A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers

The introduction of alkylated, nonporous poly-(styrene-divinylbenzene) microparticles in 1992 enabled the subsequent development of denaturing HPLC that has emerged as the most sensitive screening method for mutations to date. Denaturing HPLC has provided unprecedented insight into human origins and prehistoric migrations, accelerated the cloning of genes involved in mono- and polygenic traits, and facilitated the mutational analysis of more than a hundred candidate genes of human disease. A significant step toward increased sample-throughput and information content was accomplished by the recent introduction of monolithic poly(styrene-divinylbenzene) capillary columns. They have enabled the construction of capillary arrays amenable to multiplex analysis of fluorescent dye-labeled nucleic acids by laser-induced fluorescence detection. Hyphenation of denaturing HPLC with electrospray ionization mass spectrometry, on the other hand, has allowed the direct elucidation of the chemical nature of DNA variation and determination of phase of multiple alleles on a chromosome.     

Mass spectrometric identification of novel posttranslational modification sites in Huntingtin

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway

The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase LpxC that removes an acetate from the nitrogen at the 2' '-position of UDP-3-O-acyl-N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aquifex aeolicus, a heat-stable orthologue that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of k(cat)/K(M) and k(cat) for the deacetylation of the substrate UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc are both bell-shaped. The ascending acidic limb (pK(1)) was fitted to 6.1 +/- 0.2 for k(cat) and 5.7 +/- 0.2 for k(cat)/K(M). The descending basic limb (pK(2)) was fitted to 8.0 +/- 0.2 for k(cat) and 8.4 +/- 0.2 for k(cat)/K(M). The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general base for deprotonation and activation of the zinc-bound water.     

An Ion-channel Modulator from the Saliva of the Brown Ear Tick has a Highly Modified Kunitz/BPTI Structure

Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 Å resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by β-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the ”Kunitz head” by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a β-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.     

Influence of hydrological connectivity of riverine wetlands on nitrogen removal via denitrification

Wetland ecosystems in agricultural areas often become progressively more isolated from main water bodies. Stagnation favors the accumulation of organic matter as the supply of electron acceptors with water renewal is limited. In this context it is expected that nitrogen recycling prevails over nitrogen dissipation. To test this hypothesis, denitrification rates, fluxes of dissolved oxygen (SOD), inorganic carbon (DIC) and nitrogen and sediment features were measured in winter and summer 2007 on 22 shallow riverine wetlands in the Po River Plain (Northern Italy). Fluxes were determined from incubations of intact cores by measurement of concentration changes or isotope pairing in the case of denitrification. Sampled sites were eutrophic to hypertrophic; 10 were connected and 12 were isolated from the adjacent rivers, resulting in large differences in nitrate concentrations in the water column (from <5 to 1,133 μM). Benthic metabolism and denitrification rates were investigated by two overarching factors: season and hydrological connectivity. SOD and DIC fluxes resulted in respiratory quotients greater than one at most sampling sites. Sediment respiration was coupled to both ammonium efflux, which increased from winter to summer, and nitrate consumption, with higher rates in river-connected wetlands. Denitrification rates measured in river-connected wetlands (35–1,888 μmol N m^?2 h^?1) were up to two orders of magnitude higher than rates measured in isolated wetlands (2–231 μmol N m^?2 h^?1), suggesting a strong regulation of the process by nitrate availability. These rates were also significantly higher in summer (9–1,888 μmol N m^?2 h^?1) than in winter (2–365 μmol N m^?2 h^?1). Denitrification supported by water column nitrate (D_W) accounted for 60–100% of total denitrification (Dtot); denitrification coupled to nitrification (D_N) was probably controlled by limited oxygen availability within sediments. Denitrification efficiency, calculated as the ratio between N removal via denitrification and N regeneration, and the relative role of denitrification for organic matter oxidation, were high in connected wetlands but not in isolated sites. This study confirms the importance of restoring hydraulic connectivity of riverine wetlands for the maintenance of important biogeochemical functions such as nitrogen removal via denitrification.     

Photolabeling study of the ligand binding domain of natriuretic peptide receptor A: development of a model

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are loop-shaped peptidic hormones that have multiple actions on body fluid homeostasis. Their physiological effects are mediated through the activation of their receptor, natriuretic peptide receptor A (NPRA). This receptor is a member of the membrane guanylyl cyclase family and catalyzes cyclic guanosine monophosphate (cGMP) production following its activation. To map the binding site of human NPRA, we applied the methionine proximity assay method to this receptor. We photolabeled NPRA mutants, presenting a single methionine in the binding domain of the receptor, and used benzoylphenylalanine- (Bpa-) substituted peptides at positions 0, 3, 18, 26, and 28 of the ligand. We identified that the N-terminus of the peptide is interacting with the region between Asp(177) and Val(183) of the receptor. Arg(3) is interacting in the vicinity of Phe(172). Leu(18) binds close to Val(116). Phe(26) binds in the vicinity of His(195), and the C-terminal Tyr(28) is located close to Met(173). We next proceeded with photolabeling of a dual Bpa-substituted peptide and showed that the N-terminus and Leu(18) interact with opposite receptor subunits. On the basis of our results, a molecular model of peptide-bound NPRA was developed by homology modeling with the C-type natriuretic peptide- (CNP-) bound natriuretic peptide receptor C (NPRC) crystal structure. The model has been validated by molecular dynamics simulations. Our work provides a rational basis for interpreting and predicting natriuretic peptide binding to the human NPRA.     

Habitat utilization by sympatric European mink<Emphasis Type="Italic">Mustela lutreola</Emphasis> and polecats<Emphasis Type="Italic">Mustela putorius</Emphasis> in south-western France

The European minkMustela lutreola Linnaeus, 1761 and the European polecatMustela putorius Linnaeus, 1758 are related species sympatric in southwestern France. The European mink is rapidly disappearing whereas the polecat maintains good populations. Seasonal habitat use of both species was compared in the Landes de Gascogne region to identify if some vulnerability factors of the European mink were associated with habitats occupied by this mustelid. Potential habitats were mapped using a satellite picture and 12 main types of habitats were defined. Animal locations were recorded by radiotracking 9 European mink and 14 polecats from March 1996 to August 1999. Resting animals were located by triangulation, and, when possible, resting places were described. Animals in activity were tracked by continuous monitoring. Data collected revealed a strong preference of European mink for flooded habitats, particularly open marshes, flooded woodlands and moorlands. They seldom left the corridor of the riparian forest and their resting places were mainly in flooded environments, above ground (under herbs or bushes) or in cavities between tree roots. European polecats were less tightly linked to wetlands. Most of their locations were in the pine forests outside the valleys and their resting places were mainly in burrows. The strong specialisation of European mink in aquatic habitats is probably one of the main reasons for its decline because wetlands suffer drastic damages throughout all of its range. Maintaining adequate water levels is crucial for satisfying habitat requirements of mink.     

SMN-mediated assembly of RNPs: a complex story

Although many RNA-protein complexes or ribonucleoproteins (RNPs) assemble spontaneously in vitro, little is known about how they form in the environment of a living cell. Insight into RNP assembly has come unexpectedly from functional analyses of the survival motor neuron (SMN) protein, a gene product that is affected in the neuromuscular disease spinal muscular atrophy. These studies show that the assembly of spliceosomal U-rich small nuclear RNPs in vivo depends on the activity of two large protein complexes, one of which contains the SMN protein. These complexes might also facilitate the assembly of other cellular RNPs.