Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.     

Net P-removal deterioration in enriched PAO sludge subjected to permanent aerobic conditions

Recently, some research in the field of enhanced biological phosphorus removal (EBPR) has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. This can occur, for example, in a full scale wastewater treatment plant during heavy rainfall periods when the anaerobic hydraulic retention time is temporarily shortened. To study this situation that could induce EBPR failure, the operation of a sequencing batch reactor (SBR) working under alternating anaerobic-aerobic conditions with an enriched EBPR population (50% Candidatus Accumulibacter phosphatis and less than 1% Candidatus Competibacter phosphatis) was shifted to strict aerobic operation. Seven cycle studies were performed during the 11 days of aerobic operation. Net P-removal was observed in this aerobic SBR during the first 4 days of operation but the system could not achieve net-P removal after this period, although the microbial composition, in terms of percentage of Accumulibacter and Competibacter, did not change significantly. The observed changes in the different compounds analysed (phosphorus, acetate, glycogen and PHB) as well as in the OUR profile indicate that metabolic changes are produced for the adaptation of PAO to aerobic conditions.     

Structural and ultrastructural features of boar bulbourethral glands

The morphological features of boar bulbourethral glands were examined by light and transmission microscopy. Bulbourethral glands are compound tubuloalveolar glands surrounded by a capsule of dense connective tissue and arranged in multiple lobules formed by endpieces and excretory ducts. Endpieces and excretory ducts are both lined by a single epithelium of mucous cells with a basal nucleus. Epithelial cells accumulate secretory granules containing neutral and carboxylated acid mucosubstances and a small amount of sulphated acid mucosubstances. The ultrastructure of epithelial cells varies according to the secretory cycle. In initial stages, the cells show a columnar shape and secretory granules unevenly distributed in the cytoplasm. As the synthesis of mucosubstances progresses, the amount of the secretory granules increases and the cellular shape becomes pyramidal. Secretory granules can contain inclusions and present differences among them according to their different phases of formation. In pyramidal cells, secretory products are released into the lumen by a merocrine mechanism.     

Inhibitors of sphingolipid metabolism enzymes

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery of small chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools and therapeutic agents.     

Inhibitors of sphingolipid metabolism enzymes

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery of small chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools and therapeutic agents.     

Crystalline structure of poly(hexamethylene adipate). Study on the morphology and the enzymatic degradation of single crystals

The crystalline structure of polyester 6 6 was studied by means of X-ray and electron diffraction and real-space electron microscopy. An orthorhombic unit cell containing eight chain segments with a quasi planar zigzag conformation was derived. The chain axis projection could be defined by a small rectangular cell containing only two molecular segments. Simulation of electron diffraction patterns indicates that molecular segments were arranged with azimuthal angles close to +/-46 degrees . X-ray diffraction patterns suggested that the large dimensions of the unit cell were a consequence of a slight shift between neighboring chains that improved the electrostatic interactions. Chain-folded lamellar crystals were obtained by isothermal crystallization of dilute n-hexanol or n-octanol solutions. The crystalline habit was studied, and the influence of temperature was evaluated. A regular folding surface was observed by using polyethylene decoration techniques. Lamellar crystals were easily degraded with different lipases. A preferential enzymatic attack was, in some cases, observed to occur in the crystal edges, giving rise to highly irregular borders with a fringed texture.     

Cheap and Long‐Life Reusable Polymer for Asymmetric Organozinc Catalysis Based on Camphor‐Derived Hydroxyamides

Polystyrene grafted with a chiral zinc‐complexing camphor‐derived N,N‐disubstituted hydroxyamide is proposed as a new type of functional polymer of high reusability for the development of sustainable organozinc‐catalyzed asymmetric reactions. The main goal of this new functional polymer is the ease of the hydroxyamide‐moiety preparation (cheap chiral ligand obtained straightforwardly from an enantiopure starting material coming from the chiral pool), as well as its chemical robustness when compared with other related zinc‐complexing functional groups. The latter allows the polymer to be active after multiple applications, without significant loss of its catalytic activity. This fact is exemplified by the design and preparation of a polymer functionalized with a bis(hydroxyamide) proved previously as active in the homogeneous enantioselective addition of diethylzinc to aldehydes. The result is a cheap functional polymer with a very high reusability (the enantioselectivity and chemical yield are maintained practically constant after 20 applications). Additionally, a methodology for the multicycle use of these functional polymers is presented. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Accumulated bending energy elicits neutral sphingomyelinase activity in human red blood cells

We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A(2). The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypo-and hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A(2) activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stress-induced ceramide synthesis and apoptosis.     

Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9

Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5-6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.     

Determination of sphingosine-1-phosphate lyase activity by gas chromatography coupled to electron impact mass spectrometry

Sphingosine-1-phosphate lyase (SGPL1) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldolic cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. In this article we report on an easy and sensitive procedure to determine SPL activity. The assays uses C17-sphinganine-1-phosphate as substrate and the aldehyde product, pentadecanal, is quantified as its pentafluorobenzyloxime derivative by GC/MS. Derivatization of pentadecanal is performed as a one-step reaction, and the oxime product is directly injected for GC/MS analysis without any further purification. Acquisition in selected ion monitoring mode allows very high sensitivity, with a limit of detection of 281fmol. The assay is linear with both protein concentration and incubation time up to 20μg and 40min, respectively. The K(m) value obtained (6μM) is similar to that for the natural substrate sphingosine-1-phosphate. Using this method, FTY720 and deoxypyridoxine phosphate inhibited SPL with similar potencies to those reported.