Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.     

Modulation of reconstituted pig kidney Na+/K(+)-ATPase activity by cholesterol in endogenous lipid vesicles: role of lipid domains

Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na+/K+-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 mol %) predicted by the superlattice or regular distribution model. Fluorescence parameters associated with the membrane probes also showed abrupt changes with peaks, coincident with the cholesterol concentrations associated with the peaks in the enzyme activity, while parameters associated with the protein probes also showed slight but abrupt changes resulting in dips at the same cholesterol concentrations. Notably, the IR amide I band maximum also showed spectral shifts, characterized by a frequency variation pattern with peaks at the same cholesterol concentrations. Overall, these results indicate that the lipid phase had slightly lower hydration, at or near the two critical cholesterol concentrations predicted by the superlattice theory. However, in the protein domains monitored there was a slight but significant hydration increase along with increased peptide backbone flexibility at these cholesterol concentrations. We propose that in the vicinity of the critical mole fractions, where superlattice formation can occur, minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. These changes, in turn, induce small changes in the protein's structure and dynamics, therefore acting to fine-tune the enzyme.     

<Emphasis Type="Italic">Agave manantlanicola</Emphasis> (Agavaceae), a new species from western Mexico

Agave manantlanicola, a new endemic species from the high altitude mountains of the Sierra de Manantlán in western Mexico, is described and illustrated. The species belongs to the subgenus Littaea and Gentry’s Amolae group. The new species is compared to other morphologically similar species from western Mexico, including Agave attenuata subsp. attenuata, A. attenuata subsp. dentata, A. bakeri, A. chazaroi, and A. vazquezgarciae. Information about its distribution, habitat, and conservation status is included.     

The effect of strobilurins on leaf gas exchange, water use efficiency and ABA content in grapevine under field conditions

Strobilurins are one of the most important classes of agricultural fungicides. In addition to their anti-fungal effect, strobilurins have been reported to produce simultaneous effects in plant physiology. This study investigated whether the use of strobilurin fungicide improved water use efficiency in leaves of grapevines grown under field conditions in a Mediterranean climate in southern Spain. Fungicide was applied three times in the vineyard and measurements of leaf gas exchange, plant water status, abscisic acid concentration in sap ([ABA]), and carbon isotope composition in leaves were performed before and after applications. No clear effect on stomatal conductance, leaf water potential and intrinsic water use efficiency was found after three fungicide applications. ABA concentration was observed to increase after fungicide application on the first day, vanishing three days later. Despite this transient effect, evolution of [ABA] matched well with the evolution of leaf carbon isotope ratio, which can be used as a surrogate for plant water use efficiency. Morning stomatal conductance was negatively correlated to [ABA]. Yield was enhanced in strobilurin treated plants, whereas fruit quality remained unaltered.     

Anthracene Removal using Activated Soil Reactors

The aim of this study was to evaluate anthracene removal using activated soil reactors, previously inoculated, under both aerobic and anaerobic conditions. In the reactors, the soil was maintained at 60% moisture (weight basis), room temperature, in the dark, and under constant agitation at 100 rpm. Two experiments were run during and after acclimatization to evaluate anthracene removal under both aerobic and anaerobic conditions. The first one took place during inoculum acclimatization using three different concentrations of anthracene (50, 100, and 500 mg anthracene/L per day) during 90 days. The second experiment took place after acclimatization (during 132 days). The results of anthracene removal were compared with controls in which no additional inoculum was added. During the two experiments, the behavior of pH, chemical oxygen demand (COD), and biogas production was evaluated. Results indicate that the bacterial community adapted for removal of anthracene became enriched through the acclimatization process. Anthracene biodegradation occurred in the soil model with both types of reactors (aerobic and anaerobic), but the rates and extent of biodegradation in the aerobic reactor were higher (95%) than those in anaerobic conditions (74%). Microbial activity also contributed to enhancing bioremediation in the soil by reducing anthracene sorption.     

Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).     

Deficient dopamine D2 receptor function causes renal inflammation independently of high blood pressure

Renal dopamine receptors participate in the regulation of blood pressure. Genetic factors, including polymorphisms of the dopamine D(2) receptor gene (DRD2) are associated with essential hypertension, but the mechanisms of their contribution are incompletely understood. Mice lacking Drd2 (D(2)-/-) have elevated blood pressure, increased renal expression of inflammatory factors, and renal injury. We tested the hypothesis that decreased dopamine D(2) receptor (D(2)R) function increases vulnerability to renal inflammation independently of blood pressure, is an immediate cause of renal injury, and contributes to the subsequent development of hypertension. In D(2)-/- mice, treatment with apocynin normalized blood pressure and decreased oxidative stress, but did not affect the expression of inflammatory factors. In mouse RPTCs Drd2 silencing increased the expression of TNFα and MCP-1, while treatment with a D(2)R agonist abolished the angiotensin II-induced increase in TNF-α and MCP-1. In uni-nephrectomized wild-type mice, selective Drd2 silencing by subcapsular infusion of Drd2 siRNA into the remaining kidney produced the same increase in renal cytokines/chemokines that occurs after Drd2 deletion, increased the expression of markers of renal injury, and increased blood pressure. Moreover, in mice with two intact kidneys, short-term Drd2 silencing in one kidney, leaving the other kidney undisturbed, induced inflammatory factors and markers of renal injury in the treated kidney without increasing blood pressure. Our results demonstrate that the impact of decreased D(2)R function on renal inflammation is a primary effect, not necessarily associated with enhanced oxidant activity, or blood pressure; renal damage is the cause, not the result, of hypertension. Deficient renal D(2)R function may be of clinical relevance since common polymorphisms of the human DRD2 gene result in decreased D(2)R expression and function.     

Phylogeography and molecular evolution of potato virus Y

Potato virus Y (PVY) is an important plant pathogen, whose host range includes economically important crops such as potato, tobacco, tomato, and pepper. PVY presents three main strains (PVY(O), PVY(N) and PVY(C)) and several recombinant forms. PVY has a worldwide distribution, yet the mechanisms that promote and maintain its population structure and genetic diversity are still unclear. In this study, we used a pool of 77 complete PVY genomes from isolates collected worldwide. After removing the effect of recombination in our data set, we used bayesian techniques to study the influence of geography and host species in both PVY population structure and dynamics. We have also performed selection and covariation analyses to identify evolutionarily relevant amino acid residues. Our results show that both geographic and host-driven adaptations explain PVY diversification. Furthermore, purifying selection is the main force driving PVY evolution, although some indications of positive selection accounted for the diversification of the different strains. Interestingly, the analysis of P3N-PIPO, a recently described gene in potyviruses, seems to show a variable length among the isolates analyzed, and this variability is explained, in part, by host-driven adaptation.