Escherichia coli

Nucleic Acids Res . 2004 Mar 18;32(5):e51.
Improving the sensitivity and specificity of gene expression analysis in highly related organisms through the use of electronic masks; Nagpal S et al.; DNA microarrays are powerful tools for comparing gene expression profiles from closely related organisms . However, a single microarray design is frequently used in these studies . Therefore, the levels of certain transcripts can be grossly underestimated due to sequence differences between the transcripts and the arrayed DNA probes . Here, we seek to improve the sensitivity and specificity of oligonucleotide microarray-based gene expression analysis by using genomic sequence information to predict the hybridization efficiency of orthologous transcripts to a given microarray . To test our approach, we examine hybridization patterns from three Escherichia coli strains on E.coli K-12 MG1655 gene expression microarrays . We create electronic mask files to discard data from probes predicted to have poor hybridization sensitivity and specificity to cDNA targets from each strain . We increased the accuracy of gene expression analysis and identified genes that cannot be accurately interrogated in each strain using these microarrays . Overall, these studies provide guidelines for designing effective electronic masks for gene expression analysis in organisms where substantial genome sequence information is available.

J Biol Chem, 2004 May 21, 279(21), 22258 - 66 Epub 2004 Mar 18.
A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain; Tremuth L et al.; The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits . Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site . Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP) . Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments . The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions . Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively . Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G . Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions . Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation.

J Biol Chem, 2004 Jun 11, 279(24), 25058 - 65 Epub 2004 Mar 18.
Characterization of recombinant, membrane-attached full-length prion protein; Eberl H et al.; An abnormal isoform, PrP(Sc), of the normal cellular prion protein (PrP(C)) is the major component of the causative agent of prion diseases . Both isoforms were found to possess the same covalent structures, including a C-terminal glycosylphosphatidylinositol anchor, but different secondary and tertiary structures . In this study, a variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment . Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism . Moreover, limited proteolysis experiments failed to detect changes in the stability of liposome-bound PrP relative to soluble PrP . These data suggest that the requirement of raft localization for the PrP(C) to PrP(Sc) conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membranebound PrP(C) but caused by other factors, e.g . increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors . The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with recombinant PrP on the surface of membranes . In addition, our results indicate that the three-dimensional structure of mammalian PrP(C) in membranes is identical to that of recombinant PrP in solution.

Ultrason Sonochem, 2004 Apr, 11(2), 57 - 60
Inactivation of Escherichia coli by ultrasonic irradiation; Furuta M et al.; Ultrasonic inactivation of Escherichia coli XL1-Blue has been investigated by high-intensity ultrasonic waves from horn type sonicator (27.5 kHz) utilizing the "squeeze-film effect" . The amplitude of the vibration face contacting the sample solution was used as an indication of the ultrasonic power intensity . The inactivation of the E . coli cells by ultrasonic irradiation shows pseudo first-order behavior . The inactivation rate constant gradually increased with increasing amplitude of the vibration face and showed rapid increase above 3 microm (p-p) . In contrast, the H2O2 formation was not observed below 3 microm (p-p), indicating that the ultrasonic shock wave might be more important than indirect effect of OH radicals formed by ultrasonic cavitation in this system . The optimal thickness of the squeeze film was determined as 2 mm for the E . coli inactivation . More than 99% of E . coli cells was inactivated within 180-s sonication at the amplitude of 3 microm (p-p) and 2 mm of the thickness of the squeeze film.

Eur J Biochem, 2004 Apr, 271(7), 1299 - 309
Escherichia coli thioredoxin inhibition by cadmium: two mutually exclusive binding sites involving Cys32 and Asp26; Rollin-Genetet F et al.; Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd(2+) led us to investigate the thioredoxin-cadmium interaction properties . We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site . At pH 8 or 7.5 two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 x 10(6) m(-1) and 1 x 10(6) m(-1) . For both sites, a proton was released upon Cd(2+) binding . One mole of Cd(2+) per mole of reduced thioredoxin was measured by mass spectrometry at these pH values, demonstrating that the two binding sites were partially occupied and mutually exclusive . Cd(2+) binding at either site totally inhibited the thiol-disulfide transferase activity of Trx . The absence of Cd(2+) interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd(2+) supported the role of Cys32 at the first site . The fluorescence profile of Cd(2+)-bound thioredoxin differed, however, from that of oxidized thioredoxin, indicating that Cd(2+) was not coordinated with Cys32 and Cys35 . From FTIR spectroscopy, we inferred that the second site might involve Asp26, a buried residue that deprotonates at a rather high and unusual pK(a) for a carboxylate (7.5/9.2) . The pK(a) of the two residues Cys32 and Asp26 have been shown to be interdependent {Chivers, T . P . (1997) Biochemistry36, 14985-14991} . A mechanism is proposed in which Cd(2+) binding at the solvent-accessible thiolate group of Cys32 induces a decrease of the pK(a) of Asp26 and its deprotonation . Conversely, interaction between the carboxylate group of Asp26 and Cd(2+) at a second binding site induces Cys32 deprotonation and thioredoxin inhibition, so that Cd(2+) inhibits thioredoxin activity not only by binding at the Cys32 but also by interacting with Asp26.

Eur J Biochem, 2004 Apr, 271(7), 1258 - 65
The C-terminal domain of Escherichia coli Hfq increases the stability of the hexamer; Arluison V et al.; The Hfq (Host factor 1) polypeptide is a nucleic acid binding protein involved in the synthesis of many polypeptides . Hfq particularly affects the translation and the stability of several RNAs . In an earlier study, the use of fold recognition methods allowed us to detect a relationship between Escherichia coli Hfq and the Sm topology . This topology was further validated by a series of biophysical studies and the Hfq structure was modelled on an Sm protein . Hfq forms a beta-sheet ring-shaped hexamer . As our previous study predicted a large number of alternative conformations for the C-terminal region, we have determined whether the last 19 C-terminal residues are necessary for protein function . We find that the C-terminal truncated protein is fully capable of binding a polyadenylated RNA (K(d) of 120 pm vs . 50 pm for full-length Hfq) . This result shows that the functional core of E . coli Hfq resides in residues 1-70 and confirms previous genetic studies . Using equilibrium unfolding studies, however, we find that full-length Hfq is 1.8 kcal x mol(-1) more stable than its truncated variant . Electron microscopy analysis of both truncated and full-length proteins indicates a structural rearrangement between the subunits upon truncation . This conformational change is coupled to a reduction in beta-strand content, as determined by Fourier transform infra-red . On the basis of these results, we propose that the C-terminal domain could protect the interface between the subunits and stabilize the hexameric Hfq structure . The origin of this C-terminal domain is also discussed.

Eur J Biochem, 2004 Apr, 271(7), 1250 - 7
Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism; Amichot M et al.; Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane (DDT) resistance in the RDDT(R) strain of Drosophila melanogaster . To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL . All CYP6A2 variants were less stable than the CYP6A2wt protein . Two activities enhanced in the RDDT(R) strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism . Testosterone was hydroxylated at the 2beta position with little quantitative variation among the variants . In contrast, metabolism of DDT was strongly affected by the mutations . The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid . The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra . Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2 . These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley . Thus this area, including helix J, is important for the structure and activity of CYP6A2 . Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.

Biochem J, 2004 Jul 1, 381(Pt 1), 137 - 46
The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron centre; Urich T et al.; The SOR (sulphur oxygenase reductase) is the initial enzyme in the sulphur-oxidation pathway of Acidianus ambivalens . Expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferric ions were present in the refolding solution . Wild-type, recombinant and refolded SOR possessed indistinguishable properties . Conformational stability studies showed that the apparent unfolding free energy in water is approx . 5 kcal x mol(-1) (1 kcal=4.184 kJ), at pH 7 . The analysis of the quaternary structures showed a ball-shaped assembly with a central hollow core probably consisting of 24 subunits in a 432 symmetry . The subunits form homodimers as the building blocks of the holoenzyme . Iron was found in the wild-type enzyme at a stoichiometry of one iron atom/subunit . EPR spectroscopy of the colourless SOR resulted in a single isotropic signal at g=4.3, characteristic of high-spin ferric iron . The signal disappeared upon reduction with dithionite or incubation with sulphur at elevated temperature . Thus both EPR and chemical analysis indicate the presence of a mononuclear iron centre, which has a reduction potential of -268 mV at pH 6.5 . Protein database inspection identified four SOR protein homologues, but no other significant similarities . The spectroscopic data and the sequence comparison led to the proposal that the Acidianus ambivalens SOR typifies a new type of non-haem iron enzyme containing a mononuclear iron centre co-ordinated by carboxylate and/or histidine ligands.

Parasitology, 2004 Feb, 128(Pt 2), 209 - 21
Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression; Dabrowska M et al.; The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T . pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T . spiralis thymidylate synthase gene expression . The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight . The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed . The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E . coli . As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs . 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs . 54.7 microM) . With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Biofizika, 2004 Jan-Feb, 49(1), 32 - 7
{Level of hydrogen peroxide in electrochemically activated solutions and study of its effect on growth of Escherichia coli}; Miroshnikov AI et al.; The formation of hydrogen peroxide in catholytes and anolytes of electrochemically activated solutions: bidistilled water and solutions of sodium chloride and nutrition medium M9 was studied . The concentration of hydrogen peroxide was determined by the method of enhanced chemiluminescence in a system peroxidase-luminol-p-iodophenol . It was shown that the concentration of hydrogen peroxide depends on the ionic content of the solution and varies from a few fractions of a micromole in catholytes of bidistilled water and sodium chloride solutions (10(-5) divided by 10(-2) M) to 20-25 microM in catholytes of medium M9 . The concentration of H2O2 in anolytes of various solutions was 15-20 times lower than in the corresponding catholytes and was equal to a few nanomoles in bidistilled water and a few micromoles in medium M9 . The biological activity of the catholyte of medium M9 was determined from changes in the growth of E . coli cells . It was found that this catholyte stimulates the cell growth . The stimulating effect was 20-25% and did not change after the decomposition of hydrogen peroxide in the catholyte by catalase . The addition of H2O2 at the corresponding concentration to the inactivated nutrient medium produced no stimulating effect . These data suggest that hydrogen peroxide formed in the catholyte of nutrient medium M9 does not affect its biological activity.

Nature, 2004 Mar 18, 428(6980), 319 - 23
Protein-only transmission of three yeast prion strains; King CY et al.; Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein . Here we demonstrate the protein-only nature of prion strains in a yeast model, the {PSI} genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae . Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains . Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein . De novo generation of strain-specific {PSI} infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts . The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy . Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids . The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

J Biol Chem, 2004 May 14, 279(20), 20692 - 8 Epub 2004 Mar 17.
Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity; Kikuma T et al.; The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism . Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized . To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity . The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain . We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA . In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP . These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.

J Biol Chem, 2004 Jun 4, 279(23), 24236 - 45 Epub 2004 Mar 17.
Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C: use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C; Nilsson M et al.; Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins . This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils . One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life . The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors . The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C . In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers . The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C . In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C . It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.

J Biol Chem, 2004 May 21, 279(21), 21966 - 75 Epub 2004 Mar 17.
Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study; Nuscher B et al.; A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of alpha-synuclein (alphaS) . The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein . The present study investigates the lipid binding and the coil-helix transition of alphaS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy . Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values . The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface . The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding . There was no heat release and folding of alphaS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the alphaS-membrane interaction . The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone . We speculate that binding and helix folding of alphaS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes . This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes.

J Bacteriol, 2004 Apr, 186(7), 2147 - 55
Cell death in Escherichia coli dnaE(Ts) mutants incubated at a nonpermissive temperature is prevented by mutation in the cydA gene; Strauss B et al.; Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40 degrees C in Luria-Bertani medium . Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density . Cells survive at 40 degrees C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids . Deletion of recA or sulA has no effect . We isolated suppressors which survived for long periods at 40 degrees C but did not form colonies . The suppressors protected against hydroxyurea-induced killing . Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene . The DNA content of dnaE mutants increased about eightfold in 4 h at 40 degrees C, as did the DNA content of the suppressed strains . The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs . Transformation activity also increased . 4',6'-diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40 degrees C, indicating that there was segregation of the newly formed DNA . We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation . These observations support the view that E . coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis.

J Bacteriol, 2004 Apr, 186(7), 2123 - 33
Plasmid evolution and interaction between the plasmid addiction stability systems of two related broad-host-range IncQ-like plasmids; Deane SM et al.; Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin . This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC . As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmids are likely to encounter each other . We investigated the relative efficiencies of the two stability systems and whether they had evolved apart sufficiently for each pas to stabilize a plasmid in the presence of the other . The three-component pTF-FC2 pas was more efficient at stabilization of a heterologous tester plasmid than the two component pas of pTC-F14 in Escherichia coli host cells (+/- 92% and +/- 60% after 100 generations, respectively) . The PasA antidote of each pas was unable to neutralize the PasB toxin of the other plasmid . The pas proteins of each plasmid autoregulated their own expression as well as that of the pas of the other plasmid . The pas of pTF-FC2 was more effective at repressing the pas operon of pTC-F14 than the pas of pTC-F14 was able to repress itself or the pas of pTF-FC2 . This increased efficiency was not due to the PasC of pTF-FC2 . The effect of this stronger repression was that pTF-FC2 displaced pTC-F14 when the two plasmids were coresident in the same E . coli host cell . Plasmid curing resulted in the arrest of cell growth but did not cause cell death, and plasmid stability was not influenced by the E . coli mazEF genes.

J Bacteriol, 2004 Apr, 186(7), 2091 - 8
Protective role for H-NS protein in IS1 transposition; Rouquette C et al.; The transposase (InsAB') of the insertion element IS1 can create breaks in DNA that lead to induction of the SOS response . We have used the SOS response to InsAB' to screen for host mutations that affect InsAB' function and thus point to host functions that contribute to the IS1 transposition mechanism . Mutations in the hns gene, which codes for a DNA binding protein with wide-ranging effects on gene expression, abolish the InsAB'-induced SOS response . They also reduce transposition, whether by simple insertion or cointegrate formation, at least 100-fold compared with the frequency seen in hns+ cells . Examination of protein profiles revealed that in an hns-null mutant, InsAB' is undetectable under conditions where it constitutes the most abundant protein in hns+ cells . Likewise, brief labeling of the hns cells with {35S}methionine revealed very small amounts of InsAB', and this was undetectable after a short chase . Transcription from the promoters used to express insAB' was essentially unaltered in hns cells, as was the level of insAB' mRNA . A mutation in lon, but not in ftsH or clpP, restored InsAB' synthesis in the hns strain, and a mutation in ssrA partially restored it, implying that the absence of H-NS leads to a problem in completing translation of insAB' mRNA and/or degradation of nascent InsAB' protein.

J Bacteriol, 2004 Apr, 186(7), 2085 - 90
Effect of D-lactate on the physiological activity of the ArcB sensor kinase in Escherichia coli; Rodriguez C et al.; The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to the respiratory growth conditions . Under anoxic growth conditions ArcB autophosphorylates and transphosphorylates ArcA, which in turn represses or activates its target operons . The anaerobic metabolite D-lactate has been shown to stimulate the in vitro autophosphorylating activity of ArcB . In this study, the in vivo effect of D-lactate on the kinase activity of ArcB was assessed . The results demonstrate that D-lactate does not act as a direct signal for activation of ArcB, as previously proposed, but acts as a physiologically significant effector that amplifies ArcB kinase activity.

J Bacteriol, 2004 Apr, 186(7), 2068 - 73
Biochemical characterization of a dihydromethanopterin reductase involved in tetrahydromethanopterin biosynthesis in Methylobacterium extorquens AM1; Caccamo MA et al.; During growth on one-carbon (C1) compounds, the aerobic alpha-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H4MPT) derivative dephospho-H4MPT as a C1 carrier in addition to tetrahydrofolate . The enzymes involved in dephospho-H4MPT biosynthesis have not been identified in bacteria . In archaea, the final step in the proposed pathway of H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase . A gene encoding a dihydrofolate reductase homolog has previously been reported for M . extorquens and assigned as the putative H2MPT reductase gene (dmrA) . In the present work, we describe the biochemical characterization of H2MPT reductase (DmrA), which is encoded by dmrA . The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration . Purified DmrA catalyzed the NAD(P)H-dependent reduction of H2MPT with a specific activity of 2.8 micromol of NADPH oxidized per min per mg of protein at 30 degrees C and pH 5.3 . Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8 . While the existence of an H2MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea . Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase . This may be a consequence of different electron donors, NAD(P)H versus reduced F420, used, respectively, in bacteria and methanogenic archaea.

J Bacteriol, 2004 Apr, 186(7), 2061 - 7
A DNA adenine methyltransferase of Escherichia coli that is cell cycle regulated and essential for viability; Kossykh VG et al.; DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp . strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT . The yhdJ gene was cloned, and the enzyme was overexpressed and purified . Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT . This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for "cell cycle-regulated methyltransferase") . The CcrM DNA adenine methyltransferase is required for viability in E . coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans . The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off . Overexpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population . Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology . Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.

Artif Cells Blood Substit Immobil Biotechnol, 2004 Feb, 32(1), 67 - 75
Encapsulation of enzymes in liposomes: high encapsulation efficiency and control of substrate permeability; Chaize B et al.; Enzyme encapsulation into liposomes is a promising technique to stabilize and prevent them from denaturation and proteolysis . We demonstrate this using acetylcholinesterase which is the main target for pesticides . In order to achieve a reasonable encapsulation yield, we analyzed the parameters involved in each step of various encapsulation procedures . The only encapsulation method which did not denature the protein was the lipid film hydration technique, however the encapsulation efficiency was usually low . The efficiency could be increased up to more than 40% by induction of a specific interaction between the enzyme and the lipid surface . Once encapsulated, the enzyme encountered another problem: the permeability barrier of the lipid membrane drastically diminished the activity of the enzyme entrapped in the liposome by reducing the entrance rate of the substrate molecules and then reducing the substrate concentration inside the liposome . To solve this problem, we controlled the permeability of the liposome wall by reconstituting a porin from Escherichia coli . We succeeded to recover the full functionality of the enzyme, while retaining the protection against denaturation and proteolytic enzymes.

Genetika, 2004 Jan, 40(1), 15 - 25
{Structural-functional analysis of the promoter region of Escherichia coli udp gene}; Ovcharova IV et al.; Effect of mutations in the -10 and -35 regions of the udp gene promoter on the nature of its regulation by CytR and CRP proteins was studied . In studies of expression of mutant promoters, competition between RNA polymerase and the CytR repressor for the promoter region of the udp gene was shown . In the presence of the improved -10 region, the introduction of a substitution 15C-->T (that is the presence of the elongated Pribnow block) resulted in the CRP-independent transcription of the udp gene promoter . The binding site CRP2 was shown to be indispensable for the maximum promoter activation by the transcription-activating cAMP-CRP complex . Both positive (cAMP-CRP complex) and negative (CytR) regulation of the promoter was virtually fully abolished after the introduction of mutations leading to the creation of canonical sequences in -10 and -35 promoter regions.

Arch Insect Biochem Physiol, 2004 Apr, 55(4), 200 - 14
Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis; Brandt KS et al.; Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification . Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors . The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids . The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin . Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences . RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes . Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems . Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells .

J Mol Recognit, 2004 Mar-Apr, 17(2), 85 - 94
The substrate recognition mechanisms in chaperonins; Gomez-Puertas P et al.; Chaperonins are a family of proteins devoted to assisting the folding of other proteins . They are large oligomers assembled into ring structures that enclose a cavity in which folding takes place . For this process to occur, the chaperonin must first recognize and interact with the unfolded polypeptide, then undergo a conformational change upon nucleotide binding that results in the closure of the cavity which in turn mediates the folding reaction inside the cavity . Although this general mechanism seems to apply to every chaperonin studied so far, there exist two different modes of interaction between the chaperonin and the substrate . The first occurs mainly through the interaction between the exposed hydrophobic residues of the unfolded polypeptides and those of the chaperonin substrate binding site, as elucidated for the chaperonin GroEL from E . coli . The second type of mechanism has been described so far only for the cytosolic chaperonin CCT (Chaperonin Containing TCP-1) and here the interaction seems to be of a more specific nature, involving charged and polar residues in both the chaperonin and the substrate, which interacts with CCT in a structured, quasi-native conformation .

J Gene Med, 2004 Mar, 6(3), 328 - 36
Gene transfection into fetal sheep airways in utero using guanidinium-cholesterol cationic lipids; Luton D et al.; BACKGROUND: Over the last several years, we have developed a novel class of cationic lipids, cholesterol derivatives characterized by polar head groups with guanidinium functions . We have in particular shown that bis(guanidinium)-tren-cholesterol/dioleoylphosphatidylethanolamine (BGTC/DOPE) cationic liposomes can mediate efficient gene transfection into the mouse airways in vivo via direct intratracheal administration or intranasal instillation . As prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we have explored in the present work the feasibility of BGTC-mediated gene transfection into the respiratory tract of fetal sheep in utero . METHODS: Thus, BGTC/DOPE liposomes were complexed with plasmids expressing the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene and the resulting lipoplexes were administered to fetal sheep at 70 days of gestation via surgical replacement of the airway fluid by the transfection mixture followed by tracheal occlusion . The fetal lungs and tracheas were harvested at 72 h and examined for CAT expression and evidence of toxicity . RESULTS: CAT expression was detected in both lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked plasmid DNA . Immunohistochemical analysis showed that airway epithelial cells and some mesenchymal cells were transfected . Pulmonary histopathology of varied severity was however observed under our transfection conditions and manifested as focal epithelial and mesenchymal lesions . CONCLUSIONS: These results show that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium . They also invite the development of optimized BGTC-based formulations and administration conditions with a view to future prenatal gene transfer experiments involving therapeutic genes .

J Biol Chem, 2004 May 28, 279(22), 22848 - 56 Epub 2004 Mar 16.
Identification of a novel rat microsomal vitamin D3 25-hydroxylase; Yamasaki T et al.; Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity . Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation . Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases . However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates . In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes . The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively . The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450 . The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3 . CYP2J3 was heterologously expressed in Escherichia coli . Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases . Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism . These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.

J Biol Chem, 2004 Jun 18, 279(25), 26417 - 24 Epub 2004 Mar 16.
Initial events in the photocycle of photoactive yellow protein; Kort R et al.; The light-induced isomerization of a double bond is the key event that allows the conversion of light energy into a structural change in photoactive proteins for many light-mediated biological processes, such as vision, photosynthesis, photomorphogenesis, and photo movement . Cofactors such as retinals, linear tetrapyrroles, and 4-hydroxy-cinnamic acid have been selected by nature that provide the essential double bond to transduce the light signal into a conformational change and eventually, a physiological response . Here we report the first events after light excitation of the latter chromophore, containing a single ethylene double bond, in a low temperature crystallographic study of the photoactive yellow protein . We measured experimental phases to overcome possible model bias, corrected for minimized radiation damage, and measured absorption spectra of crystals to analyze the photoproducts formed . The data show a mechanism for the light activation of photoactive yellow protein, where the energy to drive the remainder of the conformational changes is stored in a slightly strained but fully cis-chromophore configuration . In addition, our data indicate a role for backbone rearrangements during the very early structural events.

Cancer Res, 2004 Mar 15, 64(6), 2062 - 9
Evidence of antiangiogenic and antimetastatic activities of the recombinant disintegrin domain of metargidin; Trochon-Joseph V et al.; Metargidin, a transmembrane protein of the adamalysin family, and integrins, e.g., alpha5beta1 and alphav, are preferentially expressed on endothelial cells on angiogenesis . Furthermore, metargidin interacts with these integrins via its disintegrin domain . In this study, recombinant human disintegrin domain (RDD) was produced in Escherichia coli by subcloning its cDNA into the pGEX-2T vector, and the effect of purified RDD on different steps of angiogenesis was evaluated . At concentrations of 2-10 micro g/ml, RDD exhibited inhibitory activities in a variety of in vitro functional assays, including endothelial cell proliferation and adhesion on the integrin substrates fibronectin, vitronectin, and fibrinogen . RDD (10 micro g/ml) totally abrogated endothelial cell migration and blocked most capillary formation in a three-dimensional fibrin gel . To test RDD efficacy in vivo, the RDD gene inserted into pBi vector containing a tetracycline-inducible promoter was electrotransferred into nude mouse muscle . RDD was successfully synthesized by muscle cells in vivo as shown by immunolabeling and Western blotting . In addition, 78% less MDA-MB-231 tumor growth, associated with strong inhibition of tumor angiogenesis, was observed in athymic mice bearing electrotransferred RDD . Moreover, in the presence of RDD, 74% fewer B16F10 melanoma lung metastases were found in C57BL/6 mice . Taken together, these results identified this RDD as a potent intrinsic inhibitor of angiogenesis, tumor growth, and metastasis, making it a promising tool for use in anticancer treatment.

Mol Cell Endocrinol, 2004 Feb 27, 215(1-2), 161 - 4
Putative F-G loop is involved in association with the membrane in P450scc (P450 11A1); Pikuleva IA; Cytochrome P450scc (P450 11A1) catalyzes the conversion of cholesterol to pregnenolone, the first step in overall steroid hormone biosynthesis in steroidogenic tissues . On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F-G loop of P450 11A1 and mutated amino acid residues in this region . Wild type and mutant P450s 11A1 were expressed in E . coli and compared with respect to subcellular distribution and turnover number . About 30% of the wild type P450 was found in the bacterial cytosol indicating loose association of the enzyme with the membrane . The N210S/V211M and L218R/F219Y replacements increased the fraction of P450 in the bacterial cytosol 1.5-1.7-fold and the latter mutant also showed an almost two-fold increase of the turnover number . These data indicate that the putative F-G loop is the site of attachment to the membrane in P450 11A1 and changes in the enzyme-membrane interactions may affect the rate of catalysis.

Mol Cell Endocrinol, 2004 Feb 27, 215(1-2), 143 - 8
Characterization of the adrenal-specific antigen IZA (inner zone antigen) and its role in the steroidogenesis; Min L et al.; Inner zone antigen (IZA) is a protein specifically expressed in the zona fasciculata and reticularis of the adrenal cortex . The cDNA encoding IZA was found to be identical to that encoding the previously reported putative membrane-associated progesterone receptor (MPR) and the TCDD-induced 25kDa protein (25-Dx) . From its structure, MPR was classed as a member of a protein family containing a haem-binding domain, and progesterone was proposed to be a ligand of this domain . Indeed, when GST-tagged IZA was expressed in Escherichia coli and purified, the purified GST-IZA had a brown colour with maximum absorbance at 400 nm . The addition of dithionate shifted the absorbance peak to 420 nm, suggesting a haem-binding function . The possible role of IZA in steroidogenesis has been addressed, and the inhibition of adrenal steroidogenesis by the addition of an anti-IZA monoclonal antibody has been reported . When COS-7 cells were transformed with plasmids for appropriate steroidogenic enzymes in the presence or absence of an IZA expression plasmid and tested for their steroidogenic activities, 21-hydroxylation of progesterone was found to be specifically activated by IZA overexpression, suggesting the involvement of IZA in progesterone metabolism . Taken together, the available evidence suggests that IZA may have an important role in the functions of the adrenal zona fasciculata and reticularis.

Biochim Biophys Acta, 2004 Mar 17, 1671(1-3), 79 - 86
Effect of secretagogues and pH on intestinal transport in guanylin-deficient mice; Charney AN et al.; The small and large intestine secrete guanylin, a peptide homologous to heat stable enterotoxin (STa) elaborated by enterotoxigenic Escherichia coli . Guanylin's role in intestinal electrolyte transport was investigated in guanylin-deficient knockout mice and heterozygous littermate controls . Segments of mid-jejunum, distal ileum, and proximal and distal colon were studied in Ussing chambers in HCO3- Ringer under short circuit conditions . We found that (1) under basal conditions, all segments in control and knockout mice absorb Na+, and the knockout mouse proximal colon secretes Cl-; (2) all segments except the jejunum of knockout mice respond by increasing absorption in response to reductions in pH from 7.6 to 7.1; (3) all segments exhibit decreased absorption in response to 1 mM cAMP; (4) the jejunum and ileum of knockout and control mice, and the proximal colon of control mice (but not knockout mice) respond to the mucosal addition of 50 nM STa with decreases in absorption; and (5) mucosal guanylin caused similar decreases in proximal colon absorption in control and guanylin-deficient mice . These findings suggest that guanylin deficiency causes basal Cl- secretion and reduced responsiveness to STa in mouse proximal colon . The effectiveness of guanylin in this segment suggests a difference in the intestinal secretory actions of STa and guanylin.

Bioorg Med Chem Lett, 2004 Apr 5, 14(7), 1633 - 6
Biosynthesis of vitamin B6: direct identification of the product of the PdxA-catalyzed oxidation of 4-hydroxy-l-threonine-4-phosphate using electrospray ionization mass spectrometry; Banks J et al.; PdxA (E.C . 1.1.1.262) catalyzes a key step in the biosynthesis of vitamin B(6): the nicotinamide-dependent oxidation of 4-hydroxy-l-threonine-4-phosphate (HTP) to a product tentatively identified as 3-amino-1-hydroxyacetone 1-phosphate (AHAP) . To date, the evidence for the formation of AHAP, while self-consistent, has been largely circumstantial, and does not exclude the possibility that the actual product of the enzyme-catalyzed oxidation of HTP might be 2-amino-3-oxo-4-hydroxybutyric acid 4-phosphate which could undergo rapid, non-enzyme-catalyzed decarboxylation once released from the protein . Use of negative ion electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometric analysis (MS-MS) confirms that AHAP is the product of the PdxA-catalyzed reaction.

Biologicals, 2004 Mar, 32(1), 37 - 47
Human serum inactivates non-glycosylated but not glycosylated granulocyte colony stimulating factor by a protease dependent mechanism: significance of carbohydrates on the glycosylated molecule; Carter CR et al.; It has previously been reported that the biological activity of the human hematopoetic cytokine granulocyte colony stimulating factor (G-CSF) was reduced following incubation with human serum . The mechanism of action of serum has remained elusive although a number of possible mechanisms have been suggested including inactivation due to binding to the serum protein alpha(2)-macroglobulin (alpha(2)M) and degradation by serum proteases . The aim of this study was to clarify the conditions required by serum to reduce the biological activity of the cytokine and to define the mechanism involved . It has also been noted that G-CSF obtained from a CHO expression system (and therefore considered a glycosylated molecule) was resistant to serum inactivation unlike G-CSF obtained from an E . coli expression system (considered to be non-glycosylated) . We used an enzymatic approach to remove the carbohydrate residues from glycosylated G-CSF and tested this material for its stability in serum . We additionally used a mutated G-CSF lacking glycosylation sites . We concluded that glycosylation was important in protecting against serum inactivation . We observed that serum reduced the biological activity of non-glycosylated G-CSF in a dose, and temperature dependent manner and deduced that the mechanism of action was dependent upon alpha(2)M bound serum protease enzymes.

J Endotoxin Res, 2004, 10(1), 25 - 31
Characteristic biological activities of lipopolysaccharides from Sinorhizobium and Mesorhizobium; Tsukushi Y et al.; The biological actions of lipopolysaccharides (LPSs) from Sinorhizobium meliloti, Mesorhizobium loti and Escherichia coli were compared . In biological activities including lethality, production of tumor necrosis factor (TNF)-alpha and nitric oxide (NO), adjuvant action and Limulus activity, LPS from S . meliloti exhibited stronger actions than LPS from M . loti, but had a weaker action than LPS from E . coli . On the other hand, M . loti LPS showed a higher activity to activate human complement than S . meliloti LPS . Further, there was a significant difference in polymyxin B binding between S . meliloti LPS and M . loti LPS, suggesting a difference in the lipid A structure . LPSs from S . meliloti and M . loti seem to exhibit characteristic biological actions that may be dependent on the difference in the lipid A structure.

Hum Mutat . 2004 Apr;23(4):396.
Biochemical characterization of two GALK1 mutations in patients with galactokinase deficiency; Sangiuolo F et al.; Galactokinase (GALK1) deficiency is an autosomal recessive disorder, which causes cataract formation in children not maintained on a lactose-free diet . Galactokinase deficiency results from mutation in the GALK1 gene mapped on 17q24 . Since GK1 cDNA was cloned about 20 mutations (prevalently deletions and missense) have been reported to date . Most of these reported mutations are confined to single families, and only one of them, P28T, has been referred as the founder Romani mutation . In this paper we report two novel missense mutations in GALK1 gene, identified in two unrelated patients with galactokinase deficiency . One mutation, g.575G>A, substitutes a valine for a methionine at amino acid 32 (p.V32M), while the other mutation, g.2839G>A, results in the arginine to glutamine substitution p.R239Q (GenBank sequence L76927) . Biochemical studies demonstrate that these mutations led to a drastic modification in GALK activity when individual mutant cDNAs were expressed in an E . coli system . These findings indicate the pathogeneticity of these mutations causing GALK deficiency .

Eur J Pediatr Surg, 2004 Feb, 14(1), 70 - 2
Tubo-ovarian abscess in a sexually inactive adolescent patient; Arda IS et al.; Tubo-ovarian abscess as a complication of acute salpingitis or salpingo-oophoritis is very uncommon in pre-menarchal and/or sexually inactive girls . It is generally the result of a blood-borne or genitourinary infection . Early diagnosis and treatment are essential to prevent future sequelae causing infertility . Laparoscopic surgery which minimises postoperative complications should be the first option in the treatment of TOA.

Curr Genet, 2004 Jun, 45(6), 371 - 7 Epub 2004 Mar 13.
A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides; Velayos A et al.; Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised . The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases . By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity . In addition, a M . circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.

Intensive Care Med, 2004 Aug, 30(8), 1652 - 9 Epub 2004 Mar 13.
Effects of the angiotensin-converting enzyme inhibitor perindopril on endothelial injury and hemostasis in rabbit endotoxic shock; Wiel E et al.; OBJECTIVE: To assess the effects of the angiotensin-converting enzyme (ACE) inhibitor (ACEI) perindopril on prolonged endothelial cell dysfunction in a rabbit endotoxic model . DESIGN: Randomized, controlled, interventional trial . SETTING: University animal laboratory . SUBJECTS: A total of 65 male New Zealand White rabbits, randomly assigned to one of eight groups . INTERVENTIONS: Endotoxic shock was induced by a single lipopolysaccharide (LPS, serotype O55:B5) bolus (0.5 mg.kg(-1), i.v., Escherichia coli endotoxin) . Coagulation factors and expression of monocyte tissue factor (TF) were determined by functional assay . Endothelium-dependent vascular relaxation was assessed by in vitro vascular reactivity . Immunohistochemical staining (CD31) was performed to assess endothelial injury of the abdominal aorta . These parameters were studied 5 days (D5) after the onset of endotoxic shock . Rabbits were randomized to receive perindopril (1 mg kg(-1) day(-1) orally) alone, or with N(G)-nitro-L-arginine methyl ester (L-NAME; 15 mg kg(-1) day(-1) orally), or L-NAME alone initiated 7 days before the onset of endotoxic shock and maintained for 5 days afterward . MEASUREMENTS AND RESULTS: Perindopril prevented altered endothelium-dependent relaxation to acetylcholine induced by LPS injection (E(max)=75.6+/-3.7 vs 42.3+/-9.4% in LPS group, p<0.05) . This effect was inhibited by co-treatment with L-NAME . Perindopril had no effect on either LPS-induced endothelial histological injury or monocyte TF expression . CONCLUSION: These data suggest that perindopril can prevent endothelial dysfunction in endotoxin-induced shock through an NO-dependent mechanism.

Proc Natl Acad Sci U S A, 2004 Mar 23, 101(12), 4024 - 9 Epub 2004 Mar 15.
CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata; Helvig C et al.; The molecular analysis of insect hormone biosynthesis has long been hampered by the minute size of the endocrine glands producing them . Expressed sequence tags from the corpora allata of the cockroach Diploptera punctata yielded a new cytochrome P450, CYP15A1 . Its full-length cDNA encoded a 493-aa protein that has only 34% amino acid identity with CYP4C7, a terpenoid omega-hydroxylase previously cloned from this tissue . Heterologous expression of the cDNA in Escherichia coli produced >300 nmol of CYP15A1 per liter of culture . After purification, its catalytic activity was reconstituted by using phospholipids and house fly P450 reductase . CYP15A1 metabolizes methyl (2E,6E)-3,7,11-trimethyl-2,6-dodecatrienoate (methyl farnesoate) to methyl (2E,6E)-(10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate {juvenile hormone III, JH III} with a turnover of 3-5 nmol/min/nmol P450 . The enzyme produces JH III with a ratio of approximately 98:2 in favor of the natural (10R)-epoxide enantiomer . This result is in contrast to other insect P450s, such as CYP6A1, that epoxidize methyl farnesoate with lower regio- and stereoselectivity . RT-PCR experiments show that the CYP15A1 gene is expressed selectively in the corpora allata of D . punctata, at the time of maximal JH production by the glands . We thus report the cloning and functional expression of a gene involved in an insect-specific step of juvenile hormone biosynthesis . Heterologously expressed CYP15A1 from D . punctata or its ortholog from economically important species may be useful in the design and screening of selective insect control agents.

Proc Natl Acad Sci U S A, 2004 Mar 23, 101(12), 4071 - 6 Epub 2004 Mar 15.
Analytic models for mechanotransduction: gating a mechanosensitive channel; Wiggins P et al.; Analytic estimates for the forces and free energy generated by bilayer deformation reveal a compelling and intuitive model for MscL channel gating analogous to the nucleation of a second phase . We argue that the competition between hydrophobic mismatch and tension results in a surprisingly rich story that can provide both a quantitative comparison with measurements of opening tension for MscL when reconstituted in bilayers of different thickness, and qualitative insights into the function of the MscL channel and other transmembrane proteins.

J Biol Chem, 2004 May 14, 279(20), 20678 - 84 Epub 2004 Mar 15.
Interference of mRNA function by sequence-specific endoribonuclease PemK; Zhang J et al.; In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin . The pemI-pemK system is an addiction module present on plasmid R100 . It helps to maintain the plasmid by post-segregational killing in E . coli population . Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E . coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis . Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK . PemK cleaves only single-stranded RNA preferentially at the 5' or 3' side of the A residue in the "UAH" sequences (where H is C, A, or U) . Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E . coli . The pemK homologue genes have been identified on the genomes of a wide range of bacteria . We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.

J Biol Chem, 2004 Jun 4, 279(23), 23933 - 41 Epub 2004 Mar 15.
Isolation and characterization of the early nodule-specific protein homologue (Hev b 13), an allergenic lipolytic esterase from Hevea brasiliensis latex; Arif SA et al.; Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis latex appeared to have been resolved with the discovery of a 43-kDa allergenic latex protein that was a homologue to patatin . However, the low to moderate prevalence of sensitization to the protein, designated Hev b 7, among latex-allergic patients could not adequately explain the frequent observations of the 42-46-kDa allergen . This led to the hypothesis that another, more allergenic protein of a similar molecular mass existed in Hevea latex . We report the isolation and purification of a 42.98-kDa latex glycoprotein showing homology to the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago truncatula, and Glycine max . The protein is allergenic, being recognized by immunoglobulin E (IgE) in sera from latex-allergic patients . The IgE epitope resides on the carbohydrate moiety of the protein, and the presence of a similar carbohydrate component on potato tuber patatin enables the latter to inhibit IgE binding to the ENSP homologue . The cDNA encoding the ENSP homologue was isolated by reverse transcription-PCR and cloned . The protein predicted from the cDNA sequence has 391 amino acids, the first 26 of which constitute a putative signal peptide . The deduced molecular mass of the mature protein is 40.40 kDa, while its isoelectric point is estimated at 5.0 . The discrepancy between the predicted and observed molecular mass might be due to glycosylation, for which three N-sites on the protein are predicted . The purified protein showed lipase and esterase activities and may be involved in plant defense.

J Biol Chem, 2004 May 21, 279(21), 22548 - 57 Epub 2004 Mar 15.
Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol; Ravanel S et al.; The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles . In the present work we show that Arabidopsis contains three functional isoforms of vitamin B(12)-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor . One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment . Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells . The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle . Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol . In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process . This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine . The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities . Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.

J Biol Chem, 2004 May 21, 279(21), 21759 - 65 Epub 2004 Mar 15.
Human 1-D-myo-inositol-3-phosphate synthase is functional in yeast; Ju S et al.; We have cloned, sequenced, and expressed a human cDNA encoding 1-d-myo-inositol-3-phosphate (MIP) synthase (hINO1) . The encoded 62-kDa human enzyme converted d-glucose 6-phosphate to 1-d-myo-inositol 3-phosphate, the rate-limiting step for de novo inositol biosynthesis . Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37 degrees C and exhibited K(m) values of 0.57 mm and 8 microm for glucose 6-phosphate and NAD(+), respectively . NH(4)(+) and K(+) were better activators than other cations tested (Na(+), Li(+), Mg(2+), Mn(2+)), and Zn(2+) strongly inhibited activity . Expression of the protein in the yeast ino1Delta mutant lacking MIP synthase (ino1Delta/hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion . MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Delta/hINO1 was grown in the presence of a therapeutically relevant concentration of the anti-bipolar drug valproate (0.6 mm) . However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect . Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.

J Biol Chem, 2004 May 7, 279(19), 20529 - 38 Epub 2004 Mar 15.
Activation of the redox-regulated chaperone Hsp33 by domain unfolding; Graf PC et al.; The molecular chaperone Hsp33 in Escherichia coli responds to oxidative stress conditions with the rapid activation of its chaperone function . On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer . While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown . To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33 . These proteins were studied by a combination of biochemical and NMR spectroscopic techniques . We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, alpha-helical structure . This appears to conceal both the substrate-binding site as well as the dimerization interface . Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33 . This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric . Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers . This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress.

Biochim Biophys Acta, 2004 Mar 11, 1697(1-2), 271 - 8
Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase; Delbaere LT et al.; Phosphoenolpyruvate carboxykinase (PCK) catalyzes the conversion of oxaloacetate (OAA) to PEP and carbon dioxide with the subsequent conversion of nucleoside triphosphate to nucleoside diphosphate (NDP) . The 1.9 A resolution structure of Escherichia coli PCK consisted of a 275-residue N-terminal domain and a 265-residue C-terminal domain with the active site located in a cleft between these domains . Each domain has an alpha/beta topology and the overall structure represents a new protein fold . Furthermore, PCK has a unique mononucleotide-binding fold . The 1.8 A resolution structure of the complex of ATP/Mg(2+)/oxalate with PCK revealed a 20 degrees hinge-like rotation of the N- and C-terminal domains, which closed the active site cleft . The ATP was found in the unusual syn conformation as a result of binding to the enzyme . Along with the side chain of Lys254, Mg(2+) neutralizes charges on the P beta and P gamma oxygen atoms of ATP and stabilizes an extended, eclipsed conformation of the P beta and P gamma phosphoryl groups . The sterically strained high-energy conformation likely lowers the free energy of activation for phosphoryl transfer . Additionally, the gamma-phosphoryl group becomes oriented in-line with the appropriate enolate oxygen atom, which strongly supports a direct S(N)2-type displacement of this gamma-phosphoryl group by the enolate anion . In the 2.0 A resolution structure of the complex of PCK/ADP/Mg(2+)/AlF(3), the AlF(3) moiety represents the phosphoryl group being transferred during catalysis . There are three positively charged groups that interact with the fluorine atoms, which are complementary to the three negative charges that would occur for an associative transition state.

Biochemistry, 2004 Mar 23, 43(11), 3273 - 9
Regulatory role of the C-terminus of the epsilon subunit from the chloroplast ATP synthase; Nowak KF et al.; The ATP synthases from chloroplasts and Escherichia coli are regulated by several factors, one of which is the epsilon subunit . This small subunit is also required for ATP synthesis . Thylakoid membranes reconstituted with CF1 lacking the epsilon subunit (CF1-epsilon) exhibit no ATP synthesis and very high ATP hydrolysis . Either native or recombinant epsilon restores ATP synthesis and inhibits ATP hydrolysis . Previously, we showed that truncated epsilon, lacking the last 45 C-terminal amino acids, restored ATP synthesis to membranes reconstituted with CF1-epsilon but was not an efficient inhibitor of ATP hydrolysis . In this paper, we show that this truncated epsilon is unable to inhibit ATP hydrolysis when Mg(2+) is the divalent cation present, both for the enzyme in solution and on the thylakoid membrane . In addition, the rate of reduction of the disulfide bond of the gamma subunit by dithiothreitol is not decreased by truncated epsilon, although full-length epsilon greatly impedes reduction . Thylakoid membranes can synthesize ATP at the expense of proton gradients generated by pH transitions in the dark . Our reconstituted membranes are able to produce a limited amount of ATP under these "acid-bath" conditions, with approximately equal amounts produced by the membranes containing wild-type epsilon and those containing truncated epsilon . However, the membranes containing truncated epsilon exhibit much higher background ATP hydrolysis under the same acid-bath conditions, leading to the conclusion that, without the C-terminus of epsilon, the CF1CFo is unable to check unwanted ATP hydrolysis.

Biochemistry, 2004 Mar 23, 43(11), 3214 - 21
In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA; Hirao I et al.; Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation . To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool . Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain . Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E . coli 16S rRNA . The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence . The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.

Biochemistry, 2004 Mar 23, 43(11), 3111 - 9
Structure of the functional fragment of auxilin required for catalytic uncoating of clathrin-coated vesicles; Gruschus JM et al.; The three-dimensional structure of the C-terminal 20 kDa portion of auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR . Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process . This 20 kDa auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically . The tertiary structure consists of six helices, where the first three are unique to auxilin and believed to be important in the catalytic uncoating of clathrin . The last three helices correspond to the canonical J-domain of Hsp40 proteins . The first helix, helix 1, which contains a conserved FEDLL motif believed to be necessary for clathrin binding, is transient and not packed against the rest of the structure . Helix 1 is joined to helix 2 by a flexible linker . Helix 2 packs loosely against the J-domain surface, whereas helix 3 packs tightly and makes critical contributions to the J-domain core . A long insert loop, also unique to the auxilin J-domain, is seen between helix 4 and helix 5 . Comparison with a previously reported structure of auxilin containing only helices 3-6 shows a significant difference in the invariant HPD segment of the J-domain . The region where helix 1 is located corresponds to the expected region of the unstructured G/F-rich domain seen in DnaJ, i.e., the canonical N-terminal J-domain protein . In contrast, the location of helix 1 differs from the substrate binding regions of two other Hsp40 proteins, Escherichia coli Hsc20 and viral large T antigen . The variety of biological functions performed by Hsp40 proteins such as auxilin, as well as the observed differences in the structure and function of their substrate binding regions, supports the notion that Hsp40 proteins act as target-specific adaptors that recruit their more general Hsp70 partners to specific biological roles.

Arch Pharm Res, 2004 Feb, 27(2), 199 - 205
Potent inhibition of human cytochrome P450 1 enzymes by dimethoxyphenylvinyl thiophene; Lee SK et al.; Cytochrome P450 (P450) 1 enzymes such as P450 1A1, 1A2, and 1B1 are known to be involved in the oxidative metabolism of various procarcinogens and are regarded as important target enzymes for cancer chemoprevention . Previously, several hydroxystilbene compounds were reported to inhibit P450 1 enzymes and were rated as candidate chemopreventive agents . In this study, we investigated the inhibitory effect of 2-{2-(3,5-dimethoxyphenyl)vinyl}-thiophene (DMPVT), produced from the chemical modification of oxyresveratrol, on the activities of P450 1 enzymes . The inhibitory potential by DMPVT on the P450 1 enzyme activity was evaluated with the Escherichia coli membranes of the recombinant human cytochrome P450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P450 reductase . DMPVT significantly inhibited ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 61, 11, and 2 nM for 1A1, 1A2, and 1B1, respectively . The EROD activity in DMBA-treated rat lung microsomes was also significantly inhibited by DMPVT in a dose-dependent manner . The modes of inhibition by DMPVT were non-competitive for all three P450 enzymes . The inhibition of P450 1B1-mediated EROD activity by DMPVT did not show the irreversible mechanism-based effect . The loss of EROD activity in P450 1B1 with DMPVT incubation was not blocked by treatment with the trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol . Taken together, the results suggested DMPVT to be a strong noncompetitive inhibitor of human P450 1 enzymes that should be considered as a good candidate for a cancer chemopreventive agent in humans.

Methods Mol Biol, 2004, 260, 83 - 96
Tn5 as a molecular genetics tool: In vitro transposition and the coupling of in vitro technologies with in vivo transposition; Reznikoff WS et al.; The development of in vitro transposition technologies have provided many powerful tools for the molecular genetics research laboratory . In this chapter we describe some of these tools with a focus on the Tn5 transposition system . Tn5 technologies are particularly useful because the Tn5 transposition system has simple requirements, is efficient, random in target recognition, and robust . In particular we will describe the use of in vitro Tn5 transposition in transposon tagging and in the generation of nested deletions . We will also describe a unique in vitro/in vivo technology in which Tn5 inserts can be generated in a wide spectrum of bacterial species through the electroporation of preformed tranposase-transposon DNA complexes.

J Cell Sci, 2004 Mar 15, 117(Pt 8), 1553 - 66
CRM1 and Ran are present but a NES-CRM1-RanGTP complex is not required in Balbiani ring mRNP particles from the gene to the cytoplasm; Zhao J et al.; Messenger RNA is formed from precursors known as pre-mRNA . These precursors associate with proteins to form pre-mRNA-protein (pre-mRNP) complexes . Processing machines cap, splice and polyadenylate the pre-mRNP and in this way build the mRNP . These processing machines also affect the export of the mRNP complexes from the nucleus to the cytoplasm . Export to the cytoplasm takes place through a structure in the nuclear membrane called the nuclear pore complex (NPC) . Export involves adapter proteins in the mRNP and receptor proteins that bind to the adapter proteins and to components of the NPC . We show that the export receptor chromosomal region maintenance protein 1 (CRM1), belonging to a family of proteins known as importin-beta-like proteins, binds to gene-specific Balbiani ring (BR) pre-mRNP while transcription takes place . We also show that the GTPase known as Ran binds to BR pre-mRNP, and that it binds mainly in the interchromatin . However, we also show using leptomycin B treatment that a NES-CRM1-RanGTP complex is not essential for export, even though both CRM1 and Ran accompany the BR mRNP through the NPC . Our results therefore suggest that several export receptors associate with BR mRNP and that these receptors have redundant functions in the nuclear export of BR mRNP.

J Cell Sci, 2004 Mar 15, 117(Pt 8), 1547 - 52
The EBP50-moesin interaction involves a binding site regulated by direct masking on the FERM domain; Finnerty CM et al.; Members of the ezrin-radixin-moesin (ERM) protein family serve as regulated microfilament-membrane crosslinking proteins that, upon activation, bind the scaffolding protein ERM-phosphoprotein of 50 kDa (EBP50) . Here we report a 3.5 A resolution diffraction analysis of a complex between the active moesin N-terminal FERM domain and a 38 residue peptide from the C terminus of EBP50 . This crystallographic result, combined with sequence and structural comparisons, suggests that the C-terminal 11 residues of EBP50 binds as an alpha-helix at the same site occupied in the dormant monomer by the last 11 residues of the inhibitory moesin C-terminal tail . Biochemical support for this interpretation derives from in vitro studies showing that appropriate mutations in both the EBP50 tail peptide and the FERM domain reduce binding, and that a peptide representing just the C-terminal 14 residues of EBP50 also binds to moesin . Combined with the recent identification of the I-CAM-2 binding site on the ERM FERM domain (Hamada, K., Shimizu, T., Yonemura, S., Tsukita, S., and Hakoshima, T . (2003) EMBO J . 22, 502-514), this study reveals that the FERM domain contains two distinct binding sites for membrane-associated proteins . The contribution of each ligand to ERM function can now be dissected by making structure-based mutations that specifically affect the binding of each ligand.

Genetics, 2004 Feb, 166(2), 681 - 92
Distinct signatures for mutator sensitivity of lacZ reversions and for the spectrum of lacI/lacO forward mutations on the chromosome of nondividing Escherichia coli; Bharatan SM et al.; A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature . Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells . The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5 . In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate . Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5 . Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants . This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state.

Ann Rheum Dis, 2004 Apr, 63(4), 386 - 94
Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity; Haugbro K et al.; BACKGROUND: The presence of "anti-DNA antibodies in abnormal titres" is a well established criterion for SLE classification, but there is no agreement on the performance of this test . OBJECTIVE: To study the correlation between clinical findings and five different solid and solution phase anti-DNA antibody assays . METHODS: 158 consecutively collected ANA positive sera were studied in a double blind fashion . Anti-DNA antibodies were determined by different solid phase assays (ssDNA-, dsDNA- specific ELISA, EliA anti-dsDNA assay, Crithidia luciliae assay), and by an experimental solution phase anti-DNA assay using biotinylated pUC18 plasmid, human, calf thymus, and E coli DNA . Antibody affinity was determined by surface plasmon resonance . Clinical data were obtained independently of the laboratory analyses and later related to the anti-dsDNA findings . RESULTS: Anti-dsDNA antibodies were most frequently detected by ELISA, but were not specific for SLE as they were present in up to 30% of other disease groups . Those detected by the Crithidia luciliae assay were predictive for SLE, while antibodies binding in solution phase ELISA using the pUC18 correlated strongly with the Crithidia luciliae assay . Surface plasmon resonance analysis showed that antibody binding to pUC18 was not due to higher relative affinity for dsDNA in general, but apparently to specificity for that plasmid DNA . Serum samples from three patients with lupus nephritis were positive in both pUC18 solution phase and Crithidia luciliae assays . CONCLUSIONS: Assay principle selection is decisive for the detection of clinically significant anti-DNA antibodies . Revision of the anti-DNA antibody criterion in the SLE classification may be needed.

Am J Respir Crit Care Med, 2004 Jul 15, 170(2), 126 - 32 Epub 2004 Mar 12.
The role of Toll-like receptor 4 in environmental airway injury in mice; Hollingsworth JW 2nd et al.; Inhalation of toxins commonly found in air pollution contributes to the development and progression of asthma and environmental airway injury . In this study, we investigated the requirement of toll-like receptor 4 (TLR4) in mice for pulmonary responses to three environmental toxins: aerosolized lipopolysaccharide, particulate matter (residual oil fly ash), and ozone . The physiologic and biologic responses to these toxins were evaluated by the extent of airway responsiveness, neutrophil recruitment to the lower respiratory tract, changes in inflammatory cytokines, and the concentration of protein in the lavage fluid . Genetically engineered, TLR4-deficient mice (C57BL/6(TLR4-/-)) were unresponsive to inhaled lipopolysaccharide, except for minimal increases in some inflammatory cytokines . In contrast, C57BL/6(TLR4-/-) mice did not differ from wild-type mice in their airway response to instilled residual oil fly ash or acute ozone exposure; however, we found that, despite a robust inflammatory response, C57BL/6(TLR4-/-) mice are protected against the development of airway hyperresponsiveness after subchronic ozone exposure . These data demonstrate in the mouse that the requirement of TLR4 for pulmonary inflammation depends on the nature of the toxin and appears specific to toxin and exposure conditions.

Biochem Biophys Res Commun, 2004 Apr 2, 316(2), 540 - 4
Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli; Bhattacharya J et al.; The superoxide dismutase (SOD) gene (slr 1516) from the cyanobacterium Synechocystis sp . PCC 6803 was cloned and overexpressed in Escherichia coli BL 21 (DE3) using the pET-20b(+) expression vector . E . coli cells transformed with pET-SOD overexpressed the protein in cytosol, upon induction by isopropyl beta-D-thiogalactopyranoside (IPTG) . The recombinant protein was purified to near homogeneity by gel filtration and ion-exchange chromatography . The SOD activity of the recombinant protein was sensitive to hydrogen peroxide and sodium azide, confirming it to be FeSOD . The pET-FeSOD transformed E . coli showed significantly higher SOD activity and tolerance to paraquat-mediated growth inhibition compared to the empty vector transformed cells . Based on these results it is suggested that overexpression of FeSOD gene from a heterologous source like Synechocystis sp . PCC 6803 may provide protection to E . coli against superoxide radical-mediated oxidative stress mediated by paraquat.

Gene, 2004 Mar 17, 328, 95 - 102
AtPARN is an essential poly(A) ribonuclease in Arabidopsis; Chiba Y et al.; Deadenylation is the first and rate-limiting step in the degradation of many mRNAs in a wide-range of organisms from yeast to higher eukaryotes . It can also play a regulatory role in early development . In this study, we examined the Arabidopsis homolog of poly(A) ribonuclease (PARN), a deadenylase first identified in mammals and absent from yeast . Consistent with the conservation of domains and residues important for catalytic activity, Arabidopsis PARN (AtPARN) expressed in Escherichia coli has poly(A) degradation activity in vitro . Protein localization experiments in plant cells indicate that AtPARN resides in both the nucleus and cytoplasm . To address the importance of the enzyme in vivo, we identified three independent T-DNA insertion mutants of AtPARN which interrupt the gene at different positions between the ATG and the stop codon . All three alleles cause lethality prior to seed germination, indicating that AtPARN is an essential gene first required during early development . Although homologous genes have yet to be inactivated in any other organism, our observations argue for the critical importance of PARN and suggest that it may be essential in many other multicellular eukaryotes.

Free Radic Biol Med, 2004 Apr 1, 36(7), 911 - 8
Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli; Leinweber B et al.; Although large amounts of wild-type human Cu,Zn superoxide dismutase (SOD) are easily expressed in Escherichia coli, the amyotrophic lateral sclerosis-associated mutants have a strong propensity to aggregate into inclusion bodies . The alanine to valine mutation at the fourth codon (A4V) is responsible for a rapidly progressive disease course and is particularly prone to aggregation when expressed in E . coli . We found that A4V SOD remained soluble when expressed at 18 degrees C, but >95% A4V SOD aggregated in inclusion bodies when expressed at 23 degrees C or above . The SOD aggregates dissolved with 4 M urea, suggesting that intermolecular hydrophobic interactions were predominantly responsible for making SOD insoluble . Many of the urea-solubilized subunits were cross-linked via disulfide bridges . Fully active mutant SOD could be produced by dialyzing urea away in the presence of beta-mercaptoethanol and subsequently adding copper plus zinc, providing a fast procedure for purifying hundreds of milligrams of protein . Extensive rinsing removed most contaminating E . coli proteins from A4V SOD inclusion bodies except for a 37 kDa protein identified as outer membrane protein F using MALDI ToF/ToF mass spectrometry . Our results indicate that metal-deficient ALS-mutant SOD folds into stable apo conformation able to rebind metals . At high protein concentrations, SOD forms aggregates through hydrophobic interactions between subunits that seem to act as a kinetic snare to entrap additional proteins.

Arch Biochem Biophys, 2004 Apr 1, 424(1), 33 - 43
The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism; Liu J et al.; Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 --> Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography . In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities . Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue . Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models.

J Mol Biol, 2004 Mar 26, 337(3), 731 - 41
Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli; Zhao K et al.; Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved from bacteria to human, and have been implicated to play important roles in gene regulation, metabolism and longevity . cobB is a bacterial sirtuin that deacetylates acetyl-CoA synthetase (Acs) at an active site lysine to stimulate its enzymatic activity . Here, we report the structure of cobB bound to an acetyl-lysine containing non-cognate histone H4 substrate . A comparison with the previously reported archaeal and eukaryotic sirtuin structures reveals the greatest variability in a small zinc-binding domain implicated to play a particularly important role in substrate-specific binding by the sirtuin proteins . Comparison of the cobB/histone H4 complex with other sirtuin proteins in complex with acetyl-lysine containing substrates, further suggests that contacts to the acetyl-lysine side-chain and beta-sheet interactions with residues directly C-terminal to the acetyl-lysine represent conserved features of sirtuin-substrate recognition . Isothermal titration calorimetry studies were used to compare the affinity of cobB for a variety of cognate and non-cognate acetyl-lysine-bearing peptides revealing an exothermic reaction with relatively little discrimination between substrates . In contrast, similar studies employing intact acetylated Acs protein as a substrate reveal a binding reaction that is endothermic, suggesting that cobB recognition of substrate involves a burial of hydrophobic surface and/or structural rearrangement involving substrate regions distal to the acetyl-lysine-binding site . Together, these studies suggest that substrate-specific binding by sirtuin proteins involves contributions from the zinc-binding domain of the enzyme and substrate regions distal to the acetyl-lysine-binding site.

J Mol Biol, 2004 Mar 26, 337(3), 597 - 610
Nucleotide binding to DNA gyrase causes loss of DNA wrap; Heddle JG et al.; DNA gyrase negatively supercoils DNA in a reaction coupled to the binding and hydrolysis of ATP . Limited supercoiling can be achieved in the presence of the non-hydrolysable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (ADPNP) . In order to negatively supercoil DNA, gyrase must wrap a length of DNA around itself in a positive sense . In previous work, the effect of ADPNP on the gyrase-DNA interaction has been assessed but has produced conflicting results; the aim of this work was to resolve this conflict . We have probed the wrapping of DNA around gyrase in the presence and in the absence of ADPNP using direct observation by atomic force microscopy (AFM) . We confirm that gyrase indeed generates a significant curvature in DNA in the absence of nucleotide and we show that the addition of ADPNP leads to a complete loss of wrap . These results have been corroborated using a DNA relaxation assay involving topoisomerase I . We have re-analysed previous hydroxyl-radical footprinting and crystallography data, and highlight the fact that the gyrase-DNA complex is surprisingly asymmetric in the absence of nucleotide but is symmetric in the presence of ADPNP . We suggest a revised model for the conformation of DNA bound to the enzyme that is fully consistent with these AFM data, in which a closed loop of DNA is stabilised by the enzyme in the absence of ADPNP and is lost in the presence of nucleotide.

J Mol Biol, 2004 Mar 26, 337(3), 561 - 71
Histone-like protein HU from Deinococcus radiodurans binds preferentially to four-way DNA junctions; Ghosh S et al.; The histone-like protein HU from Escherichia coli is involved in DNA compaction and in processes such as DNA repair and recombination . Its participation in these events is reflected in its ability to bend DNA and in its preferred binding to DNA junctions and DNA with single-strand breaks . Deinococcus radiodurans is unique in its ability to reconstitute its genome from double strand breaks incurred after exposure to ionizing radiation . Using electrophoretic mobility shift assays (EMSA), we show that D.radiodurans HU (DrHU) binds preferentially only to DNA junctions, with half-maximal saturation of 18 nM . In distinct contrast to E.coli HU, DrHU does not exhibit a marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able to mediate circularization of linear duplex DNA . These unexpected properties identify DrHU as the first member of the HU protein family not to serve an architectural role and point to its potential participation in DNA recombination events . Our data also point to a mechanism whereby differential target site selection by HU proteins is achieved and suggest that the substrate specificity of HU proteins should be expected to vary as a consequence of their individual capacity for inducing the required DNA bend.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 101 - 5
Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase; White EL et al.; The gene for dihydrofolate reductase of Mycobacterium tuberculosis was amplified by polymerase chain reaction (PCR) from M . tuberculosis H37Rv strain genomic DNA . The protein was expressed in inclusion bodies in high yield in Escherichia coli under the control of the T7 promoter . Active enzyme was obtained by refolding from guanidine HCl and after a single chromatography step the sample was > 99% homogeneous with a specific activity of approximately 15.5 micromol min(-1) mg(-1) . Mass spectrometry analysis confirmed the expected mass of 17.6 kDa . Gel filtration of the enzyme indicated that it was a monomer . Steady-state kinetic parameters were determined and the effect of pH and KCl on the enzyme examined . Methotrexate and trimethoprim inhibited the enzyme.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 51 - 9
Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate; Lim JH et al.; The proteins secreted by Mycobacterium tuberculosis are an important target for vaccine development . To identify the antigens from M . tuberculosis culture filtrate (CF) that strongly stimulate T-cells, the CF was fractionated by ion-exchange chromatography and then non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mini-whole gel elution . Each fraction was screened for its ability to induce interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells isolated from healthy tuberculin reactors . The protein bands that strongly induced IFN-gamma production were subjected to N-terminal sequencing . Two new proteins, a 17-kDa protein (Rv0164, MTSP17) and an 11-kDa (Rv3204, MTSP11) protein, were identified . The recombinant MTSP17 (rMTSP17) and rMTSP11 induced significant production of IFN-gamma and interleukin (IL)-12p40 in peripheral blood mononuclear cells from healthy tuberculin reactors . Interestingly, IL-12p40 production in response to rMTSP11 was significantly higher than that in response to rMTSP17 or the three components of the antigen 85 complex . These results suggest that MTSP11 antigen should be further evaluated as a component of a subunit vaccine.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 45 - 9
Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry; Bahl MI et al.; A method was developed to study plasmid stability in Escherichia coli cells, which utilised the high speed analysis properties of flow cytometry . To discriminate between plasmid-harbouring cells and plasmid-free cells a plasmid-encoded Lac repressor protein was used to regulate the expression of a chromosomally inserted green fluorescent protein gene in the host cells . Flow cytometric analysis enabled detection and quantification of plasmid-free cells due to their green fluorescent phenotype . The reported system offers real-time analysis in combination with a very low detection level of plasmid loss in bacterial populations . This could be useful in future investigations of plasmid stability and population selection in bacterial communities.

Immunol Lett, 2004 Feb 15, 91(2-3), 163 - 70
Isolation of two distinct populations of recombinant antibody molecules specific for rat malonyl-CoA decarboxylase from a semi-synthetic human scFv display library using Ex-phage system; Baek HJ et al.; A semi-synthetic human scFv phage display library by randomizing amino acid residues at CDR3H was constructed using pIGT3 phagemid vector . Recombinant phages were rescued by super-infecting the JS5 E . coli library stock with Ex-phage, the mutant M13KO7 helper phage containing amber mutations at gIII . The library was composed of 2 x 10(8) independent clones, and selected for the specific binders against malonyl-CoA decarboxylase (MCD) by panning . Five soluble scFv clones specific for MCD were finally identified and classified into two groups based on the difference in their binding pattern to MCD . Two clones (M4 and M8) showed good binding reactivity to MCD in ELISA but not in Western blot, whereas, the rest three clones (M23, M28 and M41) reacted to the antigen in Western blot but not in ELISA implying they bound to somewhat different epitopes on MCD . DNA sequencing analysis of M4, M8, M23 and M28 showed that VH of all clones were belonged to VH3 subgroup . On the other hand, M4 and M8 utilized VLkappa subgroup I, and M23 and M28 used VLkappa subgroup IV, suggesting that difference in binding pattern between M4/M8 and M23/M28 against MCD might come from the different VL gene utilization . In conclusion, human monoclonal scFv antibodies specific for MCD were successfully isolated and we demonstrated that distinct populations of recombinant antibodies specific to the target antigen could be isolated by Ex-phage system.

Anal Chem, 2004 Mar 15, 76(6), 1804 - 9
Improved mass analysis of oligoribonucleotides by 13C, 15N double depletion and electrospray ionization FT-ICR mass spectrometry; Xiong Y et al.; 13C, 15N doubly depleted 32-ribonucleotide was synthesized enzymatically by in vitro transcription from nucleoside triphosphates isolated from E . coli grown in a minimal medium containing 12C, 14N-enriched glucose and ammonium sulfate . Following purification and desalting by reversed-phase HPLC, buffer exchange with Microcon YM-3, and ethanol precipitation, electrospray ionization Fourier transform ion cyclotron resonance mass spectra revealed greatly enhanced abundance of monoisotopic ions (by a factor of approximately 100) and a narrower isotopic distribution with higher signal-to-noise ratio . The abrupt onset and high magnitude of the monoisotopic species promise to facilitate accurate mass measurement of RNA's.

Anal Chem, 2004 Mar 15, 76(6), 1720 - 5
Activation of phosphorothionate pesticides based on a cytochrome P450 BM-3 (CYP102 A1) mutant for expanded neurotoxin detection in food using acetylcholinesterase biosensors; Schulze H et al.; A novel enzymatic in vitro activation method for phosphorothionates has been developed to allow their detection with acetylcholinesterase (AChE) biosensors . Activation is necessary because this group of insecticides shows nearly no inhibitory effect toward AChE in their pure nonmetabolized form . In contrast, they exert a strong inhibitory effect on AChE after oxidation as it takes place by metabolic activation in higher organisms . Standard chemical methods to oxidize phosphorothionates showed inherent disadvantages that impede their direct use in food analysis . In contrast, a genetically engineered triple mutant of P450 BM-3 (CYP102 A1) could convert the two frequently used insecticides parathion and chlorpyrifos into their oxo variants as was confirmed by GC/MS measurements . The wild-type protein was unable to do so . In the case of chlorpyrifos, the enzymatic activation was as good as the chemical oxidation . In the case of parathion, the P450 activation was more efficient than the oxidation by NBS but neither activation method yielded an AChE inhibition that was as high as with paraoxon . The application of the method to infant food in combination with a disposable AChE biosensor enabled detection of chlorpyrifos and parathion at concentrations down to 20 microg/kg within an overall assay time of 95 min.

Anal Chem, 2004 Mar 15, 76(6), 1611 - 7
Amperometric detection of nucleic acid at femtomolar levels with a nucleic acid/electrochemical activator bilayer on gold electrode; Xie H et al.; Cationic redox polymers containing osmium-bipyridine complexes strongly interact with anionic enzymes, such as glucose oxidase and peroxidases, and electrochemically "activate" the enzymes . On the basis of these observations, attempts were made to develop an ultrasensitive nucleic acid biosensor . A mixed monolayer of single-stranded oligonucleotide capture probe and 16-mercaptohexadecanoic acid was formed on a gold electrode through self-assembly . Following hybridization with a complementary nucleic acid and glucose oxidase labeled oligonucleotide detection probe, a cationic redox polymer (electrochemical activator) overcoating was applied to the electrode through layer-by-layer electrostatic self-assembly . The formation of an anionic-cationic bilayer brought the glucose oxidase in electrical contact with the redox polymer, making the bilayer an electrocatalyst for the oxidation of glucose . Thus, nucleic acid molecules were quantified amperometrically at femtomolar levels . The effect of experimental variables on the amperometric response was investigated and optimized to maximize the sensitivity and speed up the assay time . A detection limit of 1.0 fmol/L in 1.0-microL droplets and a linear current-concentration relationship up to 800 fmol/L were attained following a 30-min hybridization . The biosensor was applied to the detection of the 16S gene in a mixture of Escherichia coli 16S + 32S rRNA and a full-length rat housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), of a RT-PCR product.

Curr Microbiol, 2004 Jan, 48(1), 32 - 8
Identification and initial characterization of a putative Mycoplasma gallinarum leucine aminopeptidase gene; Wan X et al.; Aminopeptidases (APN) may play a role in host colonization of M . gallinarum . Characterization of endogenous APN activity suggests that the leucine APN (LAP) of M . gallinarum is a metallo-aminopeptidase activated by Mn2+ and is present in the cytosol and possibly associated with the inner leaflet of the membrane . A 1.36-kb open reading frame (ORF) identified from overlapping genomic phage clones showed 68% nucleotide identity and 51% amino acid identity with the M . salivarium LAP gene . This ORF is expressed as a 1.5-kb monocistronic transcript and is present as a single copy in M . gallinarum . This gene sequence was modified to account for codon usage, and expression in E . coli produced a 51-kDa protein, which compares well with the product predicted from the ORF . This ORF is a strong candidate for contributing the LAP activity of M . gallinarum protein extracts.

Cell Death Differ, 2004 Jul, 11(7), 747 - 59
E2FBP1/hDril1 modulates cell growth through downregulation of promyelocytic leukemia bodies; Fukuyo Y et al.; Promyelocytic leukemia nuclear bodies (PML-NBs) comprise multiple regulatory factors and play crucial roles in the maintenance of cellular integrity, while unregulated activation of PML-NBs induces death and premature senescence . Hence, the function of PML-NBs must be directed properly; however, the mechanism that regulates PML-NBs remains unclear . In this paper, we show that PML-NBs are disintegrated by an AT-rich interaction domain family protein E2FBP1/hDril1 through specific desumoylation of promyelocytic leukemia protein (PML) in vivo and in vitro . RNA interference-mediated downregulation of E2FBP1/hDril1 results in hyperplasis of PML-NBs and consequent commitment to PML-dependent premature senescence . Thus, the function of E2FBP1/hDril1 is required for maintenance of survival potential of the cells . Our data suggest a novel mechanism to govern cellular integrity through the modulation of nuclear depots.

Methods Mol Biol, 2004, 252, 483 - 91
RNAi expression vectors in mammalian cells; Miyagishi M et al.; RNA interference (RNAi) is a recently developed technique for gene silencing by introducing dsRNA into cells, and it is shown to work in mammalian cells when siRNAs are used . Several groups have developed vector-based siRNA expression systems that can induce RNAi in living cells . These vector systems use a pol III promoter, such as U6 or H1, and are classified into two groups based on the form of expressed RNAs: tandem-type and hairpin-type . Here, we describe how to generate these siRNA expression vectors and outline the experimental procedure for suppressing the expression of a reporter gene by transient transfection of a siRNA expression vector.

Methods Mol Biol, 2004, 252, 471 - 82
RNA interference (RNAi) with RNase III-prepared siRNAs; Yang D et al.; Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells . Because different siRNAs of the same gene have varying silencing capacities, several different siRNAS typically must be screened to obtain a region that will effectively silence the gene of interest . However, RNA interference with synthetic siRNA is inefficient and cost-intensive, especially for large, functional genomic studies . Here, we describe the use of E . coli endoribonuclease III to cleave double-stranded RNA (dsRNA) into esiRNA (endoribonuclease-prepared siRNA) that can target multiple sites within an mRNA . EsiRNA mediates effective RNA interference with no apparent nonspecific effects in cultured mammalian cells . Since the whole gene can be used at once, screening for an active siRNA for an individual gene is eliminated . Because of its simplicity and potency, this approach is useful for large-scale analysis of mammalian gene function.

Methods Mol Biol, 2004, 252, 385 - 98
General design and construction of RNase P ribozymes for gene-targeting applications; Zou H et al.; RNase P ribozyme, such as M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate . When covalently linked with a guide sequence, the M1 ribozyme can function as a sequence-specific endonuclease, M1GS RNA, and cleave any target RNA sequences that basepair with the guide sequence . Using the mRNA coding for the major transcription regulatory protein ICP4 of herpes simplex virus 1 (HSV-1) as the model target, we describe in this chapter the general design and construction of M1GS ribozymes for gene-targeting applications . Specifically, methods are described in detail to determine ideal target regions of an mRNA for M1GS ribozymes and to construct highly active RNase P ribozymes that target these regions . Extensive protocols for in vitro synthesis of the ribozymes and for the cleavage assay of the ribozyme activity are also included . These methods are intended to provide general guidelines for the design and construction of M1GS ribozymes for gene-targeting applications.

Proc Natl Acad Sci U S A, 2004 Mar 23, 101(12), 4083 - 8 Epub 2004 Mar 11.
Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol; Mitra K et al.; A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer . Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid/cholesterol bilayers . Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol . The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 +/- 0.4 A, 39.5 +/- 0.4 A, 35.6 +/- 0.6 A, and 42.5 +/- 0.3 A, respectively . After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 A . Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness . Protein-depleted membranes demonstrated changes in thickness of up to 5 A, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes . A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings.

J Virol, 2004 Apr, 78(7), 3387 - 97
Nonnucleoside inhibitor binding affects the interactions of the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase with DNA; Peletskaya EN et al.; Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit . Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA . HIV-1 RT is quite flexible . There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs) . Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP) . Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking . This finding suggests that the fingers of p66 are closer to an extended template in the "open" configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP . NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template . Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74 . The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding . These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

J Biol Chem, 2004 May 21, 279(21), 21689 - 94 Epub 2004 Mar 11.
Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function; Neely A et al.; Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization . They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits . The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity . Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified . The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit . However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria . We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies . The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain) . Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA . We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.

Am J Physiol Heart Circ Physiol, 2004 Aug, 287(2), H691 - 4 Epub 2004 Mar 11.
LPS-induced microvascular leukocytosis can be assessed by blue-field entoptic phenomenon; Kolodjaschna J et al.; Administration of low doses of Escherichia coli endotoxin {a lipopolysaccharide (LPS)} to humans enables the study of inflammatory mechanisms . The purpose of the present study was to investigate whether the blue-field entoptic technique may be used to quantify the increase in circulating leukocytes in the ocular microvasculature after LPS infusion . In addition, combined laser Doppler velocimetry and retinal vessel size measurement were used to study red blood cell movement . Twelve healthy male volunteers received 20 IU/kg iv LPS as a bolus infusion . Outcome parameters were measured at baseline and 4 h after LPS administration . In the first protocol (n = 6 subjects), ocular hemodynamic effects were assessed with the blue-field entoptic technique, the retinal vessel analyzer, and laser Doppler velocimetry . In the second protocol (n = 6 subjects), white blood cell (WBC) counts from peripheral blood samples and blue-field entoptic technique measurements were performed . LPS caused peripheral blood leukocytosis and increased WBC density in ocular microvessels (by 49%; P = 0.036) but did not change WBC velocity . In addition, retinal venous diameter was increased (by 9%; P = 0.008), but red blood cell velocity remained unchanged . The LPS-induced changes in retinal WBC density and leukocyte counts were significantly correlated (r = 0.87) . The present study indicates that the blue-field entoptic technique can be used to assess microvascular leukocyte recruitment in vivo . In addition, our data indicate retinal venous dilation in response to endotoxin.

Structure (Camb), 2004 Mar, 12(3), 417 - 28
Cofactor-induced conformational rearrangements establish a catalytically competent active site and a proton relay conduit in FabG; Price AC et al.; beta-Ketoacyl-acyl carrier protein reductase (FabG) is a key component in the type II fatty acid synthase system . The structures of Escherichia coli FabG and the FabG{Y151F} mutant in binary complexes with NADP(H) reveal that mechanistically important conformational changes accompany cofactor binding . The active site Ser-Tyr-Lys triad is repositioned into a catalytically competent constellation, and a hydrogen bonded network consisting of ribose hydroxyls, the Ser-Tyr-Lys triad, and four water molecules creates a proton wire to replenish the tyrosine proton donated during catalysis . Also, a disordered loop in FabG forms a substructure in the complex that shapes the entrance to the active site . A key observation is that the nicotinamide portion of the cofactor is disordered in the FabG{Y151F}.NADP(H) complex, and Tyr151 appears to be necessary for high-affinity cofactor binding . Biochemical data confirm that FabG{Y151F} is defective in NADPH binding . Finally, structural changes consistent with the observed negative cooperativity of FabG are described.

EMBO J, 2004 Apr 7, 23(7), 1474 - 82 Epub 2004 Mar 11.
Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein; Graille M et al.; Colicins are toxins secreted by Escherichia coli in order to kill their competitors . Colicin D is a 75 kDa protein that consists of a translocation domain, a receptor-binding domain and a cytotoxic domain, which specifically cleaves the anticodon loop of all four tRNA(Arg) isoacceptors, thereby inactivating protein synthesis and leading to cell death . Here we report the 2.0 A resolution crystal structure of the complex between the toxic domain and its immunity protein ImmD . Neither component shows structural homology to known RNases or their inhibitors . In contrast to other characterized colicin nuclease-Imm complexes, the colicin D active site pocket is completely blocked by ImmD, which, by bringing a negatively charged cluster in opposition to a positively charged cluster on the surface of colicin D, appears to mimic the tRNA substrate backbone . Site-directed mutations affecting either the catalytic domain or the ImmD protein have led to the identification of the residues vital for catalytic activity and for the tight colicin D/ImmD interaction that inhibits colicin D toxicity and tRNase catalytic activity.

J Biomol NMR, 2004 Jun, 29(2), 175 - 85
Binding ability of a HHP-tagged protein towards Ni2+ studied by paramagnetic NMR relaxation: the possibility of obtaining long-range structure information; Jensen MR et al.; The binding ability of a protein with a metal binding tag towards Ni(2+) was investigated by longitudinal paramagnetic NMR relaxation, and the possibility of obtaining long-range structure information from the paramagnetic relaxation was explored . A protein with a well-defined solution structure (Escherichia coli thioredoxin) was used as the model system, and the peptide His-His-Pro (HHP) fused to the N-terminus of the protein was used as the metal binding tag . It was found that the tag forms a stable dimer complex with the paramagnetic Ni(2+) ion, where each metal ion binds two HHP-tagged protein molecules . However, it was also found that additional sites in the protein compete with the HHP-tag for the binding of the metal ion . These binding sites were identified as the side chain carboxylate groups of the aspartic and glutamic acid residues . Yet, the carboxylate groups bind the Ni(2+) ions considerably weaker than the HHP-tag, and only protons spatially close to the carboxylate sites are affected by the Ni(2+) ions bound to these groups . As for the protons that are unaffected by the carboxylate-bound Ni(2+) ions, it was found that the long-range distances derived from the paramagnetic relaxation enhancements are in good agreement with the solution structure of thioredoxin . Specifically, the obtained long-range paramagnetic distance constraints revealed that the dimer complex is asymmetric with different orientations of the two protein molecules relative to the Ni(2+) ion.

J Biol Chem, 2004 May 14, 279(20), 21500 - 10 Epub 2004 Mar 10.
Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity; Wlodawer A et al.; Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53) . This enzyme is active at low pH and in elevated temperatures . In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity . Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges . Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme . All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen . A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur . The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.

J Biol Chem, 2004 May 28, 279(22), 23302 - 10 Epub 2004 Mar 10.
240s loop interactions stabilize the T state of Escherichia coli aspartate transcarbamoylase; Alam N et al.; Here the functional and structural importance of interactions involving the 240s loop of the catalytic chain for the stabilization of the T state of aspartate transcarbamoylase were tested by replacement of Lys-244 with Asn and Ala . For the K244A and K244N mutant enzymes, the aspartate concentration required to achieve half-maximal specific activity was reduced to 8.4 and 4.0 mm, respectively, as compared with 12.4 mM for the wild-type enzyme . Both mutant enzymes exhibited dramatic reductions in homotropic cooperativity and the ability of the heterotropic effectors to modulate activity . Small angle x-ray scattering studies showed that the unligated structure of the mutant enzymes, and the structure of the mutant enzymes ligated with N-phosphonacetyl-L-aspartate, were similar to that observed for the unligated and N-phosphonacetyl-L-aspartateligated wild-type enzyme . A saturating concentration of carbamoyl phosphate alone has little influence on the small angle x-ray scattering of the wild-type enzyme . However, carbamoyl phosphate was able to shift the structure of the two mutant enzymes dramatically toward R, establishing that the mutations had destabilized the T state of the enzyme . The x-ray crystal structure of K244N enzyme showed that numerous local T state stabilizing interactions involving 240s loop residues were lost . Furthermore, the structure established that the mutation induced additional alterations at the subunit interfaces, the active site, the relative position of the domains of the catalytic chains, and the allosteric domain of the regulatory chains . Most of these changes reflect motions toward the R state structure . However, the K244N mutation alone only changes local conformations of the enzyme to an R-like structure, without triggering the quaternary structural transition . These results suggest that loss of cooperativity and reduction in heterotropic effects is due to the dramatic destabilization of the T state of the enzyme by this mutation in the 240s loop of the catalytic chain.

Clin Diagn Lab Immunol, 2004 Mar, 11(2), 372 - 8
Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis; Schultz M et al.; Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine . This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo . Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water . EcN was administered from day -2 to day +7 . Chronic colitis was induced by transfer of CD4(+) CD62L(+) T lymphocytes from BALB/c mice in SCID mice . EcN was administered three times/week from week 1 to week 8 after cell transfer . Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon {IFN-gamma}, interleukin 5 {IL-5}, IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay . Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4 . Intestinal contents and homogenized MLN were cultured, and the number of E . coli-like colonies was determined . EcN was identified by repetitive extragenic palindromic (REP) PCR . EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-gamma, 32,477 +/- 6,377 versus 9,734 +/- 1,717 {P = 0.004}; IL-6, 231 +/- 35 versus 121 +/- 17 {P = 0.02}) but had no effect on the mucosal inflammation . In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 +/- 0.2 versus 1.9 +/- 0.3 {P = 0.02}) and reduced the secretion of proinflammatory cytokines . Translocation of EcN and resident E . coli into MLN was observed in the chronic colitis model but not in healthy controls . Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis . In this model, preexisting colitis was necessary for translocation of EcN and resident E . coli into MLN.

Clin Diagn Lab Immunol, 2004 Mar, 11(2), 320 - 4
Molecular cloning and characterization of tropomyosin, a major allergen of Chironomus kiiensis, a dominant species of nonbiting midges in Korea; Jeong KY et al.; Chironomids are widely and abundantly distributed in the vicinity of standing waters . Larvae of Chironomus and some other genera are known to contain hemoglobins, which have been described as a major allergen, and the adults that have no hemoglobins also have been reported to contain allergens . In this study, we tried to establish the role of chironomid allergy and characterize the allergen of Chironomus kiiensis adults . Skin tests using C . kiiensis adult extracts were performed on patients with allergic symptoms . A cDNA library of C . kiiensis adults was screened with C . kiiensis immune mouse sera to identify allergens, and results were confirmed using skin test-positive human sera . Recombinant allergen was expressed in Escherichia coli and purified by affinity chromatography using nickel-nitrilotriacetic acid agarose to investigate its allergenic properties . Out of 275 allergic patients 14.2% showed a positive reaction to C . kiiensis adult crude extracts in the skin test . The tropomyosin was cloned by immunoscreening and expressed in Escherichia coli . C . kiiensis tropomyosin has a high homology at the amino acid level with tropomyosins which were previously known to be allergens in various arthropods (Periplaneta americana, 86.3%; Panulirus stimpson, 78.9%; Dermatophagoides pteronyssinus, 76.5%) . Specific immunoglobulin E antibodies reacting to recombinant tropomyosin were detected in 17 (81%) of 21 patients whose skin test results were positive . Cross-reactivity against house dust mites and other insects was noticed with mouse anti-recombinant tropomyosin immune serum . C . kiiensis adults were shown to be an important source of inhalant allergens in Korea . Molecular cloning of C . kiiensis tropomyosin was performed and IgE reactivity was demonstrated using skin test-positive human sera . Recombinant tropomyosin will be useful for further studies or clinical applications.

FEBS Lett, 2004 Mar 12, 561(1-3), 3 - 10
A hypothesis to explain division site selection in Escherichia coli by combining nucleoid occlusion and Min; Norris V et al.; The positioning of the site of cell division in Escherichia coli results, it is generally believed, from the operation of nucleoid occlusion in combination with the Min system . Nucleoid occlusion prevents division over the nucleoids and directs it by default to the mid-cell region between segregating nucleoids or to polar regions while the Min system prevents division in polar regions . Unresolved questions include how these systems interact to control the earliest known event in division, the assembly at the membrane of the tubulin-like protein, FtsZ, and, more importantly, what exactly constitutes a division site . Evidence exists that (1) the coupled transcription, translation and insertion of proteins into membrane (transertion), can structure the cytoplasmic membrane into phospholipid domains, (2) the MinD protein can convert vesicles into tubes and (3) a variety of membranous structures can be observed at mid-cell . These data support a model in which transertion from the segregating daughter chromosomes leads to the formation of a distinct proteolipid domain between them at mid-cell; the composition of this domain allows phospholipid tubes to extend like fingers into the cytoplasm; these tubes then become the substrate for the dynamic assembly and disassembly of FtsZ which converts them into the invaginating fold responsible for division; the Min system inhibits division at unwanted sites and times by removing these tubes especially at the cell poles.

Biochem Biophys Res Commun, 2004 Feb 27, 315(1), 10 - 5
The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer; Nakamura A et al.; The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer) . Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein . This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones . The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.

Biosystems, 2004 Feb, 73(2), 89 - 97
The relationship between synonymous codon usage and protein structure in Escherichia coli and Homo sapiens; Gu W et al.; The role of silent position in the codon on the protein structure is an interesting and yet unclear problem . In this paper, 563 Homo sapiens genes and 417 Escherichia coli genes coding for proteins with four different folding types have been analyzed using variance analysis, a multivariate analysis method newly used in codon usage analysis, to find the correlation between amino acid composition, synonymous codon, and protein structure in different organisms . It has been found that in E . coli, both amino acid compositions in differently folded proteins and synonymous codon usage in different gene classes coding for differently folded proteins are significantly different . It was also found that only amino acid composition is different in different protein classes in H . sapiens . There is no universal correlation between synonymous codon usage and protein structure in these two different organisms . Further analysis has shown that GC content on the second codon position can distinguish coding genes for different folded proteins in both organisms.

J Am Chem Soc, 2004 Mar 17, 126(10), 3072 - 80
Substrate affinities for membrane transport proteins determined by 13C cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy; Patching SG et al.; We have devised methods in which cross-polarization magic-angle spinning (CP-MAS) solid-state NMR is exploited to measure rigorous parameters for binding of (13)C-labeled substrates to membrane transport proteins . The methods were applied to two proteins from Escherichia coli: a nucleoside transporter, NupC, and a glucuronide transporter, GusB . A substantial signal for the binding of methyl {1-(13)C}-beta-d-glucuronide to GusB overexpressed in native membranes was achieved with a sample that contained as little as 20 nmol of GusB protein . The data were fitted to yield a K(D) value of 4.17 mM for the labeled ligand and 0.42 mM for an unlabeled ligand, p-nitrophenyl beta-d-glucuronide, which displaced the labeled compound . CP-MAS was also used to measure binding of {1'-(13)C}uridine to overexpressed NupC . The spectrum of NupC-enriched membranes containing {1'-(13)C}uridine exhibited a large peak from substrate bound to undefined sites other than the transport site, which obscured the signal from substrate bound to NupC . In a novel application of a cross-polarization/polarization-inversion (CPPI) NMR experiment, the signal from undefined binding was eliminated by use of appropriate inversion pulse lengths . By use of CPPI in a titration experiment, a K(D) value of 2.6 mM was determined for uridine bound to NupC . These approaches are broadly applicable to quantifying binding of substrates, inhibitors, drugs, and antibiotics to numerous membrane proteins.

Plant Mol Biol, 2003 Nov, 53(5), 715 - 31
Arabidopsis MBD proteins show different binding specificities and nuclear localization; Scebba F et al.; Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control . In plants DNA methylation affects both symmetric and asymmetric cytosines by means of different DNA-methyltransferases . In vertebrates these modifications are interpreted by a group of proteins (methylated DNA-binding domain proteins, MBDs) able to specifically bind methylated CpG . In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is still to be completed . Here we present the characterization of six different MBDs from Arabidopsis . As judged by semi-quantitative RT-PCR, their expression proved to be differentially modulated in different organs . All the corresponding polypeptides, expressed in Escherichia coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only two of them (namely MBD5, 6) were able to specifically bind methylated CpG oligonucleotides . A third protein, AtMBD11, showed a strong affinity for DNA independently from the level of methylation . Moreover we were able to differentiate MBD5 and 6, despite their high homology, for their ability to recognize methylated asymmetrical sites . The binding specificity of these three AtMBD proteins was tested not only on arbitrarily chosen probes but also on the Arabidopsis E2F recognition sequence containing a single CpG site . Protoplasts transient expression experiments of GFP-fusion proteins showed for AtMBD5 and AtMBD6 a heterochromatic localization which was affected by 5-azacytidine treatment . These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificity and might therefore have different roles in methylation-mediated transcriptional silencing.

Plant Mol Biol, 2003 Nov, 53(5), 633 - 45
Members of the aquaporin family in the developing pea seed coat include representatives of the PIP, TIP, and NIP subfamilies; Schuurmans JA et al.; Water and nutrients required by developing seeds are mainly supplied by the phloem and have to be released from a maternal parenchyma tissue before being utilized by the filial tissues of embryo and endosperm . To identify aquaporins that could be involved in this process four full-length cDNAs were cloned and sequenced from a cDNA library of developing seed coats of pea (Pisum sativum L.) . The cDNA of PsPIP1-1 appeared to be identical to that of clone 7a/TRG-31, a turgor-responsive gene cloned previously from pea roots . PsPIP1-1, PsPIP2-1, and PsTIP1-1, or their possible close homologues, were also expressed in cotyledons of developing and germinating seeds, and in roots and shoots of seedlings, but transcripts of PsNIP-1 were only detected in the seed coat . In mature dry seeds, high hybridization signals were observed with the probe for PsPIP1-1, but transcripts of PsPIP2-1, PsTIP1-1, and PsNIP-1 were not detected . Functional characterization after heterologous expression in Xenopus oocytes showed that PsPIP2-1 and PsTIP1-1 are aquaporins whereas PsNIP-1 is an aquaglyceroporin . PsNIP-1, like several other NIPs, contains a tryptophan residue corresponding with Trp-48 in GlpF (the glycerol facilitator of Escherichia coli) that borders the selectivity filter in the permeation channel . It is suggested that PsPIP1-1 and/or its possible close homologues could play a role in water absorption during seed imbibition, and that PsPIP2-1, possibly together with PsPIP1-1, could be involved in the release of phloem water from the seed coat symplast, which is intimately connected with the release of nutrients for the embryo.

Protein Sci, 2004 Apr, 13(4), 1124 - 33 Epub 2004 Mar 09.
Sites of interaction between SecA and the chaperone SecB, two proteins involved in export; Randall LL et al.; SecB, a small tetrameric cytosolic chaperone in Escherichia coli, facilitates the export of precursor poly-peptides by maintaining them in a nonnative conformation and passing them to SecA, which is a peripheral member of the membrane-bound translocation apparatus . It has been proposed by several laboratories that as SecA interacts with various components along the export pathway, it undergoes conformational changes that are crucial to its function . Here we report details of molecular interactions between SecA and SecB, which may serve as conformational switches . One site of interaction involves the final C-terminal 21 amino acids of SecA, which are positively charged and contain zinc . The C terminus of each subunit of the SecA dimer makes contact with the flat beta-sheet that is formed by each dimer of the SecB tetramer . Here we demonstrate that a second interaction exists between the extreme C-terminal alpha-helix of SecB and a site on SecA, as yet undefined but different from the C terminus of SecA . We investigated the energetics of the interactions by titration calorimetry and characterized the hydrodynamic properties of complexes stabilized by both interactions or each interaction singly using sedimentation velocity centrifugation.

Protein Sci, 2004 Apr, 13(4), 992 - 9 Epub 2004 Mar 09.
Mimicking the action of folding chaperones in molecular dynamics simulations: Application to the refinement of homology-based protein structures; Fan H et al.; A novel method for the refinement of misfolded protein structures is proposed in which the properties of the solvent environment are oscillated in order to mimic some aspects of the role of molecular chaperones play in protein folding in vivo . Specifically, the hydrophobicity of the solvent is cycled by repetitively altering the partial charges on solvent molecules (water) during a molecular dynamics simulation . During periods when the hydrophobicity of the solvent is increased, intramolecular hydrogen bonding and secondary structure formation are promoted . During periods of increased solvent polarity, poorly packed regions of secondary structures are destabilized, promoting structural rearrangement . By cycling between these two extremes, the aim is to minimize the formation of long-lived intermediates . The approach has been applied to the refinement of structural models of three proteins generated by using the ROSETTA procedure for ab initio structure prediction . A significant improvement in the deviation of the model structures from the corresponding experimental structures was observed . Although preliminary, the results indicate computationally mimicking some functions of molecular chaperones in molecular dynamics simulations can promote the correct formation of secondary structure and thus be of general use in protein folding simulations and in the refinement of structural models of small- to medium-size proteins.

J Exp Biol, 2004 Mar, 207(Pt 8), 1387 - 98
The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme; Hirayama M et al.; Full-length cDNA clones encoding two types of hemopexin-like protein, mWap65-1 and mWap65-2, were isolated from the HNI inbred line of medaka Oryzias latipes . The deduced amino acid sequence of mWap65-2 resembled mammalian hemopexins more closely than that of mWap65-1 . Histidine residues required for the high affinity of hemopexins for hemes were conserved in mWap65-2, but not in mWap65-1 . Surprisingly, mWap65-1, but not mWap65-2, showed heme-binding ability as revealed by hemin-agarose affinity chromatography, even though mWap65-1 lacked the essential histidine residues . Furthermore, RT-PCR analysis of different tissues demonstrated that the transcripts of mWap65-2 were restricted to liver, whereas those of mWap65-1 were found in various tissues including liver, eye, heart and brain . Quantitative RT-PCR revealed that transcripts of mWap65-2 were expressed earlier than those of mWap65-1 during ontogeny . However, the accumulated mRNA levels of both mWap65-1 and mWap65-2 did not differ significantly in fish acclimated to either 10 degrees C or 30 degrees C for 5 weeks . These characteristics suggest that the two proteins have different physiological functions and that mWap65-2 is not a hemopexin.

J Biol Chem, 2004 May 14, 279(20), 21489 - 99 Epub 2004 Mar 09.
Immunological characterization of tristetraprolin as a low abundance, inducible, stable cytosolic protein; Cao H et al.; Tristetraprolin (TTP) is a zinc finger protein that can bind to AU-rich elements within certain mRNAs, resulting in deadenylation and destabilization of those mRNAs . Its physiological targets include the mRNAs encoding the cytokines tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor . TTP was originally identified on the basis of its massive but transient increase in mRNA levels following mitogen stimulation of fibroblasts . It has been difficult to reconcile this transient mRNA profile with the presumed continuing "need" for TTP protein, for example, to reverse the effects of lipopolysaccharide (LPS)-stimulated TNF secretion . To investigate this and other questions concerning endogenous TTP protein in cells and tissues, we raised a high titer rabbit antiserum against full-length mouse TTP . TTP could be detected on immunoblots of mouse cytosolic tissue extracts; it was most highly expressed in spleen, but its concentration in that tissue was only about 1.5 nm . TTP could be detected readily in splenic macrophages and stromal cells from LPS-injected rats . In both LPS-treated RAW 264.7 macrophages and fetal calf serum-treated mouse embryonic fibroblasts, TTP protein was stable after induction, with minimal degradation occurring for several hours after treatment of the cells with cycloheximide . The biosynthesis of TTP was accompanied by large changes in electrophoretic mobility consistent with progressive phosphorylation . Confocal microscopy revealed that TTP accumulated in a vesicular pattern in the cytosol of the LPS-stimulated RAW 264.7 cells, and was occasionally seen in the cytosol of unstimulated dividing cells . Gel filtration of the endogenous protein suggested that its predominant structure was monomeric . TTP appears to be a low abundance, cytosolic protein in unstimulated cells and tissues, but once induced is relatively stable, in contrast to its very labile mRNA.

J Biol Chem, 2004 May 7, 279(19), 19481 - 5 Epub 2004 Mar 09.
Mechanism of stimulation of ribosomal promoters by binding of the +1 and +2 nucleotides; Lew CM et al.; The rate of transcription of Escherichia coli ribosomal RNA promoters is central to adjusting the cellular growth rate to nutritional conditions . The +1 initiating nucleotide and ppGpp are regulatory effectors of these promoters . The data herein show that in vitro transcription is also regulated by the +2 nucleotide . Both the +1 and +2 nucleotides act by driving polymerase into an altered conformation rather than by increasing the lifetime of transcription complexes . The unique design of the ribosomal promoters may stabilize a distorted state of polymerase that is relieved by the binding of the two nucleotides required for transcription initiation.

DNA Repair (Amst), 2004 Apr 1, 3(4), 369 - 78
The possible roles of the DNA helicase and C-terminal domains in RECQ5/QE: complementation study in yeast; Nakayama M et al.; DmRECQ5/QE is a member of the RECQ5 subfamily, which shares homology with the Escherichia coli RecQ DNA helicase . Although the DNA helicase activity of RECQ5/QE has been characterized in vitro, the in vivo function of RECQ5/QE was essentially unknown . To investigate the cellular role of RECQ5, the potential of RECQ5/QE was evaluated by substitution of the only RecQ-like helicase, Sgs1, in budding yeast . RECQ5/QE can complement several phenotypes of sgs1, including the synthetic growth defect with srs2, the hypersensitivity to hydroxyurea and methyl methanesulfonate, and the elevated frequency of homologous recombination and sister chromatid exchange (SCE), but poorly complemented the suppression of slow growth in top3 . These data suggested that RECQ5/QE exhibits an evolutionarily conserved RecQ function in vivo . The RECQ5/QE domain necessary for the yeast complementation was determined . The helicase domain and helicase activity were required to complement both the sgs1srs2 and sgs1top3 phenotypes . In contrast, the C-terminal domain was dispensable for complementing the sgs1srs2 phenotype, but was required for the sgs1top3 phenotype . These results suggested that the RECQ5/QE helicase activity is important for cellular function and that the C-terminal domain has a specific function in the absence of Top3.

Life Sci, 2004 Apr 2, 74(20), 2515 - 26
Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro anti-inflammatory activity; Diaz AM et al.; Five phenylpropanoid glycosides isolated from Scrophularia scorodonia L . (Scrophulariaceae), namely angoroside A (1), angoroside C (2), angoroside D (3), acteoside (4) and isoacteoside (5), had been evaluated as potential inhibitors of some macrophage functions involved in the inflammatory process . These compounds have been tested in two experimental systems: ionophore-stimulated mouse peritoneal macrophages and human platelets serve as source of COX-1 and 5-LOX, and mouse peritoneal macrophages stimulated with E . coli LPS are the means of testing for COX-2, NO and TNF-alpha activity . None of compounds assayed had a significant effect on LTC(4)-release from calcium ionophore-stimulated mouse peritoneal macrophages . However, the release of PGE(2) by mouse peritoneal macrophages stimulated with calcium ionophore was inhibited by most of these compounds . In the TXB(2)-release assay, acteoside (4), angoroside A (1) and angoroside C (2) showed a significant effect . These five compounds, except angoroside C (2) significantly inhibited LPS-induced PGE(2), NO and TNF-alpha in a concentration-dependent manner . In LPS-stimulated macrophages, the phenylpropanoid glycoside angoroside C (2) only had activity on NO . These results indicate that the pharmacology of these compounds may participate in the anti-inflammatory effect of Scrophularia scorodonia.

Vet Immunol Immunopathol, 2004 Apr, 98(3-4), 175 - 84
Cloning of bovine MAIL and its mRNA expression in white blood cells of Holstein cows; Yamaji D et al.; Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is known as an IkappaB protein induced after administration of bacterial lipopolysaccharide (LPS) to mice . In the present study, we cloned bovine MAIL cDNA and examined its mRNA expression in white blood cells isolated from Holstein cows . Bovine MAIL had more than 80% amino acid identities with murine and human MAILs, highly conserved ankyrin-repeat motifs and PEST-like sequences . Bovine MAIL mRNA was undetectable in isolated peripheral white blood cells, but rapidly induced (<1h) after stimulation by LPS and lipid A in vitro in a dose-dependent manner . The lipid A-induced MAIL mRNA expression was found in polymorphonuclear cells, monocytes/macrophages and total lymphocytes, but not in T-lymphocytes . MAIL mRNA was also induced in vivo in peripheral blood leukocytes of cows after intramammary injection of Escherichia coli derived from coliform mastitis . Thus, bovine MAIL, as rodent MAILs, is induced by inflammatory stimuli in specific immune cells in vitro and in vivo, suggesting a role in inflammatory responses to bacterial infection in cattle.

Mol Microbiol, 2004 Mar, 51(6), 1745 - 55
Induction of the sufA operon encoding Fe-S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant-responsive factor; Lee JH et al.; A promoter (sufAp), inducible by various oxidants, directs transcription of the sufABCDSE operon encoding an alternative Fe-S cluster assembly system in Escherichia coli . Superoxide generators and H2O2 induced expression of sufA-lacZ even in DeltasoxRS and DeltaoxyR mutants, suggesting participation of an additional regulator(s) in oxidant induction of the sufA operon . Through deletion and linker scanning mutagenesis, we found three cis-acting oxidant-responsive elements (OREs) . ORE-I lies between -236 and -197 nucleotides from the transcription start site, overlapping extensively with the OxyR binding site reported previously . ORE-II (-156 to -127) was found to be the site of IHF action . ORE-III (-56 to -35) had no predictable binding sites for known regulators . Gel mobility shift assays with a 50 bp DNA probe containing ORE-III revealed the presence of an ORE-III-specific factor that binds only when cells are treated with oxidants . S1 mapping analysis revealed that phenazine methosulphate (PMS) and H2O2 induced sufA expression by more than 40-fold . In a DeltaoxyR mutant, sufA was still induced more than 10-fold . Fur, a ferric uptake regulator that negatively regulates this operon in response to iron availability, did not mediate the oxidant induction . Deletion of the suf operon caused cells to be more sensitive to superoxide-generating agents without affecting sensitivity to H2O2 . From these results, we propose that the oxidant induction of the sufA operon is mediated through OxyR, IHF, plus an unidentified oxidant-responsive factor, and that the suf gene products are needed to defend cells against oxidative stress caused by superoxide generators.

Mol Microbiol, 2004 Mar, 51(6), 1705 - 17
Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM-yoeB toxin-antitoxin system; Christensen SK et al.; In Escherichia coli, the Lon ATP-dependent protease is responsible for degradation of several regulatory proteins and for the elimination of abnormal proteins . Previous studies have shown that the overproduction of Lon is lethal . Here, we showed that Lon overproduction specifically inhibits translation through at least two different pathways . We have identified one of the pathways as being the chromosomal yefM-yoeB toxin-antitoxin system . The existence of a second pathway is demonstrated by the observation that the deletion of the yefM-yoeB system did not completely suppress lethality and translation inhibition . We also showed that the YoeB toxin induces cleavage of translated mRNAs and that Lon overproduction specifically activates YoeB-dependent mRNAs cleavage . Indeed, none of the other identified chromosomal toxin-antitoxin systems (relBE, mazEF, chpB and dinJ-yafQ) was involved in Lon-dependent lethality, translation inhibition and mRNA cleavage even though the RelB and MazE antitoxins are known to be Lon substrates . Based on our results and other studies, translation inhibition appears to be the key element that triggers chromosomal toxin-antitoxin systems . We propose that under Lon overproduction conditions, translation inhibition is mediated by Lon degradation of a component of the YoeB-independent pathway, in turn activating the YoeB toxin by preventing synthesis of its unstable YefM antidote.

Mol Microbiol, 2004 Mar, 51(6), 1691 - 704
Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system; Livny J et al.; Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B . The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release . To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system . The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase . Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant . Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB . (ii) Variations in cellular physiology (e.g . RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced . (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages . (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H-19B, usually both prophages in the same cell are induced.

Mol Microbiol, 2004 Mar, 51(6), 1589 - 600
Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically; Bach T et al.; In rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs) . All origins within a cell are initiated essentially in synchrony and only once per cell cycle . Immediate re-initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC . Here, GATC sites in oriC were changed to GTTC . This reduced the sequestration to essentially the level found in SeqA-less cells . The mutant origins underwent re-initiation, showing that the GATC sites in oriC are required for sequestration . Each re-initiation eventually gave rise to a cell containing an extra nucleoid . The three-nucleoid cells displayed one asymmetrically placed FtsZ-ring and divided into a two-nucleoid cell and a one-nucleoid cell . The three nucleoid-cells thus divided into three daughters by two consecutive divisions . The results show that extra rounds of replication cause extra daughter cells to be formed prematurely . The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.

J Pineal Res, 2004 Apr, 36(3), 146 - 54
Melatonin prevents lipopolysaccharide-induced hyporeactivity in rat; d'Emmanuele di Villa Bianca R et al.; Melatonin (MT) is the principal secretory product of the pineal gland and its role as an immumo-modulator is well established . Recent evidence shows that MT exerts protective effects in septic shock, hemorrhagic shock and inflammation . Lipopolysaccharide (LPS), from Escherichia coli, administered to animals directly stimulates a number of cells and systems to produce various inflammatory mediators . LPS-induced septic shock is characterized by hypotension and vascular hyporeactivity to contracting agents . In particular, the reactive oxygen species such as superoxide and nitric oxide (NO) contribute to the pathophysiology of septic shock . In this study, we demonstrate that MT pretreatment prevents the hyporeactivity to phenylephrine in vivo and in aorta rings collected from rats treated with the endotoxin . The beneficial effect of MT seems related to its antioxidant properties and with inhibition of inducible nitric oxide synthase (iNOS) protein expression, reduction of NO production and nitrotyrosine formation, in aorta, preventing vascular, and endothelial injury . Additionally, we first demonstrate, that MT inhibited nuclear enzyme poly (ADP-ribose) synthetase activation in vascular tissue . The current study underlined the protective effect of MT on the vascular dysfunction associated with septic shock, data that could support the clinical use of MT in human endotoxemia.

J Thromb Haemost, 2004 Mar, 2(3), 499 - 506
Shiga toxin binds to activated platelets; Ghosh SA et al.; Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)-producing Escherichia coli . Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis . However, whether Shiga toxin directly activates platelets is controversial . The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin . Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid-citrate-dextrose (ACD) plasma . Platelet binding of Stx was significantly higher in EDTA-washed preparations relative to ACD-derived platelets . Binding of Stx was also increased with ACD-derived platelets when activated with thrombin (1 U mL-1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin . EDTA-exposed platelets lost their normal discoid shape and were larger . P-selectin (CD62P) exposure was significantly increased in EDTA-washed preparations relative to ACD-derived platelets, suggesting platelet activation . Taken together, these results suggest that direct binding of Stx occurs only on 'activated' platelets rather than on resting platelets . The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.

Eur J Biochem, 2004 Mar, 271(5), 932 - 40
Furanocoumarin biosynthesis in Ammi majus L . Cloning of bergaptol O-methyltransferase; Hehmann M et al.; Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as bergapten or xanthotoxin, as the final products of their furanocoumarin biosynthesis, and the rate of accumulation depends on environmental and other cues . Distinct O-methyltransferase activities had been reported to methylate bergaptol to bergapten and xanthotoxol to xanthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus . Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L . cells treated with a crude fungal elicitor . The translated polypeptide of 38.7 kDa, composed of 354 amino acids, revealed considerable sequence similarity to heterologous caffeic acid 3-O-methyltransferases (COMTs) . For homologous comparison, COMT was cloned from A . majus plants and shown to share 64% identity and about 79% similarity with the BMT sequence at the polypeptide level . Functional expression of both enzymes in Escherichia coli revealed that the BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas the COMT activity remained stable . Furthermore, the recombinant AmBMT, which was most active in potassium phosphate buffer of pH 8 at 42 degrees C, showed narrow substrate specificity for bergaptol (Km SAM 6.5 micro m; Km Bergaptol 2.8 micro m) when assayed with a variety of substrates, including xanthotoxol, while the AmCOMT accepted 5-hydroxyferulic acid, esculetin and other substrates . Dark-grown A . majus cells expressed significant BMT activity which nevertheless increased sevenfold within 8 h upon the addition of elicitor and reached a transient maximum at 8-11 h, whereas the COMT activity was rather low and did not respond to the elicitation . Complementary Northern blotting revealed that the BMT transcript abundance increased to a maximum at 7 h, while only a weak constitutive signal was observed for the COMT transcript . The AmBMT sequence thus represents a novel database accession specific for the biosynthesis of psoralens.

Eur J Biochem, 2004 Mar, 271(6), 1087 - 93
Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae; Yamashita S et al.; Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea . A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains . In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae . The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized . The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues . Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN . Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.

Cell Microbiol, 2004 Apr, 6(4), 345 - 53
The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells; Dowling AJ et al.; Photorhabdus bacteria produce a number of toxins to kill their insect hosts . The expression of one of these, Makes caterpillars floppy (Mcf), is sufficient to allow Escherichia coli to persist within and kill caterpillars . Mcf causes shedding of the insect midgut epithelium and destructive blebbing of haemocytes suggesting it may trigger apoptosis . To investigate this hypothesis, here we examine the effects of E . coli-expressed Mcf on the mammalian cell lines COS-7, NIH 3T3 and HeLa cells . Cells treated with Mcf show apoptotic nuclear morphology, active caspase-3, DNA laddering after 6 h, and the presence of cleaved PARP after 16 h . These effects are prevented by the apoptosis inhibitor zVAD-fmk . Transfection of cells with constructs expressing only the NH2-terminal 1280 amino acids of Mcf, as a fusion with Myc, also triggered cell destruction . The expressed fusion protein was concentrated into the Golgi apparatus before cell death . These results confirm that the novel insecticidal toxin Mcf induces apoptosis but the precise intracellular pathway remains obscure.

J Enzyme Inhib Med Chem, 2003 Dec, 18(6), 475 - 83
Isoform-selectivity of PKC inhibitors acting at the regulatory and catalytic domain of mammalian PKC-alpha, -betaI, -delta, -eta and -zeta; Saraiva L et al.; The aim of the present study was to compare the potency of a series of widely used PKC inhibitors acting either at the regulatory (NPC 15437, tamoxifen and D-sphingosine) or at the catalytic domain (Ro 32-0432, chelerythrine and rottlerin) on individual mammalian PKC isoforms of the classical (alpha and betaI), novel (delta and eta) and atypical (zeta) PKC families, using the yeast phenotypic assay, in order to determine their isoform-selectivity . The PKC inhibitors studied presented differences in their ability to reduce the effect of the appropriate PKC activator (estimated as EC50 ratios) which was interpreted as an index of PKC inhibitory potency . In general, the more marked inhibition was observed on novel PKC isoforms, particularly on PKC-eta . This study indicates promising isoform-selectivity of some PKC inhibitors, namely NPC 15437 for PKC-eta or rottlerin for both novel PKC isoforms . It also suggests that the PKC domain involved in the inhibition does not seem to be relevant for the potency and isoform-selectivity of PKC inhibitors.

J Perinat Med, 2004, 32(1), 37 - 41
Cervical immunoglobulin A and altered vaginal flora in pregnant women with threatened preterm delivery; Glasow S et al.; The aim of our study was to investigate the cervical immunoglobulin A concentration in women with threatened preterm delivery . Immunoglobulin A concentration in the cervical mucus of 80 women with symptoms of preterm delivery was measured using radial immunodiffusion . The results were compared with those of 60 healthy pregnant women . Concentrations of immunoglobulins in maternal serum were also measured . There was no significant difference of cervical immunoglobulin A (IgA) concentration between women with threatened preterm delivery and controls: 53.98 (0.0-189.7) mg/l vs . 61.7 (1.4-400.9; p<0.4) mg/l (median, range) . The median of cervical IgA levels in the group of threatened preterm delivery did not differ significantly between patients delivered preterm (n=34) or at term (n=46): 38.3 (0.0-187.9) vs . 65.7 (1.4-189.7; p<0.2) mg/l . Women with a normal vaginal flora showed a significantly higher cervical IgA concentration than those with a pathological colonization: 72.7 (0.0-187.9) vs . 42.5 (0.0-189.7) mg/l . Patients with a pathological vaginal smear and preterm delivery had the lowest IgA levels (35.0; 0.0-187.9 mg/l) . Measurement of cervical IgA concentration does not differentiate between women who deliver before or at term.

Biotechnol Bioeng, 2004 Apr 5, 86(1), 19 - 26
Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis; Jewett MC et al.; Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis . This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change . To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein . The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein . By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds .

Eur Phys J E Soft Matter, 2003 Dec, 12(4), 605 - 15
Probing complex RNA structures by mechanical force; Harlepp S et al.; RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations . We find that force-induced unfolding pathways cannot usually be interpreted by solely invoking successive openings of native helices . Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices . This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli's 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching . Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements . Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called "kinetic traps", thereby capturing -at the single molecular level- the hallmark of RNA folding/unfolding dynamics.

J Biol Chem, 2004 Jun 4, 279(23), 24283 - 90 Epub 2004 Mar 08.
Sequence of interactions in receptor-G protein coupling; Herrmann R et al.; Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes . The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated . We employed time-resolved near infrared light scattering to study the order in which the Galpha and Ggamma C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes . We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive . The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e . myristoylation of the Galpha N terminus and/or farnesylation of the Ggamma C terminus) . A holo-G protein with a high affinity Galpha C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis . We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gbetagamma subunit, and (ii) the Galpha C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gbetagamma subunit . The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state.

J Biol Chem, 2004 May 21, 279(21), 22490 - 7 Epub 2004 Mar 07.
Global co-ordination of protein translocation by the SecA IRA1 switch; Vrontou E et al.; SecA, the dimeric ATPase subunit of protein translocase, contains a DEAD helicase catalytic core that binds to a regulatory C-terminal domain . We now demonstrate that IRA1, a conserved helix-loop-helix structure in the C-domain, controls C-domain conformation through direct interdomain contacts . C-domain conformational changes are transmitted to the DEAD motor and alter its conformation . These interactions establish DEAD motor/C-domain conformational cross-talk that requires a functional IRA1 . IRA1-controlled binding/release cycles of the C-domain to the DEAD motor couple this cross-talk to protein translocation chemistries, i.e . DEAD motor affinities for ligands (nucleotides, preprotein signal peptides, and SecYEG, the integral membrane component of translocase) and ATP turnover . IRA1-mediated global co-ordination of SecA catalysis is essential for protein translocation.

J Biol Chem, 2004 May 21, 279(21), 21824 - 32 Epub 2004 Mar 07.
A binding site for highly sulfated heparan sulfate is identified in the N terminus of the circumsporozoite protein: significance for malarial sporozoite attachment to hepatocytes; Ancsin JB et al.; Circumsporozoite protein (CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria . An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate (HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse . To better understand this efficient targeting process, we set out to identify and characterize the HS binding site(s) of CSP . We synthesized a series of peptides corresponding to five regions of Plasmodium falciparum CSP containing basic residues, a common requirement of HS binding sites, and screened them for heparin and HS binding activity . Only one of these peptides (Pf 2), which contains a motif we have named region I-plus, demonstrated both high affinity heparin/HS binding activity and the ability to block the binding of recombinant CSP to heparin-Sepharose 4B . Analysis by isothermal titration calorimetry revealed that region I-plus has a binding constant of K(d) = 5.0 microm and a stoichiometry of n = 7.8 binding sites/heparin chain . Heparin binding was dependent on the amino acid sequence of region I-plus, and the binding sites on heparin/HS are contained within a decasaccharide . Furthermore, HS oligosaccharides rich in sulfate and iduronic acid content (heparin-like) are required for efficient binding . Because liver HS is exceptionally high in both these components relative to the HS of other organs, the HS structural requirements for efficient region I-plus/HS binding are consistent with this peptide sequence functioning to target sporozoites to the liver for attachment to hepatocytes . Finally, the region I-plus heparin/HS binding site was also discovered for two other species that infect humans, Plasmodium malariae and Plasmodium vivax, further supporting the existence of a HS binding domain in the N-terminal portion of CSP.

Chest, 2004 Mar, 125(3), 1071 - 6
HIV-1 infection does not impair human alveolar macrophage phagocytic function unless combined with cigarette smoking; Elssner A et al.; OBJECTIVE: Macrophages are an important reservoir for the HIV and contribute to innate lung defense by their ability to phagocytose, digest, and process invading pathogens . We hypothesized that HIV-1 infection may lead to a defect in the phagocytic activity of alveolar macrophages . DESIGN: In order to test this hypothesis, the phagocytic activity of alveolar macrophages from asymptomatic HIV-1 seropositive subjects was compared to healthy seronegative control subjects . Macrophages from one cohort were fed with Escherichia coli and from another cohort with opsonized sheep RBCs (SRBCs), and the phagocytic index was determined at different time intervals . SETTING: A tertiary-care, urban, university-based referral center . PARTICIPANTS: Asymptomatic HIV-1 seropositive subjects and healthy seropositive control subjects recruited from local community . RESULTS: No differences were found in the phagocytic activity between alveolar macrophages from the first cohort of eight seropositive and nine seronegative subjects . Although not statistically significant, there was a trend toward a lower phagocytic activity of HIV-positive smokers compared to HIV-positive nonsmokers . Opsonized phagocytic capacity (using opsonized SRBCs) was further analyzed in a second set of five HIV-positive subjects and five healthy control subjects . Whereas HIV status did not affect opsonized SRBC uptake, a history of smoking was associated with a statistically significant depression in phagocytic index . CONCLUSIONS: Although there is no significant impairment of phagocytic capacity in HIV-positive subjects compared to HIV-negative control subjects, cigarette smoking produces a significant depression in phagocytic activity that is amplified in HIV-positive smokers.

Appl Environ Microbiol, 2004 Mar, 70(3), 1874 - 81
Nocardia sp . carboxylic acid reductase: cloning, expression, and characterization of a new aldehyde oxidoreductase family; He A et al.; We have cloned, sequenced, and expressed the gene for a unique ATP- and NADPH-dependent carboxylic acid reductase (CAR) from a Nocardia species that reduces carboxylic acids to their corresponding aldehydes . Recombinant CAR containing an N-terminal histidine affinity tag had K(m) values for benzoate, ATP, and NADPH that were similar to those for natural CAR, and recombinant CAR reduced benzoic, vanillic, and ferulic acids to their corresponding aldehydes . car is the first example of a new gene family encoding oxidoreductases with remote acyl adenylation and reductase sites.

Appl Environ Microbiol, 2004 Mar, 70(3), 1545 - 54
Morphological and physiological changes induced by high hydrostatic pressure in exponential- and stationary-phase cells of Escherichia coli: relationship with cell death; Manas P et al.; The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment . The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min . Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells . In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium . Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane . In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments . The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.

Appl Environ Microbiol, 2004 Mar, 70(3), 1425 - 33
Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea; Pritchett MA et al.; A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed . Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type . Reintroduction of the hpt gene into a Delta hpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M . acetivorans chromosome . We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants . Thus, the method can be repeated many times in the same cell line . The method was validated by construction of Delta proC Delta hpt mutants, which were recovered at a frequency of 22% . Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts . Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M . acetivorans proC locus . In vitro assay of beta-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina: A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed approximately 300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.

Appl Environ Microbiol, 2004 Mar, 70(3), 1297 - 306
Novel pathway of salicylate degradation by Streptomyces sp . strain WA46; Ishiyama D et al.; A novel salicylate-degrading Streptomyces sp., strain WA46, was identified by UV fluorescence on solid minimal medium containing salicylate; trace amounts of gentisate were detected by high-pressure liquid chromatography when strain WA46 was grown with salicylate . PCR amplification of WA46 DNA with degenerate primers for gentisate 1,2-dioxygenase (GDO) genes produced an amplicon of the expected size . Sequential PCR with nested GDO primers was then used to identify a salicylate degradation gene cluster in a plasmid library of WA46 chromosomal DNA . The nucleotide sequence of a 13.5-kb insert in recombinant plasmid pWD1 (which was sufficient for the complete degradation of salicylate) showed that nine putative open reading frames (ORFs) (sdgABCDEFGHR) were involved . Plasmid pWD1 derivatives disrupted in each putative gene were transformed into Streptomyces lividans TK64 . Disruption of either sdgA or sdgC blocked salicylate degradation; constructs lacking sdgD accumulated gentisate . Cell extracts from Escherichia coli DH5 alpha transformants harboring pUC19 that expressed each of the sdg ORFs showed that conversions of salicylate to salicylyl-coenzyme A (CoA) and salicylyl-CoA to gentisyl-CoA required SdgA and SdgC, respectively . SdgA required CoA and ATP as cofactors, while NADH was required for SdgC activity; SdgC was identified as salicylyl-CoA 5-hydroxylase . Gentisyl-CoA underwent spontaneous cleavage to gentisate and CoA . SdgA behaved as a salicylyl-CoA ligase despite showing amino acid sequence similarity to an AMP-ligase . SdgD was identified as a GDO . These results suggest that Streptomyces sp . strain WA46 degrades salicylate by a novel pathway via a CoA derivative . Two-dimensional polyacrylamide gel electrophoresis and reverse transcriptase-PCR studies indicated that salicylate induced expression of the sdg cluster.

J Biotechnol, 2004 Mar 18, 108(3), 271 - 8
Production of the kringle fragments of human apolipoprotein(a) by continuous lactose induction strategy; Lim HK et al.; A novel lactose induction strategy for the production of rhLK68, the kringle fragments of human apolipoprotein(a) (apo(a)) as a novel anti-angiogenic protein, was investigated . A scale-up of the production was accompanied by a decrease in expression level, and severe aggregation occurred during the solubilization of rhLK68 from the inclusion body during a conventional single introduction of lactose . To overcome this problem, a continuous induction strategy was applied where lactose was mixed with glycerol and fed continuously in a dissolved oxygen (DO)-stat manner . With the sub-optimal feed medium consisted of 1:50 of lactose/glycerol (w/w), the expression level reached 16% of the total cellular protein, which was 1.6-fold higher than that obtained from the conventional lactose induction . Moreover, the solubilization yield of rhLK68 from the inclusion body increased from 30 +/- 5 to 85 +/- 3% compared to the conventional single introduction of lactose . This result suggests that the continuous lactose induction strategy beneficially influenced the expression level of rhLK68 and the quality of its inclusion body.

J Biotechnol, 2004 Mar 18, 108(3), 219 - 26
Molecular cloning and expression of a mammalian homologue of a translationally controlled tumor protein (TCTP) gene from Penaeus monodon shrimp; Bangrak P et al.; White spot syndrome, caused by white spot syndrome virus (WSSV), is a deadly disease of shrimps, causing a catastrophic loss in shrimp industries worldwide . In order to investigate molecular response of shrimp haemocyte to WSSV infection, we performed subtraction hybridization of mRNAs from healthy and WSSV-infected haemocyte . One of the genes that were severely down-regulated in moribund WSSV-infected-haemocyte was translationally controlled tumor protein (TCTP) (or fortilin) . Strikingly, while there was a slight difference in the amount of TCTP message between normal and early WSSV-infected shrimps, shrimps that exhibited severe symptoms uniformly had very little TCTP in their haemocyte . Taken together with the fact that TCTP functions as an anti-apoptotic protein in mammals, our data suggest that TCTP in shrimp protects WSSV-infected shrimps from death.

Clin Exp Allergy, 2004 Mar, 34(3), 354 - 62
Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients; Lee CS et al.; BACKGROUND: House dust mites are regarded as important indoor allergens . While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned . We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11 . OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients . METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR . The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli . The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema . RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein . The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins . The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%) . A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative . CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.

Biochemistry, 2004 Mar 16, 43(10), 2941 - 6
Transposon Tn7 protein TnsD binding to Escherichia coli attTn7 DNA and its eukaryotic orthologs; Chakrabarti A et al.; Transposon Tn7 inserts itself into the attTn7 target DNA sequence at the 3' end of the Escherichia coli glmS gene with high specificity and efficiency . This site in the E . coli genome displays amino acid conservation and nucleotide similarity with orthologous sequences in Archaebacteria and eukaryotes . On the basis of the high degree of nucleotide similarity, particularly with eukaryotes, we examined the interactions of a set of 20-bp duplex DNA sequences with the Tn7 protein TnsD . The protein was overexpressed in the IPTG-inducible vector pET14b-TnsD in E . coli BL21(DE3)-RIL-Codon-Plus, and purified by nickel chelation and ion exchange chromatography . Changes in the conformation of DNA duplexes upon interaction with TnsD were monitored by circular dichroism (CD) spectroscopy . TnsD binding to and dissociation from immobilized DNA duplexes were monitored by total internal reflectance (TIR) . CD and TIR results were analyzed to calculate k(on), k(off), and K(D) values . The 20-bp DNA duplex corresponding to attTn7 at the 3' end of E . coli glmS displayed strong affinity for TnsD protein, with K(D) approximately 20 nM . Eukaryotic orthologs of attTn7 from yeast and mammalian GFPT1 displayed lower affinity, with K(D) approximately 500 nM . Mutant DNA sequences with a single central mismatch did not display any detectable interaction with TnsD . The physical studies validate our biological observation of Tn7 transposition into a plasmid containing the mammalian attTn7 ortholog sequence {Cleaver, S . H., and Wickstrom, E . (2000) Gene 254, 37-44}, and suggest that 1-2 amino acid substitutions in TnsD might be sufficient to permit binding to mammalian orthologs that is as strong as wild-type TnsD binding to attTn7.

Biochemistry, 2004 Mar 16, 43(10), 2738 - 46
Insights into signal transduction involving PAS domain oxygen-sensing heme proteins from the X-ray crystal structure of Escherichia coli Dos heme domain (Ec DosH); Park H et al.; The X-ray crystal structure of the Escherichia coli (Ec) direct oxygen sensor heme domain (Ec DosH) has been solved to 1.8 A using Fe multiple-wavelength anomalous dispersion (MAD), and the positions of Met95 have been confirmed by selenomethionine ((Se)Met) MAD . Ec DosH is the sensing part of a larger two-domain sensing/signaling protein, in which the signaling domain has phosphodiesterase activity . The asymmetric unit of the crystal lattice contains a dimer comprised of two differently ligated heme domain monomers . Except for the heme ligands, the monomer heme domains are identical . In one monomer, the heme is ligated by molecular oxygen (O(2)), while in the other monomer, an endogenous Met95 with S --> Fe ligation replaces the exogenous O(2) ligand . In both heme domains, the proximal ligand is His77 . Analysis of these structures reveals sizable ligand-dependent conformational changes in the protein chain localized in the FG turn, the G(beta)-strand, and the HI turn . These changes provide insight to the mechanism of signal propagation within the heme domain following initiation due to O(2) dissociation.

Yonsei Med J, 2004 Feb 29, 45(1), 129 - 34
Efficacy of the merozoite surface protein 1 of Plasmodium vivax as an antigen for ELISA to diagnose malaria; Kim YM et al.; Malaria is still a major health problem in Thailand and its incidence is currently rising in Korea . To identify a useful antigen for the diagnosis of malaria patients, a cDNA expression library from malaria parasites was constructed and screened out immunologically . One clone was selected in view of its predominant reactivity with the patient sera . The recombinant malaria parasite antigen (Pv30) with 27 kDa as a C-terminal His-tag fusion protein that was produced in Escherichia coli was identified through immunoblot analysis . The deduced amino acid sequence had the sequence homology with the merozoite surface protein 1 (MSP1) genes of Plasmodium falciparum and P . yoelii, each by 41% and 42%, respectively . Measurement of serum IgG and IgM antibody to Pv30 by enzyme-linked immunosorbent assay (ELISA) was evaluated as a serodiagnostic test for malaria patients in Thailand (endemic area) and Korea (recently reemerging area) . The sensitivity of P . vivax, P . falciparum, and P . malariae was 96.3% (26 /27), 90.6% (29/32), and 100% (6/6), respectively, and the specificity was 63.5% (40/63) in Thailand samples . The sensitivity of P . vivax was 98.8% (88/89), and the specificity was 96.6% (86/89) in Korean samples . Pv30 appears to be a good and reliable recombinant antigen for serodiagonosis of malaria in a nonendemic area.

Electrophoresis, 2004 Mar, 25(6), 936 - 45
Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides; Cargile BJ et al.; In this work, we demonstrate the potential use of immobilized pH gradient isoelectric focusing as a first dimension in shotgun proteomics . The high resolving power and resulting reduction in matrix ionization effects due to analyzing peptides with almost the exact same physiochemical properties, represents a significant improvement in performance over traditional strong cation-exchange first-dimensional analysis associated with the shotgun proteomics approach . For example, using this technology, we were able to identify more than 6000 peptides and > 1200 proteins from the cytosolic fraction of Escherichia coli from approximately 10 microg of material analyzed in the second-dimensional liquid chromatography-tandem mass spectrometry experiment . Sample loads on the order of 1 mg can be resolved to 0.25 isoelectric point (pI) units, which make it possible to analyze organisms with significantly larger genomes/proteomes . Accurate pI prediction can then be employed using currently available algorithms to very effectively filter data for peptide/protein identification, and thus lowering the false-positive rate for cross-correlation-based peptide identification algorithms . By simplifying the protein mixture problem to tryptic peptides, the effect of specific amino acids on pI prediction can be evaluated as a function of their position in the peptide chain.

Magn Reson Med, 2004 Mar, 51(3), 616 - 20
Novel NMR approach to assessing gene transfection: 4-fluoro-2-nitrophenyl-beta-D-galactopyranoside as a prototype reporter molecule for beta-galactosidase; Cui W et al.; Gene therapy holds great promise for the treatment of diverse diseases . However, widespread implementation is hindered by difficulties in assessing the success of transfection in terms of spatial extent, gene expression, and longevity of expression . The development of noninvasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression . 4-Fluoro-2-nitrophenyl-beta-D-galactopyranoside (PFONPG) is a novel prototype NMR-sensitive molecule, which is highly responsive to the action of beta-galactosidase (beta-gal), the product of the lacZ gene . The molecule is stable in solution and with respect to wild-type cells, but the enzyme causes very rapid liberation of the aglycone, accompanied by color formation and a 19F NMR chemical shift of 5-10 ppm, depending on pH . Since the product is pH-sensitive, this opens the possibility for direct pH determinations at the site of enzyme activity . Molecular and 19F NMR characteristics of PFONPG in solution, blood, and prostate tumor cells are presented . This prototype molecule facilitates a novel approach for assaying gene activity in vivo .

Nat Struct Mol Biol, 2004 Apr, 11(4), 308 - 15 Epub 2004 Mar 07.
Regulation of the p300 HAT domain via a novel activation loop; Thompson PR et al.; The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells . However, the molecular events that regulate p300 HAT activity are poorly understood . We evaluated autoacetylation of the p300 HAT protein domain to determine its function . Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity . This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif . This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation . We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.

Mol Biotechnol, 2004 Mar, 26(3), 221 - 4
An inverse PCR technique to rapidly isolate the flanking DNA of dictyostelium insertion mutants; Keim M et al.; Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum . In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site . For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA . This is a time-consuming process that requires Southern blot analysis of the mutant DNA . In addition, plasmid rescue requires transformation into highly competent Escherichia coli . Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination . We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.

Mol Biotechnol, 2004 Mar, 26(3), 179 - 86
Development of a genetic system in chitinase-producing streptomyces and the application of an allosamidin-insensitive chitinase gene to homologous overexpression; Kawachi R et al.; A transformation system for Streptomyces sp . AJ9463 strain (allosamidin producer) was successfully developed using protoplasts and a PEG-mediated method . To prepare protoplasts, the concentration of glycine and sucrose in YEME medium were optimized to 0.5% (w/v) and 34.0% (w/v), respectively . When the protoplasts of Streptomyces sp . AJ9463 were transformed with pUWL-KS, transformants could be obtained at a high efficiency of 7.0 x 10(4) transformants per microg DNA . To ensure that the transformation system worked properly, we then constructed a constitutive expression vector pYK1, in which the ermE* promoter drives transcription of the allosamidin-insensitive chitinase gene, chiIS . Although no transformant could be obtained by the genetic system using pYK1 isolated from Escherichia coli DH5alpha, pYK1 isolated from the methylase-deficient mutant E . coli SCS110, could be introduced into Streptomyces sp . AJ9463 . This indicates that Streptomyces sp . AJ9463 has a methylation-specific restriction system, and that the chiIS and/or ermE* promoter region of pYK1 includes a restriction site of its endonuclease . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that pYK1 in Streptomyces sp . AJ9463 started to obviously express ChiIS from 14-h . Moreover, the pYK1-introduced strain gave a five-fold higher chitinase activity than the wild-type, suggesting that this system can be widely applied for the overexpression and gene functional analysis.

Proc Natl Acad Sci U S A, 2004 Mar 16, 101(11), 3759 - 64 Epub 2004 Mar 02.
Proteomic analysis of thioredoxin-targeted proteins in Escherichia coli; Kumar JK et al.; Thioredoxin, a ubiquitous and evolutionarily conserved protein, modulates the structure and activity of proteins involved in a spectrum of processes, such as gene expression, apoptosis, and the oxidative stress response . Here, we present a comprehensive analysis of the thioredoxin-linked Escherichia coli proteome by using tandem affinity purification and nanospray microcapillary tandem mass spectrometry . We have identified a total of 80 proteins associated with thioredoxin, implicating the involvement of thioredoxin in at least 26 distinct cellular processes that include transcription regulation, cell division, energy transduction, and several biosynthetic pathways . We also found a number of proteins associated with thioredoxin that either participate directly (SodA, HPI, and AhpC) or have key regulatory functions (Fur and AcnB) in the detoxification of the cell . Transcription factors NusG, OmpR, and RcsB, not considered to be under redox control, are also associated with thioredoxin.

J Clin Microbiol, 2004 Mar, 42(3), 1082 - 8
Molecular cloning, expression, and serological evaluation of an 8-kilodalton subunit of antigen B from Echinococcus multilocularis; Mamuti W et al.; Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E . multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively . The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH(2)-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen . Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E . multilocularis . The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron . The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE) . Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively . By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients . Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases . There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.

J Clin Microbiol, 2004 Mar, 42(3), 1069 - 74
Evaluation of recombinant fragments of Entamoeba histolytica Gal/GalNAc lectin intermediate subunit for serodiagnosis of amebiasis; Tachibana H et al.; We have recently identified a 150-kDa surface antigen of Entamoeba histolytica as an intermediate subunit (Igl) of galactose- and N-acetyl-D-galactosamine-inhibitable lectin, which is a cysteine-rich protein consisting of 1,101 amino acids (aa) and containing multiple CXXC motifs in amino acid sequences . In the present study, full-length Igl except for the signal sequences (aa 14 to 1088) and three fragments of Igl-the N-terminal part (aa 14 to 382), the middle part (aa 294 to 753), and the C-terminal part (aa 603 to 1088)-were prepared in Escherichia coli, and the reactivity of these recombinant proteins with sera from patients with amebiasis was examined by means of enzyme-linked immunosorbent assay (ELISA) . Sera from 57 symptomatic patients with amebic liver abscess or amebic colitis, sera from 15 asymptomatic cyst passers, sera from 40 individuals with other protozoan infections, and sera from 50 healthy controls were used . The sensitivity and specificity of the recombinant full-length Igl in the ELISA were 90 and 94%, respectively . When three fragments were used as antigens in the ELISA, the sensitivities were 56% in the N terminus, 92% in the middle part, and 97% in the C terminus . The specificities of the three antigens were 96% in the N terminus and 99% in both the middle and C-terminal fragments . These results demonstrate that Igl is well recognized in not only symptomatic but also asymptomatic patients with E . histolytica infection and that the carboxyl terminus of Igl is an especially useful antigen for the serodiagnosis of amebiasis.

J Clin Microbiol, 2004 Mar, 42(3), 1058 - 63
Enteroaggregative Escherichia coli virulence factors are found to be associated with infantile diarrhea in Brazil; Zamboni A et al.; We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Brazilian infants . Most EAEC strains harbor a plasmid (pAA) from which a DNA fragment has been used as a probe (EAEC probe) . To better understand the characteristics of EAEC in Brazil, 109 strains carrying and lacking the EAEC probe sequence were tested for the presence of pAA plasmid-borne and chromosomal factors . Common virulence factors of probe-positive and probe-negative isolates included the presence of the Pet, EAST-1, Shf, Irp2, ShET1/Pic, and Hly virulence markers . The presence of AggR or one other virulence factor (AAF/I, AAF/II, AAF/III, or Aap) was predominantly identified only in probe-positive strains . In EAEC probe-positive strains, the virulence marker Aap was found significantly more frequently (P = 0.023) in isolates from children with diarrhea (22%) than in isolates from controls (3%) . EAST-1 and Shf were the markers most frequently detected (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (P = 0.003 and P = 0.020, respectively) . Furthermore, our data suggest that AggR can be used as an important genetic marker for EAEC probe-positive strains.

J Clin Microbiol, 2004 Mar, 42(3), 954 - 62
Phenotypic and genotypic analyses of enterohemorrhagic Escherichia coli O145 strains from patients in Germany; Sonntag AK et al.; Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome . However, there have been few genetic analyses of this EHEC group . We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002 . Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H(-)) . One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains . The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain . Each of the 117 strains with the fliC-RFLP(H28) pattern harbored eae gamma, whereas the three strains with the fliC-RFLP(H25) pattern possessed eae beta . Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified . Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles . These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types . The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens.

J Biol Chem, 2004 May 21, 279(21), 22002 - 9 Epub 2004 Mar 05.
A natively unfolded toxin domain uses its receptor as a folding template; Anderluh G et al.; Natively unfolded proteins range from molten globules to disordered coils . They are abundant in eukaryotic genomes and commonly involved in molecular interactions . The essential N-terminal translocation domains of colicin toxins from Escherichia coli are disordered bacterial proteins that bind at least one protein of the Tol or Ton family . The colicin N translocation domain (ColN-(1-90)), which binds to the C-terminal domain of TolA (TolA-(296-421)), shows a disordered far-UV CD spectrum, no near-UV CD signal, and non-cooperative thermal unfolding . As expected, TolA-(296-421) displays both secondary structure in far-UV CD and tertiary structure in near-UV CD . Furthermore it shows a cooperative unfolding transition at 65 degrees C . CD spectra of the 1:1 complex show both increased secondary structure and colicin N-specific near-UV CD signals . A new cooperative thermal transition at 35 degrees C is followed by the unchanged unfolding behavior of TolA-(296-421) . Fluorescence and surface plasmon resonance confirm that the new unfolding transition accompanies dissociation of ColN-(1-90) . Hence upon binding the disordered structure of ColN-(1-90) converts to a cooperatively folded domain without altering the TolA-(296-421) structure.

J Biol Chem, 2004 May 7, 279(19), 19839 - 45 Epub 2004 Mar 05.
Photoreactive bicyclic amino acids as substrates for mutant Escherichia coli phenylalanyl-tRNA synthetases; Bentin T et al.; Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions . The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm . Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation . These amino acids were also tested for in vitro charging of tRNA(Phe) and for protein mutagenesis via the phenylalanyl-tRNA synthetase variant alphaA294G that is able to facilitate in vivo protein synthesis using a range of para-substituted phenylalanine analogues . The results demonstrate that benzofuranylalanine, but not benzotriazolylalanine, is a substrate for phenylalanine tRNA synthetase alphaA294G, and matrix-assisted laser desorption ionization time-of-flight analysis showed it to be incorporated into a model protein with high efficiency . The in vivo incorporation into a target protein of a bicyclic phenylalanine analogue, as described here, demonstrates the applicability of phenylalanine tRNA synthetase variants in expanding the scope of protein engineering.

Mol Biochem Parasitol, 2004 Apr, 134(2), 245 - 55
Human malaria parasite orotate phosphoribosyltransferase: functional expression, characterization of kinetic reaction mechanism and inhibition profile; Krungkrai SR et al.; Plasmodium falciparum, the causative agent of the most lethal form of human malaria, relies on de novo pyrimidine biosynthesis . A gene encoding orotate phosphoribosyltransferase (OPRT), the fifth enzyme of the de novo pathway catalyzing formation of orotidine 5'-monophosphate (OMP) and pyrophosphate (PP(i)) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, was identified from P . falciparum (pfOPRT) . The deduced amino acid sequence for pfOPRT was compared with OPRTs from other organisms and found to be most similar to that of Escherichia coli . The catalytic residues and consensus sequences for substrate binding in the enzyme were conserved among other organisms . The pfOPRT was exceptional in that it contained a unique insertion of 20 amino acids and an amino-terminal extension of 66 amino acids, making the longest amino acid sequence (281 amino acids with a predicted molecular mass of 33kDa) . The cDNA of the pfOPRT gene was cloned, sequenced and functionally expressed in soluble form . The recombinant pfOPRT was purified from the E . coli lysate by two steps, nickel metal-affinity and gel-filtration chromatography . From 1l E . coli culture, 1.2-1.5mg of pure pfOPRT was obtained . SDS-PAGE revealed that the pfOPRT had a molecular mass of 33kDa and analytical gel-filtration chromatography showed that the enzyme activity eluted at approximately 67kDa . Using dimethyl suberimidate to cross-link neighboring subunits of the pfOPRT, it was confirmed that the native enzyme exists in a dimeric form . The steady state kinetics of initial velocity and product inhibition studies indicate that the enzyme pfOPRT follows a random sequential kinetic mechanism . Compounds aimed at the pfOPRT nexus may act against the parasite through at least two mechanisms: by directly inhibiting the enzyme activity, or be processed to an inhibitor of thymidylate synthase . This study provides a working system with which to investigate new antimalarial agents targeted against P . falciparum OPRT.

Methods, 2004 Apr, 32(4), 406 - 15
Peptides as modulators of enzymes and regulatory proteins; Troitskaya LA et al.; There is currently great interest in the development of methods to modulate the function of diverse classes of target proteins with chemicals (agonists or antagonists) . These would be valuable reagents for biomedical research and some might serve as potential drug leads . Traditionally, most chemicals that modulate protein function have been enzyme inhibitors isolated in functional screens specific for the enzyme of interest . However, recent efforts from many laboratories have suggested that relatively simple binding assays may provide a more convenient and general route to chemical modulators . We review here this work with a particular emphasis on peptide modulators.

Methods, 2004 Apr, 32(4), 349 - 62
Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions; Fetchko M et al.; The characterization of protein-protein interactions provides the foundation for further studies concerning protein complex function and regulation . Since the advent of the yeast two-hybrid assay, many additional genetic systems based upon the principle of protein fragment complementation have been designed . One such system, the split-ubiquitin membrane yeast two-hybrid system (MbYTH), is able to analyze the interaction status between two integral membrane proteins . This ability of the MbYTH system augments genetic analysis of protein interactions by covering for the inherent limitation of the yeast two-hybrid system when studying membrane protein interactions . Herein, we provide a description of the MbYTH method and detailed protocols in order to monitor protein interactions and discover novel interacting partners using the MbYTH system.

Anal Biochem, 2004 Mar 15, 326(2), 190 - 9
Mutant rat trypsin selectively cleaves tyrosyl peptide bonds; Pal G et al.; A double mutant of rat trypsinogen (Asp189Ser, DeltaAsp223) was constructed by site-directed mutagenesis . The recombinant protein was produced in Escherichia coli under the control of a periplasmic expression vector . The purified and enterokinase-activated enzyme was characterized by synthetic fluorogenic tetrapeptide and natural polypeptide substrates and by a recently developed method . In case of this latter method the specificity profile of the enzyme was examined by simultaneous digestion of a mixture of oligopeptide substrates each differing only at the P(1) site residue, and the results were analyzed by high-performance liquid chromatography . All these assays unanimously demonstrated that the recombinant proteinase lacks trypsin-like activity but acquired a rather unique selectivity: it preferentially hydrolyses peptide bonds C-terminal to tyrosyl residues . This narrow specificity should be useful in peptide-analytical applications such as sequence-specific fragmentation of large proteins prior to sequencing.

J Mol Biol, 2004 Mar 19, 337(2), 337 - 54
Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium; Caradoc-Davies TT et al.; Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate . Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity . We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate . These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft . However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed . In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site . This overall change in conformation results in compression of the active site cleft . Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP . Enzyme activity shows a twofold dependence on potassium ion concentration . The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation . The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.

J Mol Biol, 2004 Mar 19, 337(2), 273 - 83
The Escherichia coli YadB gene product reveals a novel aminoacyl-tRNA synthetase like activity; Campanacci V et al.; In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame products of unknown function, we have determined the structure of YadB at 1.5A using molecular replacement . The YadB protein is 298 amino acid residues long and displays 34% sequence identity with E.coli glutamyl-tRNA synthetase (GluRS) . It is much shorter than GluRS, which contains 468 residues, and lacks the complete domain interacting with the tRNA anticodon loop . As E.coli GluRS, YadB possesses a Zn2+ located in the putative tRNA acceptor stem-binding domain . The YadB cluster uses cysteine residues as the first three zinc ligands, but has a weaker tyrosine ligand at the fourth position . It shares with canonical amino acid RNA synthetases a major functional feature, namely activation of the amino acid (here glutamate) . It differs, however, from GluRSs by the fact that the activation step is tRNA-independent and that it does not catalyze attachment of the activated glutamate to E.coli tRNAGlu, but to another, as yet unknown tRNA . These results suggest thus a novel function, distinct from that of GluRSs, for the yadB gene family.

J Mol Biol, 2004 Mar 19, 337(2), 263 - 72
The location and the significance of a cross-link between the sarcin/ricin domain of ribosomal RNA and the elongation factor-G; Chan YL et al.; During translocation peptidyl-tRNA moves from the A-site to the P-site and mRNA is displaced by three nucleotides in the 3' direction . This reaction is catalyzed by elongation factor-G (EF-G) and is associated with ribosome-dependent hydrolysis of GTP . The molecular basis of translocation is the most important unsolved problem with respect to ribosome function . A critical question, one that might provide a clue to the mechanism of translocation, is the precise identity of the contacts between EF-G and ribosome components . To make the identification, a covalent bond was formed, by ultraviolet irradiation, between EF-G and a sarcin/ricin domain (SRD) oligoribonucleotide containing 5-iodouridine . The cross-link was established, by mass spectroscopy and by Edman degradation, to be between a tryptophan at position 127 in the G domain in EF-G and either one of two 5-iodouridine nucleotides in the sequence UAG2655U in the SRD . G2655 is a critical identity element for the recognition of the factor's ribosomal binding site . The site of the cross-link provides the first direct evidence that the SRD is in close proximity to the EF-G catalytic center . The proximity suggests that the SRD RNA has a role in the activation of GTP hydrolysis that leads to a transition in the conformation of the factor and to its release from the ribosome.

Peptides, 2004 Jan, 25(1), 37 - 43
Optimal designing of beta-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide; Onishi K et al.; Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2-7), into three homologous sites in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis . The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo . In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases . Then, fourth RPLKPW was also introduced into the extension domain of the protein . The newly modified protein, which was produced in E . coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration . Furthermore, we produced an extension domain that corresponds to residues 1-143 of the modified alpha' subunit containing four RPLKPW sequences by introducing a termination codon . The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.

Exp Hematol, 2004 Mar, 32(3), 277 - 81
Diphtheria toxin fused to variant interleukin-3 provides enhanced binding to the interleukin-3 receptor and more potent leukemia cell cytotoxicity; Liu TF et al.; Chemoresistance is a common cause of treatment failure in patients with acute myeloid leukemia (AML) . We generated a diphtheria toxin (DT) fusion protein composed of the catalytic and translocation domains of DT (DT388) fused to interleukin-3 (IL-3) . IL-3 receptors (IL-3R) are overexpressed on blasts from many AML patients . DT388IL-3 showed cytotoxicity to leukemic blasts in vitro and in vivo and minimal damage to normal tissues in nonhuman primate models . However, only a fraction of patient leukemic samples were sensitive to the agent . To enhance the potency and specificity of the DT388IL-3 molecule, we constructed variants with altered residues in the IL-3 moiety . Two of these variants, DT388IL-3{K116W} and DT388IL-3{Delta125-133}, were produced and partially purified from Escherichia coli with excellent yields . They showed enhanced binding to the human IL-3R and greater cytotoxicity to human leukemia cell lines relative to wild-type DT388IL-3 . Interestingly, the results support a previously hypothesized model for interaction of the C-terminal residues of IL-3 with a hydrophobic patch on the alpha-subunit of IL-3R . Rational modification of the targeting domain based on structural analysis can produce a fusion toxin with increased ability to kill tumor cells . One or both of these variant fusion proteins merit further development for therapy of chemotherapy refractory AML.

Protein Expr Purif, 2004 Apr, 34(2), 317 - 23
Efficient production of recombinant human transcription factor IIE; Moon WJ et al.; Transcription factor IIE (TFIIE) is a general initiation and promoter escape factor for RNA polymerase II composed of p56 (TFIIE-alpha) and p34 (TFIIE-beta) subunits . Our laboratories experienced difficulty producing adequate quantities of recombinant human TFIIE-alpha for in vitro studies using available clones . We therefore re-engineered the TFIIE subunit production vectors and tested various Escherichia coli host strains to optimize expression . We report a much-improved system for production of pure, soluble, and active TFIIE complex for in vitro studies.

Protein Expr Purif, 2004 Apr, 34(2), 296 - 301
Production and enhanced biological activity of a novel GHRH analog, hGHRH with an N-terminal Pro-Pro extension; Tang SS et al.; Growth Hormone Releasing Hormone (GHRH) is one of the most important hormones in life . Because of its potential clinical importance, its short half-life, and its expensive chemical synthesis, an analog of hGHRH with a prolonged half-life and better activity has been studied for clinical application, especially for the treatment of muscle wasting, type II diabetes, or sleep disorders . The Pro-Pro-hGHRH(1-44) peptide has better activity . The fusion partner gene with 127 amino acid residues of the C-terminus from l-asparaginase was recombined with asp-pro-pro-hGHRH(1-44) gene synthesized by PCR method to form a fusion protein with the unique acid labile linker Asp-Pro . The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3) . The Pro-Pro-hGHRH(1-44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25, and Sephadex G-25 column chromatography . The fold of the purification was about 88 times and the yield was 1.1% of the total protein weight of the inclusion body . The peptide molecular mass of 5235.25 Da was determined by ESI mass spectroscopy . Its purity was determined by SDS-PAGE . In the study of the activity, we measured GH release of rat pituitary by using the antiserum kit against human GH . The peptide doses of 0.01, 0.1, 1.0, 7.72, and 20.9 microg/ml used, respectively, released the GH values of 0.1+/-0.1, 12.5+/-7.3, 16.6+/-5.8, 49.8+/-7.6, and 79.5+/-5.7 ng/ml whereas their blank controls, respectively, were 0.5+/-0.8, 4.1+/-2.6, 3.1+/-3.1, 4.7+/-1.8, and 1.2+/-0.3 ng/ml . The activity results of all dose groups except 0.01 microg/ml Pro-Pro-hGHRH(1-44) group and hGHRH(1-40) group showed that there were significant differences between GH released by the peptide and that by its blank control . With the increase of dosage, the differences were more significant . hGHRH(1-40) showed no measured GH release when the dose was up to 2 microg/ml . The activity results show that the Pro-Pro-hGHRH(1-44) peptide is a potential GH releasing analog.

Protein Expr Purif, 2004 Apr, 34(2), 280 - 3
Prevention of aggregation after refolding by balanced stabilization-destabilization: production of the Arabidopsis thaliana protein APG8a (At4g21980) for NMR structure determination; Chae YK et al.; The gene coding for APG8a (At4g21980), a protein from Arabidopsis thaliana, is involved in the autophagy process . The protein is an interesting candidate for structure determination by NMR spectroscopy . Toward this end, APG8a fused to an N-terminal His-tag has been expressed in Escherichia coli under a T7 expression system, refolded in vitro, and kept soluble by slight destabilization . The expressed protein appeared in both the soluble and the insoluble fractions . The whole-cell lysate was denatured by the addition of guanidinium chloride . The protein was immobilized on nickel-agarose resin and refolded by stepwise decrement of the denaturant . The elution buffer was 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, 300 mM NaCl, and 1 M imidazole . After the removal of imidazole by ultrafiltration, the His-tag was cleaved with biotinylated thrombin . The protein product was kept in 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, and 300 mM NaCl . The protein was found to aggregate extensively over time if any one of the three ingredients (sodium chloride, urea, or glycerol) was omitted . The yield of the protein was around 20 mg/L Luria-Bertani culture medium . The (1)H-(15)N NMR correlation spectrum of (15)N-labeled APG8a showed the characteristic signature of a folded protein; thus, the solutes appear to have no deleterious effect on the sample . These solution conditions kept the protein soluble and unaggregated for at least 2 days (enough time for NMR data collection) . This approach of balanced stabilization-destabilization may offer a general approach for structural investigations of proteins that tend to aggregate.

Protein Expr Purif, 2004 Apr, 34(2), 270 - 9
Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions; Corisdeo S et al.; Several different vector designs are currently being used to display and express Fab molecules in Escherichia coli, but their relative efficiency in phage display and protein expression cannot be compared from the published data . We systematically investigated which vector design most effectively displays and expresses Fab molecules in E . coli using, as a model system, a human Fab against tetanus toxoid (tt) . Three different vectors were used in this study: pFab1 where the VL-CL and VH-CH1 genes were driven by two promoters in two separate expression cassettes, and pFab2 and pFab3 that both contain one dicistronic expression cassette with two translation initiation sites and either VH-CH1 before VL-CL or VL-CL before VH-CH1, respectively . The display of tt-Fab on the surface of phage and the expression of tt-Fab protein in E . coli were compared for the aforementioned vectors . Our results showed that the pFab3 vector was most effective in Fab display . A 10-fold increase in the expression of secreted Fab was observed in pFab3 when compared with vectors pFab1 and pFab2 . Further experiments were conducted using pFab3 to optimize expression levels using different strains of E . coli and various culture conditions . The highest expression of tt-Fab was obtained using the pFab3 vector in host strain JM105 with an induction temperature at 37 degrees C and IPTG concentration of 0.1 mM.

Protein Expr Purif, 2004 Apr, 34(2), 261 - 9
Expression, purification, and characterization of human malonyl-CoA decarboxylase; Zhou D et al.; The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct . Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD . The apo-hMCD enzymes were solubilized and purified to homogeneity . Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330-520 microM and a turnover rate kcat of 13-28s(-1) . For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4-10); whereas the full-length hMCD appeared to be stable only at pH >/= 8.5 . Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD.

Protein Expr Purif, 2004 Apr, 34(2), 249 - 56
Single-step purification and refolding of recombinant mouse and human myelin oligodendrocyte glycoprotein and induction of EAE in mice; Linares D et al.; The extracellular domain of human and rat MOG (ED-MOG) induces experimental autoimmune encephalomyelitis (EAE) when injected into susceptible animals . EAE is a T cell-mediated disease of the central nervous system commonly used as an animal model for human multiple sclerosis . Here, we describe a straightforward procedure for the purification and refolding of mouse and human ED-MOG overexpressed in Escherichia coli as inclusion bodies . Following solubilization and purification using Ni-NTA resin chromatography under denaturing conditions, a column-based refolding proceeded in renaturation buffer supplemented with a glutathione redox buffer system . Using this approach up to 33 mg of highly pure soluble proteins was obtained per liter of expression culture . The ability of purified proteins to induce EAE was evaluated in three strains of mice . We believe that the strategy described here would facilitate researchers to carry out encephalitogenic as well as structure-function studies of this autoantigen . Additionally, we show for the first time that mouse ED-MOG induces severe disease in mice.

Protein Expr Purif, 2004 Apr, 34(2), 243 - 8
Coenzyme precursor-assisted cooperative overexpression of an active pyridoxine 4-oxidase from Microbacterium luteolum; Yoshikane Y et al.; The overexpression system of the active pyridoxine 4-oxidase from Microbacterium luteolum was developed . When chaperonin GroEL/ES genes in plasmid pKY206 were coexpressed, the pyridoxine 4-oxidase gene cloned in the vector pTrc99A was overexpressed in Escherichia coli JM109 cultured in LB medium containing 50microM riboflavin, the precursor of coenzyme (FAD) of the enzyme, under the cold stress at 23 degrees C . The crude extract from the cotransformant cells showed 88-fold higher specific activity than that from M . luteolum . The chaperonins, cold stress, and the riboflavin cooperatively served to increase the soluble form of the enzyme . A significant correlation between the specific activity and percentage of the soluble form in the total expressed enzyme was found . The overexpressed pyridoxine 4-oxidase was easily purified to homogeneity with two steps of the conventional column chromatography.

Protein Expr Purif, 2004 Apr, 34(2), 215 - 22
Reconstitution of an E box-binding Myc:Max complex with recombinant full-length proteins expressed in Escherichia coli; Farina A et al.; The c-Myc oncoprotein (Myc) is a DNA sequence-specific transcription factor that regulates transcription of a wide variety of genes involved in the control of cell growth, proliferation, differentiation, and apoptosis and its deregulated expression is implicated in many types of human cancer . Myc has an N-terminal transcription activation domain (TAD) that interacts with various coactivators and a C-terminal basic-helix-loop-helix-leucine zipper (bHLHZip) domain required for E box-specific DNA-binding and heterodimerization with its obligatory bHLHZip protein partner Max . The analysis of the mechanisms by which the Myc:Max complex regulates transcription at the molecular level in vitro has been hampered by the difficulty in obtaining highly pure recombinant Myc:Max heterodimers that contain full-length Myc with its complete TAD domain and that have sequence-specific DNA-binding activity . Here, we describe a simple method to reconstitute recombinant Myc:Max complexes from highly purified full-length proteins expressed in Escherichia coli that are soluble and highly active in E box-specific DNA-binding in vitro . The reconstituted Myc:Max complexes are stable and lack Max:Max homodimers . This procedure should facilitate the characterization of the DNA-binding and transcription activation functions of full-length Myc:Max complexes in vitro and in particular the role of Myc TAD-interacting cofactors and Myc:Max post-translational modifications.

Protein Expr Purif, 2004 Apr, 34(2), 202 - 7
An efficient method for the overexpression and purification of active tyrosinase from Streptomyces castaneoglobisporus; Kohashi PY et al.; The melanin-synthesizing gene operon cloned from Streptomyces castaneoglobisporus HUT6202 consists of two genes, designated tyrC and orf378, which encode apotyrosinase (TYRC) and its activator protein (ORF378), respectively . We have suggested that ORF378 may facilitate the incorporation of Cu(II) into apotyrosinase to express tyrosinase activity . To overproduce ORF378 and TYRC in Escherichia coli BL21(DE3)-pLysS, tyrC, and orf378 were independently but not polycistronically placed under the control of a T7 promoter in a vector, pET-21a(+) . His(6)-tagged TYRC and His(6)-tagged ORF378 were simultaneously overproduced in an E . coli strain harboring a plasmid, designated pET-mel2, and the two proteins were co-purified with a Ni(II)-bound affinity column . Gel filtration analysis revealed that the two proteins form a heterodimer complex . The complexed protein was retrieved at a high efficiency (11 mg/L) . To obtain an active TYRC, which is a Cu(II)-bound form of tyrosinase, we constructed pET-mel3 that carries orf378 without His(6)-tag and His(6)-tagged tyrC . After the cell-free extract from E . coli harboring pET-mel3 was subjected to Cu(II)-bound affinity column chromatography, His(6)-tagged TYRC, eluted from the column, exhibited the tyrosinase activity . The k(cat) and K(m) values for l-3,4-dihydroxyphenylalanine (l-DOPA) of His(6)-tagged TYRC, which catalyzes the oxidation of l-DOPA to dopaquinone, were 880+/-80s(-1) and 8.1+/-0.9 mM, respectively.

Protein Expr Purif, 2004 Apr, 34(2), 197 - 201
Expression of ricin A chain and ricin A chain-KDEL in Escherichia coli; Zhan J et al.; Ricin and its A chains can be used to conjugate with monoclonal antibodies to prepare immunotoxins . Ricin A chain (RTA) and its modification RTA-KDEL (ER-retrieval signal) were expressed with the pKK223.3 system in Escherichia coli under control of a tac promoter . The recombinant proteins can be purified by one-step affinity chromatography on a column of Blue-Sepharose 6B . The toxicities of RTA and its mutant RTA-KDEL were evaluated by the MTT assay in HeLa, MCF, and ECV-304 cells following fluid-phase endocytosis . RTA-KDEL was somewhat more cytotoxic than RTA itself in the different cell lines . The results suggest that rRTA-KDEL may be useful for the synthesis of more potent immunotoxins.

Protein Expr Purif, 2004 Apr, 34(2), 190 - 6
A general procedure for the purification of human beta-secretase expressed in Escherichia coli; Sardana V et al.; Expression and purification of human beta-secretase (BACE1) in bacteria have been plagued with issues concerning solubility, inhomogeneous N-terminus, and lack of enzymic activity . Several forms of the mature human BACE1 have been expressed in Escherichia coli with different N-terminal extensions and without the C-terminus transmembrane domain . Although each of the proteins expresses in inclusion bodies, a generalized protocol has been developed to solubilize, refold, and purify these BACE1 variants . The resultant proteins are homogeneous and monodispersed in solution . Each possesses a unique N-terminus . Activity assays using the peptide substrate 7-methoxycoumarin-4-yl-SEVNLDAEFK-2,4-dinitrophenyl-RR, corresponding to the beta-secretase cleavage sequence in the amyloid precursor protein with the Swedish mutations of N(670)L(671) substituting for the residues K(670)M(671), reveal a kcat and KM of 9.3 min(-1) and 55 microM, respectively.

Protein Expr Purif, 2004 Apr, 34(2), 183 - 9
Production and purification of melanoma gp100 antigen and polyclonal antibodies; Gelbart Y et al.; gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells . Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma . To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides . High yield was achieved in an Escherichia coli expression system . The protein was purified by AKTA Prime using anionic-columns . Polyclonal antibodies developed in chicken against HR-gp100 were efficient at detecting gp100 in melanoma cells, as determined by Western blot analysis and by immunohistochemistry . HR-gp100 can be used to develop a vaccine against melanoma . Antibodies to HR-gp100 may be used to detect tumors of melanocytic origin or to determine the level of gp100 expression in tumors prior to immunotherapy with the protein or one of its peptides.

Protein Expr Purif, 2004 Apr, 34(2), 167 - 75
Expression and purification of recombinant cytoplasmic domain of human erythrocyte band 3 with hexahistidine tag or chitin-binding tag in Escherichia coli; Ding Y et al.; The cytoplasmic domain of erythrocyte band 3 (cdb3) serves as a center of membrane organization in the erythrocytes by its interaction with multiple proteins including ankyrin, protein 4.1, protein 4.2, hemoglobin, and several glycolytic enzymes . In this paper, human cdb3 was cloned into three different expression vectors controlled by T7 polymerase promoter and induced with isopropyl beta-D-thiogalactopyranoside in Escherichia coli . Two of the fusion proteins containing hexahistidine tag in the N-terminal or C-terminal were purified by immobilized metal affinity column chromatography . The third recombinant cdb3 containing the affinity chitin-binding tag was purified using chitin beads followed by specific self-cleavage, which released cdb3 according to the mechanism of protein splicing . The molecular weights of purified recombinant proteins were verified by mass spectrometry . The pH-dependent properties of the intrinsic tryptophan fluorescence of the three kinds of recombinant cdb3 were compared with that of the cdb3 extracted from the erythrocytes, showing that there were no significant differences between them . Using pull-down assay combined with Western blot analysis, the interaction between recombinant cdb3 and protein 4.2 C3 fragment was verified . These demonstrated that the recombinant proteins were both structurally and functionally active . The typical yields of cdb3 purified with hexahistidine tag in the N-terminal, C-terminal, and cleaved from chitin bead were 10.6, 9.6, and 1.5 mg from 1L culture medium, respectively.

Curr Opin Plant Biol, 2004 Apr, 7(2), 171 - 81
Posttranslational modification of therapeutic proteins in plants; Gomord V et al.; Plants have emerged as an alternative to current systems for the production of therapeutic proteins . The advantages of plants for the low-cost and large-scale production of safe and biologically active mammalian proteins have been documented recently . A major advantage of transgenic plants over production systems that are based on yeast or Escherichia coli is their ability to perform most of the posttranslational modifications (PTMs) that are required for the bioactivity and pharmacokinetics of recombinant therapeutic proteins . Furthermore, recent advances in the control of PTMs in transgenic plants have made it possible for plants to perform, at least to some extent, human-like modifications of recombinant proteins . Hence, plants have become a suitable alternative to animal cell factories for the production of therapeutic proteins.

J Clin Periodontol, 2003 Dec, 30(12), 1091 - 6
Increased release of IL-12p70 by monocytes after periodontal therapy; Fokkema SJ et al.; OBJECTIVES: It has been suggested that periodontal inflammation may result in an altered immune response . The peripheral immune capacity in periodontitis patients can be investigated using lipopolysaccharide (LPS)-stimulated whole blood cell cultures (WBCC), known to reflect the behavior of monocytes in particular . A previous study in our laboratory revealed that monocytes in the stimulated cultures from periodontitis patients behaved functionally different compared with controls . The present study investigated whether this different response of periodontitis patients' monocytes is intrinsic or acquired . MATERIAL AND METHODS: The release of inflammatory mediators was measured in Escherichia coli LPS-stimulated WBCC from 12 periodontitis patients before and after periodontal therapy . In addition, the total leukocyte and leukocyte differentiation counts were also determined in the patients before and after therapy . RESULTS: The levels of interleukin (IL)-12p70 in cell culture supernatants increased two times and those of prostaglandin E2 showed a trend towards reduction after therapy, whereas the levels of IL-1beta, IL-6, IL-8, IL-10, IL-12p40 and tumor necrosis factor-alpha did not change . The total number of white blood cells was decreased after periodontal therapy . CONCLUSIONS: After periodontal therapy, the functional phenotype of the peripheral blood monocytes from patients was reconstituted, resembling that of subjects without periodontitis.

J Plant Physiol, 2004 Jan, 161(1), 87 - 94
Cloning, characterization and tissue specific expression of a sucrose synthase gene from tropical epiphytic CAM orchid Mokara Yellow; Li CR et al.; A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic CAM orchid Mokara Yellow . The cDNA is 2748bp in length containing an open reading frame of 2447bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa . The deduced amino acid sequence of M . Yellow sucrose synthase (Msus1) shares more than 80% identity with those from other monocotyledonous plants . The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli . Northern blot analysis showed that the expression pattern of Msus1 mRNA is tissue specific with highest levels in strong sinks such as expanding leaves and root tips, but not detectable in mature leaves and flowers . Incubation with sugars resulted in a significant increase in the steady-state Msus1 mRNA levels in shoots of seedlings.

Plant Physiol, 2004 Mar, 134(3), 969 - 78 Epub 2004 Mar 04.
A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family; Jeong J et al.; Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants . These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3 . The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined . We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1 . AgDCAT1 mRNA was detected only in the nodules and not in other plant organs . Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface . The AgDCAT1 substrate was determined by its heterologous expression in two systems . Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant . Using the E . coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate . It also transported succinate, fumarate, and oxaloacetate . To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.

J Biol Chem, 2004 May 14, 279(20), 21257 - 65 Epub 2004 Mar 04.
The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis; Domenech P et al.; To study the role of MmpL8-mediated lipid transport in sulfatide biogenesis, we insertionally inactivated the mmpL8 gene in Mycobacterium tuberculosis . Characterization of this strain showed that the synthesis of mature sulfolipid SL-1 was interrupted and that a more polar sulfated molecule, termed SL-N, accumulated within the cell . Purification of SL-N and structural analysis identified this molecule as a family of 2,3-diacyl-alpha,alpha'-D-trehalose-2'-sulfates . This structure suggests that transport and biogenesis of SL-1 are coupled and that the final step in sulfatide biosynthesis may be the extra-cellular esterification of two trehalose 6-positions with hydroxyphthioceranic acids . To assess the effect of the loss of this anionic surface lipid on virulence, we infected mice via aerosol with the MmpL8 mutant and found that, although initial replication rates and containment levels were identical, compared with the wild type, a significant attenuation of the MmpL8 mutant strain in time-to-death was observed . Early in infection, differential expression of cytokines and cytokine receptors revealed that the mutant strain less efficiently suppresses key indicators of a Th1-type immune response, suggesting an immunomodulatory role for sulfatides in the pathogenesis of tuberculosis.

Arch Biochem Biophys, 2004 Mar 15, 423(2), 277 - 87
Mechanistic studies with N-benzyl-1-aminobenzotriazole-inactivated CYP2B1: differential effects on the metabolism of 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, and benzphetamine; Kent UM et al.; Mechanistic studies with N-benzyl-1-aminobenzotriazole (BBT)-inactivated cytochrome P450 2B1 were conducted to determine which step(s) in the reaction cycle had been compromised . Stopped-flow studies, formation of the oxy-ferro intermediate, and analysis of products suggested that the reductive process was slower with the BBT-modified enzyme . The reduced rate of reduction alone could not account for the loss in 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) O-deethylation or testosterone hydroxylation activity . Surprisingly, the ability of the BBT-modified enzyme to generate formaldehyde from benzphetamine was much less affected . Benzphetamine metabolite analysis by electrospray ionization-mass spectrometry showed that the BBT-modified enzyme had a slightly greater propensity towards aromatic hydroxylation together with reduced levels of N-demethylation and little change in the N-debenzylation of benzphetamine . Orientation of substrates within the active site of the BBT-inactivated enzyme may be affected such that the more flexible benzphetamine can be metabolized, whereas metabolism of rigid, planar molecules such as EFC and testosterone is hindered.

Arch Biochem Biophys, 2004 Mar 15, 423(2), 266 - 76
Mutagenesis and molecular dynamics suggest structural and functional roles for residues in the N-terminal portion of the cytochrome P450 2B1 I helix; Scott EE et al.; To investigate their potential roles in ligand access, binding, and subsequent metabolism, residues in the N-terminal portion of the cytochrome P450 2B1 I helix were mutated to alanine and phenylalanine . Of the 18 mutants from E286 to S294 only 7 yielded holoprotein in an Escherichia coli expression system . Substitutions at positions 289, 290, 292, and 294 caused >/= 2-fold changes in kcat and/or Km for two or more of the 2B1 substrates examined, testosterone, 7-ethoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and benzphetamine . I290 substitutions had the largest effects on steady-state parameters for three substrates and increased benzphetamine affinity . Steered molecular dynamics simulations of testosterone egress along the I helix identified hydrophobic interactions with I290, L293, and S294 and water bridges to E286 and S294 . Sensitivity of holoprotein formation to substitution and effects on substrate binding and metabolism suggest structural and functional roles for residues in the N-terminus of the cytochrome P450 2B1 I helix.

Arch Biochem Biophys, 2004 Mar 15, 423(2), 247 - 52
Specificity of the MAP kinase ERK2 for phosphorylation of tyrosine hydroxylase; Royo M et al.; Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of tyrosine hydroxylase . The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31 . As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of tyrosine hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized . Complete phosphorylation results in incorporation of 2mol of phosphate into each subunit of tyrosine hydroxylase . The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two . Phosphorylation of S8A tyrosine hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.

J Mol Biol, 2004 Mar 12, 337(1), 147 - 55
The mechanism of nucleic acid melting by a CspA family protein; Phadtare S et al.; Cold-shock proteins of the CspA family help bacterial cells to acclimate to low temperatures . Some Csps bind single-stranded nucleic acids and destabilize nucleic acid secondary structures in vitro, and act as transcription antiterminators in vivo and in vitro . Nucleic acid melting by Escherichia coli CspE is critical for its ability to support low-temperature survival of the cell . Here, we explore the molecular mechanism of nucleic acid melting using CspE mutants harboring substitutions in surface-exposed residues critical for this function . Analysis of the mutants identifies two intermediates of the melting pathway and shows that CspE Phe17 and Phe30 act at the earliest stages of melting, while His32 acts later and is necessary for the propagation of melting . The results allow us to orient a CspE molecule relative to the melting substrate and to put forward a mechanistic model of nucleic acid melting by Csps.

Microb Pathog, 2004 Apr, 36(4), 189 - 96
Escherichia coli Shiga toxin 1 and TNF-alpha induce cytokine release by human cerebral microvascular endothelial cells; Eisenhauer PB et al.; Infection with Shiga toxin (Stx)-producing Escherichia coli can lead to development of hemolytic uremic syndrome (HUS) . Patients with severe HUS often exhibit central nervous system (CNS) pathology, which is thought to involve damage to brain endothelium, a component of the blood-brain barrier . We hypothesized that this neuropathology occurs when cerebral endothelial cells of the blood-brain barrier, sensitized by exogenous TNF-alpha and stimulated by Stx1, produce and release proinflammatory cytokines . This was tested by measuring changes in cytokine mRNA and protein expression in human brain endothelial cells (hBEC) in vitro when challenged by TNF-alpha and/or Stx . High doses of Stx1 alone were somewhat cytotoxic to hBEC; Stx1-treated cells produced increased amounts of IL-6 mRNA and secreted this cytokine . IL-1beta and TNF-alpha mRNA, but not protein, were increased, and IL-8 secretion increased without an observed increase in mRNA . Cells pretreated with TNF-alpha were more sensitive to Stx1, displaying greater Stx1-induction of mRNA for TNF-alpha, IL-1beta, and IL-6, and secretion of IL-6 and IL-8 . These observations suggest that in the pathogenesis of HUS, Stx can induce cytokine release from hBEC, which may contribute toward the characteristic CNS neuropathology.

Acta Pharmacol Sin, 2004 Mar, 25(3), 372 - 7
cDNA cloning, sequence analysis, and recombinant expression of akitonin beta, a C-type lectin-like protein from Agkistrodon acutus; Zha XD et al.; AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production . METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template . The PCR products were cloned into the plasmid pGEM-T and sequenced . The deduced protein sequence was analyzed with some bioinformatic programs . A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells . RESULTS: A novel cDNA sequence encoding akitonin beta was found and accepted by GenBank (accession number AF387100) . Akitonin beta consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins . It was predicted to be a platelet antagonist . Upon induction with arabinose rAkitonin beta expressing in E coli was achieved at a high level (superior to 150 mg/L) . The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro . CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.

Acta Pharmacol Sin, 2004 Mar, 25(3), 306 - 12
Effects of phytoestrogen genistein on myocardial ischemia/reperfusion injury and apoptosis in rabbits; Ji ES et al.; AIM: To study the effect of genistein (GST) on rabbit heart ischemia/reperfusion (I/R) injury . METHODS: Rabbit heart I/R injury was induced by occluding the left anterior descending coronary artery for 45 min and reperfusing for 180 min . GST (1.0 mg/kg) was intravenously injected 5 min before heart ischemia . Hemodynamic data, infarct size, and cardiomyocytic apoptosis were measured . The pathologic changes of I/R myocardium were observed . RESULTS: During the I/R, heart rate, mean arterial blood pressure, myocardial oxygen consumption, left ventricular (LV) -dp/dtmax and +dp/dtmax were decreased progressively . The infarct size was occupied 60.23 %+/-3.97 % (% of area at risk) in vehicle +I/R group while GST reduced the infarct size to 39.62 %+/-4.30 % (P<0.01) . DNA ladder pattern in myocardium was revealed by agarose gel electrophoresis in vehicle +I/R group while was not found in GST+I/R group . Apoptotic cardiomyocytes were sparse within ischemic myocardium at risk in GST+I/R group as compared with that in vehicle +I/R group (TUNEL stain) . Apoptosis rate in ischemic myocardium from vehicle +I/R and GST+I/R groups detected by flow cytometry were 15.33 %+/-1.31 % and 3.88 %+/-0.33 %, respectively . Fas and Bax protein expressions in ischemic myocardium of vehicle +I/R group were higher than that in GST+I/R group (P<0.01) . Bcl-2/Bax ratio in vehicle +I/R group was lower than that in nonischemic myocardium (P<0.01), while in GST+I/R group, the Bcl-2/Bax ratio was higher than that in vehicle +I/R group (P<0.01) . CONCLUSION: GST reduced infarct size and apoptosis of myocytes in I/R rabbit heart.

Hybrid Hybridomics, 2004 Feb, 23(1), 19 - 22
Monoclonal antibody against mouse CAR following genetic immunization; Carson SD et al.; To broaden our repertoire of monoclonal antibodies against CAR (coxsackievirus and adenovirus receptor), we inoculated mice with an expression vector containing the cDNA encoding human CAR extracellular and transmembrane sequence, and boosted the response by inoculation with soluble human CAR protein produced in E . coli . Of the hybridomas obtained following this immunization protocol, one secreted IgG with exceptional reactivity against mouse CAR . Since CAR has been shown to form dimers, expression of human CAR in cells that express mouse CAR may have stimulated the host immune system to recognize endogenous CAR in heterodimers.

DNA Cell Biol, 2004 Feb, 23(2), 111 - 7
Human lectin-like oxidized low-density lipoprotein receptor-1 functions as a dimer in living cells; Xie Q et al.; Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a unique scavenger receptor that plays important roles in atherogenesis and has been thought to function as a monomer . Using coimmunoprecipitation studies, we demonstrate that human LOX-1 (hLOX-1) forms constitutive homo-interactions in vivo . Western blot analysis of cell lysates under nonreducing or reducing conditions revealed one clear immunoreactive species corresponding to the size of a putative receptor dimer or a monomer, respectively, consistent with the presence of disulfide-linked hLOX-1 complexes . Site-directed mutagenesis studies indicated that cysteine 140 has a key role in the formation of these disulfide-linked hLOX-1 dimers . Eliminating this intermolecular disulfide bond markedly impairs the recognition of Escherichia coli by hLOX-1 . Furthermore, these dimers can act as a "structural unit" to form noncovalently associated oligomers, as demonstrated by a membrane-impermeant crosslinker, which resulted in immunoreactive species corresponding to the sizes of putative tetramers and hexamers . These results provide the first evidence for the existence of hLOX-1 dimers/oligomers.

Biochemistry (Mosc), 2004 Feb, 69(2), 188 - 94
Mechanisms of protective functions of Escherichia coli polyamines against toxic effect of paraquat, which causes superoxide stress; Tkachenko AG; The toxic effect of paraquat, mainly caused by production of superoxide radicals, results in the induction of polyamine synthesizing enzymes and their products in cells of exponentially growing E . coli cultures . The activity of ornithine decarboxylase increases approximately twofold and the activity of lysine decarboxylase increases 1.4-fold . Unlike cadaverine, putrescine and spermidine stimulate expression of the soxRS regulon genes, and this depends on the polyamine concentration and is specific for different genes of the regulon . Of six genes studied, the maximum (to 130%), minimum (about 40%), and average (60-80%) stimulation was observed for the stress induction of nfo (endonuclease IV), sodA (superoxide dismutase), and the soxS gene of the transcriptional regulator, respectively . Addition of paraquat to exponentially growing E . coli culture results in oscillations of the topological state of DNA . Putrescine prevents the drop in the extent of DNA supercoiling caused by the damaging effect of free radicals during the first minutes of stress and increases it during the restoration (the peak of the transcriptional activity of the soxRS regulon genes) . These effects are due to properties of putrescine as a DNA protector and modulator of its topological state . The ability of putrescine to decrease the mutation rate under conditions of superoxide stress induced by addition of paraquat is shown by the example of rifampicin resistance.

Biotechnol Lett, 2004 Jan, 26(2), 121 - 5
Rational design of a protein-based molecular device consisting of blue fluorescent protein and zinc protoporphyrin IX incorporated into a cytochrome b562 scaffold; Takeda S et al.; To make a single molecular photo-device, it is essential to control the exact orientation of two types of proteins . We made a chimeric protein in which cytochrome b562 was linked to the N-terminus of enhanced green fluorescent protein, cytb562-EGFP . Within cytb562-EGFP, the excitation energy of EGFP was transferred to the cytochrome b562 cofactor fixed proximally to EGFP . Cytb562-EGFP was engineered so that iron protoporphyrin IX was substituted by zinc protoporphyrin IX to make it a suitable cofactor for photo-induced electron transfer . The photosensitizer pigment was optimized and the EGFP was replaced by a blue fluorescent mutant that gave 15% higher energy transfer efficiency.

Mol Plant Microbe Interact, 2004 Mar, 17(3), 235 - 44
The typA gene is required for stress adaptation as well as for symbiosis of Sinorhizobium meliloti 1021 with certain Medicago truncatula lines; Kiss E et al.; In this article, we describe the typA gene of Sinorhizobium meliloti, the orthologue of typA/bipA genes found in a wide range of bacteria . We found that typA was required for survival of S . meliloti under certain stress conditions, such as growth at low temperature or low pH and in the presence of sodium dodecyl sulfate (SDS) . The cold-sensitive phenotype of both Escherichia coli bipA and S . meliloti typA mutants were cross-complemented, indicating that the two genes are functionally equivalent . typA was indispensable for symbiosis on Medicago truncatula Jemalong and F83005.5 and contributes to the full efficiency of symbiosis on other host plant lines such as DZA315.16 or several cultivars of M . sativa . Hence, the symbiotic requirement for typA is host dependent . Interestingly, the symbiotic defect was different on Jemalong and F83005.5 plants, thus indicating that typA is required at a different stage of the symbiotic interaction.

Sens Actuators B Chem, 2004 Jan 1, 97(1), 81 - 9
Phosphate binding protein as the biorecognition element in a biosensor for phosphate; Salins LL et al.; This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi) . The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm . The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge . The Pi binding site is located deep within the cleft between the two domains . In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner . The resultant conformational change constitutes the basis of the sensor development . A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR) . The mutant was expressed, from plasmid pSD501, in the periplasmic space of E . coli and purified in a single chromatographic step on a perfusion anion-exchange column . Site-specific labeling was achieved by attaching the fluorophore, N-{2-(1-maleimidyl)ethyl}-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety . Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi . Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample . The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample . It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes . The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi . In all three cases the detection limits for the analyte were in the sub-microMolar range . It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins . c2003 Elsevier B.V . All rights reserved.

Clin Infect Dis, 2004 Mar 15, 38(6), e41 - 5 Epub 2004 Feb 27.
Asymptomatic bacteriuria in women with diabetes: influence of metabolic control; Bonadio M et al.; We screened 228 women with diabetes for bacteriuria during the period of January 1997 through December 2000 at Pisa General Hospital (Pisa, Italy) . A control group of 146 women without diabetes was also evaluated . The frequency of significant bacteriuria was 17.5% (40 of 228) among women with diabetes and 18.5% (27 of 146) among women in the control group . Seven (13.5%) of 52 and 33 (18.8%) of 176 women with type 1 and in type 2 diabetes, respectively, had significant bacteriuria . The presence of higher glycated hemoglobin levels was the only significant risk factor for significant bacteriuria in women with type 2 diabetes . A similar frequency of bacteriuria in women with and women without diabetes was found . Severe impairment of metabolic control of type 2 diabetes increases the risk of acquiring asymptomatic bacteriuria.

Nucleic Acids Res, 2004 Mar 03, 32(4), 1518 - 26 Print 2004.
Efficiency and pattern of UV pulse laser-induced RNA-RNA cross-linking in the ribosome; Shapkina T et al.; Escherichia coli ribosomes were irradiated with a KrF excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10-40 mJ for a 1 cm2 area, corresponding to fluences of 4.5 to 18 x 10(9) W m(-2), to determine strand breakage yields and the frequency and pattern of RNA-RNA cross- linking in the 16S rRNA . Samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample . The yield of strand breaks per photon was intensity dependent, with values of 0.7 to 1.3 x 10(-3) over the incident intensity range studied . The yield for RNA-RNA cross-linking was 3 x 10(-4) cross-links/photon at the intensity of 4.5 x 10(9) W m(-2), an approximately 4-fold higher yield per photon than obtained with a transilluminator . The cross-link yield/photon decreased at higher light intensities, probably due to intensity-dependent photoreversal . The pattern of cross-linking was similar to that observed with low intensity irradiation but with four additional long-range cross-links not previously seen in E.coli ribosomes . Cross- linking frequencies obtained with one laser pulse are more correlated to internucleotide distances than are frequencies obtained with transilluminator irradiation.

J Biochem (Tokyo), 2004 Jan, 135(1), 101 - 7
Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene; Hatakeyama T et al.; CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata . This lectin shows very high N-acetylgalactosamine-binding specificity . We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells . Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps . The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity . rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay . Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+) . Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures . Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity . These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I . Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I.

J Biochem (Tokyo), 2004 Jan, 135(1), 17 - 24
Mass spectrometry on segment-specific hydrogen exchange of dihydrofolate reductase; Yamamoto T et al.; To address the effects of local structures on structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the backbone-fluctuation map was determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) coupled with H/D exchange and pepsin digestion . H/D exchange kinetics was examined at 15 degrees C with 18 identified digestion fragments covering almost the entire amino acid sequence of DHFR . These fragments exhibited significant variations in the first-order rate constant of proton exchange, k(ex) (0.47-0.71 min(-1)), the fraction of deuterium incorporation at the initial stage, D(o) (0.20-0.60), the fraction of deuterium incorporation at infinite time, D(infinity) (0.75-0.97), and the number of protons protected from exchange, P (0.4-4.7), relative to the corresponding values for the whole DHFR molecule (k(ex) = 0.51 min(-1), D(o) = 0.41, D(infinity) = 0.85, and P = 20.7) . H/D exchange was very fast in the fragment comprising residues 5-28 (Met20 loop), which participates in substrate uptake, and reasonably fast in disordered and hydrophobic fragments, but slow in beta-strand-rich fragments . These results indicate that the local structures contribute differently to the fluctuation of the DHFR molecule, and that mass spectrometry coupled with H/D exchange and protease digestion is a useful tool for detecting segment-dependent protein fluctuation.

J Biol Chem, 2004 Apr 30, 279(18), 18107 - 10 Epub 2004 Mar 03.
Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold; Kaya Y et al.; TruD, a recently discovered novel pseudouridine synthase in Escherichia coli, is responsible for modifying uridine13 in tRNA(Glu) to pseudouridine . It has little sequence homology with the other 10 pseudouridine synthases in E . coli which themselves have been grouped into four related protein families . Crystal structure determination of TruD revealed a two domain structure consisting of a catalytic domain that differs in sequence but is structurally very similar to the catalytic domain of other pseudouridine synthases and a second large domain (149 amino acids, 43% of total) with a novel alpha/beta fold that up to now has not been found in any other protein.

J Biol Chem, 2004 May 7, 279(19), 20501 - 10 Epub 2004 Mar 03.
Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236; Gruber K et al.; The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily . Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme . We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure . Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme . However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex . Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH . All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e . by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base . These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues . Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.

Hum Reprod, 2004 Mar, 19(3), 715 - 22 Epub 2004 Jan 29.
Blocking of the placental immune-modulatory ferritin activates Th1 type cytokines and affects placenta development, fetal growth and the pregnancy outcome; Nahum R et al.; BACKGROUND: Placenta immunomodulatory ferritin (PLIF) cDNA was recently cloned from the human placenta, where it is expressed in syncytiotrophoblast and decidual mononuclear cells . PLIF and its subcloned bioactive domain (C48), expressed in Escherichia coli, are immunosuppressive proteins and induce pronounced IL-10 production in vitro and in vivo . METHODS AND RESULTS: PLIF serum level, measured by enzyme-linked immunosorbent assay, was elevated in pregnant mice throughout gestation and declined towards delivery . Blocking of PLIF activity by vaccination of mice with C48 prior to mating inhibited pregnancy development . Passive transfer of anti-C48 immunoglobulin (Ig) starting at 3.5-12.5 days post coitum (dpc) resulted in high rate of embryo resorption . Furthermore, treatment with anti-C48 Ig resulted in placental and embryonal growth restriction . At gestation day 13.5, growth retardation was especially notable in the placentae, while at 16.5 dpc it was pronounced in the embryos . Histopathological examination revealed that experimental placentae were globally hypoplastic and the labyrinth was strikingly pale and contained less maternal blood compared with control . Immune-activated spleen cells harvested at 13.5 dpc from anti-C48 Ig-treated pregnant mice secreted in vitro increased level of Th1 cytokines (IL-2, TNF-alpha, IL-12) and decreased level of Th2 cytokines (IL-10, IL-4, IL-5, IL-6) as compared with the level of the respective cytokines secreted by spleen cells from control pregnant mice . CONCLUSION: This study provides the first in vivo evidence that PLIF plays a major role in placentation and embryonic growth.

J Proteome Res, 2004 Jan-Feb, 3(1), 120 - 5
Proteomics approach to identifying ATP-covalently modified proteins; Besant PG et al.; This study aims to investigate functionally similar proteins based on their capacity to remain bound to ATP under stringent resolving conditions . Using two-dimensional gel electrophoresis and capillary liquid chromatography on-line mass spectrometry, we have identified several mammalian and E . coli proteins that appear to covalently bind ATP . To validate this approach, we obtained commercially purified forms of proteins identified from two-dimensional protein maps and tested their capacity to bind alpha 32P phosphate labeled ATP . This proteomics approach provides an initial screening method of identifying functionally similar proteins for further scrutiny by a more traditional analysis.

J Proteome Res, 2004 Jan-Feb, 3(1), 112 - 9
Gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification; Cargile BJ et al.; Here we present the theoretical and experimental evaluation of peptide isoelectric point as a method to aid in the identification of peptides from complex mixtures . Predicted pI values were found to match closely the experimentally obtained data, resulting in the development of a unique filter that lowers the effective false positive rate for peptide identification . Due to the reduction of the false positive rate, the cross-correlation parameters Xcorr and deltaCn from the SEQUEST program can be lowered resulting in 25% more peptide identifications . This approach was successfully applied to analysis of the soluble fraction of the E . coli proteome, where 417 proteins were identified from 1022 peptides using just 20 microg of material.

Rev Esp Anestesiol Reanim, 2004 Jan, 51(1), 44 - 6
{Vertebral osteomyelitis and epidural abscess after epidural anesthesia for a cesarean section}; Veiga Sanchez AR; A 40-year-old woman underwent cesarean section under epidural anesthesia . The anesthetic procedure was carried out in strict aseptic conditions, the catheter was withdrawn 24 hours after surgery, and the patient was discharged 5 days after surgery . She was readmitted with fever, backache, and pain in the lower limbs, with signs of radiculitis but no indication of inflammation or pain at the site of puncture . Magnetic resonance imaging revealed vertebral osteomyelitis at the fifth lumbar and first sacral vertebrae and an epidural abscess with compression of the nerve root . Treatment consisted of 2 g of ceftriaxone daily for 6 weeks, rest, and measures to assure local immobilization . Symptoms gradually improved and no surgical drainage measures were needed . The cause of osteomyelitis was never ascertained . Vertebral osteomyelitis is an unusual event after epidural anesthesia and there have been few opportunities to demonstrate a relationship . Such infections appear spontaneously in immunodepressed patients who undergo diagnostic procedures and treatments that lead to bacteremias with secondary colonization of spinal structures . The topography and characteristics of the infectious lesion, the patient's susceptibility, and the anesthetic procedure and pathogenic agent may help clarify the cause of the osteomyelitis.

Proteomics, 2004 Mar, 4(3), 599 - 608
Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-to-background ratio and improved baseline resolution; Lamanda A et al.; In proteomics the ability to visualize proteins from electropherograms is essential . Here a new protocol for staining and destaining gels treated with Ruthenium II tris (bathophenantroline disulfonate) is presented . The method is compared with the silver-staining procedure of Swain and Ross, the Ruthenium II tris (bathophenantroline disulfonate) stain described by Rabilloud (Rabilloud T., Strub, S . M . Luche, S., Girardet, S . L . et al., Proteomics 2001, 1, 699-704) and the SYPRO Ruby gel stain . The method offers a better signal-to-background ratio with improved baseline resolution for both sodium dodecyl sulfate-polyacrylamide gels and two-dimensional gels.

Biopolymers, 2004, 76(1), 21 - 6
Incorporation of anthraquinonyl group into lambda-Cro repressor protein for strand- and position-specific photocleavage of double-stranded DNA; Sasaki H et al.; lambda-Cro repressor protein incorporated with a 2-anthraquinonylalanine (anqAla) at the 64th position was chemically synthesized by solid-phase method . The 64th position was selected according to previous information on various mutants of Cro incorporated with a single anqAla unit, that were synthesized through an Escherichia coli in vitro protein-synthesizing system . The 64anqAla mutant bound to a dsDNA of consensus operator sequence and underwent strand- and position-specific photocleavage of the dsDNA at the GG sequence after treatment with piperidine . The mutant also underwent position-specific self-photoscission . The self-photoscission was retarded in the presence of the dsDNA .

Br J Cancer, 2004 Mar 8, 90(5), 1084 - 92
2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT; Helsby NA et al.; An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours . Activation of the aziridinyl dinitrobenzamide CB 1954 by E . coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite . We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines) . The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines . Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line . These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours . The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites . These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

J Biol Chem, 2004 May 14, 279(20), 20836 - 49 Epub 2004 Mar 02.
Structural characterization of the SARS-coronavirus spike S fusion protein core; Tripet B et al.; The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells . It is a type 1 viral fusion protein that characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein . Previous studies have shown that the two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers), which mediates fusion of viral and host cell membranes . Here we describe the biophysical analysis of the two predicted heptad repeat regions within the severe acute respiratory syndrome coronavirus S protein . Our results show that in isolation the HR-N region forms a stable alpha-helical coiled coil that associates in a tetrameric state . The HR-C region in isolation formed a weakly stable trimeric coiled coil . When mixed together, the two peptide regions (HR-N and HR-C) associated to form a very stable alpha-helical 6-stranded structure (trimer of heterodimers) . Systematic peptide mapping showed that the site of interaction between the HR-N and HR-C regions is between residues 916-950 of HR-N and residues 1151-1185 of HR-C . Additionally, interchain disulfide bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel . Overall, the structure of the hetero-stranded complex is consistent with the structures observed for other type 1 viral fusion proteins in their fusion-competent state.

J Biol Chem, 2004 May 7, 279(19), 20002 - 8 Epub 2004 Mar 02.
Unique features of plant mitochondrial sulfhydryl oxidase; Levitan A et al.; The yeast and human mitochondrial sulfhydryl oxidases of the Erv1/Alr family have been shown to be essential for the biogenesis of mitochondria and the cytosolic iron sulfur cluster assembly . In this study we identified a likely candidate for the first mitochondrial flavin-linked sulfhydryl oxidase of the Erv1-type from a photosynthetic organism . The central core of the plant enzyme (AtErv1) exhibits all of the characteristic features of the Erv1/Alr protein family, including a redox-active YPCXXC motif, noncovalently bound FAD, and sulfhydryl oxidase activity . Transient expression of fusion proteins of AtErv1 and the green fluorescence protein in plant protoplasts showed that the plant enzyme preferentially localizes to the mitochondria . Yet AtErv1 has several unique features, such as the presence of a CXXXXC motif in its carboxyl-terminal domain and the absence of an amino-terminally localized cysteine pair common to yeast and human Erv1/Alr proteins . In addition, the dimerization of AtErv1 is not mediated by its amino terminus but by its unique CXXXXC motif . In vitro assays with purified protein and artificial substrates demonstrate a preference of AtErv1 for dithiols with a defined space between the thiol groups, suggesting a thioredoxin-like substrate.

J Bacteriol, 2004 Mar, 186(6), 1879 - 89
DNA interaction and phosphotransfer of the C4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli; Abo-Amer AE et al.; The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C(4)-dicarboxylates and citrate . The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C(4)-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function . For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase . The two kinase-domain-containing fragments were autophosphorylated by {gamma-(32)P}ATP . The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C(4)-dicarboxylate sensing . Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly . No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins . The DNA-binding specificity of DcuR was studied by use of the pure protein . It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize . DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 micro M for untreated DcuR and < or =1 to 2 microM for the acetylphosphate-treated form . The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif {tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences} to be identified . The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

J Bacteriol, 2004 Mar, 186(6), 1861 - 8
Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site; Song L et al.; The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays . The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of alpha-helix 5 that forms a coiled-coil motif . To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue alpha-helix 5 of MerR . The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro . In vitro MBD bound ca . 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR . Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR . In vivo, cells overexpressing MBD accumulated 70 to 100% more (203)Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth . Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo . We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step.

J Bacteriol, 2004 Mar, 186(6), 1720 - 8
Regulation of expression of the divergent ulaG and ulaABCDEF operons involved in LaAscorbate dissimilation in Escherichia coli; Campos E et al.; The ula regulon, responsible for the utilization of L-ascorbate in Escherichia coli, is formed by two divergently transcribed operons, ulaG and ulaABCDEF . The regulon is negatively regulated by a repressor of the DeoR family which is encoded by the constitutive gene ulaR located downstream of ulaG . Full repression of the ula regulon requires simultaneous interaction of the repressor with both divergent promoters and seems to be dependent on repressor-mediated DNA loop formation, which is helped by the action of integration host factor . Two operator sites have been identified in each promoter . Lack of either of the two sets of operators partially relieved the repression of the other operon; thus, each promoter is dependent on the UlaR operator sites of the other promoter to enhance repression . Electrophoretic mobility shift assays with purified UlaR protein and promoter deletion analyses revealed a conserved sequence, present in each of the four operators, acting as a UlaR binding site . Glucose represses the ula regulon via at least two mechanisms, one dependent on cyclic AMP (cAMP)-cAMP receptor protein (CRP) and the other (possibly inducer exclusion) independent of it . Glucose effects mediated by other global regulators cannot be ruled out with the present information . Changes in cAMP-CRP levels affected only the expression of the ulaABCDEF operon.

Int Immunopharmacol, 2004 Feb, 4(2), 223 - 34
Chloroquine protects mice from challenge with CpG ODN and LPS by decreasing proinflammatory cytokine release; Hong Z et al.; Bacterial DNA (bDNA) and lipopolysaccharide (LPS) are potent activators of immune cells such as monocytes and macrophages, which contribute to systemic inflammatory response syndrome (SIRS) and sepsis . To date, no effective anti-sepsis drugs have been developed for clinical use . Chloroquine (CQ), a diprotic weak base traditionally used for treating malaria, was recently shown to decrease cytokine release from macrophages induced by LPS and CpG oligonucleotide (CpG ODN) . In the present study, Escherichia coli DNA (EC DNA), CpG ODN and LPS were used to induce SIRS/sepsis in animal models . We found that 30 mg/kg of CQ could protect mice from lethal challenge by CpG ODN and LPS, and 25 mg/kg of CQ could decrease serum TNF-alpha and IL-6 in rats injected with sublethal doses of CpG ODN and LPS . In addition, treatment of murine macrophage ANA-1 cells with 2 mM CQ potently inhibited the release of TNF-alpha, IL-6 and IL-12 induced by CpG ODN and LPS . In human peripheral blood mononuclear cells (hPBMC), 100-200 microM CQ almost completely abrogated release of both TNF-alpha and IL-6 induced by CpG ODN and LPS, whereas IL-6 release induced by EC DNA was not significantly affected by 50 microM CQ . Furthermore, CQ reduced the expression of TLR9 and TLR4 mRNA and the activation of NFkappaB and AP-1 stimulated by CpG ODN and LPS in ANA-1 cells . Flow cytometry and confocal microscopy revealed that CQ increased the accumulation of CpG ODN within ANA-1 cells without influence on its uptake, suggesting that the delayed degradation of CpG ODN was associated with the reduction of proinflammatory cytokine release from the cells . Our results demonstrated that CQ-mediated protection of lethal challenge by CpG ODN and LPS was associated with the reduction of proinflammatory cytokine release.

Plant J, 2004 Mar, 37(6), 828 - 38
The poplar K+ channel KPT1 is associated with K+ uptake during stomatal opening and bud development; Langer K et al.; To gain insights into the performance of poplar guard cells, we have measured stomatal conductance and aperture, guard cell K+ content and K+-channel activity of the guard cell plasma membrane in intact poplar leaves . In contrast to Arabidopsis, broad bean and tobacco grown under same conditions, poplar stomata operated just in the dynamic range - any change in conductance altered the rate of photosynthesis . In response to light, CO2 and abscisic acid (ABA), the stomatal opening velocity was two to five times faster than that measured for Arabidopsis thaliana, Nicotiana tabacum and Vicia faba . When stomata opened, the K+ content of guard cells increased almost twofold, indicating that the very fast stomatal opening in this species is mediated via potassium uptake . Following impalement of single guard cells embedded in their natural environment of intact leaves with triple-barrelled microelectrodes, time-dependent inward and outward-rectifying K+-channel-mediated currents of large amplitude were recorded . To analyse the molecular nature of genes encoding guard cell K+-uptake channels, we cloned K+-transporter Populustremula (KPT)1 and functionally expressed this potassium channel in a K+-uptake-deficient Escherichia coli mutant . In addition to guard cells, this K+-transporter gene was expressed in buds, where the KPT1 gene activity strongly correlated with bud break . Thus, KPT1 represents one of only few poplar genes associated with bud flush.

Acta Anaesthesiol Scand, 2004 Feb, 48(2), 198 - 204
The angiotensin II receptor blocker candesartan improves survival and mesenteric perfusion in an acute porcine endotoxin model; Laesser M et al.; BACKGROUND: Blockade of the angiotensin II type 1 (AT1) receptor has been demonstrated to ameliorate splanchnic hypoperfusion in acute experimental circulatory failure . This study focused on hemodynamic changes and survival in pigs treated with AT1 blockade prior to or during acute endotoxinemia . METHODS: Escherichia coli lipopolysaccharide endotoxin was infused in anesthetized and mechanically ventilated pigs . Systemic, renal, mesenteric and jejunal mucosal perfusion as well as systemic oxygen and acid-base balance were monitored . The selective AT1 receptor blocker candesartan was administered prior to as well as during endotoxinemia . Control animals received the saline vehicle . RESULTS: Pre-treatment with candesartan resulted in higher survival rate (83%, 10 out of 12 animals) compared with 50% (6 of 12) in control animals and 27% (3 of 11) in animals treated during endotoxinemia . Pre-treatment with candesartan resulted in higher cardiac output, mixed venous oxygen saturation, arterial standard base-excess, portal venous blood flow during endotoxin infusion compared with controls and animals treated during endotoxinemia . No adverse effects were found on neither systemic nor renal circulation . CONCLUSION: The favorable results of AT1 receptor blockade prior to endotoxinemia are lost when blockade is established during endotoxinemia demonstrating the importance of the renin-angiotensin system and its dynamic involvement in acute endotoxinemic shock.

J Am Chem Soc, 2004 Mar 10, 126(9), 2963 - 70
Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins; Pintacuda G et al.; A novel strategy for fast NMR resonance assignment of (15)N HSQC spectra of proteins is presented . It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively (15)N-labeled samples . Comparison of sensitive undecoupled (15)N HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling . Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center . The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia coli DNA polymerase III . The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.

J Am Chem Soc, 2004 Mar 10, 126(9), 2720 - 6
Manganese(II) zero-field interaction in cambialistic and manganese superoxide dismutases and its relationship to the structure of the metal binding site; Un S et al.; The Mn(II) high-magnetic-field electron paramagnetic resonance (HFEPR) spectra of five different superoxide dismutases (SODs) were measured at 190 and 285 GHz . The native E . coli manganese SOD was found to be distinct from the other SODs by virtue of its large zero-field E-value . The two wild-type cambialistic proteins from Porphyromonas gingivalis and Rhodobacter capsulatus were also distinct . However, the Gly155Thr mutant of the P . gingivalis SOD changed the Mn(II) spectrum so that it closely resembled the spectrum of manganese reconstituted E . coli iron SOD . This observation paralleled enzyme activity measurements that show that this mutation causes the loss of activity with manganese and enhanced activity with iron indicating a conversion from a cambialistic to an iron-specific protein . The Mn(II) magnetic parameters were determined by simultaneously fitting the multifrequency data . Simulations were carried out by numerically diagonalizing the spin Hamiltonian and explicitly calculating all possible transition probabilities . The relationship between the Mn(II) zero-field interaction and structure of the metal binding site is also discussed.

J Agric Food Chem, 2004 Mar 10, 52(5), 1350 - 6
Cloning, functional expression, and characterization of cystatin in sesame seed; Shyu DJ et al.; A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds . The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli . The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay . The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin . Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K(i) values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively . Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination . Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds . It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination.

Cell Mol Biol (Noisy-le-grand), 2003, 49 Online Pub, OL453 - 9
Structural characterization of T-protein of the Escherichia coli glycine cleavage system by X-ray small angle scattering; Orun O et al.; T-protein, one of the components of the glycine cleavage complex, catalyses the formation of ammonia and methylene-tetrahydrofolate from H-protein-bound intermediate . Native T-protein of the glycine cleavage system from E . coli was efficiently purified using a combination of hydrophobic interaction, gel permeation and ion exchange chromatography . Synchrotron radiation small angle X-ray solution scattering indicates that T-protein has an extended structure in solution . A low resolution model of the protein was constructed ab initio and tentative models of the tertiary structure were built using prediction methods constrained by the scattering data.

J Environ Pathol Toxicol Oncol, 2004, 23(1), 33 - 43
Radiation-induced DNA damage: formation, measurement, and biochemical features; Cadet J et al.; Most of the reactions induced by *OH radicals (indirect effects) and by one-electron oxidation (direct effects) as the result of exposure to ionizing radiation may be described for the four main DNA nucleobases . Relevant information is now available on the formation of single and tandem base lesions implicating guanine as the most susceptible DNA component to the deleterious effects of ionizing radiation . In contrast, there is still a paucity of information on the radiation-induced formation of base damage within cellular DNA . This is mostly a result of difficulties associated with the measurement of oxidized purine and pyrimidine bases that appear to be generated in very low yields . This is illustrated by the measurement of low amounts of E . coli formamidopyrimidine glycosylase- and endonuclease-III-sensitive sites in the DNA of neoplastic monocytes upon exposure to gamma rays (48 and 53 per 10(9) bases and per Gy, respectively) using a modified comet assay (the overall number of strand breaks and alkali-labile sites was estimated to be 130 per 10(9) bases and per Gy) . More specifically, the level of several radiation-induced modified bases, including thymine glycols, 5-formyluracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroadenine, together with related formamidopyrimidine derivatives was assessed using the suitable HPLC-MS/MS method . Information is also provided on the substrate specificity of DNA repair enzymes and the mutagenic potential of base lesions using site-specific modified oligonucleotides as the probes.

Biol Pharm Bull, 2004 Mar, 27(3), 303 - 7
Efficient expression of modified human papillomavirus 16 e6/e7 fusion protein and the antitumor efficacy in a mouse model; Zhou X et al.; Infection with human papillomavirus, particularly type 16 (HPV16), is highly associated with the development of cervical intraepithelial neoplasia and cervical cancer . The two early viral oncogenes, E6 and E7, are selectively retained and constitutively expressed in tumor cells and are therefore attractive immunotherapeutic targets . Thus a vaccine strategy based on recombinant HPV16 E6/E7 fusion protein represents an efficient approach against HPV16-associated tumors . Although the expression level of HPV16 E6/E7 fusion protein was presumed to be low, direct experimental proof in vivo was lacking . To enhance the expression level and investigate its antitumor efficacy in vivo, we constructed a modified HPV16 E6/E7 fusion gene with three point-mutations and expressed it in Escherichia coli . The encoded protein, denoted mE6(1-120)/mE7(1-60), comprises 120 N-terminus amino acids of E6 and 60 N-terminus amino acids of E7 plus a histine tag, was purified on an affinity column, and subsequently characterized by Western blotting . Immunization of mice with mE6(1-120)/mE7(1-60) completely protected them against subsequent challenge and rechallenge with TC-1 tumor cells expressing HPV16 E6 and E7 proteins . In the therapeutic experiments, most mice eliminated the preexisting tumors and had a long-term protection . Consistent with the results of in vivo experiments, the splenocytes from immunized mice elicited cytotoxic T lymphocytes and specifically lysed TC-1 cells in vitro . More importantly, the expression level of mE6(1-120)/mE7(1-60) was significantly improved, meeting the necessary quantity required for a vaccine clinical trial . In conclusion, these data provide a scientific basis for the use of modified mE6(1-120)/mE7(1-60) in future human trials.

Chem Pharm Bull (Tokyo), 2004 Mar, 52(3), 377 - 9
Hydrophobic scale: a second parameter to elucidating various specific ligand-protein interactions; Numao N et al.; In the sequence Fourier analysis (SFA) of specific interactions such as those between fibroblast growth factors (FGFs)/FGF receptors (FGFRs), bone morphogenetic proteins (BMPs)/BMP receptors (BMPRs), or tumor repressor protein p53/mouse double minute 2 homolog (MDM2), the characteristic frequency peak(s) could be observed with the hydrophobic scale for 20 amino acids as well as 4 nucleotides as the physicochemical parameter, but not successfully with the absolute electronegativity scale . This result implies that these two independent scales should be appropriately selected in various specific ligand-protein interactions, though the critical difference has to be determined.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 561 - 3 Epub 2004 Feb 25.
Crystallization and preliminary crystallographic analysis of the Escherichia coli water channel AqpZ; Daniels BV et al.; AqpZ is a 24 kDa integral membrane protein that facilitates water movement across the plasma membrane of Escherichia coli . In this study, the first crystallization and preliminary X-ray analysis of AqpZ are described . AqpZ was overexpressed and purified with a yield of 13 mg of purified AqpZ per litre of cell culture . The purified AqpZ was shown to be a monodisperse species consisting of tetrameric protein-detergent complexes . A crystallization condition for producing diffraction-quality crystals was identified . Initial X-ray analysis indicated that the diffraction limit of AqpZ extended to 3.6 A . Crystals were found to belong to space groups P4(1)22 or P4(3)22, with unit-cell parameters a = b = 119.04, c = 380.23 A.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 558 - 60 Epub 2004 Feb 25.
Crystallization and preliminary X-ray diffraction analysis of Wza outer-membrane lipoprotein from Escherichia coli serotype O9a:K30; Beis K et al.; A novel integral membrane lipoprotein, Wza, from Escherichia coli serotype O9a:K30 has been purified and crystallized . Wza is required for the surface expression of the serotype K30 group 1 capsular polysaccharide of E . coli; closely related homologues are found in other bacteria that produce extracellular polysaccharides . The Wza crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 94.6, b = 215.5, c = 218.5 A . A data set to 3.0 A with 99.8% completeness and an R(merge) of 10.5% has been collected from a single crystal.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 534 - 6 Epub 2004 Feb 25.
Expression, purification and preliminary crystallographic analysis of phosphoribosyl isomerase (PriA) from Streptomyces coelicolor; Wright H et al.; The priA gene encoding the enzyme phosphoribosyl isomerase from Streptomyces coelicolor, a novel bifunctional enzyme involved in both histidine and tryptophan biosynthesis, was heterologously expressed and purified in Escherichia coli as an N-terminal His-tag fusion . The purified recombinant enzyme was crystallized using the hanging-drop method in 1.50 M ammonium sulfate and 100 mM sodium citrate pH 4.8 . Crystals were obtained of up to 0.05 x 0.05 x 0.3 mm in size . A full data set to 2 A resolution was collected at the ESRF beamline ID14-1 and space group P3(1,2)21 was assigned, with unit-cell parameters a = 65.1, c = 104.7 A.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 531 - 3 Epub 2004 Feb 25.
Crystallization and preliminary X-ray analysis of Aes, an acetyl-esterase from Escherichia coli; Gerber K et al.; Aes belongs to the family of hormone-sensitive lipases and has acetyl-esterase activity . It is also known to control maltose uptake through interaction with MalT, the central regulator of the Escherichia coli maltose system . Aes was crystallized as an N-terminally His(6)-tagged protein both in the native form and with selenomethionine substitution . Crystals grew in both cases in space group R32 to dimensions of about 0.2 x 0.15 x 0.05 mm (native His(6)-Aes) and about 0.5 x 0.3 x 0.1 mm (SeMet-His(6)-Aes) . A native data set has been obtained at 2.4 A resolution; the selenomethionine-substituted Aes crystals diffracted to 3.0 A resolution.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 524 - 7 Epub 2004 Feb 25.
Cloning, purification, crystallization and X-ray analysis of the Escherichia coli pyrimidine nucleoside hydrolase YeiK; Giabbai B et al.; The E . coli yeiK gene product is homologous to members of the nucleoside hydrolase family of enzymes, the physiological role of which in bacteria is still unknown . Here, the cloning, expression in milligram quantities and enzymatic characterization of YeiK as a pyrimidine-specific nucleoside hydrolase is reported . Crystals of YeiK diffract X-rays to a resolution of 1.7 A and belong to the triclinic crystal system in space group P1, with unit-cell parameters a = 44.81, b = 85.71, c = 90.68 A, alpha = 112.95, beta = 101.95, gamma = 85.92 degrees.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 507 - 11 Epub 2004 Feb 25.
Structure of Escherichia coli YfdW, a type III CoA transferase; Gogos A et al.; Crystal structures are reported for free and coenzyme A (CoA) bound forms of the YfdW protein from Escherichia coli, a representative type III CoA transferase . The structures reveal a two-domain protomer with interdomain connections forming a ring-like structure with a large central hole . Two protomers associate to form a highly intertwined dimer in which the hole of each ring is filled by the partner molecule . Each protomer binds a single CoA molecule and these CoA-binding sites are distant from one another in the dimer.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 432 - 8 Epub 2004 Feb 25.
Likelihood-enhanced fast rotation functions; Storoni LC et al.; Experiences with the molecular-replacement program Beast have shown that maximum-likelihood rotation targets are more sensitive to the correct orientation than traditional targets . However, this comes at a high computational cost: brute-force rotation searches can take hours or even days of computation time on current desktop computers . Series approximations to the full likelihood target have been developed that can be computed by fast Fourier transforms in minutes . These likelihood-enhanced targets are more sensitive to the correct orientation than the Crowther fast rotation function and they take advantage of information from partial solutions . The likelihood-enhanced rotation targets have been implemented in the program Phaser.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 412 - 21 Epub 2004 Feb 25.
The role of solvent transport in cryo-annealing of macromolecular crystals; Juers DH et al.; Macromolecular crystals are usually cooled to approximately 100 K for X-ray diffraction experiments in order to diminish lattice damage arising from the ionizing radiation . Such cooling often produces lattice disorder, but this disorder can sometimes be substantially reduced by cycling the crystal between low and higher temperatures (called annealing) . Here, two related aspects of cryocooling and annealing are investigated using crystals of beta-galactosidase and thermolysin . Firstly, as has been reported with other systems, there is an optimal cryoprotectant concentration above and below which diffraction is poor, with high mosaicity, diffuse scatter and low signal to noise . Measurements of the bulk density of the respective cryosolvents are consistent with the idea that at the optimal cryoprotectant concentration the contraction of the bulk solvent on cooling largely compensates for the contraction of the macromolecular lattice . Secondly, by controlling the relative humidity of the gas that contacts the crystal during the high (room) temperature phase, it is found that water is either imported into or exported out of the crystals during the melting phase of annealing . This water transport appears to change the concentration of the cryoprotectant solution and in so doing alters its thermal contraction . Thus, annealing appears to be involved, at least in part, in the tuning of the thermal contraction of the bulk solvent to best compensate for lattice contraction . Furthermore, it is found that if the cryoprotectant concentration is initially too high then annealing is more successful than if the concentration is initially too low . This result suggests that the search for optimal cryoprotectant conditions may be facilitated by equilibration of the crystal to relatively high cryoprotectant concentration followed by annealing.

J Gen Virol, 2004 Mar, 85(Pt 3), 761 - 8
RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus; Herranz MC et al.; The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport . In this study, putative RNA-binding properties of the PNRSV MP were studied . The PNRSV MP was produced in Escherichia coli using an expression vector . Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity . Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio . The different responses of the complexes to urea treatment strongly suggested that they have different structural properties . Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88 . This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated . This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates . Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships . The evolutionary implications of this observation are discussed.

J Gen Virol, 2004 Mar, 85(Pt 3), 687 - 91
Cellular casein kinase II-mediated phosphorylation of rinderpest virus P protein is a prerequisite for its role in replication/transcription of the genome; Kaushik R et al.; Phosphoprotein P of rinderpest virus (RPV), when expressed in E . coli, is present in the unphosphorylated form . Bacterially expressed P protein was phosphorylated by a eukaryotic cellular extract, and casein kinase II (CK II) was identified as the cellular kinase involved in phosphorylation . In vitro phosphorylation of P-deletion mutants identified the N terminus as a phosphorylation domain . In vivo phosphorylation of single or multiple serine mutants of P protein identified serine residues at 49, 88 and 151 as phospho-acceptor residues . The role of P protein phosphorylation in virus replication/transcription was evaluated using the RPV minigenome system and replication/transcription of a reporter gene in vivo . P protein phosphorylation was shown to be essential for in vivo replication/transcription since phosphorylation-null mutants do not support expression of a reporter gene . Transfection of increased amounts of phosphorylation-null mutant did not support minigenome replication/transcription in vivo.

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3462 - 7 Epub 2004 Mar 01.
Attractant binding alters arrangement of chemoreceptor dimers within its cluster at a cell pole; Homma M et al.; Many sensory systems involve multiple steps of signal amplification to produce a significant response . One such mechanism may be the clustering of transmembrane receptors . In bacterial chemotaxis, where a stoichiometric His-Asp phosphorelay from the kinase CheA to the response regulator CheY plays a central role, the chemoreceptors (methyl-accepting chemotaxis proteins) cluster together with CheA and the adaptor CheW, at a pole of a rod-shaped cell . This clustering led to a proposal that signal amplification occurs through an interaction between chemoreceptor homodimers . Here, by using in vivo disulfide crosslinking assays, we examined an interdimer interaction of the aspartate chemoreceptor (Tar) . Two cysteine residues were introduced into Tar: one at the subunit interface and the other at the external surface of the dimer . Crosslinked dimers and higher oligomers (especially a deduced hexamer) were detected and their abundance depended on CheA and CheW . The ligand aspartate significantly reduced the amounts of higher oligomers but did not affect the polar localization of Tar-GFP . Thus, the binding of aspartate alters the rate of collisions between Tar dimers in assembled signaling complexes, most likely due to a change in the relative positions or trajectories of the dimers . These collisions could occur within a trimer-ofdimers predicted by crystallography, or between such trimers . These results are consistent with the proposal that the interaction of chemoreceptor dimers is involved in signal transduction.

Microbiology, 2004 Mar, 150(Pt 3), 687 - 95
Temperature-dependent processing of the cspA mRNA in Rhodobacter capsulatus; Jager S et al.; The expression of genes for cold-shock proteins is proposed to be regulated primarily at the post-transcriptional level by increase of mRNA stability after transition to low temperatures . Destabilization of the Escherichia coli cold-induced cspA transcript at 37 degrees C as well as stabilization upon cold shock is known to depend on the unusually long (159 nt) 5'-untranslated region . Determination of the cspA mRNA 5'-end from Rhodobacter capsulatus revealed a shorter distance between the start of transcription and the start codon for translation . The cspA mRNA of R . capsulatus was shown to be stabilized at low temperatures to a greater extent than other investigated transcripts . To address the mechanism of decay of the cspA transcript, it was incubated with purified degradosome of R . capsulatus . Endoribonucleolytic in vitro cleavage in the 5'-untranslated region as reported for the cspA transcript of E . coli in vivo was not observed . Instead, the data indicated that the cspA mRNA decay in R . capsulatus is mediated by endoribonucleolytic cleavages within the cspA coding region.

Microbiology, 2004 Mar, 150(Pt 3), 527 - 38
Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin; Cleary J et al.; Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic 'attaching and effacing' (A/E) lesions on small intestinal enterocytes . EPEC adhesins that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and intimin . BFP are involved in bacteria-bacteria interaction and microcolony formation but their role in cell adhesion remains unclear; EspA filaments are components of the EPEC type III secretion system but since they interact directly with host cells they may also function as adhesins; intimin is the well characterized intimate EPEC adhesin which binds the translocated intimin receptor, Tir . However, other uncharacterized host cell receptors have been implicated in intimin-mediated adhesion . In this study, the role of BFP, EspA filaments and intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in bfpA, espA and eae (intimin gene) to differentiated human intestinal Caco-2 cells . E2348/69 (bfpA(+) espA(+) eae(+)) adhered rapidly (<10 min) to the brush border of Caco-2 cells and subsequently produced microcolonies and typical A/E lesions . Non-intimate brush border adhesion of double mutant strain UMD880 (bfpA(+) espA(-) eae(-)) also occurred rapidly, whereas adhesion of strain UMD886 (bfpA(-) espA(+) eae(-)) occurred later in the infection (>1 h) and with much lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively . Strain UMD883 (bfpA(-) espA(-) eae(+)), which is unable to translocate Tir, was non-adherent although this strain was able to form intimate attachment and A/E lesions when co-cultured with strain CVD206 (bfpA(+) espA(+) eae(-)) which supplied Tir to the membrane . Single mutant strains CVD206 (bfpA(+) espA(+) eae(-)) and UMD872 (bfpA(+) espA(-) eae(+)) showed adherence characteristics of strain UMD880 (bfpA(+) espA(-) eae(-)), whilst triple mutant strain UMD888 (bfpA(-) espA(-) eae(-)) was totally non-adherent . These results support an adhesive role for BFP and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as initial attachment factors in atypical EPEC which lacks BFP . The study found no evidence for an independent host cell intimin receptor or for other adhesive factors able to support bacterial adherence.

Mol Cell Biol, 2004 Mar, 24(6), 2286 - 95
Replication stalling at Friedreich's ataxia (GAA)n repeats in vivo; Krasilnikova MM et al.; Friedreich's ataxia (GAA)n repeats of various lengths were cloned into a Saccharymyces cerevisiae plasmid, and their effects on DNA replication were analyzed using two-dimensional electrophoresis of replication intermediates . We found that premutation- and disease-size repeats stalled the replication fork progression in vivo, while normal-size repeats did not affect replication . Remarkably, the observed threshold repeat length for replication stalling in yeast (approximately 40 repeats) closely matched the threshold length for repeat expansion in humans . Further, replication stalling was strikingly orientation dependent, being pronounced only when the repeat's homopurine strand served as the lagging strand template . Finally, it appeared that length polymorphism of the (GAA)n . (TTC)n repeat in both expansions and contractions drastically increases in the repeat's orientation that is responsible for the replication stalling . These data represent the first direct proof of the effects of (GAA)n repeats on DNA replication in vivo . We believe that repeat-caused replication attenuation in vivo is due to triplex formation . The apparent link between the replication stalling and length polymorphism of the repeat points to a new model for the repeat expansion.

J Biol Chem, 2004 May 21, 279(21), 22693 - 703 Epub 2004 Mar 01.
Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1; Kakuda S et al.; The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules . Its structure is characterized by a terminal sulfated glucuronyl acid . The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme . We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli . Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate . Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide . The asymmetric unit contains two independent molecules . Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites . The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)) . Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate . In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety . These three residues play a key role in establishing the acceptor substrate specificity.

Biochemistry, 2004 Mar 9, 43(9), 2656 - 63
In vitro effects of a C4'-oxidized abasic site on DNA polymerases; Greenberg MM et al.; Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites . The C4'-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents . This lesion accounts for approximately 40% of the DNA damage produced by bleomycin . The effect of a C4'-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3' --> 5' exonuclease activity . Both enzymes preferentially incorporated dA > dG >> dC, T opposite C4-AP . Neither enzyme is able to extend the primer past the lesion . Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites . Extension of the primer by alternative polymerases pol II, pol II exo(-), pol IV, and pol V was examined . Pol II exo(-) was most efficient . Qualitative translesion synthesis experiments showed that pol II exo(-) preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC . Thymidine incorporation opposite C4'-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template . These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the "A-rule" . The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C --> T:A transversions in cells treated with bleomycin.

Biochemistry, 2004 Mar 9, 43(9), 2405 - 11
Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate; Arjunan P et al.; Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP) . To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex . When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others . These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP . The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues . Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP . The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor . Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release . This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

Biochemistry, 2004 Mar 9, 43(9), 2359 - 72
Histone release during transcription: NAP1 forms a complex with H2A and H2B and facilitates a topologically dependent release of H3 and H4 from the nucleosome; Levchenko V et al.; Transcription through a multinucleosomal template was studied to determine why histones are released to the nascent RNA . It was first determined in competition experiments between DNA and RNA that histones H2A and H2B have a 20-fold preference for binding RNA over DNA; a preference was not seen for histones H3 and H4 . Histones H3 and H4 would preferentially bind RNA, provided they were in an octameric complex with H2A and H2B . In transcription studies with T7 RNA polymerase, H3 and H4 were transferred to the nascent RNA, provided the template was linear . If the DNA was topologically restrained, which is a condition that more closely maintains transcription-induced stresses, H3 and H4 would not release . Histones H3 and H4 would be released from this template when H2A and H2B were present, a release that was enhanced by the presence of nucleosome assembly protein-1 (NAP1) . Since a small quantity of H2A and H2B is sufficient to facilitate this transfer, it is proposed that H2A and H2B function to repeatedly shuttle H3 and H4 from the template DNA to the RNA . Cross-linked histones (dimethylsuberimidate-cross-linked octamer) were reconstituted into nucleosomes and found to be transferred to the RNA at the same frequency as un-cross-linked histones, an indication that such large complexes can be released during transcription . Transcription was carried out in the presence of Escherichia coli topoisomerase I so that positive coils would accumulate on the DNA . Histones H3 and H4 would again not be transferred from this DNA, unless H2A and H2B were present . In this instance, however, when NAP1 was present, the shuttling of H3 and H4 to the RNA caused a significant depletion of H2A and H2B from the positively coiled DNA . These results are discussed with regard to current models for transcription through nucleosomes.

Biopolymers, 2004 Mar, 73(4), 524 - 31
Fourier transform infared spectroscopy investigation of protein conformation in spray-dried protein/trehalose powders; French DL et al.; Spray drying is a way to generate protein solids (powders), which is also true for lyophilization . Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity . The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy . The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins . Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure . Trehalose reduced the magnitude of the changes in beta sheets and turns . Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns) . Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets . Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes . At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose . Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity . At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders . Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders . The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity .

Arch Virol, 2004 Mar, 149(3), 495 - 506 Epub 2003 Nov 13.
Glycoprotein gpTRL10 of human cytomegalovirus is dispensable for virus replication in human fibroblasts; Spaderna S et al.; Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which by far exceeds that of other herpesviruses . Few of these proteins have been characterized . Glycoprotein gpTRL10 represents a structural component of HCMV . The protein has no homologous counterparts in other herpesviruses . We have isolated bacterial artificial chromosomes (BACs) harboring the genome from the low passage clinical isolate PAN and constructed a deletion mutant in reading frame TRL10 . Our results show that deletion of gpTRL10 has only minimal effects on viral replication in human fibroblasts.

Planta, 2004 Jun, 219(2), 325 - 31 Epub 2004 Feb 27.
Functional analysis and regulation of the malate synthase from Chlamydomonas reinhardtii; Nogales J et al.; Malate synthase (EC 2.3.3.9, formerly EC 4.1.2.2) has been investigated in the unicellular green algae Chlamydomonas reinhardtii . The molecular characteristics and the regulation of gene expression have been investigated for the enzyme . A full-length malate synthase cDNA has been isolated, containing an open reading frame of 1,641 bp encoding a polypeptide of 546 amino acids . This protein shares the conserved signature of the malate synthase family, along with the catalytic residues essential for enzymatic activity and a C-terminal motif that matches the consensus for glyoxysome import . Functionality studies have been facilitated by heterologous expression of the malate synthase cDNA in Escherichia coli . The remarkable metabolic versatility of the alga has been used to analyse the metabolic control of malate synthase gene expression . The data strongly support the role of acetate and light as the main regulatory effectors, and the existence of cross-talk between the two signalling pathways .

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3393 - 7 Epub 2004 Feb 27.
The role of the allosteric B site in the fumarase reaction; Rose IA et al.; The role of a malate binding site in a concavity external to the more deeply situated active site has been a major mystery of the fumarase reaction . The malate, within 12 A of the active site, was bound by hydrogen bonds to two main-chain amides and to two basic residues, H129 and R126 . Mutation of the His of this so-called B site of Escherichia coli fumarase had little effect on the overall initial rate kinetics of the enzyme, which has obscured an understanding of the critical role of the site . Contrary to the WT enzyme, which is rate-limited in the recycling of free enzyme isoforms that follows product release, the enzyme with both basic residues modified is rate-limited in the product release step itself . A loss of complexity in the mutated, but still functional, step is indicated by a greatly reduced sensitivity of its rate to changes in temperature . Unlike the inhibition by glycerol shown with normal enzyme and attributed to a viscogenic effect on the recycling rate, the product-release step of the B-site mutants is accelerated by glycerol, suggestive of a structural effect on the 12-A space between the A and B sites . It is proposed that the "extra" malate represents a stage in the transfer of substrate and product between the solvent and the "buried" active site of the enzyme.

Proc Natl Acad Sci U S A, 2004 Mar 23, 101(12), 4065 - 70 Epub 2004 Feb 27.
Elastic coupling of integral membrane protein stability to lipid bilayer forces; Hong H et al.; It has been traditionally difficult to measure the thermodynamic stability of membrane proteins because fully reversible protocols for complete folding these proteins were not available . Knowledge of the thermodynamic stability of membrane proteins is desirable not only from a fundamental theoretical standpoint, but is also of enormous practical interest for the rational design of membrane proteins and for optimizing conditions for their structure determination by crystallography or NMR . Here, we describe the design of a fully reversible system to study equilibrium folding of the outer membrane protein A from Escherichia coli in lipid bilayers . Folding is shown to be two-state under appropriate conditions permitting data analysis with a classical folding model developed for soluble proteins . The resulting free energy and m value, i.e., a measure of cooperativity, of unfolding are DeltaG(u,H2O)(o)=3.4 kcal/mol and m = 1.1 kcal/mol M(-1), respectively, in a reference bilayer composed of palmitoyl-oleoyl-phosphatidylcholine (C(16:0)C(18:1)PC) and palmitoyloleoyl-phosphatidylglycerol (C(16:0)C(18:1)PG) . These values are strong functions of the lipid bilayer environment . By systematic variation of lipid headgroup and chain composition, we show that elastic bilayer forces such as curvature stress and hydrophobic mismatch modulate the free energy and cooperativity of folding of this and perhaps many other membrane proteins.

J Biol Chem, 2004 Apr 23, 279(17), 16899 - 902 Epub 2004 Feb 26.
An unsubstituted C2 hydrogen of adenine is critical and sufficient at the -11 position of a promoter to signal base pair deformation; Lee HJ et al.; The conserved A:T base pair at the -11 position of the promoters in Escherichia coli is very sensitive to substitutions . In vitro transcription with the galP1 promoter having a natural or unnatural base in either strand at position -11 showed that only a purine base with no side group at C2 in the nontemplate strand is transcriptionally potent; neither a purine with an amino group at C2 nor a pyrimidine support transcription . The amino group at C6 in the omnipresent adenine at -11 does not play any role in promoting transcription . The nature of the base, complementary or noncomplementary, at -11 in the template strand also does not influence transcription . We propose that the adenine, by becoming extrahelical, interacts with an amino acid(s) of the 2.3-2.4 region of sigma for which an unsubstituted C2 hydrogen is critical.

J Biol Chem, 2004 May 14, 279(20), 20807 - 15 Epub 2004 Feb 27.
A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb; Nie J et al.; LNX was originally cloned as a Numb PTB-binding molecule, and it was subsequently found to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of mNumb . Numb is a PTB domain-containing protein that functions as an intrinsic determinant of cell fate in asymmetric cell division . In mammals, four protein isoforms arise from alternative mRNA splicing . Here we report that while all four protein isoforms bind to LNX, only p72 and p66 Numb isoforms are ubiquitylated and degraded . The p72 and p66 Numb proteins differ from the other two isoforms by the presence of an 11-amino acid sequence insert in the PTB domain (PTBi) . We demonstrate that the isoform-specific ubiquitylation of mNumb is due to a novel interaction between the first PDZ domain (PDZ1) of LNX and the PTBi variant . Deletion of LNX PDZ1 domain resulted in loss of ubiquitylation and subsequent degradation of the PTBi form of Numb . Interestingly efficient PTBi ubiquitylation not only depends on association with the LNX PDZ1 domain but also requires binding to the canonical PTB-binding motif NPAY in LNX . Thus two distinct modes of PTBi-mediated interaction with LNX work in concert to allow the effective and specific degradation of the p72 and p66 isoforms of mNumb.

J Biol Chem, 2004 May 7, 279(19), 20076 - 87 Epub 2004 Feb 29.
The mouse formin, FRLalpha, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments; Harris ES et al.; Formins are a conserved class of proteins expressed in all eukaryotes, with known roles in generating cellular actin-based structures . The mammalian formin, FRLalpha, is enriched in hematopoietic cells and tissues, but its biochemical properties have not been characterized . We show that a construct composed of the C-terminal half of FRLalpha (FRLalpha-C) is a dimer and has multiple effects on muscle actin, including tight binding to actin filament sides, partial inhibition of barbed end elongation, inhibition of barbed end binding by capping protein, acceleration of polymerization from monomers, and actin filament severing . These multiple activities can be explained by a model in which FRLalpha-C binds filament sides but prefers the topology of sides at the barbed end (end-sides) to those within the filament . This preference allows FRLalpha-C to nucleate new filaments by side stabilization of dimers, processively advance with the elongating barbed end, block interaction between C-terminal tentacles of capping protein and filament end-sides, and sever filaments by preventing subunit re-association as filaments bend . Another formin, mDia1, does not reduce the barbed end elongation rate but does block capping protein, further supporting an end-side binding model for formins . Profilin partially relieves barbed end elongation inhibition by FRLalpha-C . When non-muscle actin is used, FRLalpha-C's effects are largely similar . FRLalpha-C's ability to sever filaments is the first such activity reported for any formin . Because we find that mDia1-C does not sever efficiently, severing may not be a property of all formins.

Biophys J, 2004 Mar, 86(3), 1807 - 12
Structural studies of the manganese stabilizing subunit in photosystem II; Svensson B et al.; Photosystem II (PSII) is the plant photosynthetic reaction center that carries out the light driven oxidation of water . The water splitting reactions are catalyzed at a tetranuclear manganese cluster . The manganese stabilizing protein (MSP) of PSII stabilizes the manganese cluster and accelerates the rate of oxygen evolution . MSP can be removed from PSII, with an accompanying decrease in activity . Either an Escherichia coli expressed version of MSP or native, plant MSP can be rebound to the PSII reaction center; MSP reconstitution reverses the deleterious effects associated with MSP removal . We have employed Fourier transform infrared (FTIR) spectroscopy and solution small angle x-ray scattering (SAXS) techniques to investigate the structure of MSP in solution and to define the structural changes that occur before and after reconstitution to PSII . FTIR and SAXS are complementary, because FTIR spectroscopy detects changes in MSP secondary structure and SAXS detects changes in MSP size/shape . From the SAXS data, we conclude that the size/shape and domain structure of MSP do not change when MSP binds to PSII . From FTIR data acquired before and after reconstitution, we conclude that the reconstitution-induced increase in beta-sheet content, which was previously reported, persists after MSP is removed from the PSII reaction center . However, the secondary structural change in MSP is metastable after removal from PSII, which indicates that this form of MSP is not the lowest energy conformation in solution.

Biophys J, 2004 Mar, 86(3), 1625 - 31
Effects of HU binding on the equilibrium cyclization of mismatched, curved, and normal DNA; Arthanari H et al.; The effects of HU, the histone-like protein from Escherichia coli, on the equilibrium cyclization of duplex DNAs have been observed as a function of protein concentration and DNA sequence . The results indicate that the presence of HU significantly enhances the extent of cyclization and increases the melting temperature, T(m), of the cyclized form of the DNA by >10 K . The stabilization of equilibrium cyclization by HU binding is at least -1.2 kcal/mol . The results are consistent with two HU homotypic dimers binding to each of the three 29-mer duplexes studied . One of the 29-mer duplexes contains a central dA tract, one contains mismatched sites, and one a conventional sequence . Stepwise or microscopic association constants, determined from the fluorescence data, range from 1.5 to 0.6 micro M(-1) . The binding affinity of the HU dimer is strongest for the mismatched duplex and lowest for the dA tract, consistent with HU dimers having a preference for flexible DNA substrates . These results demonstrate the utility of the equilibrium cyclization approach to monitor DNA-protein interactions . These results have been considered along with those previously obtained to refine a model for the interaction of HU with duplex DNA.

Biophys J, 2004 Mar, 86(3), 1357 - 72
Bridging the gap between stochastic and deterministic regimes in the kinetic simulations of the biochemical reaction networks; Puchalka J et al.; The biochemical reaction networks include elementary reactions differing by many orders of magnitude in the numbers of molecules involved . The kinetics of reactions involving small numbers of molecules can be studied by exact stochastic simulation . This approach is not practical for the simulation of metabolic processes because of the computational cost of accounting for individual molecular collisions . We present the "maximal time step method," a novel approach combining the Gibson and Bruck algorithm with the Gillespie tau-leap method . This algorithm allows stochastic simulation of systems composed of both intensive metabolic reactions and regulatory processes involving small numbers of molecules . The method is applied to the simulation of glucose, lactose, and glycerol metabolism in Escherichia coli . The gene expression, signal transduction, transport, and enzymatic activities are modeled simultaneously . We show that random fluctuations in gene expression can propagate to the level of metabolic processes . In the cells switching from glucose to a mixture of lactose and glycerol, random delays in transcription initiation determine whether lactose or glycerol operon is induced . In a small fraction of cells severe decrease in metabolic activity may also occur . Both effects are epigenetically inherited by the progeny of the cell in which the random delay in transcription initiation occurred.

Biophys J, 2004 Mar, 86(3), 1282 - 92
Influence of catabolite repression and inducer exclusion on the bistable behavior of the lac operon; Santillan M et al.; A mathematical model of the lac operon which includes all of the known regulatory mechanisms, including external-glucose-dependent catabolite repression and inducer exclusion, as well as the time delays inherent to transcription and translation, is presented . With this model we investigate the influence of external glucose, by means of catabolite repression and the regulation of lactose uptake, on the bistable behavior of this system.

Bioinformatics, 2004 Mar 1, 20(4), 538 - 46 Epub 2004 Jan 22.
A multi-algorithm, multi-timescale method for cell simulation; Takahashi K et al.; MOTIVATION: Many important problems in cell biology require the dense nonlinear interactions between functional modules to be considered . The importance of computer simulation in understanding cellular processes is now widely accepted, and a variety of simulation algorithms useful for studying certain subsystems have been designed . Many of these are already widely used, and a large number of models constructed on these existing formalisms are available . A significant computational challenge is how we can integrate such sub-cellular models running on different types of algorithms to construct higher order models . RESULTS: A modular, object-oriented simulation meta-algorithm based on a discrete-event scheduler and Hermite polynomial interpolation has been developed and implemented . It is shown that this new method can efficiently handle many components driven by different algorithms and different timescales . The utility of this simulation framework is demonstrated further with a 'composite' heat-shock response model that combines the Gillespie-Gibson stochastic algorithm and deterministic differential equations . Dramatic improvements in performance were obtained without significant accuracy drawbacks . A multi-timescale demonstration of coupled harmonic oscillators is also shown.

Free Radic Biol Med, 2004 Mar 15, 36(6), 765 - 73
Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells; Hoffmann S et al.; The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells . To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair glycosylase Fpg (mostly 7,8-dihydro-8-oxoguanine) in wild-type HeLa cells and HeLa rho0 cells . The latter are depleted of mitochondrial DNA and therefore are unable to produce ROS in the ETC . Although the levels of ROS measured by flow cytometry and redox-sensitive probes in rho0 cells were only 10-15% those of wild-type cells, steady-state levels of oxidative DNA base modifications were the same as in wild-type cells . Mitochondrial generation of ROS was then stimulated in HeLa wild-type cells using inhibitors interfering with the ETC . Although mitochondrial ROS production was raised up to 6-fold, none of the substances nor their combinations induced additional oxidative base modifications in the nuclear DNA . This was also true for glutathione-depleted cells . The results indicate that the contribution of mitochondria to the endogenously generated background levels of oxidative damage in the nuclear DNA is negligible.

Res Microbiol, 2004 Mar, 155(2), 76 - 9
Relief of catabolite repression in a cAMP-independent catabolite gene activator mutant of Escherichia coli; Karimova G et al.; We isolated and characterized a new catabolite gene activator mutant (crp*) of Escherichia coli that confers cAMP-independent expression and total relief of catabolite repression of beta-galactosidase and tryptophanase synthesis . The two mutations responsible for this phenotype change the amino acids at codon 72 from Glu to Ala and at codon 144 from Ala to Thr in the corresponding CAP* protein.

Zhonghua Jie He He Hu Xi Za Zhi, 2004 Feb, 27(2), 89 - 92
{Mycobacterium tuberculosis secreting protein Ag85B-ESAT6 fused expression and purification}; Shi CH et al.; OBJECTIVE: To investigate the fused expression of secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis, and to provide a promising preventive subunit vaccine against tuberculosis . METHODS: The gene encoding Ag85B and ESAT6 protein was amplified by PCR from genome of Mycobacterium tuberculosis H(37)Rv strain, and inserted into cloning vector P(GEM)-T-easy . After sequence analysis, and digestion by restriction endonuclease, Ag85B-ESAT6 was cloned into corresponding sites of the expression vector P(PRO) EXHT, and the recombinant plasmid was transformed into expressive strain E . coli DH5 alpha, induced with IPTG and fusion protein was purified by Ni-NTA purification system . The specific antibody titer in the sera of BALB/c mouse immunized with two fusion protein was detected by ELISA . RESULTS: The sequences of Ag85B and ESAT6 by PCR amplification were identical to those reported by GenBank . The recombinant plasmid fused expression protein of Ag85B-ESAT6 with relative molecular mass (Mr) of 37,000, which was confirmed by Western-blot analysis with specific monoclonal antibody against 6 x HismAb . The fused expression protein was insoluble . It could be purified by Ni-NTA purification system . The specific antibody titer in the sera of BALB/c mouse immunized with fusion Ag85B-ESAT6 was 1:1000 and that of mouse immunized with fusion protein ESAT6-Ag85B was 1:5000 . CONCLUSIONS: Secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis was successfully fused expressed in E . Coli DH5 alpha . It may become a new type of vaccine against tuberculosis.

J Med Entomol, 2004 Jan, 41(1), 69 - 73
Extracts of scabies mites (Sarcoptidae: Sarcoptes scabiei) modulate cytokine expression by human peripheral blood mononuclear cells and dendritic cells; Arlian LG et al.; We performed a series of experiments to determine if human peripheral blood mononuclear cells (PBMCs) from a healthy donor and dendritic cells (NHDCs) derived from these PBMCs reacted to molecules in a scabies extract . PBMCs extravasate from the circulatory system and enter tissues such as scabietic lesions, where monocytes become macrophages . Cells were cultured in medium alone or medium containing 50 microg/ml of Sarcoptes scabiei (SS) extract, 50 ng/ml E . coli lipopolysaccharide (LPS), or SS + LPS together . Supernatants were collected and assayed by enzyme-linked immunosorbent assay (ELISA) for specific cytokines . PBMCs stimulated with SS or LPS exhibited moderately upregulated production of interleukin (IL)-1beta and huge increases in secretions of IL-6, IL-8 and TNF-alpha . Cells co-stimulated with both SS and LPS generally secreted more of these cytokines than cells stimulated with either SS or LPS alone . LPS induced a small amount of IL-1alpha secretion, whereas SS did not, and neither additive resulted in the production of IL-10 . NHDCs did not produce IL-1alpha, IL-1beta, IL-6, IL-8, or IL-10 in response to stimulation with SS . These cells did produce IL-6, IL-8, and tumor necrosis factor (TNF)-alpha in response to LPS . When cells were co-stimulated with both LPS and SS, the production of IL-6 and IL-8 was significantly reduced compared with the levels secreted after LPS stimulation alone . These studies show that molecules in a whole body extract of S . scabiei modulate the function of PBMCs (probably monocytes) and dendritic cells.

Science, 2004 Feb 27, 303(5662), 1382 - 4
Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting; Young BA et al.; We determined the minimal portion of Escherichia coli RNA polymerase (RNAP) holoenzyme able to accomplish promoter melting, the crucial step in transcription initiation that provides RNAP access to the template strand . Upon duplex DNA binding, the N terminus of the beta' subunit (amino acids 1 to 314) and amino acids 94 to 507 of the sigma subunit, together comprising less than one-fifth of RNAP holoenzyme, were able to melt an extended -10 promoter in a reaction remarkably similar to that of authentic holoenzyme . Our results support the model that capture of nontemplate bases extruded from the DNA helix underlies the melting process.

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3387 - 92 Epub 2004 Feb 26.
Molecular mechanism of enantioselective proton transfer to carbon in catalytic antibody 14D9; Zheng L et al.; Catalytic antibody 14D9 catalyzes the enantioselective protonation of prochiral enol ethers with high enantioselectivity (>99% ee) and a practical turnover (k(cat) = 0.4 s(-1)), allowing for preparative scale applications . This antibody represents one of the rare examples of catalytic antibodies promoting acid-catalyzed processes . Antibody 14D9 was cloned and expressed as a chimeric Fab fragment in Escherichia coli . Crystal structures of Fab 14D9 as apo form and of its close analog 19C9 in complex with the transition state analog were determined at 2.8-A resolution . A series of site-directed mutagenesis experiments was carried out to probe the role of individual active-site amino acids . Proton transfer to carbon is catalyzed by a hydrogen bond network formed by the side chains of Asp(H101) and Tyr(L36) with a water molecule serving as a relay . The intermediate oxocarbonium ion formed during the protonation step is trapped by the same water molecule, resulting in an overall syn-addition of water to the enol ether's double bond . The enantioselectivity is caused by steric crowding at the active site, mainly because of the side chain of Phe(H84) . The 20-fold lower activity of 19C9 compared with 14D9 was traced down to residue Thr(L46), which forms a nonproductive hydrogen bond with the catalytic residue Asp(H101), which competes with the critical Asp(H101)-Tyr(L36) hydrogen bond and therefore reduces catalytic efficiency . The catalytic activity of 19C9 was restored to that of 14D9 by using either site-directed mutagenesis (Thr(L46)Ala) or chain shuffling.

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3352 - 7 Epub 2004 Feb 26.
Water in protein structure prediction; Papoian GA et al.; Proteins have evolved to use water to help guide folding . A physically motivated, nonpairwise-additive model of water-mediated interactions added to a protein structure prediction Hamiltonian yields marked improvement in the quality of structure prediction for larger proteins . Free energy profile analysis suggests that long-range water-mediated potentials guide folding and smooth the underlying folding funnel . Analyzing simulation trajectories gives direct evidence that water-mediated interactions facilitate native-like packing of supersecondary structural elements . Long-range pairing of hydrophilic groups is an integral part of protein architecture . Specific water-mediated interactions are a universal feature of biomolecular recognition landscapes in both folding and binding.

Plant Physiol, 2004 Mar, 134(3), 1135 - 45 Epub 2004 Feb 26.
Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+ uptake; Ahn SJ et al.; Potassium (K(+)) is the most abundant cation in plants and is required for plant growth . To ensure an adequate supply of K(+), plants have multiple mechanisms for uptake and translocation . However, relatively little is known about the physiological role of proteins encoded by a family of 13 genes, named AtKT/KUP, that are involved in K(+) transport and translocation . To begin to understand where and under what conditions these transporters function, we used reverse transcription-PCR to determine the spatial and temporal expression patterns of each AtKT/KUP gene across a range of organs and tested whether selected AtKT/KUP cDNAs function as K(+) transporters in Escherichia coli . Many AtKT/KUPs were expressed in roots, leaves, siliques, and flowers of plants grown under K(+)-sufficient conditions (1.75 mm KCl) in hydroponic culture . AtHAK5 was the only gene in this family that was up-regulated upon K(+) deprivation and rapidly down-regulated with resupply of K(+) . Ten AtKT/KUPs were expressed in root hairs, but only five were expressed in root tip cells . This suggests an important role for root hairs in K(+) uptake . The growth and rubidium (Rb(+)) uptake of two root hair mutants, trh1-1 (tiny root hairs) and rhd6 (root hair defective), were studied to determine the contribution of root hairs to whole-plant K(+) status . Whole-plant biomass decreased in the root hair mutants only when K(+) concentrations were low; Rb(+) (used as a tracer for K(+)) uptake rates were lower in the mutants at all Rb(+) concentrations . Seven genes encoding AtKUP transporters were expressed in E . coli (AtKT3/KUP4, AtKT/KUP5, AtKT/KUP6, AtKT/KUP7, AtKT/KUP10, AtKT/KUP11, and AtHAK5), and their K(+) transport function was demonstrated.

J Biol Chem, 2004 May 21, 279(21), 22759 - 64 Epub 2004 Feb 26.
Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Gö1; Coskun U et al.; A modified isolation procedure provides a homogeneous A(1)-ATPase from the archaeon Methanosarcina mazei Go1, containing the five subunits in stoichiometric amounts of A(3):B(3):C:D:F . A(1) obtained in this way was characterized by three-dimensional electron microscopy of single particles, resulting in the first three-dimensional reconstruction of an A(1)-ATPase at a resolution of 3.2 nm . The A(1) consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A(3) and B(3) . At the bottom of the A(3)B(3) complex, a stalk of 3.0 nm in length can be seen . The A(3)B(3) domain surrounds a large cavity that extends throughout the length of the A(3)B(3) barrel . A part of the stalk penetrates inside this cavity and is displaced toward an A-B-A triplet . To investigate further the topology of the stalk subunits C-F in A(1), cross-linking has been carried out by using dithiobis{sulfosuccinimidylpropionate} (DSP) and 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC) . In experiments where DSP was added the cross-linked products B-F, A(x)-D, A-B-D, and A(x)-B(x)-D were formed . Subunits B-F, A-D, A-B-D, and A-B-C-D could be cross-linked by EDC . The subunit-subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage . Finally, the three-dimensional organization of this A(1) complex is discussed in terms of the relationship to the F(1)- and V(1)-ATPases at a resolution of 3.2 nm.

J Biol Chem, 2004 May 7, 279(19), 20127 - 36 Epub 2004 Feb 26.
Proteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli; Hardwidge PR et al.; We present the first large scale proteomic analysis of a human cellular response to a pathogen . Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide . EPEC uses a type III secretion system (TTSS) to inject bacterial proteins into the cytosol of intestinal epithelial cells, resulting in diarrhea . We analyzed the host response to TTSS-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or TTSS-deficient EPEC . Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry . We identified over 2000 unique proteins from infected Caco-2 monolayers, of which approximately 13% are expressed differentially in the presence of TTSS-delivered EPEC effector proteins . We validated these data in silico and through immunoblotting and immunofluorescence microscopy . The identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host proteins potentially involved in EPEC-induced diarrhea . These data provide a framework for future biochemical analyses of host-pathogen interactions.

Diabetes, 2004 Mar, 53(3), 535 - 41
Stimulation of hepatocyte glucose metabolism by novel small molecule glucokinase activators; Brocklehurst KJ et al.; Glucokinase (GK) has a major role in the control of blood glucose homeostasis and is a strong potential target for the pharmacological treatment of type 2 diabetes . We report here the mechanism of action of two novel and potent direct activators of GK: 6-{(3-isobutoxy-5-isopropoxybenzoyl)amino}nicotinic acid(GKA1) and 5-({3-isopropoxy-5-{2-(3-thienyl)ethoxy}benzoyl}amino)-1,3,4-thiadiazole-2-carboxylic acid(GKA2), which increase the affinity of GK for glucose by 4- and 11-fold, respectively . GKA1 increased the affinity of GK for the competitive inhibitor mannoheptulose but did not affect the affinity for the inhibitors palmitoyl-CoA and the endogenous 68-kDa regulator (GK regulatory protein {GKRP}), which bind to allosteric sites or to N-acetylglucosamine, which binds to the catalytic site . In hepatocytes, GKA1 and GKA2 stimulated glucose phosphorylation, glycolysis, and glycogen synthesis to a similar extent as sorbitol, a precursor of fructose 1-phosphate, which indirectly activates GK through promoting its dissociation from GKRP . Consistent with their effects on isolated GK, these compounds also increased the affinity of hepatocyte metabolism for glucose . GKA1 and GKA2 caused translocation of GK from the nucleus to the cytoplasm . This effect was additive with the effect of sorbitol and is best explained by a "glucose-like" effect of the GK activators in translocating GK to the cytoplasm . In conclusion, GK activators are potential antihyperglycemic agents for the treatment of type 2 diabetes through the stimulation of hepatic glucose metabolism by a mechanism independent of GKRP.

Blood, 2004 Jun 15, 103(12), 4545 - 53 Epub 2004 Feb 26.
Induction of microparticle- and cell-associated intravascular tissue factor in human endotoxemia; Aras O et al.; The precise role of intravascular tissue factor (TF) remains poorly defined, due to the limited availability of assays capable of measuring circulating TF procoagulant activity (PCA) . As a model of inflammation-associated intravascular thrombin generation, we studied 18 volunteers receiving an infusion of endotoxin . A novel assay that measures microparticle (MP)-associated TF PCA from a number of cellular sources (but not platelets) demonstrated an 8-fold increase in activity at 3 to 4 hours after endotoxin administration (P <.001), with a return to baseline by 8 hours . TF antigen-positive MPs isolated from plasma were visualized by electron microscopy . Interindividual MP-associated TF response to lipopolysaccharide (LPS) was highly variable . In contrast, a previously described assay that measures total (cell and MP-borne) whole-blood TF PCA demonstrated a more modest increase, with a peak in activity (1.3-fold over baseline; P <.000 01) at 3 to 4 hours, and persistence for more than 24 hours . This surprisingly modest increase in whole-blood TF activity is likely explained by a profound although transient LPS-induced monocytopenia . MP-associated TF PCA was highly correlated with whole-blood TF PCA and total number of circulating MPs, and whole-blood TF PCA was highly correlated with TF mRNA levels.

FEBS Lett, 2004 Feb 27, 560(1-3), 199 - 204
Molecular characterization of a Penicillium chrysogenum exo-1,5-alpha-L-arabinanase that is structurally distinct from other arabinan-degrading enzymes; Sakamoto T et al.; The nucleotide sequence of the abnx cDNA gene, which encodes an exo-arabinanase (Abnx) of Penicillium chrysogenum 31B, was determined . Abnx was found to be structurally distinct from known arabinan-degrading enzymes based on its amino acid sequence and a hydrophobic cluster analysis . The protein in the protein database with the highest similarity to Abnx was the Neurospora crassa conserved hypothetical protein . The abnx cDNA gene product expressed in Escherichia coli catalyzed the release of arabinobiose from alpha-1,5-L-arabinan . The activity of the recombinant Abnx towards a series of arabino-oligosaccharides, as expressed by k(cat)/K(m) value, was greatest with arabinohexaose.

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