A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer

Background  

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg²⁺, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg²⁺, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.     

A Temporally Explicit Production Efficiency Model for Fuel Load Allocation in Southern Africa

Abstract We present a regional fuel load model (1 km² spatial resolution) applied in the southern African savanna region. The model is based on a patch-scale production efficiency model (PEM) scaled up to the regional level using empirical relationships between patch-scale behavior and multi-source remote sensing data (spatio-temporal variability of vegetation and climatic variables). The model requires the spatial distribution of woody vegetation cover, which is used to determine separate respiration rates for tree and grass. Net primary production, grass and tree leaf death, and herbivory are also taken into account in this mechanistic modeling approach. The fuel load model has been calibrated and validated from independent measurements taken from savanna vegetation in Africa southward from the equator. A sensitivity analysis on the effect of climate variables (incoming radiation, air temperature, and precipitation) has been conducted to demonstrate the strong role that water availability has in determining productivity and subsequent fuel load over the southern African region. The model performance has been tested in four different areas representative of a regional increasing rainfall gradient—Etosha National Park, Namibia, Mongu and Kasama, Zambia, as well as in Kruger National Park, South Africa. Within each area, we analyze model output from three different magnitudes of canopy coverage (<5, 30, and 50%). We find that fuel load ranges predicted by the model are globally in agreement with field measurements for the same year. High rainfall sustains green herbaceous production late in the dry season and delays tree leaf litter production. Effect of water on production varies across the rainfall gradient with delayed start of green material production in more arid regions.     

Coupled Diffusion of Peripherally Bound Peptides along the Outer and Inner Membrane Leaflets

Transmembrane signaling implies that peripheral protein binding to one leaflet be detected by the opposite leaflet. Therefore, protein recruitment into preexisting cholesterol and sphingolipid rich platforms may be required. However, no clear molecular picture has evolved about how these rafts in both leaflets are connected. By using planar lipid bilayers, we show that the peripheral binding of a charged molecule (poly-lysine, PLL) is detected at the other side of the bilayer without involvement of raft lipids. The diffusion coefficient, D_P, of PLL differed by a factor of √2 when PLL absorbed to one or to both leaflets of planar membranes. Fluorescence correlation spectroscopy showed that the changes of the lipid diffusion coefficient, D_M, were even more pronounced. Although D_M remained larger than D_P on PLL binding to the first membrane leaflet, D_M dropped to D_P on PLL binding to both leaflets, which indicated that the lipids sandwiched between two PLL molecules had formed a nanodomain. Due to its small area of ∼20 nm² membrane electrostriction or leaflet interaction at bilayer midplane can only make a small contribution to interleaflet coupling. The tendency of the system to maximize the area where the membrane is free to undulate seems to be more important. As a spot with increased bending stiffness, the PLL bound patch in one leaflet attracts a stiffening additive on the other leaflet. That is to say, instead of suppressing undulations in two spots, two opposing PLL molecules migrate along a membrane at matching positions and suppress these undulations in a single spot. The gain in undulation energy is larger than the energy required for the alignment of two small PLL domains in opposite leafs and their coordinated diffusion. We propose that this type of mechanical interaction between two membrane separated ligands generally contributes to transmembrane signaling.     

Multiple-Interval Mapping for Quantitative Trait Loci With a Spike in the Trait Distribution

For phenotypic distributions where many individuals share a common value—such as survival time following a pathogenic infection—a spike occurs at that common value. This spike affects quantitative trait loci (QTL) mapping methodologies and causes standard approaches to perform suboptimally. In this article, we develop a multiple-interval mapping (MIM) procedure based on mixture generalized linear models (GLIMs). An extended Bayesian information criterion (EBIC) is used for model selection. To demonstrate its utility, this new approach is compared to single-QTL models that appropriately handle the phenotypic distribution. The method is applied to data from Listeria infection as well as data from simulation studies. Compared to the single-QTL model, the findings demonstrate that the MIM procedure greatly improves the efficiency in terms of positive selection rate and false discovery rate. The method developed has been implemented using functions in R and is freely available to download and use.     

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1 complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1 PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10-day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02 microgram/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71-86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1-100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.     

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).