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 o