reaction has been added to the microtiter-adapted enzyme method of Tietze; thus NADP(+) and glucose-6-phosphate replace NADPH in the method . This modification lowers the possibility for substrate inhibition of the reductase by high levels of NADPH during the initial phase of the reaction while at the same time reducing the assay costs by 75-85% . To calculate the cellular concentration of GSH, the number of cells used for the GSH determination, estimated by counting cell nuclei of benzalkonium chloride-lysed cells with a Coulter Counter Z2, and the average cell volume, also determined with the Coulter Counter, are multiplied to give the total sample volume . The quotient of the amount of GSH found in the cells and the total sample volume yields the GSH concentration in moles/liter . The assay has been validated with respect to precision (+/-2.6%), relative accuracy (-4.2 %), linearity (r(2)=0.98), linear range (0.5-10 microM), and limit of detection (80 pmol) . Recovery was cell line dependent and ranged between 70 and 103% in the six cell lines . As an application of this method, the GSH concentrations in six human cancer cell lines were determined, without and with a 24-h preincubation with 200 microM D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis . As expected, BSO lowered GSH levels on the average 85%. Biochem Biophys Res Commun, 2003 Sep 5, 308(4), 713 - 8 Enhanced cell-mediated IFN-gamma-secreting activity against the HIV-1IIIB V3 peptide of the TAB9 multiepitope after DNA vaccine backbone engineering; Rodriguez EG et al.; The SV40t polyadenylation and splicing signals of the pAEC plasmid vectors were replaced by synthetic intron and synthetic rabbit beta globin-based termination/polyadenylation sequences, and 5, 10, and 20 copies of the 5'-AACGTT-3' CpG motif were inserted . Balb/c mice were immunized by intramuscular injection of 200 microg of each plasmid, coding for the HIV-1 multiepitope TAB9, under the control of the human cytomegalovirus promoter . After three doses of DNA, a fourth boost with plasmid DNA or a TAB9-expressing recombinant fowlpox virus rFPTAB9LZ was administered . ELISA and ELISPOT assays were conducted for antibody and IFN-gamma-secreting cell-mediated responses' evaluation against the whole TAB9 and the TAB9's IIIB V3 peptide, respectively . Serum IgG antibodies were not detected . Effector IFN-gamma-secreting responses were only detected on the animals receiving the new set of DNA constructs, alone or in combination with a recombinant virus boost, with or without in vitro re-stimulation . The response was dependent on the new transcriptional unit and influenced by the number of CpG motifs . We showed that plasmid backbone optimization based on these two factors could enhance the response against a multiepitope-based DNA vaccine . A new family of plasmid vectors is also available for evaluation with desired antigens. Clin Chim Acta, 2003 Sep, 335(1-2), 49 - 57 An ELISA for the detection of TIMP-1 based on recombinant single chain Fv fusion proteins; Wozniak G et al.; BACKGROUND: Altered serum levels of TIMP-1 are an indicator of various pathological states . To quantitate TIMP-1 in biological samples, we have previously isolated TIMP-1 specific single-chain Fv fragments (scFvs) using phage-display . In the work presented here, we have used these scFvs to establish a TIMP-1 ELISA based exclusively on recombinant scFv fusion proteins . METHODS: Two distinct TIMP-1 specific scFvs were used as the antigen binding components after being genetically fused to the N-termini of two different fusion protein constructs . One fusion protein, comprising a CL domain, serves as a coating reagent, while the second fusion protein with a modified form of bacterial alkaline phosphatase is used as a detection reagent . A double antibody sandwich-ELISA was then established and optimized . RESULTS: An ELISA for the quantitation of tissue inhibitor of metalloproteinase (TIMP)-1, based entirely on recombinant antibody fragments, was developed as an alternative to assays using polyclonal antisera or monoclonal antibodies . Its performance was shown to compare well with a conventional ELISA . Finally, TIMP-1 concentrations in the sera of sixty healthy individuals were determined . CONCLUSION: The assay described here provides a standardized, reliable and readily available means of quantitation of TIMP-1 in a large number of blood samples. J Mol Biol, 2003 Aug 29, 331(5), 1131 - 40 A proline switch controls folding and domain interactions in the gene-3-protein of the filamentous phage fd; Martin A et al.; The amino-terminal domains N1 and N2 of the gene-3-protein of phage fd form a bilobal structural and functional entity that protrudes from the phage tip . Domain N2 initiates the infection of Escherichia coli by binding to the F pilus . This binding results in the dissociation of the two domains and allows N1 to interact with the TolA receptor at the cell surface . The refolding of the N1-N2 fragment begins with the folding of domain N1, which takes a few milliseconds, followed by the folding of domain N2, which is complete within five minutes . The subsequent domain assembly is unusually slow and shows a time-constant of 6200 s at 25 degrees C . We found that the rate of this reaction is controlled by the trans to cis isomerization of the Gln212-Pro213 bond in the hinge subdomain of N2, a region that provides many interactions between N1 and N2 in the gene-3-protein . The substitution of Pro213 by Gly accelerated domain association 30-fold and revealed that the folding of the two individual domains and their assembly are indeed sequential steps in the refolding of the gene-3-protein . In the course of infection, the domains must separate to expose the binding site for TolA on domain N1 . The kinetic block of domain reassembly caused by Pro213 isomerization could ensure that after the initial binding of N2 to the F pilus the open state persists until N1 and TolA are close enough for their mutual interaction . Pro213 isomerization might thus serve as a slow conformational switch in the function of the gene-3-protein. Biochem J, 2003 Nov 15, 376(Pt 1), 277 - 83 Structural and functional roles of Cys-238 and Cys-295 in Escherichia coli phosphofructokinase-2; Baez M et al.; Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with pyrene maleimide (PM) results in a rapid inactivation of the enzyme . The loss of enzyme activity correlates with the incorporation of 2 mol of PM/mol of subunit and the concomitant dissociation of the dimeric enzyme . The two modified residues were identified as Cys-238 and Cys-295 . In the presence of the negative allosteric effector, MgATP, Cys-238 was the only modified cysteine residue . Kinetic characterization of the Cys-238-labelled Pfk-2 indicates that the enzyme is fully active, with the kinetic constants ( K(m), kcat) being almost identical to the ones obtained for the native enzyme . The modified enzyme is a monomer in the absence of ligands and, like the native enzyme, behaves as a tetramer in the presence of the nucleotide . However, in the presence of fructose-6-phosphate (fru-6-P) and ATP(-4), the enzyme behaves as a dimer, suggesting that the monomers undergo re-association in the presence of the substrates and that the active species is a dimer . Modification of Pfk-2 with eosin-5-maleimide (EM) results in the labelling of Cys-295 . This modified enzyme is inactive and is not able to bind to the allosteric effector, remaining as a dimer in its presence . Nonetheless, Cys-295-labelled Pfk-2 is able to bind to the substrate fru-6-P in an hyperbolic fashion with a K(d) value that is 6-fold higher than the one determined for the native enzyme . These are the first residues to be implicated in the activity and/or structure of the Pfk-2. J Comput Chem, 2003 Oct, 24(13), 1637 - 56 FDS: flexible ligand and receptor docking with a continuum solvent model and soft-core energy function; Taylor RD et al.; The docking of flexible small molecule ligands to large flexible protein targets is addressed in this article using a two-stage simulation-based method . The methodology presented is a hybrid approach where the first component is a dock of the ligand to the protein binding site, based on deriving sets of simultaneously satisfied intermolecular hydrogen bonds using graph theory and a recursive distance geometry algorithm . The output structures are reduced in number by cluster analysis based on distance similarities . These structures are submitted to a modified Monte Carlo algorithm using the AMBER-AA molecular mechanics force field with the Generalized Born/Surface Area (GB/SA) continuum model . This solvent model is not only less expensive than an explicit representation, but also yields increased sampling . Sampling is also increased using a rotamer library to direct some of the protein side-chain movements along with large dihedral moves . Finally, a softening function for the nonbonded force field terms is used, enabling the potential energy function to be slowly turned on throughout the course of the simulation . The docking procedure is optimized, and the results are presented for a single complex of the arabinose binding protein . It was found that for a rigid receptor model, the X-ray binding geometry was reproduced and uniquely identified based on the associated potential energy . However, when side-chain flexibility was included, although the X-ray structure was identified, it was one of three possible binding geometries that were energetically indistinguishable . These results suggest that on relaxing the constraint on receptor flexibility, the docking energy hypersurface changes from being funnel-like to rugged . A further 14 complexes were then examined using the optimized protocol . For each complex the docking methodology was tested for a fully flexible ligand, both with and without protein side-chain flexibility . For the rigid protein docking, 13 out of the 15 test cases were able to find the experimental binding mode; this number was reduced to 11 for the flexible protein docking . However, of these 11, in the majority of cases the experimental binding mode was not uniquely identified, but was present in a cluster of low energy structures that were energetically indistinguishable . These results not only support the presence of a rugged docking energy hypersurface, but also suggest that it may be necessary to consider the possibility of more than one binding conformation during ligand optimization . Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1674 - 5 Epub 2003 Aug 19. Crystallization and preliminary crystallographic studies of Escherichia coli CcmG/DsbE protein; Ouyang N et al.; CcmG/DsbE is a typical thiol/disulfide oxidoreductase, exhibiting a specific reducing activity in a highly oxidizing environment, and is involved in electron transfer during the maturation of c-type cytochromes . Escherichia coli CcmG/DsbE (residues 19-185) has been crystallized using the hanging-drop vapour-diffusion technique . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.48, b = 48.52, c = 84.78 A . X-ray data have been collected to 1.9 A resolution. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1668 - 9 Epub 2003 Aug 19. Crystallization and preliminary X-ray diffraction studies on recombinant diaminopropionate ammonia lyase from Escherichia coli; Rajaram V et al.; Diaminopropionate (DAP) ammonia lyase (a PLP-dependent enzyme; EC 4.3.1.15) catalyzes the alpha,beta-elimination reaction of both L- and D-alpha,beta-diaminopropionate to form pyruvate and ammonia . Escherichia coli DAP ammonia lyase gene was cloned and overexpressed in E . coli and the protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique . Crystals of two different morphologies were obtained, one of which belonged to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 86.01, c = 209.56 A, and the other to the monoclinic space group P2(1), with unit-cell parameters a = 87.78, b = 94.35, c = 96.02 A, beta = 109.73 degrees . The tetragonal crystals diffracted X-rays to 3.0 A resolution, while diffraction from the monoclinic form extended to 2.5 A . Complete X-ray diffraction data sets have been collected for both crystal forms. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1662 - 4 Epub 2003 Aug 19. Crystallization and preliminary X-ray analysis of inorganic polyphosphate/ATP-glucomannokinase from Arthrobacter sp . strain KM; Mukai T et al.; Inorganic polyphosphate {poly(P)}/ATP-glucomannokinase from Arthrobacter sp . strain KM phosphorylates glucose and mannose, utilizing both ATP and poly(P) as phosphoryl donors . The enzyme was overexpressed in Escherichia coli, purified and crystallized by means of the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant . The crystals were orthorhombic and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 66.2, b = 83.7, c = 103.8 A . Assuming two molecules per asymmetric unit, V(sol) is 0.49 . X-ray diffraction data to 2.83 A have been collected from a single crystal. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1642 - 4 Epub 2003 Aug 19. Purification, crystallization and preliminary X-ray studies of ClpX from Helicobacter pylori; Kim DY et al.; ClpX, a member of the HSP (heat-shock protein) 100 family, functions as a molecular chaperone and is a regulatory subunit of the ClpXP protease . To understand the chaperone and regulatory mechanisms of ClpX, Helicobacter pylori ClpX has been overexpressed in Escherichia coli and crystallized at 295 K using (NH(4))(2)HPO(4) as precipitant . X-ray diffraction data have been collected to 2.6 A resolution using a synchrotron-radiation source . The crystals belong to the hexagonal space group P6(5) or P6(1), with unit-cell parameters a = b = 78.52 (04), c = 131.51 (09) A, alpha = beta = 90, gamma = 120 degrees . The crystallographic asymmetric unit contains one molecule of ClpX, with a corresponding V(M) of 2.78 A(3) Da(-1) and a solvent content of 55.8%. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1635 - 6 Epub 2003 Aug 19. Crystallization and preliminary crystallographic study of rBmKalphaIT1, a recombinant alpha-insect toxin from the scorpion Buthus martensii Karsch; Huang Y et al.; Alpha-insect scorpion toxins are a distinct group of scorpion neurotoxins for which no crystal structures are yet available . A novel alpha-insect toxin named BmKalphaIT1 from the scorpion Buthus martensii Karsch (BmK) has been expressed as an inclusion body in Escherichia coli and purified by chromatography after renaturation . Recombinant BmKalphaIT1 (rBmKaIT1) was crystallized using the vapour-diffusion technique in hanging drops at 296 K . The crystals, which were grown in sodium phosphate, belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 30.24 (1), b = 36.51 (3), c = 57.08 (2) A . Diffraction data were collected to 2.1 A resolution using synchrotron radiation . There appears to be one rBmKalphaIT1 molecule in the asymmetric unit. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1628 - 31 Epub 2003 Aug 19. Structural genomics of the SARS coronavirus: cloning, expression, crystallization and preliminary crystallographic study of the Nsp9 protein; Campanacci V et al.; The aetiologic agent of the recent epidemics of Severe Acute Respiratory Syndrome (SARS) is a positive-stranded RNA virus (SARS-CoV) belonging to the Coronaviridae family and its genome differs substantially from those of other known coronaviruses . SARS-CoV is transmissible mainly by the respiratory route and to date there is no vaccine and no prophylactic or therapeutic treatments against this agent . A SARS-CoV whole-genome approach has been developed aimed at determining the crystal structure of all of its proteins or domains . These studies are expected to greatly facilitate drug design . The genomes of coronaviruses are between 27 and 31.5 kbp in length, the largest of the known RNA viruses, and encode 20-30 mature proteins . The functions of many of these polypeptides, including the Nsp9-Nsp10 replicase-cleavage products, are still unknown . Here, the cloning, Escherichia coli expression, purification and crystallization of the SARS-CoV Nsp9 protein, the first SARS-CoV protein to be crystallized, are reported . Nsp9 crystals diffract to 2.8 A resolution and belong to space group P6(1/5)22, with unit-cell parameters a = b = 89.7, c = 136.7 A . With two molecules in the asymmetric unit, the solvent content is 60% (V(M) = 3.1 A(3) Da(-1)). Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10471 - 6 Epub 2003 Aug 18. Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD; Katzen F et al.; The central hydrophobic domain of the membrane protein DsbD catalyzes the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli . Two cysteine residues embedded in transmembrane segments are essential for this process . Our results, based on cysteine alkylation and site-directed proteolysis, provide strong evidence that these residues are capable of forming an intramolecular disulfide bond . Also, by using a combination of two complementary genetic approaches, we show that both cysteines appear to be solvent-exposed to the cytoplasmic side of the inner membrane . These data are inconsistent with earlier topological models that place these residues on opposite sides of the membrane and permit the formulation of alternate hypotheses for the mechanism of this unusual transmembrane electron transfer. Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10465 - 70 Epub 2003 Aug 18. Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes; Llosa M et al.; Conjugative coupling proteins (CPs) are proposed to play a role in connecting the relaxosome to a type IV secretion system (T4SS) during bacterial conjugation . Here we present biochemical and genetic evidence indicating that the prototype CP, TrwB, interacts with both relaxosome and type IV secretion components of plasmid R388 . The cytoplasmic domain of TrwB immobilized in an affinity resin retained TrwC and TrwA proteins, the components of R388 relaxosome . By using the bacterial two-hybrid system, a strong interaction was detected between TrwB and TrwE, a core component of the conjugative T4SS . This interaction was lost when the transmembrane domains of either TrwB or TrwE were deleted, thus suggesting that it takes place within the membrane or periplasmic portions of both proteins . We have also analyzed the interactions with components of the related IncN plasmid pKM101 . Its CP, TraJ, did not interact with TrwA, suggesting a highly specific interaction with the relaxosome . On the other side, CPs from three different conjugation systems were shown to interact with both their cognate TrwE-like component and the heterologous ones, suggesting that this interaction is less specific . Mating experiments among the three systems confirmed that relaxosome components need their cognate CP for transfer, whereas T4SSs are interchangeable . As a general rule, there is a correlation between the strength of the interaction seen by two-hybrid analysis and the efficiency of transfer. Eur J Clin Invest, 2003 Sep, 33(9), 758 - 61 Marked increase in vascular endothelial growth factor concentrations during Escherichia coli endotoxin-induced acute inflammation in humans; Mittermayer F et al.; BACKGROUND: Bacterial endotoxins can induce the synthesis and release of vascular endothelial growth factor (VEGF), which may alter vascular permeability and cause vascular leakage . MATERIALS AND METHODS: The effect of acute systemic inflammation on VEGF concentration was measured in healthy males after an intravenous bolus infusion of Escherichia coli endotoxin (lipopolysaccharide, LPS, 20 IU kg-1) in a double-blind, placebo-controlled parallel group study . LPS administration was followed by an infusion of lepirudin (bolus 0.1 mg kg-1, continuous infusion of 0.1 mg kg-1 h-1, n = 12) or saline (n = 12) . RESULTS: Plasma VEGF increased from a mean of 15.1 pg mL-1 to 74.6 pg mL-1 5 h after LPS (P < 0.003) . Body temperature, pulse rate, leukcytes, prothrombin fragment 1 + 2 (F1 + 2) and lactoferrin increased and platelets decreased after LPS (P < 0.05) . The LPS-induced increase in VEGF was paralleled by the neutrophil cell degranulation marker lactoferrin but not by F1 + 2, and was not affected by lepirudin, which blunted F1 + 2 formation (P < 0.05) . CONCLUSIONS: Inflammation-induced activation of leukcytes rather than platelets plays a role in the marked increase in VEGF, which cannot be abrogated by antithrombotic therapy. Biochemistry, 2003 Aug 26, 42(33), 10012 - 23 Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms; Hu W et al.; In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers . Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells . Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14 . These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers . This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons . To distinguish these possibilities, we have introduced modifications with benzo{a}pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14 . The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique . We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA . In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence . Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12 . Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12 . Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells . These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene. Biochemistry, 2003 Aug 26, 42(33), 9937 - 45 Three conformational states of the p300 CH1 domain define its functional properties; Dial R et al.; Numerous transcription factors interact with the basal transcriptional machinery through the transcriptional co-activators p300 and CREB-binding protein (CBP) . The Zn(2+)-binding cysteine/histidine-rich 1 (CH1) domain of p300/CBP binds many of these transcription factors, including hypoxia-inducible factor (HIF) . We studied the structural and biophysical properties of the p300 CH1 domain alone and bound to the HIF-1 alpha C-terminal transactivation domain (TAD) to understand the diverse binding properties of CH1 . The Zn(2+)-bound CH1 domain (CH1-Zn(2+)) and the HIF-1 alpha TAD-CH1 complex (CH1-Zn(2+)-HIF-1 alpha) are similarly helical, whereas metal-free CH1 is mostly random coil . CH1-Zn(2+) undergoes noncooperative thermal denaturation, does not have a near-UV elliptical signal, and binds the hydrophobic fluorophore ANS . In contrast, the CH1-Zn(2+)-HIF-1 alpha complex undergoes cooperative thermal denaturation, does produce a near-UV signal, and does not bind ANS . Addition of Zn(2+) ions to metal-free CH1 produced one conformational change, and subsequent addition of a HIF-1 alpha TAD peptide induced a second conformational change as detected by intrinsic tryptophan fluorescence spectroscopy . The NMR (1)H-(15)N HSQC spectrum of CH1-Zn(2+) exhibits few poorly dispersed peaks with broad line widths . Removal of metal ions produces more poorly dispersed peaks with sharper line widths . Addition of a HIF-1 alpha TAD peptide to CH1-Zn(2+) produces many well-dispersed peaks with sharp line widths . Taken together, these data support three conformational states for CH1, including an unstructured metal-free domain, a partially structured Zn(2+)-bound domain with molten globule characteristics, and a stable, well-ordered HIF-1 alpha TAD-CH1 complex. Proteomics, 2003 Aug, 3(8), 1418 - 24 Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins; Deshusses JM et al.; Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps . This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels . Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies . Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins . We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins . The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented . The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension . When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased . The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins. Nat Struct Biol, 2003 Sep, 10(9), 731 - 7 Epub 2003 Aug 17. Pulling geometry defines the mechanical resistance of a beta-sheet protein; Brockwell DJ et al.; Proteins show diverse responses when placed under mechanical stress . The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function . Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic . These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude . Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension. Nat Biotechnol, 2003 Sep, 21(9), 1055 - 62 Epub 2003 Aug 17. Discovery of uncharacterized cellular systems by genome-wide analysis of functional linkages; Date SV et al.; We introduce a general computational method, applicable on a genome-wide scale, for the systematic discovery of uncharacterized cellular systems . Quantitative analysis of the coinheritance of pairs of genes among different organisms, calculated using phylogenetic profiles, allows the prediction of thousands of functional linkages between the corresponding proteins . A comparison of these functional linkages to known pathways reveals that calculated linkages are comparable in accuracy to genome-wide yeast two-hybrid screens or mass spectrometry interaction assays . In aggregate, these linkages describe the structure of large-scale networks, with the resulting yeast network composed of 3,875 linkages among 804 proteins, and the resulting pathogenic Escherichia coli network composed of 2,043 linkages among 828 proteins . The search of such networks for groups of uncharacterized, linked proteins led to the identification of 27 novel cellular systems from one nonpathogenic and three pathogenic bacterial genomes. Nat Biotechnol, 2003 Sep, 21(9), 1063 - 8 Epub 2003 Aug 17. Engineering of a macromolecular scaffold to develop specific protease inhibitors; Stoop AA et al.; The specific inhibition of serine proteases, which are crucial switches in many physiologically important processes, is of value both for basic research and for therapeutic applications . Ecotin, a potent macromolecular inhibitor of serine proteases of the S1A family, presents an attractive scaffold to engineer specific protease inhibitors because of its large inhibitor-protease interface . Using synthetic shuffling in combination with a restricted tetranomial diversity, we created ecotin libraries that are mutated at all 20 amino acid residues in the binding interface . The efficacy of these libraries was demonstrated against the serine protease plasma kallikrein (Pkal) . Competitive phage display selection yielded a Pkal inhibitor with an apparent dissociation equilibrium constant (K(i)*) of 11 pM, whereas K(i)* values for related proteases (such as Factor Xa (FXa), Factor XIa (FXIa), urokinase-type plasminogen activator (uPA), thrombin, and membrane-type serine protease 1 (MT-SP1)) were four to seven orders of magnitude higher . The adaptability of the scaffold was demonstrated by the isolation of inhibitors to two additional serine proteases, MT-SP1/matriptase and Factor XIIa. Nat Rev Mol Cell Biol, 2003 Aug, 4(8), 621 - 30 A tale of two polymers: new insights into helical filaments; Egelman EH; Many proteins function as helical polymers within the cell . Two intensively studied examples are eukaryotic actin and bacterial RecA, which belong to two different protein superfamilies . However, most other members of these superfamilies do not polymerize into helical filaments . General features of polymorphism, cooperativity and allostery that emerge from studies of eukaryotic actin and bacterial RecA raise more general issues about how conserved these filamentous structures have been during evolution. RNA, 2003 Sep, 9(9), 1098 - 107 Contribution of domain structure to the RNA 3' end processing and degradation functions of the nuclear exosome subunit Rrp6p; Phillips S et al.; The 3'-5' riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3' ends of 5.8S rRNA, sno- and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs . Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis . Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo . Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism . Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner's and Bloom's Syndrome proteins . A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3' ends of 5.8S pre-rRNA and some pre-snoRNAs . In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3'-extended, poly(A)+ snoRNAs . These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p. RNA, 2003 Sep, 9(9), 1029 - 33 Inhibition of HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli; Nickens DG et al.; A better understanding of aptamer function in bacteria would help to establish simple model systems for screening RNA-protein interactions within an intracellular context . Escherichia coli DNA polymerase I mutants (Pol I(ts)) fail to grow at 37 degrees C unless an exogenous DNA polymerase such as HIV-1 reverse transcriptase (RT) is expressed within the cell . Here, we show that four RNA aptamers that inhibit HIV-1 RT in vitro block complementation by HIV-1 RT when expressed in vivo . No other essential functions are impaired by aptamer expression at either temperature . Intracellular aptamer RNA concentrations from induced cultures were measured to range from 76 to 180 nM, which is comparable with exogenously expressed HIV-1 RT levels in these cells . RT polymerase activity was reduced to background levels in cell-free extracts prepared from cultures expressing both HIV-1 RT and the 70.28 aptamer, compared with extracts from cultures expressing HIV-1 RT alone . Intracellularly expressed RNA aptamers can thus be used to generate conditional null mutants in bacteria by titrating an essential protein. J Biol Chem, 2003 Nov 7, 278(45), 44593 - 9 Epub 2003 Aug 14. Crystal structure of human cholesterol sulfotransferase (SULT2B1b) in the presence of pregnenolone and 3'-phosphoadenosine 5'-phosphate . Rationale for specificity differences between prototypical SULT2A1 and the SULT2BG1 isoforms; Lee KA et al.; The gene for human hydroxysteroid sulfotransferase (SULT2B1) encodes two peptides, SULT2B1a and SULT2B1b, that differ only at their amino termini . SULT2B1b has a predilection for cholesterol but is also capable of sulfonating pregnenolone, whereas SULT2B1a preferentially sulfonates pregnenolone and only minimally sulfonates cholesterol . We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phosphoadenosine 5'-phosphate at 2.9 and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A . These structures reveal a different catalytic binding orientation for the substrate from a previously determined structure of hydroxysteroid sulfotransferase (SULT2A1) binding dehydroepiandrosterone . In addition, the amino-terminal helix comprising residues Asp19 to Lys26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding, covering the substrate binding pocket. J Biol Chem, 2003 Oct 31, 278(44), 42942 - 9 Epub 2003 Aug 15. Transmembrane domain II of the Na+/proline transporter PutP of Escherichia coli forms part of a conformationally flexible, cytoplasmic exposed aqueous cavity within the membrane; Pirch T et al.; The Na+/proline transporter PutP of Escherichia coli is a member of a large family of Na+/substrate symporters . Previous work on PutP suggests an involvement of the region ranging from Asp-55 to Gly-58 in binding of Na+ and/or proline (Pirch, T., Quick, M., Nietschke, M., Langkamp, M., Jung, H . (2002) J . Biol . Chem . 277, 8790-8796) . In this study, a complete Cys scanning mutagenesis of transmembrane domain II (TM II) of PutP was performed to further elucidate the role of the TM in the transport process . Strong defects of PutP function were observed upon substitution of Ala-48, Ala-53, Trp-59, and Gly-63 by Cys in addition to the previously characterized residues Asp-55, Ser-57, and Gly-58 . However, except for Asp-55 none of these residues proved essential for function . The activity of eight mutants was sensitive to N-ethylmaleimide inhibition with the sensitive positions clustering predominantly on a hydrophilic face in the cytoplasmic half of TM II . The same face was also highly accessible to the bulky sulfhydryl reagent fluorescein 5-maleimide in randomly oriented membrane vesicles, suggesting an unrestricted accessibility of the corresponding amino acid positions via an aqueous pathway . Na+ stimulated the reactivity of Cys toward fluorescein 5-maleimide at two positions while proline inhibited reaction of the sulfhydryl group at nine positions . Taken together, the results demonstrate that TM II of PutP is of particular functional importance . It is proposed that hydrophilic residues in the cytoplasmic half of TM II participate in the formation of an aqueous cavity in the membrane that allows Na+ and/or proline binding to residues located in the middle of the TM (e.g . Asp-55 and Ser-57) . In addition, the data indicate that TM II participates in Na+- and proline-induced conformational alterations. J Biol Chem, 2003 Oct 31, 278(44), 43114 - 20 Epub 2003 Aug 15. The location of NuoL and NuoM subunits in the membrane domain of the Escherichia coli complex I: implications for the mechanism of proton pumping; Holt PJ et al.; The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains . In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents . Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex . Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex . The presence of N,N-dimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits . Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide . A model of subunit arrangement in bacterial complex I based on these data is proposed . Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm . This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes. J Appl Physiol, 2003 Nov, 95(5), 2047 - 54 Epub 2003 Aug 15. Gut mucosal damage during endotoxic shock is due to mechanisms other than gut ischemia; Lobo SM et al.; Whether the gut alterations seen during sepsis are caused by microcirculatory hypoxia or disturbances in cellular metabolic pathways associated with mitochondrial respiration remains controversial . We hypothesized that hypoperfusion or hypoxia and local production of nitric oxide might play an important role in the development of gut mucosal injury during endotoxic shock and investigated their roles by using differing levels of fluid resuscitation and occlusion of the superior mesenteric artery (SMA) . Anesthetized New Zealand rabbits were allocated to group I (sham, n = 8); group II {low-dose endotoxin (LPS, Escherichia coli-055:B5, 150 microg/kg)/fluid resuscitation (12 ml x kg(-1) x h(-1)); n = 8}; group III {high-dose LPS (1 mg/kg)/fluid resuscitation (12 ml x kg(-1) x h(-1)); n = 8}; group IV {high-dose LPS (1 mg/kg)/hypovolemia (4 ml x kg-1 x h(-1) fluids); n = 8}; and group V {SMA ligation/fluid resuscitation (12 ml x kg(-1) x h(-1)); n = 4} . Luminal gut lactate concentrations and PCO2 gap increased in groups IV and V (P < 0.05), reflecting alterations in gut perfusion . Interestingly, significant histological alterations were observed in all LPS groups but not in group V . Blood and luminal gut nitrate/nitrite concentrations increased only in group IV . The mechanism of gut injury in endotoxic shock seems unrelated to hypoxia and release of nitric oxide . Gut dysfunction may occur as a result of so-called "cytopathic hypoxia." J Bacteriol, 2003 Sep, 185(17), 5324 - 7 Plasmid DNA supercoiling and survival in long-term cultures of Escherichia coli: role of NaCl; Conter A; The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied . In aerated continuously shaken cultures, E . coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl . Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl . Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added . This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity. J Bacteriol, 2003 Sep, 185(17), 5310 - 3 FNR-mediated oxygen-responsive regulation of the nrdDG operon of Escherichia coli; Boston T et al.; Transcription of the nrdDG operon, which encodes the class III nucleotide reductase, which is only active under anaerobic conditions, was strongly induced after a shift to anaerobiosis . The induction was completely dependent on the transcriptional activator FNR and was independent of the ArcA-ArcB two-component response regulator system . The nrdD transcript start site was mapped to a position immediately downstream of two FNR binding sites . Transcription of the other two nucleotide reductase operons, nrdAB and nrdEF, did not respond to oxygen conditions in a wild-type background, but nrdAB expression was increased in the fnr mutant under anaerobic conditions. J Bacteriol, 2003 Sep, 185(17), 5301 - 5 High-frequency secondary mutations after suicide-driven allelic exchange mutagenesis in extraintestinal pathogenic Escherichia coli; Johnson JR et al.; Frequent unintended secondary mutations occurred in extraintestinal pathogenic Escherichia coli strains CP9, CFT073, and RS218 during suicide plasmid-mediated, putatively specific deletions of hlyA, papG allele III, and iha . Pulsed-field gel electrophoresis and PCR analyses demonstrated genomic alterations and/or unintended loss of defined virulence genes (papG, the F7-2 papA allele, iutA, sat, hlyD, and cnf) . Caution is warranted when attributing the observed phenotypic changes to the intended mutation. J Bacteriol, 2003 Sep, 185(17), 5263 - 8 Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin; Reddy PT et al.; A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame . M . smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies . Consistent with the presence of nine acidic amino acids (16%) in M . smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M . smegmatis CAMLP . Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M . smegmatis CAMLP. J Bacteriol, 2003 Sep, 185(17), 5234 - 9 Topology of RbsC, the membrane component of the Escherichia coli ribose transporter; Stewart JB et al.; The topology of RbsC, the membrane component of the ribose transporter in Escherichia coli, has been determined by using 34 single-cysteine mutants and a modified fluorescence labeling technique designated multiplex labeling . This technique gives topology, expression, and localization information for a membrane protein from a single batch of bacterial cells . The results indicate that RbsC contains 10 transmembrane-spanning helices, with the N and C termini being in the cytosol . This topology matches predictions from the latest prediction programs and the topology of the similar, recently crystallized membrane protein BtuC. J Bacteriol, 2003 Sep, 185(17), 5175 - 81 Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1; Tanaka T et al.; We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc(2)) as an end product from chitin . Here we sought to identify enzymes in T . kodakaraensis that were involved in the further degradation of GlcNAc(2) . Through a search of the T . kodakaraensis genome, one candidate gene identified as a putative beta-glycosyl hydrolase was found in the near vicinity of the chitinase gene . The primary structure of the candidate protein was homologous to the beta-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to beta-galactosidases in family 42 . The purified protein from recombinant Escherichia coli clearly showed an exo-beta-D-glucosaminidase (GlcNase) activity but not beta-galactosidase activity . This GlcNase (GlmA(Tk)), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80 degrees C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides . The GlcNase activity was also detected in T . kodakaraensis cells, and the expression of GlmA(Tk) was induced by GlcNAc(2) and chitin, strongly suggesting that GlmA(Tk) is involved in chitin catabolism in T . kodakaraensis . These results suggest that T . kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc(2) from chitin is first deacetylated and successively hydrolyzed to glucosamine . This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism. J Bacteriol, 2003 Sep, 185(17), 5148 - 57 Cyclic AMP receptor protein-dependent activation of the Escherichia coli acsP2 promoter by a synergistic class III mechanism; Beatty CM et al.; The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation . Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2 . In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by "focusing" RNA polymerase to acsP2 . We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism . Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II . Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II . The locations of these DNA sites for CRP (centered at positions -69.5 and -122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions . In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the alpha subunit of RNA polymerase holoenzyme (alpha-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the alpha-CTD . Other surface-exposed residues in the alpha-CTD contributed to acs transcription, suggesting that the alpha-CTD may interact with at least one protein other than CRP. J Bacteriol, 2003 Sep, 185(17), 5076 - 85 Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate, nitrite, and formate: roles for NarL and NarP; Wang H et al.; Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons . To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions . Complementary patterns of gene expression were seen . Optimal fdhF-lacZ expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression . In contrast, fdnG-lacZ expression was induced 25-fold in the presence of intermediate to high nitrate concentrations . Consistent with prior reports, NarL was able to induce fdnG-lacZ expression . However, NarP could not induce expression; rather, it functioned as an antagonist of fdnG-lacZ expression under low-nitrate conditions (i.e., it was a negative regulator) . Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP . The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing . Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon. Genes Dev, 2003 Aug 15, 17(16), 2048 - 59 Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers; Remenyi A et al.; Members of the POU and SOX transcription factor families exemplify the partnerships established between various transcriptional regulators during early embryonic development . Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms . In this study, we focus on two transcription factors, Oct4 and Sox2, as their combination on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo . Using experimental high-resolution structure determination, followed by model building and experimental validation, we found that Oct4 and Sox2 were able to dimerize onto DNA in distinct conformational arrangements . We demonstrate that the DNA enhancer region of their target genes is responsible for the correct spatial alignment of glue-like interaction domains on their surface . Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners. Vaccine, 2003 Sep 8, 21(25-26), 3972 - 81 Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies, cytotoxic T lymphocytes and mucosal IgA; Marinaro M et al.; Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination . In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1-86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT . Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids . Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1-20 and 46-60 . Furthermore, their splenocytes proliferated and secreted IFN-gamma and IL-6 in response to Tat . Finally, CTLs were also elicited and they recognized an epitope localized within aa 11-40 of Tat. Vaccine, 2003 Sep 8, 21(25-26), 3885 - 900 Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice; Bertolotti-Ciarlet A et al.; Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine . Rotavirus VLPs composed of simian SA11 strain VP2 and VP6 proteins (homologous 2/6-VLPs) were produced by cloning the rotavirus simian SA11 genes 2 and 6 into a single baculovirus transfer vector (pAcAB4) . The overall yield of homologous 2/6-VLPs produced with the dual recombinant baculovirus was at least 30-fold higher than that of VLPs composed of bovine RF strain VP2 and simian SA11 strain VP6 (heterologous 2/6-VLPs), produced with single recombinant baculoviruses . Adult mice were immunized intramuscularly twice with various doses of homologous or heterologous 2/6-VLPs in QS-21, orally with or without cholera toxin (CT), or intranasally with mutant Escherichia coli heat-labile enterotoxin (LT-R192G) . Both homologous and heterologous 2/6-VLPs were immunogenic and induced protection from challenge, with those administered parenterally or intranasally affording the highest mean protection from challenge . The 2/6-VLPs did not induce serum neutralizing antibody (N-Ab) responses, but these VLPs primed for a broad heterotypic N-Ab response, which was elicited after rotavirus challenge . Heterotypic N-Ab responses were not observed in 2/6-VLP vaccinated mice that were > or =94% protected from challenge . After challenge, control mice immunized with adjuvant alone developed only homotypic serum N-Ab responses . Similar results were obtained after challenge of rabbits immunized parenterally or intranasally with heterologous 2/6-VLPs . These results suggest that 2/6-VLPs prime the immune system to enhance the production of heterotypic N-Ab responses, but the induction of heterotypic N-Abs requires that virus replication occurs after challenge . The use of 2/6-VLPs expressed from a single recombinant baculovirus simplifies production and would reduce the cost of a VLP-based vaccine. Vaccine, 2003 Sep 8, 21(25-26), 3721 - 9 Induction of immunity in swine by purified recombinant VP1 of foot-and-mouth disease virus; Wang JH et al.; VP1, a capsid protein of foot-and-mouth disease virus (FMDV), contains neutralizing epitopes of the virus . Due to its poor water solubility, recombinant Escherichia coli derived VP1 (rVP1) has previously been used mainly in a denatured form and is not well characterized . Here, using SDS to assist protein refolding and then removing SDS with a detergent removing column, we have successfully purified rVP1 in two aqueous-soluble forms, i.e . monomer and dimer . Studies showed that dimerization occurs by an inter-molecular disulfide bond between two cysteine residues at position 187 of each monomer . Heat treatment revealed that rVP1 dimer exhibited a more thermal-stable conformation than the monomeric form . Both monomeric and dimeric rVP1 reacted with anti-FMDV antibodies . Immunization studies demonstrated that vaccination of swine with either forms of rVP1 was effective in generating immune responses and protecting them from viral challenge. Vaccine, 2003 Sep 8, 21(25-26), 3565 - 74 Immunological properties of a DNA plasmid encoding a chimeric protein of herpes simplex virus type 2 glycoprotein B and glycoprotein D; Domingo C et al.; A DNA plasmid containing a chimeric sequence encoding both herpes simplex virus type 2 (HSV-2) glycoprotein B (gB) and glycoprotein D (gD) external domains (pcgDB) was used to immunize BALB/c mice against genital HSV-2 infection . To determine the efficacy of this vaccine, groups of mice immunized with the pcgDB plasmid were compared with animals immunized with plasmids corresponding to the individual proteins (pcgBt or pcgDt), administered separately or in combination (pcgBt + pcgDt) . We studied the response of the different mouse groups to viral challenge by analyzing clinical disease (vaginitis), serum antibody levels, as well as lymphoproliferative responses and cytokine production by spleen cells . Increased IFN-gamma levels correlated with prolonged survival in mice immunized with the plasmid pcgDB, relative to mice immunized with plasmids coding for the individual proteins alone or in combination . Our results show that immunization with the plasmid encoding the chimeric protein is advantageous over separate proteins . These findings may have important implications for the development of multivalent DNA vaccines against HSV and other complex pathogens. Atherosclerosis, 2003 Aug, 169(2), 225 - 33 Effects of leptin on endothelial function with OB-Rb gene transfer in Zucker fatty rats; Jin X et al.; The metabolic syndrome in association with obesity is a major clinical problem inducing hypertension, diabetes mellitus, and atherosclerosis . Leptin induces angiogenesis by its proliferative effects on endothelial cells (ECs) via OB receptor (OB-Rb) gene . We evaluated the growth of ECs and intracellular signalings in response to leptin in vitro and the angiogenic effects of leptin in the cornea in vivo with and without adenovirus-mediated transfer of the OB-Rb gene in Zucker fatty (ZF) rats as a model for the metabolic syndrome . Recombinant adenovirus vector encoding rat OB-Rb (Ad.OB-Rb) or Escherichia coli . LacZ (Ad.LacZ) was transfected into cultured ECs from Zucker lean (ZL) rats and ZF rats . Leptin increased DNA synthesis dose-dependently in ECs from ZL rats but not ZF rats . Infection with Ad.OB-Rb, but not with Ad.LacZ, improved the growth effects of leptin in ECs from ZF rats . Leptin induced phosphorylation of Janus kinase (JAK)2, signal transducer and activator of transcription (STAT)3, and extracellular signal-regulated kinase (ERK) in ECs from ZL rats but not ZF rats . Infection with Ad.OB-Rb restored phosphorylation of JAK2 and STAT3 in ECs from ZF rats . Leptin induced angiogenesis in cornea from ZL rats, but not from ZF rats . Coadministration of leptin and Ad.OB-Rb induced angiogenesis in cornea from ZF rats . Ad.LacZ did not influence the angiogenic effects of leptin . The impaired endothelial function with the leptin resistance may be one of causes of the atherosclerosis in the metabolic syndrome. Arch Biochem Biophys, 2003 Sep 1, 417(1), 87 - 95 Determinants for the interaction between Janus kinase 2 and protein phosphatase 2A; Yokoyama N et al.; We have shown that the serine/threonine protein phosphatase 2A (PP2A) associates with the Jak2 tyrosine kinase in a myeloid progenitor line . In this study, we characterized the regions of Jak2 and PP2A responsible for association and evaluated the functional consequences of association . We demonstrate that PP2A interacts with truncated forms of Jak2 containing the JH1 catalytic domain . Using GST fusion proteins, we show that the isolated JH1 and JH3 domains of Jak2 bind directly to PP2A . Jak2 contains putative PP2A binding sequences (LXXLL) in the JH1 domain (residues 1078-1082) and in the JH3 domain (residues 474-478) . Mutation of the LXXLL sequence in the JH1 domain decreased PP2A binding in vitro, while mutation of the similar JH3 sequence did not affect PP2A binding . We analyzed full-length Jak2 bearing the LXXLL mutation in Cos-7 cells for association with PP2A . The JH1 mutation impaired Jak2 activity and had a modest effect on PP2A binding . Finally, we show that a mutant form of the PP2A catalytic subunit lacking a site for phosphorylation (Y307F) binds more tightly to Jak2 than wild-type PP2A, consistent with a model where phosphorylation disrupts the Jak2-PP2A interaction. Dev Biol, 2003 Aug 15, 260(2), 339 - 51 M142.2 (cut-6), a novel Caenorhabditis elegans matrix gene important for dauer body shape; Muriel JM et al.; The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm . We have characterized the C . elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure . Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle . The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages . A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum . Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells . Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae . M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6. Int Rev Cytol, 2003, 226, 165 - 258 Growth during the cell cycle; Mitchison JM; During the cell cycle, major bulk parameters such as volume, dry mass, total protein, and total RNA double and such growth is a fundamental property of the cell cycle . The patterns of growth in volume and total protein or RNA provide an "envelope" that contains and may restrict the gear wheels . The main parameters of cell cycle growth were established in the earlier work when people moved from this field to the reductionist approaches of molecular biology, but very little is known on the patterns of metabolism . Most of the bulk properties of cells show a continuous increase during the cell cycle, although the exact pattern of this increase may vary . Since the earliest days, there have been two popular models, based on an exponential increase and linear increase . In the first, there is no sharp change in the rate of increase through the cycle but a smooth increase by a factor of two . In the second, the rate of increase stays constant through much of the cycle but it doubles sharply at a rate change point (RCP) . It is thought that the exponential increase is caused by the steady growth of ribosome numbers and the linear pattern is caused by a doubling of the structural genes during the S period giving an RCP--a "gene dosage" effect . In budding yeast, there are experiments fitting both models but on balance slightly favoring "gene dosage." In fission yeast, there is no good evidence of exponential increase . All the bulk properties, except O2 consumption, appear to follow linear patterns with an RCP during the short S period . In addition, there is in wild-type cells a minor RCP in G2 where the rate increases by 70% . In mammalian cells, there is good but not extensive evidence of exponential increase . In Escherichia coli, exponential increase appears to be the pattern . There are two important points: First, some proteins do not show peaks of periodic synthesis . If they show patterns of exponential increase both they and the total protein pattern will not be cell cycle regulated . However, if the total protein pattern is not exponential, then a majority of the individual proteins will be so regulated . If this majority pattern is linear, then it can be detected from rate measurements on total protein . However, it would be much harder at the level of individual proteins where the methods are at present not sensitive enough to detect a rate change by a factor of two . At a simple level, it is only the exponential increase that is not cell cycle regulated in a synchronous culture . The existence of a "size control" is well known and the control has been studied for a long time, but it has been remarkably resistant to molecular analysis . The attainment of a critical size triggers the periodic events of the cycle such as the S period and mitosis . This control acts as a homeostatic effector that maintains a constant "average" cell size at division through successive cycles in a growing culture . It is a vital link coordinating cell growth with periodic events of the cycle . A size control is present in all the systems and appears to operate near the start of S or of mitosis when the cell has reached a critical size, but the molecular mechanism by which size is measured remains both obscure and a challenge . A simple version might be for the cell to detect a critical concentration of a gene product. Mol Imaging, 2002 Jan-Mar, 1(1), 36 - 42 Imaging expression of cytosine deaminase-herpes virus thymidine kinase fusion gene (CD/TK) expression with {124I}FIAU and PET; Hackman T et al.; Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)-herpes simplex virus type 1 thymidine kinase (HSV1-tk) fusion gene (CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors . We assessed the efficacy of noninvasive imaging with {124I}FIAU (2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene . Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells) . The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively . A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels . Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats . PET imaging of HSV1-TK subunit activity with {124I}FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively . CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay . A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation . Knowing this relationship, the parametric images of CD subunit activity were generated . Imaging with {124I}FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials. Int Microbiol, 2003 Dec, 6(4), 263 - 7 Epub 2003 Aug 13. Colicin S8 export: extracellular and cytoplasmic colicin are different; Garcia Diaz ME et al.; The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein . Interactions with its specific receptors reflect this . Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography . Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da . We suggest that a conformational change to colicin S8 may occur related to the export process. Science, 2003 Aug 15, 301(5635), 964 - 7 An expanded eukaryotic genetic code; Chin JW et al.; We describe a general and rapid route for the addition of unnatural amino acids to the genetic code of Saccharomyces cerevisiae . Five amino acids have been incorporated into proteins efficiently and with high fidelity in response to the nonsense codon TAG . The side chains of these amino acids contain a keto group, which can be uniquely modified in vitro and in vivo with a wide range of chemical probes and reagents; a heavy atom-containing amino acid for structural studies; and photocrosslinkers for cellular studies of protein interactions . This methodology not only removes the constraints imposed by the genetic code on our ability to manipulate protein structure and function in yeast, it provides a gateway to the systematic expansion of the genetic codes of multicellular eukaryotes. J Biol Chem, 2003 Nov 7, 278(45), 44959 - 65 Epub 2003 Aug 14. ATP binding is critical for the conformational change from an open to closed state in archaeal group II chaperonin; Iizuka R et al.; Group II chaperonins, found in archaea and in eukaryotic cytosol, do not have a co-chaperonin corresponding to GroES . Instead, it is suggested that the helical protrusion extending from the apical domain acts as a built-in lid for the central cavity and that the opening and closing of the lid is regulated by ATP binding and hydrolysis . However, details of this conformational change remain unclear . To investigate the conformational change associated with the ATP-driven cycle, we conducted protease sensitivity analyses and tryptophan fluorescence spectroscopy of alpha-chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1 . In the nucleotide-free or ADP-bound state, the chaperonin, especially in the helical protrusion region, was highly sensitive to proteases . Addition of ATP and ammonium sulfate induced the transition to the relatively protease-resistant form . The fluorescence intensity of the tryptophan residue introduced at the tip of the helical protrusion was enhanced by the presence of ATP or ammonium sulfate . We conclude that ATP binding induces the conformational change from the lid-open to lid-closed form in archaeal group II chaperonin. J Biol Chem, 2003 Oct 31, 278(44), 43516 - 24 Epub 2003 Aug 14. Molecular recognition in dimerization between PB1 domains; Noda Y et al.; The PB1 (Phox and Bem 1) domain is a recently identified module that mediates formation of a heterodimeric complex with other PB1 domain, e.g . the complexes between the phagocyte oxidase activators p67phox and p40phox and between the yeast polarity proteins Bem1p and Cdc24p . These PB1 domains harbor either a conserved lysine residue on one side or an acidic OPCA (OPR/PC/AID) motif around the other side; the lysine of p67phox or Bem1p likely binds to the OPCA of p40phox or Cdc24p, respectively, via electrostatic interactions . To further understand molecular recognition by PB1 domains, here we investigate the interactions mediated by proteins presenting both the lysine and OPCA on a single PB1 domain, namely Par6, atypical protein kinase C (aPKC), and ZIP . Par6 and aPKC form a complex via the interaction of the Par6 lysine with aPKC-OPCA but not via that between the aPKC lysine and Par6-OPCA, thereby localizing to the tight junction of epithelial cells . aPKC also uses its OPCA to interact with ZIP, another protein that has a PB1 domain presenting both the lysine and OPCA, whereas aPKC binds via the conserved lysine to MEK5 in the same manner as ZIP interacts with MEK5 . In addition, ZIP can form a homotypic complex via the conserved electrostatic interactions . Thus the PB1 domain appears to be a protein module that fully exploits its two mutually interacting elements in molecular recognition to expand its repertoire of protein-protein interactions. Di Yi Jun Yi Da Xue Xue Bao, 2003 Aug, 23(8), 826 - 9 {Pathogenic mechanisms of enterotoxigenic Escherichia coli in guinea pigs}; Chen Q et al.; OBJECTIVE: To explore the pathogenic mechanisms of enterotoxigenic Escherichia coli (ETEC) in guinea pigs . METHODS: Pathological examination of the intestines was performed in ETEC-infected guinea pigs, and the intracellular free Ca2+ concentration, cytoplasmic pH, cell membrane potential and mitochondria membrane potential were determined immunohistochemically and by means of flow cytometry . RESULTS: The guinea pigs were sensitive to the human-origin ETEC, which caused pathological changes in the small intestines such as edema, hyperemia and lymphocyte infiltration, but no bacteria invasion into the epithelial cells was identified . Under transmission electron microscope, the ileal epithelial cells were shown with vacuolar degeneration and evidence of mitochondrial proliferation; heat-labile (LT) and heat-stable (ST) toxins pervaded in the ileal tissue, resulting in significant increase of intracellular free Ca2+ concentration, cytoplasmic pH value and cell membrane potential, with concomitance of significant decrease in mitochondrial membrane potential . CONCLUSIONS: ETEC disturbs the absorption and secretion functions of the intestinal epithelial cells, and induces extensive inflammation in the small intestines of guinea pigs . Besides their action on intestinal epithelial cells, LT and ST also affect the myocytes in the muscularis, and their action sites and mechanism might be similar . ETEC-induced diarrhea is correlated with excessive water excretion from the cells. Di Yi Jun Yi Da Xue Xue Bao, 2003 Aug, 23(8), 802 - 5 {Synthesis, cloning, expression and antigenicity of the therapeutic multi-epitope gene of hepatitis B virus and of its products}; Luo LM et al.; OBJECTIVE: To study the synthesis, cloning, expression and antigenicity of therapeutic multi-epitope gene of hepatitis B virus . METHODS: The therapeutic multi-epitope gene of hepatitis B virus was synthesized and cloned into the vector pWR450-1, then was expressed in E.coli and the products were purified . The immunogenicity of the expressed protein was analyzed by Western-blotting . RESULTS: Recombinant plasmid PWR/BPT was constructed successfully and the protein of multi-epitope gene of hepatitis B virus was expressed in E . coli, which showed ideal antigenicity by Western-blotting . CONCLUSIONS: The designed multi-epitope therapeutic gene of hepatitis B virus was proved to be correct and the expressed protein may be a good therapeutic vaccine. Microbes Infect, 2003 Aug, 5(10), 857 - 67 Genetically engineered enteropathogenic Escherichia coli strain elicits a specific immune response and protects against a virulent challenge; Boullier S et al.; Enteropathogenic Escherichia coli (EPEC), a major cause of severe disease with diarrhea in infants, is also involved in weaned rabbit colibacillosis . EPEC O103 is frequent in rabbit-fattening units of Western Europe . It causes high mortality and growth retardation, leading to substantial economic losses . We report here the construction by allelic exchange of an EPEC O103 strain mutated in espB and tir, two essential virulence genes . Upon live oral administration to weaned rabbits, the E22DeltaTir/EspB mutant strain efficiently colonized the intestinal tract without any adverse consequences . The rabbits were challenged with the highly pathogenic parental strain E22 . The mutant provided complete protection to rabbits and total resistance to intestinal colonization by E22 . In addition, E22DeltaTir/EspB strain induced a specific humoral response against the bacterial adhesin AF/R2 . These Abs prevent bacterial attachment to epithelial cells in vitro . These results open the way for the development of an efficient vaccine strategy against rabbit EPEC infections. Virology, 2003 Aug 1, 312(2), 359 - 68 Oligomerization and polymerization of the filovirus matrix protein VP40; Timmins J et al.; The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex . Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy . Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance . Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E . coli nucleic acids . In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40 . Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings . These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle. Mol Ecol, 2003 Sep, 12(9), 2429 - 37 Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice; Bouchard E et al.; Transgenic plants expressing resistance to herbivorous insects may