Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method

The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3-(13)C(3) N-Acetyl-D-neuraminic Acid ((13)C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/z 366→330 and 369→333 for Neu5Ac and (13)C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 μM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 μM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).     

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.     

beta-Secretase inhibitors: modification at the P4 position and improvement of inhibitory activity in cultured cells

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-570 and KMI-684 in which we replaced carboxylic acid groups at the P(1)(') position of KMI-420 and KMI-429, respectively, with tetrazole derivatives as carboxylic acid bioisosteres. These modifications improved significantly BACE1 inhibitory activity and chemical stability. In this study, the acidic tetrazole ring of the P(4) position of KMI-420 and KMI-570, respectively, was replaced with various hydrogen bond acceptor groups. We found BACE1 inhibitor KMI-574 that exhibited potent inhibitory activity in cultured cells as well as in vitro enzymatic assay.     

Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases

Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step.     

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr⁸⁰⁵, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.     

The tri–μ–hydrido–bis[(η5–C5Me5)aluminum(III)] theoretical study, the assets of sandwiched M2H3 (M of 13th group elements) stability

The stability of the tri–μ–hydrido–bis[(η⁵–C₅Me₅)aluminum], Cp*₂Al₂H₃, 1 is studied at B3LYP/6–311+G(d,p), CCSD(T)//B3LYP/6–311+G(d,p) and MP4//B3LYP/6–311+G(d,p) levels. The coordination between Al₂H₃ entity and both C₅(CH₃)₅ groups is ensured by strong electrostatic and orbital interactions. The orbital analysis of the interacting fragments shows that Al₂H₃ acceptor, which keeps its tribridged structure, implies the vacant ( \texta₁￠ ) \left( {{\text{a}}_1^\prime } \right) and five antibonding (a₂^￠￠ a_2^{\prime \prime } , e′ and e″) molecular orbitals to interact with two orbitals mixtures, b₁ and e" of the donors (C₅Me₅). When we take into account the solvent effect, the computation shows that 1 seems to be stable in condensed phase with a tribridged bond between the Al atoms [Cp*Al(μ-H)₃AlCp*], whereas in the gas phase, the monobridged Cp*AlH(μ-H)AlHCp* 4 is slightly favored (4 kcal mol⁻¹). We propose that 1 could be prepared thanks to Cp*Al (2) and Cp*AlH₂ (3) reaction in acidic medium. The experimental treatment of this type of metallocenes would contribute to the development of the organometallic chemistry of 13th group elements.      

Filtration performance of microporous ceramic supports

The use of inorganic membranes in pollution treatment is actually limited by the cost of such membranes. Advantages of inorganic membranes are their chemical, thermal and pH properties. The purpose of this work was the development of microporous ceramic materials based on clay for liquid waste processing. The supports or ceramic filters having various compositions were prepared and thermally treated at 1100 °C. The results show that, at the temperature studied, porosity varied according to the support composition from 12% for the double-layered (ceramic) support to 47% for the activated carbon- filled support with a mean pore diameter between 0.8 and 1.3 μm, respectively. Volumes of 5 l of distilled water were filtered tangentially for 3 h under an applied pressure of 3.5 and 5.5 bar. The retention of tubular supports prepared was tested with molecules of varying size (Evans blue, NaCl and Sacharose). The study of the liquid filtration and flow through these supports showed that the retention rate depends on support composition and pore diameter, and solute molecular weight. The S1 support (mixture of barbotine and 1% (w/w) activated carbon) gave a flux for distilled water of 68 L/m² h while the double-layered support resulted in a flux of 8 L/m² h for the same solution at the pressure of 3.5 bar. At a pressure of 5.5 bar an increase in the distilled water flux through the various supports was observed. It was significant for the S1 support (230 L/m h).     

DECREASE IN DYNAMIC VISCOSITY AND AVERAGE MOLECULAR WEIGHT OF ALGINATE FROM LAMINARIA DIGITATA DURING ALKALINE EXTRACTION1

Alginates are natural polysaccharides that are extracted from brown seaweeds and widely used for their rheological properties. The central step in the extraction protocol used in the alginate industry is the alkaline extraction, which requires several hours. In this study, a significant decrease in alginate dynamic viscosity was observed after 2 h of alkaline treatment. Intrinsic viscosity and average molecular weight of alginates from alkaline extractions 1–4 h in duration were determined, indicating depolymerization of alginates: average molecular weight decreased significantly during the extraction, falling by a factor of 5 between 1 and 4 h of extraction. These results suggested that reducing extraction time could enable preserving the rheological properties of the extracted alginates.     

Label-free amplified bioaffinity detection using terahertz wave technology

A new affinity biosensor based on pulsed terahertz (THz) wave technology has been used to monitor binding between biotin and avidin molecules. Amplified detection of avidin-biotin binding is obtained on supported membranes composed of biotin layers on quartz surface, which is modified with octadecanol. Agarose particles are conjugated with avidin and then applied to biotin, which is already bound to the octadecanol quartz surface, the biotin binds to the conjugate rapidly and causes an enhancement of the THz difference signal between biotin and biotin-avidin complexes by a factor greater than eight fold when compared to the same sample without agarose beads. The technique was able to detect less than 10.3 ng/cm2 avidin, thus, giving the THz system a detection capability of sub-thin solid films better than ellipsometry and reflectometry techniques. Further improvement is underway using highly refractive beads together with appropriate surface chemistry. This newly developed method is being saliently optimized for future application, including the detection of DNA hybridization and ligand-analyte affinity binding.