Inhibitory effect of unconjugated bilirubin on p-aminohippurate transport in rat kidney cortex slices

(1) The effects of unconjugated bilirubin on the accumulation of p-aminohippurate, kinetics of p-aminohippurate uptake, the efflux of pre-accumulated p-aminohippurate and water and electrolyte distribution were investigated in the rat kidney cortical slice. (2) The addition of unconjugated bilirubin to the incubation medium decreased the 60 min slice-to-medium concentration ratio of p-aminohippurate. (3) The decrease in p-aminohippurate accumulation by unconjugated bilirubin was found to be more pronounced by increasing the concentration of pigment in the medium. (4) The rate of uptake of p-aminohippurate as a function of p-aminohippurate concentration differed in aerobiosis and anaerobiosis, and unconjugated bilirubin decreased only the uptake of p-aminohippurate in aerobic conditions. (5) The efflux of pre-accumulated p-aminohippurate decreased when unconjugated bilirubin concentration in the medium was low (10–20 μM) but the efflux increased when the concentration of pigment was much higher (100 μM). (6) The addition of unconjugated bilirubin to the medium (40–100 μM) increased intracellular sodium and total tissue water content, and decreased intracellular potassium and oxygen consumption of tissue. However the slices incubated with low concentration of pigment (20 μM) did not exhibit significative changes in cellular functional parameters. (7) These findings suggest that unconjugated bilirubin impairs p-aminohippurate transport by a complex mechanism that might involve binding of pigment to sites necessary for anion transport, although effects related to pigment toxicity or to its oxidative decomposition are not excluded.     

Characterization of plasma cholinesterase activity in the Eurasian Griffon Vulture Gyps fulvus and its in vitro inhibition by carbamate pesticides

The legal and illegal use of organophosphorus and carbamate pesticides represents one of many threats to birds. The activity of the cholinesterase enzyme in plasma is used as a non‐destructive biomarker to diagnose the exposure of birds to these pesticides. Scavengers are one of the most important bird groups threatened by the use of baits poisoned with anticholinesterase pesticides. Knowledge of the characteristics of this enzyme in each bird species is crucial, as several studies indicate that more than one cholinesterase form may be present in the plasma of birds. In this study, cholinesterase activity was characterized in the plasma of the Eurasian Griffon Vulture Gyps fulvus by using several substrates and inhibitors of the enzyme, and its normal activity value was also determined. The in vitro sensitivity of Gyps fulvus plasma cholinesterase to carbamate insecticides (aldicarb, carbaryl and methomyl) was also investigated. The results indicated that propionylthiocholine iodide was the preferred substrate to determine plasma cholinesterase activity, followed by acetylcholine iodide and S‐butyrylcholine iodide, and acetylcholinesterase was the predominant enzymatic activity in Gyps fulvus plasma. Aldicarb was the most potent in vitro inhibitor of plasma cholinesterase activity in this species. However, cholinesterase enzymatic activity was significantly inhibited by all tested carbamates, providing further evidence that this biomarker is a suitable tool to monitor the exposure to these poisons in the field, highlighting its utility in conservation programmes.     

The Comparative Osmoregulatory Ability of Two Water Beetle Genera Whose Species Span the Fresh-Hypersaline Gradient in Inland Waters (Coleoptera: Dytiscidae,Hydrophilidae)

A better knowledge of the physiological basis of salinity tolerance is essential to understanding the ecology and evolutionary history of organisms that have colonized inland saline waters. Coleoptera are amongst the most diverse macroinvertebrates in inland waters, including saline habitats; however, the osmoregulatory strategies they employ to deal with osmotic stress remain unexplored. Survival and haemolymph osmotic concentration at different salinities were examined in adults of eight aquatic beetle species which inhabit different parts of the fresh—hypersaline gradient. Studied species belong to two unrelated genera which have invaded saline waters independently from freshwater ancestors; Nebrioporus (Dytiscidae) and Enochrus (Hydrophilidae). Their osmoregulatory strategy (osmoconformity or osmoregulation) was identified and osmotic capacity (the osmotic gradient between the animal’s haemolymph and the external medium) was compared between species pairs co-habiting similar salinities in nature. We show that osmoregulatory capacity, rather than osmoconformity, has evolved independently in these different lineages. All species hyperegulated their haemolymph osmotic concentration in diluted waters; those living in fresh or low-salinity waters were unable to hyporegulate and survive in hyperosmotic media (> 340 mosmol kg^-1). In contrast, the species which inhabit the hypo-hypersaline habitats were effective hyporegulators, maintaining their haemolymph osmolality within narrow limits (ca. 300 mosmol kg^-1) across a wide range of external concentrations. The hypersaline species N. ceresyi and E. jesusarribasi tolerated conductivities up to 140 and 180 mS cm^-1, respectively, and maintained osmotic gradients over 3500 mosmol kg^-1, comparable to those of the most effective insect osmoregulators known to date. Syntopic species of both genera showed similar osmotic capacities and in general, osmotic responses correlated well with upper salinity levels occupied by individual species in nature. Therefore, osmoregulatory capacity may mediate habitat segregation amongst congeners across the salinity gradient.     

Industrial and biotechnological applications of laccases: a review

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.     

Plant-dependent active biological containment system for recombinant rhizobacteria

This work describes the construction of the rhizobacterium Pseudomonas putida strain CS-4, which contains an active biological containment (ABC) system that ensures bacterial suicide in the absence of proline. Maize plants exudate proline, which allows CS-4 to colonize the rhizosphere at a similar level to that of the wild-type strain. However, when the plants are removed, the CS-4 population decreased in bulk soil at a higher rate than the wild-type strain.     

Involvement of Ethylene Biosynthesis and Signalling in the Transition from Male to Female Flowering in the Monoecious Cucurbita pepo

It is well established that ethylene is the main hormonal regulator of sexual expression in the Cucurbitaceae family, controlling not only the sexual fate of individual floral buds, but also the female flower transition, that is, the time at which the first female flower appears and therefore the number of female flowers per plant. Although sex determination of individual flower buds is known to be controlled by specific ethylene biosynthesis ACS genes in melon and cucumber, the role of ethylene genes in the control of the transition to female flowering is still unknown. We have identified two contrasting monoecious inbred lines of Cucurbita pepo, Bolognese (Bog) and Vegetable spaghetti (Veg), which differ in female flower transition but not in flower development. In Bog, which is very sensitive to ethylene, the transition to female flowering is very early, whereas in Veg, which is much less sensitive to ethylene, the transition occurs much later. In this article we compare the production of ethylene and the expression profiles of seven genes involved in the biosynthesis, perception, and signalling of ethylene in the two contrasting lines. Bog, with earlier female flower transition, showed higher ethylene production and CpACO1 expression in the apex at an earlier stage of plant development, when Bog is already producing female flowers, but Veg has not transitioned to female flowering yet. Moreover, the expression of the ethylene receptor and CTR-like genes in the apex of Veg and Bog plants indicates that these genes negatively regulate female flower transition during the earlier stages of plant development. The earlier transition to female flowering in Bog is not only associated with a higher production of ethylene in the apex but also with a premature decline of ethylene negative regulators (receptors and CTR-like) in the apex of the plant. These results provide the basis for a model that explains the regulation of female flowering transition in monoecious cucurbits.     

Sensitivity of Aedes aegypti adults (Diptera: Culicidae) to the vapors of Eucalyptus essential oils

Vapors of essential oils extracted from various species of Eucalyptus (E. gunnii, E. tereticornis, E. grandis, E. camaldulensis, E. dunnii, E. cinerea, E. saligna, E. sideroxylon, E. globulus ssp. globulus, E. globulus ssp. maidenii, E. viminalis and the hybrids E. grandis × E. tereticornis and E. grandis × E. camaldulensis) and their major components were found to be toxic to Aedes aegypti adults, the yellow fever mosquito.An aliquot of each oil was placed in a cylindrical test chamber and the number of knocked-down mosquitoes was recorded as function of time. Knockdown time 50% was then calculated. Results showed that E. viminalis had the fastest knockdown time at of 4.2 min, on the same order as dichlorvos, a standard knockdown agent. A correlation was observed between the content of 1,8-cineole in the Eucalyptus essential oils and the corresponding toxic effect.The correlation between KT₅₀ values and calculated vapor pressures of the essential oil components showed that the fumigant activity of simple organic compounds in insects is correlated with their volatility.     

Constitutive cholesterol-dependent endocytosis of melanocortin-4 receptor (MC4R) is essential to maintain receptor responsiveness to α-melanocyte-stimulating hormone (α-MSH)

Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus where it controls feeding behavior. MC4R cycles constitutively and is internalized at the same rate in the presence or absence of stimulation by the agonist, melanocyte-stimulating hormone (α-MSH). This is different from other G-protein-coupled receptors, such as β(2)-adrenergic receptor (β(2)AR), which internalizes more rapidly in response to agonist stimulation. Here, it is found that in immortalized neuronal Neuro2A cells expressing exogenous receptors, constitutive endocytosis of MC4R and agonist-dependent internalization of β(2)AR were equally sensitive to clathrin depletion. Inhibition of MC4R endocytosis by clathrin depletion decreased the number of receptors at the cell surface that were responsive to the agonist, α-MSH, by 75%. Mild membrane cholesterol depletion also inhibited constitutive endocytosis of MC4R by ～5-fold, while not affecting recycling of MC4R or agonist-dependent internalization of β(2)AR. Reduced cholesterol did not change the MC4R dose-response curve to α-MSH, but it decreased the amount of cAMP generated per receptor number indicating that a population of MC4R at the cell surface becomes nonfunctional. The loss of MC4R function increased over time (25-50%) and was partially reversed by mutations at putative phosphorylation sites (T312A and S329A). This was reproduced in hypothalamic GT1-7 cells expressing endogenous MC4R. The data indicate that constitutive endocytosis of MC4R is clathrin- and cholesterol-dependent. MC4R endocytosis is required to maintain MC4R responsiveness to α-MSH by constantly eliminating from the plasma membrane a pool of receptors modified at Thr-312 and Ser-329 that have to be cycled to the endosomal compartment to regain function.     

Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis

The inhibitory effect of the main inhibitors (acetic acid, furfural and 5-hydroxymethylfurfural) formed during steam explosion of wheat straw was studied through ethanol fermentations of model substrates and hydrolysates from wheat straw by Pichia stipitis. Experimental results showed that an increase in acetic acid concentration led to a reduction in ethanol productivity and complete inhibition was observed at 3.5 g/L. Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect. Fermentations of the whole slurry from steam exploded wheat straw were completely inhibited by a synergistic effect due to the presence of 1.5 g/L acetic acid, 0.15 g/L furfural and 0.05 g/L HMF together with solid fraction. When using only the solid fraction from steam explosion, hydrolysates presented 0.5 g/L of acetic acid, whose fermentations have submitted promising results, providing an ethanol yield of 0.45 g ethanol/g sugars and the final ethanol concentration reached was 12.2 g/L (10.9 g ethanol/100 g DM).     

shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation,Secretion and Toxicity

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson’s disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.