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.     

Use of Radiation Hybrid panels to map genetic loci

The mapping of genetic loci within organisms has been accelerated by the advent of Radiation Hybrid (RH) panels. These panels are available for humans and non-humans including mice, baboon, rat, and canine. This article contains a general protocol for the use of the Genebridge 4 whole genome RH panel to map a human locus. This protocol may also be adjusted to suit the other RH panels currently available.     

The organization and structure of nerve and muscle in the jellyfish Cyanea capillata (coelenterata; scyphozoa)

The perirhopalial tissue and swimming muscle of Cyanea were examined with light microscopical and electron microscopical techniques. The perirhopalial tissue is a thin, triangular septum found on the subumbrellar surface of the animal. It separates part of the gastric canal system from the surrounding seawater, and is bound on two sides by radial muscle bands and on the third, the shorter side, by a rhopalium and the margin of the bell. The ectoderm of the perirhopalial tissue is composed of large, somewhat cuboidal, vacuolated, myoepithelial cells. The muscle tails of these cells form a single layer of radial, smooth muscle. Neurons of the “giant fiber nerve net” (GFNN), which form an extensive net over the perirhopalial tissue, lie at the base of the vacuolated portion of the myoepithelial cells. These neurons are visible in living tissue. The morphology of individual GFNN neurons was examined following intracellular injection of the fluorescent dye Lucifer Yellow. The neurons are usually bipolar and free of branches. At the electron microscope level, one usually finds that the GFNN neurons contain large vacuoles. The other characteristic feature of these cells is that they form symmetrical, or nonpolarized, synapses; that is, synaptic vesicles are found on both sides of the synapse. The swimming muscle is striated and composed of myoepithelial cells. Each myoepithelial cell has several muscle tails, and those of adjacent cells are linked to gether by desmosomes. The endoderm of the perirhopalial tissue also was examined. This investigation of the organization and ultrastructure of the perirhopalial tissue and surrounding muscle was undertaken to provide essential background information for an ongoing physiological study of the GFNN neurons and their synapses.     

Genetic interaction between Non-MHC T- and B-cell alloantigens in response to rous sarcomas in chickens

Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6₃ regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line RPRL 100, although these lines are identical for the major histocompatibility B complex. They differ, however, at three independent autosomal loci: Ly-4 and Th-1 determine the surface alloantigens of partly overlapping subsets of T lymphocytes, and Bu-1 determines a surface alloantigen of B lymphocytes. The association of genotypes at these loci with quantitative variation in their ability to regress Rous sarcomas was tested in segregating F₄ generation progeny derived from crosses of lines 100 and 6₃. The Ly-4 and Bu-1 genotypes showed association with Rous sarcoma regression, but the Th-1 genotype did not. Chickens of the Ly-4 ^a/Ly-4 ^a, Bu-1 ^b/Bu-1 ^b and Ly-4 ^b/Ly-4 ^b, Bu-1 ^a/Bu-1 ^a genotypes had a significantly higher regressor ability than the other two double homozygous genotypes. These results indicate that higher regression is associated with (1) interaction between the Ly-4 and Bu-1 loci, and (2) complementation between either the line 6 Ly-4 ^a allele and the line 100 Bu-1 ^b allele, or the line 100 Ly-4 ^b allele and the line 6 Bu-1 ^a allele.     

Procedure using voltage-sensitive spin-labels to monitor dipole potential changes in phospholipid vesicles: The estimation of phloretin-induced conductance changes in vesicles

Summary Voltage-sensitive membrane potential probes were used to monitor currents resulting from positive or negative charge movement across small and large unilamellar phosphatidylcholine (PC) vesicles. Positive currents were measured for the paramagnetic phosphonium ion or for K⁺-valinomycin. Negative currents were indirectly measured for the anionic proton carriers CCCP and DNP by monitoring transmembrane proton currents. Phloretin, a compound that is believed to decrease dipole fields in planar bilayers, increases positive currents and decreases negative currents when added to egg PC vesicles. In these vesicles, positive currents are increased by phloretin addition to a much larger degree than CCCP currents are reduced. This asymmetry, with respect to the sign of the charge carrier, is apparently not the result of changes in the membrane dielectric constant. It is most easily explained by deeper binding minima at the membrane-solution interface for the CCCP anion, when compared to the phosphonium. The measured asymmetry and the magnitudes of the current changes are consistent with the predictions of a point dipole model. The use of potential-sensitive probes to estimate positive and negative currents, provides a methodology to monitor changes in the membrane dipole potential in vesicle systems.     

Effect of phenobarbital and 3-amino-1,2,4-triazole on the metabolism and biliary excretion of 3,4-benzpyrene in the rat

The effects of 3-amino-1,2,4-triazole and phenobarbital, alone and in combination, on the metabolism and biliary excretion of 3,4-benzpyrene have been investigated in Wistar and Sprague-Dawley rats of both sexes. Although phenobarbital induced and aminotriazole inhibited metabolism, considerable variation with species and sex was observed. The effects of these drugs on liver weight, microsomal protein and bile flow also varied among these groups of rats. it is apparent that there was no quantitative relationship between the pattern of excretion of benzpyrene metabolites and bile flow, microsomal protein and liver weight.     

Determination of human factor VIII (AHG-associated protein)-antigen in endothelial cells from Xenopus laevis (XTH cells)

Summary AHG-associated protein (AHG-a.p.), the antigen of the blood-clotting factor VIII complex, is a specific endothelial cell marker. Primary (p-XTH) and established (XTH-2) endothelial cells from the hearts of Xenopus laevis tadpoles were assayed for the presence of this marker by means of immunological cross-reaction (recognition of common antigenic sites) with antiserum against human AHG-a.p. Radial imtnunodiffusion and rocket immunoelectrophoresis proved to be insufficiently sensitive, whereas immunofluorescence and a newly evaluated ELISA technique gave positive results. The very high sensitivity of the ELISA (less than 1/240000 of the AHG-a.p. in 0.1 ml human standard plasma can be detected) and the removal of interfering proteins by gel filtration also revealed the presence of AHG-a.p. in the fetal calf serum used in the culture medium; earlier investigations into this subject by a one-step radioimmunoassay had reported negative results. Specially adapted XTH-2 cells were grown in a proteinand serum-free hydrolysate medium in order to demonstrate the presence of a Xenopus-derived antigen that was immunoreactive with the anti-human AHG-a.p.