Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K-0.5 values for Na+ with minor alterations in K-0.5 values for K+ and N-H-4(+), causing a decrease in maximal velocities under saturating Na+, K+ and NH4+ concentrations. Phosphorylation measurements in the presence of 20 mu M ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na+, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na+, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na+ at the Na+-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments. (c) 2008 Elsevier Inc. All rights reserved.

POLYETHYLENE GLYCOL ADDITION DOES NOT IMPROVE EXOGENOUS SURFACTANT FUNCTION IN AN EXPERIMENTAL MODEL OF MECONIUM ASPIRATION SYNDROME

Meconium (MEC) is a potent inactivator of pulmonary surfactant. The authors studied the effects of polyethylene glycol addition to the exogenous surfactant over the lung mechanics and volumes. Human meconium was administrated to newborn rabbits. Animals were ventilated for 20 minutes and dynamic compliance, ventilatory pressure, and tidal volume were recorded. Animals were randomized into 3 study groups: MEC group (without surfactant therapy); S100 group (100 mg/kg surfactant); and PEG group (100 mg/kg porcine surfactant plus 5% PEG). After ventilation, a pulmonary pressure-volume curve was built. Histological analysis was carried out to calculate the mean alveolar size (Lm) and the distortion index (DI). Both groups treated with surfactant showed higher values of dynamic pulmonary compliance and lower ventilatory pressure, compared with the MEC group (P .05). S100 group had a larger maximum lung volume, V30, compared with the MEC group (P .05). Lm and DI values were smaller in the groups treated with surfactant than in the MEC group (P .05). No differences were observed between the S100 and PEG groups. Animals treated with surfactant showed significant improvement in pulmonary function as compared to nontreated animals. PEG added to exogenous surfactant did not improve lung mechanics or volumes.

A crucial role for TNF-alpha in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5

Background and purpose: Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP-2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice. Experimental approach: Neutrophil recruitment was assessed in immunized mice challenged with methylated bovine serum albumin, KC/CXCL1, LIX/CXCL5 or tumour necrosis factor (TNF)-alpha. Cytokine and chemokine levels were determined in peritoneal exudates and in supernatants of macrophages and mast cells by elisa. CXCR2 and intercellular adhesion molecule 1 (ICAM-1) expression was determined using immunohistochemistry and confocal microscopy. Key results: Antigen challenge induced dose- and time-dependent neutrophil recruitment and production of KC/CXCL1, LIX/CXCL5 and TNF-alpha, but not MIP-2/CXCL2, in peritoneal exudates. Neutrophil recruitment was inhibited by treatment with reparixin (CXCR1/2 antagonist), anti-KC/CXCL1, anti-LIX/CXCL5 or anti-TNF-alpha antibodies and in tumour necrosis factor receptor 1-deficient mice. Intraperitoneal injection of KC/CXCL1 and LIX/CXCL5 induced dose- and time-dependent neutrophil recruitment and TNF-alpha production, which were inhibited by reparixin or anti-TNF-alpha treatment. Macrophages and mast cells expressed CXCR2 receptors. Increased macrophage numbers enhanced, while cromolyn sodium (mast cell stabilizer) diminished, LIX/CXCL5-induced neutrophil recruitment. Macrophages and mast cells from immunized mice produced TNF-alpha upon LIX/CXCL5 stimulation. Methylated bovine serum albumin induced expression of ICAM-1 on mesenteric vascular endothelium, which was inhibited by anti-TNF-alpha or anti-LIX/CXCL5. Conclusion and implications: Following antigen challenge, CXCR2 ligands are produced and act on macrophages and mast cells triggering the production of TNF-alpha, which synergistically contribute to neutrophil recruitment through induction of the expression of ICAM-1.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle. (C) 2010 Elsevier B.V. All rights reserved.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 X 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. (C) 2010 Elsevier Inc. All rights reserved.

Bovine sperm cells viability during incubation with or without exogenous DNA

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 10(6)/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.99% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Spontaneous generation and survival of blood dendritic cells in mononuclear cell culture without exogenous cytokines

Studies on purified blood dendritic cells (DCs) are hampered by poor viability in tissue culture. We, therefore, attempted to study some of the interactions/relationships between DCs and other blood cells by culturing unseparated peripheral blood mononuclear cell (PBMC) preparations in vitro. Flow cytometric techniques were used to undertake a phenotypic and functional analysis of DCs within the cultured PBMC population. We discovered that both the CD11c(+) and CD11c(-) CD123(hi) DC subsets maintained their viability throughout the 3-day culture period, without the addition of exogenous cytokines. This viability was accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts increased (for CD123hi and CD11c+ subsets) after overnight culture of PBMCs. Single-cell lineage depletion experiments demonstrated the rapid and spontaneous emergence of new in vitro generated DCs from CD14(+)/CD16(+) PBMC radioresistant precursors, additional to the preexisting ex vivo DC population. Unlike monocyte-derived DCs, blood DCs increased dextran uptake with culture and activation. Finally, DCs obtained after culture of PBMCs for 3 days were as effective as freshly isolated DCs in stimulating an allogeneic mixed leukocyte reaction. (C) 2002 by The American Society of Hematology.

Exogenous Slit2 does not affect ureteric branching or nephron formation during kidney development

In an attempt to elucidate the role of Slit2 invertebrate kidney development, the effect of adding exogenous human Slit2 protein (hSlit2) to developing murine metanephric kidney explants was examined. To confirm the activity of the recombinant Slit2 protein, neurons from 8 day old chick sympathetic nerve chain dorsal root ganglia were cultured with hSlit2 protein, which induced significant neurite branching and outgrowth. Using kidney explants as a model system, metanephric development in the presence of hSlit2 protein was examined. Addition of hSlit2 up to a final concentration of 1 mug/ml had no detectable effect on the formation of nephrons or on branching morphogenesis of the ureteric tree after 2 or 4 days in culture, as assessed via immunofluorescence for the markers WT1 and calbindin 28K respectively. Similarly, maturation of the nephrogenic mesenchyme occurred in a phenotypically normal fashion. In situ analysis of the Slit receptors, Robot and Robot, the vasculogenic markers VEGFA and Flk-1, and the stromal cell marker BF2 displayed no difference in comparison to controls.

Exogenous induction of ovarian activity and ovulation and transfer of fresh embryos of domestic cat (Felis catus)

The objective of the present study was the exogenous stimulation of ovarian activity and definition of embryo collection, and transfer protocols, in the domestic cat for potential application in non-domestic endangered species. Sixteen adult queens and two adult male reproducers kept in the experimental cat house at the Morphology sector at the Veterinary Department (DVT), UFV, were used in this study. All the queens received a single application of 150 IU Equine Chorionic Gonadotropin (eCG) in the post estrus to induce ovarian activity and 80 to 84 hours later, received a single application of 100 UI Human Chorionic Gonadotropin (hCG) to induce ovulation. After hCG application, only the donor queens were naturally mated. The receptor queens received extra stimulus for induction of ovulation through manipulation of an intravaginal swab. Five to six days after hCG application, the donor queens were subjected to a laparotomy for embryo collection that was performed by trans-horn uterine washing. On average, six embryos were surgically inovulated. They were classified as type I and III compact morula and blastocysts in four receptor queens. Three animals presented pregnancy confirmed by ultrasound at day 36 and two of these animals gave birth to litters of two and four offsprings, respectively, at 66 and 63 days after induction of ovulation. Except for one still birth, all the offspring developed normally.

Action of auxin inhibitors on growth and grain yield of soybean

Soybean genotypes grown in sub-tropical climate may exhibit lodging. The plant lodging is influenced by soil type and fertility level, sowing date, latitude and altitude of the location, plant population and conditions of crop development. Plant regulators and herbicides are able to avoid or reduce plant lodging. This study aimed to verify the effects of the growth regulators TIBA and daminozide on vegetative growth and yield of soybean cultivar CD 214 RR. The experiment was carried out at a field in randomized block design with four replications in a factorial scheme. The A factor was represented by the combination of regulators TIBA and daminozide and its concentrations, and the Factor B was seven times of evaluation of injury and plant height or eight times of evaluation of lodging. In the range of doses used, the application of daminozide resulted in greater injury to soybean plants than TIBA. The smaller plant height was achieved by the application of 6 g ha-1 of TIBA and 1200 g ha-¹ of daminozide. Treatments with daminozide (100 g ha-¹) and TIBA (10 g ha-1) stood out due to the reduced lodging of soybean plants. Grain weight increased linearly when the levels of TIBA increased. There was a negative correlation between lodging and grain yield and a positive correlation between plant height and lodging. There was also a negative correlation between injury caused by the application of plant regulators and lodging.

Contribution of different vegetable types to exogenous nitrate and nitrite exposure

This study reports the levels of nitrate and nitrite of 34 vegetable samples, including different varieties of cabbage, lettuce, spinaches, parsley and turnips, collected in several locations of an intensive agricultural area (Modivas, Vila do Conde, northern Portugal). Nitrate levels ranged between 54 and 2440 mg NO-3 kg-1, while nitrite levels ranged between 1.1 and 57 mg NO-2 kg-1. The maximum residue levels established for nitrate in spinach and lettuce samples were not exceeded. Nitrate and nitrite levels reported in the literature for the same type of samples are reviewed, as well as the contribution of vegetables to nitrate and nitrite dietary exposure of populations.

Different doses of exogenous surfactant for treatment of meconium aspiration syndrome in newborn rabbits

OBJECTIVE: To evaluate the effects of 2 different doses of exogenous surfactant on pulmonary mechanics and on the regularity of pulmonary parenchyma inflation in newborn rabbits. METHOD: Newborn rabbits were submitted to tracheostomy and randomized into 4 study groups: the Control group did not receive any material inside the trachea; the MEC group was instilled with meconium, without surfactant treatment; the S100 and S200 groups were instilled with meconium and were treated with 100 and 200 mg/kg of exogenous surfactant (produced by Instituto Butantan) respectively. Animals from the 4 groups were mechanically ventilated during a 25-minute period. Dynamic compliance, ventilatory pressure, tidal volume, and maximum lung volume (P-V curve) were evaluated. Histological analysis was conducted using the mean linear intercept (Lm), and the lung tissue distortion index (SDI) was derived from the standard deviation of the means of the Lm. One-way analysis of variance was used with a = 0.05. RESULTS: After 25 minutes of ventilation, dynamic compliance (mL/cm H2O · kg) was 0.87 ± 0.07 (Control); 0.49 ± 0.04 (MEC*); 0.67 ± 0.06 (S100); and 0.67 ± 0.08 (S200), and ventilatory pressure (cm H2O) was 9.0 ± 0.9 (Control); 16.5 ± 1.7 (MEC*); 12.4 ± 1.1 (S100); and 12.1 ± 1.5 (S200). Both treated groups had lower Lm values and more homogeneity in the lung parenchyma compared to the MEC group: SDI = 7.5 ± 1.9 (Control); 11.3 ± 2.5 (MEC*), 5.8 ± 1.9 (S100); and 6.7 ± 1.7 (S200) (*P < 0.05 versus all the other groups). CONCLUSIONS: Animals treated with surfactant showed significant improvement in pulmonary mechanics and more regularity of the lung parenchyma in comparison to untreated animals. There was no difference in results after treatment with either of the doses used.

Official Central Bank Interventions in the Foreign Exchange Markets: A DCC Approach with Exogenous Variables

This paper assesses the impact of official central bank interventions (CBIs) on exchange rate returns, their volatility and bilateral correlations. By exploiting the recent publication of intervention data by the Bank of England, this study is able to investigate fficial interventions by a total number of four central banks, while the previous studies have been limited to three (the Federal Reserve, Bundesbank and Bank of Japan). The results of the existing literature are reappraised and refined. In particular, unilateral CBI is found to be more successful than coordinated CBI. The likely implications of these findings are then discussed.

PKS2: A link between phototropin signalling and auxin transport - a study on how plants sense and respond to light.

The blue light photoreceptors phototropins (phot1 and phot2 in Arabidopsis thaliana (L.)) carry out various light responses of great adaptive value that optimize plant growth. These processes include phototropism (the bending of an organ induced by unequal light distribution), chloroplast movements, stomatal opening, leaf flattening and solar tracking. The biochemical pathways controlling these important blue light responses are just starting to be elucidated. The PHYTOCHROME KINASE SUBSTRATE (PKS1-4) proteins - the subject of this research - have recently been identified as novel phototropism signalling components. PKS1 (the founding member of this family) interacts in a same complex in vivo with phot1 and the important phot1 signalling element NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3). This suggested that the PKS may act as early components of phot signalling. This work further investigates the role of this protein family during phototropin signalling Genetic experiments clearly showed that the PKS do not control chloroplast movements or stomatal opening. However, PKS2 plays a critical role with NPH3 during leaf flattening and solar tracking. Epistasis data indicated that both proteins act in phot1 and phot2 pathways, which is consistent with their in vivo interaction with both phototropins. Because phototropism, leaf flattening and solar tracking are developmental processes regulated by the hormone auxin, the role of PKS2 and NPH3 during auxin homeostasis was also investigated. Interestingly, PKS2 loss-of-function restores leaf flattening in the auxin transporter mutant aux1. Moreover, PKS2 and NPH3 are found in a same complex with AUX1 in vivo. Taken together, these results suggest that PKS2 may act with NPH3 as a connecting point between phot signalling and auxin transport. Further experiments were performed to explore the molecular mode of action of PKS2 and NPH3 in this process. The significance of these results is discussed.

Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling.

Plant growth is strongly influenced by the presence of neighbors that compete for light resources. In response to vegetational shading shade-intolerant plants such as Arabidopsis display a suite of developmental responses known as the shade-avoidance syndrome (SAS). The phytochrome B (phyB) photoreceptor is the major light sensor to mediate this adaptive response. Control of the SAS occurs in part with phyB, which controls protein abundance of phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) directly. The shade-avoidance response also requires rapid biosynthesis of auxin and its transport to promote elongation growth. The identification of genome-wide PIF5-binding sites during shade avoidance revealed that this bHLH transcription factor regulates the expression of a subset of previously identified SAS genes. Moreover our study suggests that PIF4 and PIF5 regulate elongation growth by controlling directly the expression of genes that code for auxin biosynthesis and auxin signaling components.