Role of vasopressin in the modifications of renal function during aging]

In the course of aging, the renal concentrating ability is markedly reduced. This defect may result from an inappropriate synthesis of antidiuretic hormone in the central nervous system or may be due to an impaired renal response to vasopressin. The two hypotheses have been studied in vivo in rats and in vitro in mice. The results of these studies indicated that: 1) dehydration induces a comparable release of vasopressin along the hypothalamo-hypophysial axis in 10, 20 and 30 month-old rats; 2) there is no change with age of the number of nephrons, single nephron filtration rate or transport capacity of Henle's loop of cortical nephrons which could account for an impaired renal response to vasopressin; 3) the reduced concentrating ability of the kidney appears to be linked to a decreased response of the medullary thick ascending limb of Henle's loop which in part is responsible for the cortico-papillary gradient of solutes within the kidney.     

Evidence for permanent water channels in the basolateral membrane of an ADH-sensitive epithelium

Summary The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (P _d) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by perforating the apical membrane with amphotericin B. The addition of this antibiotic increasedP _d from 1.12±0.10×10^–4 cm/sec (n=7) to 4.08±0.33×10^–4 cm/sec (n=7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl₂ (10^–3 m) decreasedP _d by 52% andpara-chloromercuribenzoic acid (pCMB) (1.4×10^–4 m) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52±0.23 kcal/mol (n=3), in the control situation, to 9.99±0.91 kcal/mol (n=4) in the presence of 1.4×10^–4 m pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders.pCMB (0.5 or 1.4×10^–4 m) was found to inhibit water exit by 91±2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present.     

Varying foraging patterns in response to competition? A multicolony approach in a generalist seabird

Reducing resource competition is a crucial requirement for colonial seabirds to ensure adequate self‐ and chick‐provisioning during breeding season. Spatial segregation is a common avoidance strategy among and within species from neighboring breeding colonies. We determined whether the foraging behaviors of incubating lesser black‐backed gulls (Larus fuscus) differed between six colonies varying in size and distance to mainland, and whether any differences could be related to the foraging habitats visited. Seventy‐nine incubating individuals from six study colonies along the German North Sea coast were equipped with GPS data loggers in multiple years. Dietary information was gained by sampling food pellets, and blood samples were taken for stable isotope analyses. Foraging patterns clearly differed among and within colonies. Foraging range increased with increasing colony size and decreased with increasing colony distance from the mainland, although the latter might be due to the inclusion of the only offshore colony. Gulls from larger colonies with consequently greater density‐dependent competition were more likely to forage at land instead of at sea. The diets of the gulls from the colonies furthest from each other differed, while the diets from the other colonies overlapped with each other. The spatial segregation and dietary similarities suggest that lesser black‐backed gulls foraged at different sites and utilized two main habitat types, although these were similar across foraging areas for all colonies except the single offshore island. The avoidance of intraspecific competition results in colony‐specific foraging patterns, potentially causing more intensive utilization of terrestrial foraging sites, which may offer more predictable and easily available foraging compared with the marine environment.     

Interaction between lithification and resource availability in the microbialites of Río Mesquites,Cuatro Ciénegas,México

Lithified microbial structures (microbialites) have been present on Earth for billions of years. Lithification may impose unique constraints on microbes. For instance, when CaCO₃ forms, phosphate may be captured via coprecipitation and/or adsorption and potentially rendered unavailable for biological uptake. Therefore, the growth of microbes associated with CaCO₃ may be phosphorus‐limited. In this study, we compared the effects of resource addition on biogeochemical functions of microbial communities associated with microbialites and photoautotrophic microbial communities not associated with CaCO₃ deposition in Río Mesquites, Cuatro Ciénegas, México. We also manipulated rates of CaCO₃ deposition in microbialites to determine whether lithification reduces the bioavailability of phosphorus (P). We found that P additions significantly increased rates of gross primary production (F_2,13 = 103.9, P < 0.001), net primary production (F_2,13 = 129.6, P < 0.0001) and ecosystem respiration (F_2,13 = 6.44, P < 0.05) in the microbialites, while P addition had no effect on photoautotrophic production in the non‐CaCO₃‐associated microbial communities. Growth of the non‐CaCO₃‐associated phototrophs was only marginally stimulated when nitrogen and P were added simultaneously (F_1,36 = 3.98, P = 0.053). In the microbialites, resource additions led to some shifts in the abundance of Proteobacteria, Bacteroidetes and Cyanobacteria but mostly had little effect on bacterial community composition. Ca²⁺ uptake rates increased significantly with organic carbon additions (F_1,13 = 8.02, P < 0.05). Lowering of CaCO₃ deposition by decreasing calcium concentrations in the water led to increased microbial biomass accumulation rates in terms of both organic carbon (F_4,48 = 5.23, P < 0.01) and P (F_6,48 = 13.91, P < 0.001). These results provide strong evidence in support of a role of lithification in controlling P limitation of microbialite communities.     

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids ¹. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes ². Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10⁸ TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line ^{3, 4}. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.     

Low-dose aspirin prevents age-related endothelial dysfunction in a mouse model of physiological aging

The age-related impairment of endothelium-dependent vasodilatation contributes to increased cardiovascular risk in the elderly. For primary and secondary prevention, aspirin can reduce the incidence of cardiovascular events in this patient population. The present work evaluated the effect of low-dose aspirin on age-related endothelial dysfunction in C57B/J6 aging mice and investigated its protective antioxidative effect. Age-related endothelial dysfunction was assessed by the response to acetylcholine of phenylephrine-induced precontracted aortic segments isolated from 12-, 36-, 60-, and 84-wk-old mice. The effect of low-dose aspirin was examined in mice presenting a decrease in endothelial-dependent relaxation (EDR). The effects of age and aspirin treatment on structural changes were determined in mouse aortic sections. The effect of aspirin on the oxidative stress markers malondialdehyde and 8-hydroxy-2'-deoxyguanosine (8-OhdG) was also quantified. Compared with that of 12-wk-old mice, the EDR was significantly reduced in 60- and 84-wk-old mice (P < 0.05); 68-wk-old mice treated with aspirin displayed a higher EDR compared with control mice of the same age (83.9 +/- 4 vs. 66.3 +/- 5%; P < 0.05). Aspirin treatment decreased 8-OHdG levels (P < 0.05), but no significant effect on intima/media thickness ratio was observed. The protective effect of aspirin was not observed when treatment was initiated in older mice (96 wk of age). It was found that low-dose aspirin is able to prevent age-related endothelial dysfunction in aging mice. However, the absence of this effect in the older age groups demonstrates that treatment should be initiated early on. The underlying mechanism may involve the protective effect of aspirin against oxidative stress.     

Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles

Solute reflection coefficients, sigma i, of rat kidney brush-border membrane vesicles were determined by the comparison of water flows induced by equiosmolal gradients of sucrose and NaCl, KCl or mannitol. The values of 0.53 for sigma NaCl and 0.56 for sigma KCl when compared with 0.92 for sigma mannitol suggested some interactions between salt and water pathways. Altering the membrane proteins with 0.4 mM HgCl2 decreased the osmotic water permeability of the vesicles by 70 to 80% and brought sigma NaCl and sigma KCl to a value not different from 1. This argued in favor of water protein pathways in the luminal membrane of kidney proximal cells which are partly accessible to NaCl and KCl.