Ecology of aquatic Ranunculus communities under the Mediterranean climate

The Iberian Peninsula encompasses more than 80% of the species richness of European aquatic ranunculi. The floristic diversity of the phytocoenosis characterised by aquatic Ranunculus and the main physical–chemical factors of the water were studied in 43 localities of the central Iberian Peninsula. Four aquatic Ranunculus communities are found in most of the aquatic environments. These are species-poor and have an uneven distribution: three species of Batrachium are heterophyllous and their communities are distributed in different aquatic ecosystems on silicated substrates; one species is homophyllous and its community occurs in various aquatic ecosystems with carbonated waters. In the Mediterranean climate, Ranunculus species are present in different habitats, as shown by the results of all the statistical analyses. Ranunculus trichophyllus communities occur in base-rich waters with a high buffering capacity (2273.44 ± 794.57 mg CaCO₃ L⁻¹) and a high concentration of cations (Ca²⁺, 121 ± 33.12 mg L⁻¹; Mg²⁺, 71.64 ± 82.77 mg L⁻¹), nitrates (2.89 ± 4.80 mg L⁻¹), ammonium (2.19 ± 1.36 mg L⁻¹) and sulphates (216.25 ± 218.54 mg L⁻¹). Ranunculus penicillatus communities are found in flowing waters with a high concentration of phosphates (0.48 ± 0.6 mg L⁻¹) and intermediate buffering capacity (683.66 ± 446.76 mg CaCO₃ L⁻¹). Both Ranunculus pseudofluitans and Ranunculus peltatus communities grow in waters with low buffering capacity (R. pseudofluitans, 385.91 ± 209.2 mg CaCO₃ L⁻¹; R. peltatus, 263.3 ± 180.36 mg CaCO₃ L⁻¹), and a low concentration of cations (R. pseudofluitans: Ca²⁺, 12.57 ± 9.42 mg L⁻¹; Mg²⁺, 3.42 ± 1.67 mg L⁻¹; R. peltatus: Ca²⁺, 15 ± 18.26 mg L⁻¹; Mg²⁺, 6.26 ± 8.89 mg L⁻¹) and nutrients (R. pseudofluitans: nitrates, 0.23 ± 0.2 mg L⁻¹; phosphates, 0.09 ± 0.1 mg L⁻¹; R. peltatus: nitrates, 0.19 ± 0.21 mg L⁻¹; phosphates, 0.09 ± 0.12 mg L⁻¹); the first in flowing waters, the latter in still waters.     

Kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart

An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (− TΔΔS = 5.1 ± 0.5 kcal/mol, at 20 °C). However, this unfavorable entropy term is largely offset by enthalpic gains (ΔΔH = − 3.5 ± 0.4 kcal/mol), presumably arising from tightening of non-covalent interactions throughout the monomeric complex. Stopped-flow kinetic measurements further reveal that the catalytic molten globule binds and releases ligands significantly faster than its natural counterpart, demonstrating that partial structural disorder can speed up molecular recognition. These results illustrate how structural plasticity may strongly perturb the thermodynamics and kinetics of transition-state recognition while negligibly affecting catalytic efficiency.     

Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter,Niemann–Pick type C1

Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1^−/−), and subsequently in Cav-1^−/− mice that also lacked the lysosomal cholesterol transporter Niemann–Pick C1 (Npc1) (Cav-1^−/−:Npc1^−/−). In 50-day-old Cav-1^−/− mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1^+/+ controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1^−/−:Npc1^−/− mice (0.356 ± 0.022) markedly exceeded that in their Cav-1^+/+:Npc1^+/+ controls (0.137 ± 0.009), as well as in their Cav-1^−/−:Npc1^+/+ (0.191 ± 0.013) and Cav-1^+/+:Npc1^−/− (0.213 ± 0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74 ± 0.17, 0.71 ± 0.05, 0.96 ± 0.05 and 3.12 ± 0.43, respectively, with the extra cholesterol in the Cav-1^−/−:Npc1^−/− and Cav-1^+/+:Npc1^−/− mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1^−/−:Npc1^−/− mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.     

Kinetics and bioreactor studies of immobilized invertase on polyurethane rigid adhesive foam

A new support, polyurethane rigid adhesive foam (PRAF), which can be used to cover internal surface of metallic tubes, was used to immobilize invertase for application in an enzymatic bioreactor. The kinetic parameters were: K_m - 46.5 ± 1.9 mM (PRAF-invertase) and 61.2 ± 0.1 mM (free enzyme) and V_max 42.0 ± 4.3 U/mg protein/min (PRAF-invertase) and 445.3 ± 24.0 U/mg protein/min (free invertase). The PRAF-invertase derivative maintained 50.1% of initial activity (69.17 U/g support) for 8 months (4 °C) and was not observed microbial contamination. The bioreactor showed the best production of inverted sugar syrup using up-flow rate (0.48 L/h) with average conversion of 10.64 ± 1.5% h⁻¹ at feeding rate (D) of 104 h⁻¹. The operational inactivation rate constant (k_opi) and half-life were 1.92 × 10⁻⁴ min⁻¹ and 60 h (continue use). The PRAF spray support looks promising as a new alternative to produce immobilized derivatives on reactor surfaces.     

Screening of boldenone and methylboldenone in bovine urine using disposable electrochemical immunosensors

Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone-bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG-horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3′,5,5′-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9 ± 4.3 pg ml⁻¹ for boldenone and 120.2 ± 8.2 pg ml⁻¹ for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC₅₀ = 1.0 ± 0.3 ng ml⁻¹ (n = 3, N = 3), R² = 0.969, R.S.D. = 9.6% for boldenone and 1.5 ± 0.3 ng ml⁻¹, 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of α-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.     

Comparative pharmacokinetics of baicalin and wogonoside by liquid chromatography–mass spectrometry after oral administration of Xiaochaihu Tang and Radix scutellariae extract to rats

The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC–MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C₁₈ column (150 mm × 4.6 mm, i.d.: 5 μm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/z 447 for baicalin, m/z 461 for wogonoside and m/z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156 μg mL⁻¹ for baicalin and 0.08832 to 4.416 μg mL⁻¹ for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155 μg mL⁻¹ for baicalin and 0.04416 μg mL⁻¹ for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t_1/2 of baicalin was 3.60 ± 0.90 and 5.64 ± 1.67, the C_max1 was 1.64 ± 0.99 and 5.66 ± 2.02, the t_max1 was 0.13 ± 0.05 and 0.20 ± 0.07, the C_max2 was 2.43 ± 0.46 and 3.18 ± 1.66, and the t_max2 were 6.40 ± 1.67 and 5.66 ± 2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t_1/2 of wogonoside was 4.97 ± 1.68 and 7.71 ± 1.55, the C_max1 was 1.39 ± 0.83 and 1.45 ± 0.37, the t_max1 was 0.21 ± 0.20 and 0.17 ± 0.01, the C_max2 was 1.90 ± 0.55 and 1.42 ± 0.70, and the t_max2 was 5.60 ± 1.67 and 5.20 ± 1.79, respectively. A significant difference (p < 0.05) was observed for t_1/2, and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased.     

Cytoskeletal and developmental alterations in Ceratophyllum demersum induced by microcystin-LR, a cyanobacterial toxin

The aim of the present work was the investigation of microtubule organization related to developmental processes of Ceratophyllum demersum, a submergent aquatic macrophyte, as affected by microcystin-LR (MCY-LR), a cyanobacterial toxin. We studied the time- and dose-dependent effects of the cyanotoxin in a concentration range of 0.01-20 μg mL⁻¹ (0.01-20.1 μM) at exposure times of 2-16 d. At short term (4 d) of MCY-LR exposure we observed the inhibition of C. demersum shoot tip elongation. This phenomenon was already observed at 0.01 μg mL⁻¹ MCY-LR (reduction of shoot tip length to 56 ± 3.6% of controls) and correlated with the induction of cortical microtubule (CMT) reorientation from transverse to longitudinal known to induce radial expansion of meristematic cells instead of normal elongation. Concomitantly we detected the increase of the percentage of cells in early mitosis in shoot tip meristems, from 1.17 ± 0.2% for controls to 1.93 ± 0.3 at 0.01 μg mL⁻¹ MCY-LR and 6.19 ± 0.5 at 10 μg mL⁻¹ MCY-LR. Cyanotoxin exposure induced the inhibition of general shoot elongation that was more pronounced than inhibition of the increase of leaf whorl number or fresh weight in the treatment period and this was observable at as short as 2-4 d of 2.5 μg mL⁻¹ MCY-LR exposure. This observation further proved that the MCY-LR-induced inhibition of cell elongation is responsible mainly for growth inhibition in C. demersum. Concomitantly with developmental and growth changes MCY-LR decreased protein and chlorophyll content at 16 d of exposure: at 20 μg mL⁻¹ of cyanotoxin, protein content was reduced to 53.3 ± 2.8%, while total chlorophyll content to 46.53 ± 2.7% of controls. This is the first study showing that MCY-LR inhibits the growth of C. demersum through cytoskeletal reorganization. This plant proved to be a powerful model system for toxicological as well as plant cell biology studies.     

Portal vein cross-sectional area and flow and orthostatic tolerance: a 90-day bed rest study.

The objective of this study was to evaluate the changes in the portal vein cross-sectional area (PV CSA) and flow during a stand test associated with orthostatic intolerance. Eighteen subjects underwent a 90-day head-down tilt (HDT) bed rest at 6 degrees: 9 controls (Con) and 9 with flywheel exercise countermeasures (CM). At post-HDT, nine subjects (5 CM, 4 Con) were tolerant, and nine were intolerant. The PV CSA was measured by echography. We found that at HDT day 85, the PV CSA at rest had increased less in the CM subjects than in the Con (+12 vs. +27% from pre-HDT supine; P < 0.05), whereas it increased similarly in tolerant and intolerant subjects (23 and 16%, respectively). Two days after the HDT, there was a decrease in the PV CSA supine compared with the pre-HDT PV CSA supine that was similar for all groups (Con: -11%, CM: -21%; tolerant: -10%, intolerant: -16%; P < 0.05). The PV CSA decreased significantly less from supine to standing in the Con than in the CM group (-2 vs. -10% compared with the pre-HDT stand test; P < 0.05). The PV CSA also decreased significantly from supine to standing compared with the pre-HDT stand test in the tolerant group but not in the intolerant group (-20 vs. +2%; P < 0.05). From these findings, we conclude the following. 1) Because the portal vein is the only output from the splanchnic vascular area, we suggest that the lower reduction in the PV CSA and flow associated with orthostatic intolerance was related to a lower splanchnic arterial vasoconstriction. 2) The flywheel exercise CM helped to reduce the distention of the splanchnic network at rest and to maintain partially the splanchnic vasoconstriction, but it did not reduce the orthostatic intolerance.     

Morphological and physiological adaptive traits of Mediterranean narrow endemic plants: The case of Centaurea gymnocarpa (Capraia Island,Italy)

A case study on Centaurea gymnocarpa Moris & De Not., a narrow endemic species, was carried out by analyzing its morphological, anatomical, and physiological traits in response to natural habitat stress factors under Mediterranean climate conditions. The results underline that the species is particularly adapted to the environment where it naturally grows. At the plant level, the above-ground/below-ground dry mass (1.73 ± 0.60) shows its investment predominately in the above-ground structure with a resulting total leaf area per plant of 1399 ± 94 cm². The senescent attached leaves at the base of the plant contribute to limit leaf transpiration by shading soil around the plant. Moreover, the dense C. gymnocarpa leaf pubescence, leaf rolling, the relatively high leaf mass area (LMA = 12.3 ± 1.3 mg cm⁻²) and leaf tissue density (LTD = 427 ± 44 mg cm⁻³) contribute to limit leaf transpiration, also postponing leaf death under dry conditions. At the physiological level, a relatively low respiration/photosynthesis ratio (R/P_N) in spring results from high R [2.26 ± 0.59 μmol (CO₂) m⁻² s⁻¹] and P_N [12.3 ± 1.5 μmol (CO₂) m⁻² s⁻¹]. The high photosynthetic nitrogen use efficiency [PNUE = 15.5 ± 0.4 μmol (CO₂) g⁻¹ (N) s⁻¹] shows the large amount of nitrogen (N) invested in the photosynthetic machinery of new leaves, associated to a high chlorophyll content (Chl = 35 ± 5 SPAD units). On the contrary, the highest R/P_N ratio (1.75 ± 0.19) in summer is due to a significant P_N decrease and increase of R in response to drought. The low PNUE [1.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] in this season is indicative of a greater N investment in leaf cell walls which may contribute to limit transpiration. On the contrary, the low R/P_N ratio (0.05 ± 0.02) in winter is resulting from the limited enzyme activity of the respiratory apparatus [R = 0.23 ± 0.08 μmol (CO₂) m⁻² s⁻¹] while the low PNUE [3.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] suggests that low temperatures additionally limit plant production. The experiment of the imposed water stress confirms that the C. gymnocarpa growth capability is in conformity with the severe conditions of its natural habitat, likewise as it may be the case with others narrow endemic species that have occupied niches with similar extreme conditions.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

Distribution and residual activity of two insecticidal proteins, avidin and aprotinin, expressed in transgenic tobacco plants, in the bodies and frass of Spodoptera litura larvae following feeding

To understand how a major cosmopolitan pest responds to two very different insecticidal proteins and to determine whether herbivorous insects and their frass could be environmental sources of recombinant proteins from transgenic plants, Spodoptera litura (Fab.) (Lepidoptera, Noctuidae) larvae were fed on tobacco leaves expressing either the biotin-binding protein, avidin, or the protease inhibitor, aprotinin. Control larvae received non-transgenic tobacco. Samples of larvae were taken after 5, 6 or 7 days’ feeding and frass was collected after two 24-h periods at 6 and 7 days. Insects in all treatments grew significantly during the experiment, but the avidin-fed larvae were significantly smaller than the others on Day 7. Avidin was found in all samples of avidin-fed larvae (7.0±0.86 ng mg⁻¹, n=45), at a lower level than in their frass (31.9±5.08 ng mg⁻¹, n=30), and these frass levels were lower than those of the the leaves fed to the larvae (69.0±6.71 ng mg⁻¹, n=45). All of the avidin detected in these samples was capable of binding biotin. On average, between 10 and 28% of avidin was recovered with the methods used, whereas almost full recovery of aprotinin was effected. Aprotinin levels in larvae (8.2±0.53 ng mg⁻¹, n=45) were also lower than aprotinin levels in frass (77.4±6.9 ng mg⁻¹, n=30), which were somewhat lower than those in the leaves fed to the larvae (88.6±2.51 ng mg⁻¹, n=45). Approximately half the trypsin-binding ability of aprotinin was lost in larvae, and in frass, aprotinin had lost about 90% of its ability to bind trypsin.     

The effect of flow turbulence on plant growth and several growth regulators in Egeria densa Planchon

The effects of turbulence velocity on Egeria densa Planchon was studied for 12 weeks using mechanically oscillating grid-generated turbulence without mean flow. The root-mean-square of the turbulence velocity fluctuations (u′) ranged from 1.62 ± 0.44 to 2.86 ± 0.8 cm s⁻¹ (high turbulence), 1.36 ± 0.2 to 1.86 ± 0.78 cm s⁻¹ (medium turbulence) and 0.67 ± 0.12 to 0.81 ± 0.16 cm s⁻¹ (low turbulence). The control was subjected to gentle manual mixing once a day. Shoot elongation was significantly reduced with increasing turbulence intensity, and the endogenous indole acetic acid (IAA) concentration was significantly decreased with increasing turbulence intensity and exposure time. The plants exposed to high turbulence showed a 64.6% decrease in endogenous IAA concentration compared to the control, while it was decreased only 26.9% in plants exposed to low turbulence. IAA and cytokinin catabolism was increased, and there was an increase in the hydrogen peroxide concentration of the tissues, which triggered peroxidase activity. The total chlorophyll and chlorophyll a content decreased with the time of exposure. Although the flow turbulence negatively affected plant growth and metabolism, all of the plants survived for the experimental period.     

Timing and success of reproductive stages in the seagrass Zostera noltii

The timing and success of sexual reproduction of the seagrass Zostera noltii was investigated at the Ria Formosa lagoon, Portugal. Thirty plants were tagged and monitored individually through time to determine in situ the development time of each maturation stage, from the emergence of the flowers to the production of seeds. The overall process of flowering and fruiting lasted 47 ± 4 days, during which formation and maturation of the fruits was the most time-consuming stage (27 ± 2 days). Spathe success, i.e. the percentage of spathes that produced seeds, was 22 ± 4% while spathe mortality was 34 ± 6%. A considerable percentage of spathes (37 ± 7%) was lost through leaf detachment, but some of these may still add to the reproductive success of the species by contributing to the species recruitment within the source meadow or elsewhere. Meadow seed production (MSP) of Z. noltii was estimated to be 312 ± 66 seeds m⁻², whereas the potential seed production of the studied meadow was 2623 seeds m⁻². Under laboratory conditions, 70% of Z. noltii seeds germinated within 26 days, but only 10% reached the seedling stage due to the high mortality of germlings. Fertility, defined as the probability of a seed to originate a new plant, was estimated to be 14 × 10⁻⁴, which is higher than what is expected for most seagrasses.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

Uncoupling protein 2 − 866G/A and uncoupling protein 3 − 55C/T polymorphisms in young South African Indian coronary artery disease patients

Background

Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Promoter polymorphisms of these genes have been linked to chronic diseases including heart disease and type II diabetes mellitus in several populations. This is the first investigation of the UCP2 − 866G/A rs659366 and UCP3 − 55C/T rs1800849 polymorphisms in young South African (SA) Indians with coronary artery disease (CAD).

Methods

A total of 300 subjects were recruited into this study of which 100 were SA Indian males with CAD, 100 age- (range 24–45 years), gender- and race-matched controls and 100 age-matched black SA males. The frequency of the UCP2 − 866G/A and UPC3 − 55C/T genotypes was assessed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP).

Results

The heterozygous UCP2 − 866G/A and homozygous UCP3 − 55C/C genotypes occurred at highest frequency in CAD patients (60% and 64%, respectively) compared to SA Indian controls (52% and 63%) and SA Black controls (50% and 58%). The UCP2 − 886G/A (OR = 1.110; 95% CI = 0.7438–1.655; p = 0.6835) and UCP3 − 55C/T (OR = 0.788; 95% CI = 0.482–1.289; p = 0.382) polymorphisms did not influence the risk of CAD.The rare homozygous UCP3 − 55T/T genotype was associated with highest fasting glucose (11.87 ± 3.7 mmol/L vs. C/C:6.11 ± 0.27 mmol/L and C/T:6.48 ± 0.57 mmol/L, p = 0.0025), HbA1c (10.05 ± 2.57% vs. C/C:6.44 ± 0.21% and C/T:6.76 ± 0.35%, p = 0.0006) and triglycerides (6.47 ± 1.7 mmol/L vs. C/C:2.33 ± 0.17 mmol/L and C/T:2.06 ± 0.25 mmol/L, p < 0.0001) in CAD patients.

Conclusion

The frequency of the UCP2 − 866G/A and UCP3 − 55C/T polymorphisms was similar in our SA Indian and SA Black groups. The presence of the UCP2 − 866G/A and UCP3 − 55C/T polymorphisms does not influence the risk of CAD in young South African Indian CAD patients.     

Towards a more objective assessment of equine personality using behavioural and physiological observations from performance test training

Current definitions of horse personality traits are rather vague, lacking clear, universally accepted guidelines for evaluation in performance tests. Therefore, the aim of the present study was to screen behavioural and physiological measurements taken during riding for potential links with scores the same horses received in the official stallion performance test for rideability and personality traits. Behaviour, heart rate (HR) and HR variability from thirty-six stallions participating in a performance test were recorded repeatedly during their performance test training. Using the coefficient of determination, regression analysis revealed that about 1/3 of variation (ranging between r = 0.26 (“constitution” (i.e. fitness, health)) and r = 0.46 (rideability)) in the personality trait scores could be explained by selecting the three most influential behaviour patterns per trait. These behaviour patterns included stumbling (with all traits except character), head-tossing (temperament, rideability), tail-swishing (willingness to work), involuntary change in gait (character) and the rider's use of her/his hands (constitution, rideability), voice (temperament) or whip (constitution). Subsequent mixed model analysis revealed a significant (P < 0.05) influence of the behaviour pattern “horse-induced change in gait” on character (−0.98 ± 0.31 scores per additional occurrence of change in gaits), of head-tossing (−0.25 ± 0.08 scores) and rider's use of voice (−0.51 ± 0.25; P = 0.0594) on temperament, and of stumbling on each of the following: willingness to work (−2.5 ± 1.2), constitution (−2.5 ± 1.2 scores; P = 0.0516) and rideability scores (−3.3 ± 1.4). In addition, constitution scores tended (P = 0.0889) to increase with higher low frequency/high frequency heart rate variation ratios (LF/HF), indicating a shift towards sympathetic dominance and thus a higher stress load in horses with higher scores for constitution. Rideability scores from the training phase were also significantly influenced by head-tossing (−0.5 ± 0.1), and in addition rideability scores from the final test were influenced by the training rider, ranging between average estimated rideability scores of 6.8 ± 0.4 for one training rider and 8.36 ± 0.3 scores for another training rider. Horses ridden with their nose-line predominantly behind the vertical received higher scores for rideability (8.3 ± 0.3) than horses ridden with their nose-line at the vertical (7.7 ± 0.2). These findings indicate that either judges perceive horses to have a better rideability when they readily offer a more extreme poll flexion, or that riders make use of horses’ better rideability by imposing a more extreme poll flexion. Several of the above described associations, but also of the non-existing links (e.g. no association between shying or heart rate and temperament) between behaviour patterns and scores for personality traits are rather surprising, warranting further investigation regarding the underlying causes of these relationships. Some of these behaviour patterns should be considered when redesigning the current guidelines for evaluation of personality traits during breeding horse performance tests, ultimately leading to improved genetic selection for equine personality traits. However, ethical implication of defining aversive behaviour such as head-tossing as an indicator of, for example, poor temperament, should not be neglected when devising new guidelines: such aversive behaviour may in fact be an indication of inadequate training techniques rather than poor horse personality.     

Nitrogen uptake responses of Gracilaria vermiculophylla (Ohmi) Papenfuss under combined and single addition of nitrate and ammonium

The ammonium (NH₄⁺) and nitrate (NO₃⁻) uptake responses of tetrasporophyte cultures from a Portuguese population of Gracilaria vermiculophylla were studied. Thalli were incubated at 5 nitrogen (N) levels, including single (50 μM of NH₄⁺ or NO₃⁻) and combined addition of each of the N sources. For the combined additions, the experimental conditions attempted to simulate 2 environments with high N availability (450 μM NO₃⁻ + 150 μM NH₄⁺; 250 μM NO₃⁻ + 50 μM NH₄⁺) and the mean N concentrations occurring at the estuarine environment of this population (30 μM NO₃⁻ + 5 μM NH₄⁺). The uptake kinetics of NH₄⁺ and NO₃⁻ were determined during a 4 h time-course experiment with N deprived algae. The experiment was continued up to 48 h, with media exchanges every 4 h. The uptake rates and efficiency of the two N sources were calculated for each time interval. For the first 4 h, G. vermiculophylla exhibited non-saturated uptake for both N sources even for the highest concentrations used. The uptake rates and efficiency calculated for that period (V_0-4 h), respectively, increased and decreased with increasing substrate concentration. NO₃⁻ uptake rates were superior, ranging from 1.06 ± 0.1 to 9.65 ± 1.2 μM g(dw)⁻¹ h⁻¹, with efficiencies of 19% to 53%. NH₄⁺ uptake rates were lower (0.32 ± 0.0 to 5.75 ± 0.08 μM g(dw)⁻¹ h⁻¹) but G. vermiculophylla removed 63% of the initial 150 μM and 100% at all other conditions. Uptake performance of both N sources decreased throughout the duration of the experiment and with N tissue accumulation. Both N sources were taken up during dark periods though with better results for NH₄⁺. Gracilaria vermiculophylla was unable to take up NO₃⁻ at the highest concentration but compensated with a constant 27% NH₄⁺ uptake through light and dark periods. N tissue accumulation was maximal at the highest N concentration (3.9 ± 0.25% dw) and superior under NH₄⁺ (3.57 ± 0.2% dw) vs NO3 (3.06 ± 0.1% dw) enrichment. The successful proliferation of G. vermiculophylla in estuarine environments and its potential utilization as the biofilter component of Integrated Multi-Trophic Aquaculture (IMTA) are discussed.     

Hypericin biosynthesis in Hypericum hookerianum Wight and Arn: Investigation on biochemical pathways using metabolite inhibitors and suppression subtractive hybridization

The biochemical pathway to hypericin biosynthesis is presumed to be polyketide synthase (PKS) mediated, but it has not been experimentally validated, and no alternate route (chorismate/o-succinylbenzoate pathway) has been analyzed. We report here our earlier developed auxin inducible culture systems of Hypericum hookerianum as a model, to study the metabolic pathway to hypericin synthesis. Inhibitors of the alternate pathway at varying concentrations showed steady synthesis of total hypericins with means of 2.80 ± 0.22, 18.75 ± 0.01; 16.39 ± 3.75, 29.60 ± 1.90 (mevinolin) 2.53 ± 0.10, 18.12 ± 0.56; 0.14 ± 0.01, 14.28 ± 1.11 (fosmidomycin) and 2.7 ± 0.35, 18.75 ± 0.61; 0.14 ± 0.01, 12.80 ± 1.09 mg g⁻¹ DW (glyphosate) in the control and auxin-induced shoot and shoot-forming callus cultures, respectively. SSH analysis classified the differentially expressed sequences into protein synthesis (38%), modification (20%), electron transport (9%) and remaining as unclassified (11%) and unknown proteins (22%). Functional annotation of sequences indicates the presence of additional protein components besides PKS activity. Our results demonstrate direct biochemical and molecular evidence of PKS hypothesis of hypericin biosynthesis for the first time.     

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.