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Joshua R Edwards Evangelos A Diamantakos Jacob D Peuler Peter C Lamar Walter C Prozialeck 《BMC physiology》2007,7(1):1
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. 相似文献4.
Joshua D. Doyle Jennifer E. Stencel-Baerenwald Courtney A. Copeland Jillian P. Rhoads Judy J. Brown Kelli L. Boyd James B. Atkinson Terence S. Dermody 《PLoS pathogens》2015,11(3)
Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses. 相似文献
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Effect of DNA superhelicity on transcription termination 总被引:1,自引:0,他引:1
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Ryan P. Bourbour Breanna L. Martinico Megan M. Crane Angus C. Hull Joshua M. Hull 《Ecology and evolution》2019,9(3):1452-1457
Complex coevolutionary relationships among competitors, predators, and prey have shaped taxa diversity, life history strategies, and even the avian migratory patterns we see today. Consequently, accurate documentation of prey selection is often critical for understanding these ecological and evolutionary processes. Conventional diet study methods lack the ability to document the diet of inconspicuous or difficult‐to‐study predators, such as those with large home ranges and those that move vast distances over short amounts of time, leaving gaps in our knowledge of trophic interactions in many systems. Migratory raptors represent one such group of predators where detailed diet studies have been logistically challenging. To address knowledge gaps in the foraging ecology of migrant raptors and provide a broadly applicable tool for the study of enigmatic predators, we developed a minimally invasive method to collect dietary information by swabbing beaks and talons of raptors to collect trace prey DNA. Using previously published COI primers, we were able to isolate and reference gene sequences in an open‐access barcode database to identify prey to species. This method creates a novel avenue to use trace molecular evidence to study prey selection of migrating raptors and will ultimately lead to a better understanding of raptor migration ecology. In addition, this technique has broad applicability and can be used with any wildlife species where even trace amounts of prey debris remain on the exterior of the predator after feeding. 相似文献
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Aerosol bolus inspirations were used to assess lung injury in 15 isolated dog lungs exposed to low (0-375 units) or high doses (600-1,200 units) of papain. Effective air space size (EAD) was determined from aerosol deposition during a 5-s breath hold. Convective mixing was assessed by the spreading of the expired bolus with respect to expired volume, quantified by a coefficient of dispersion (CD) equal to the square root of the difference in the variances of the expired and inspired boluses divided by the volumetric penetration of the bolus. After exposure, CD measured with deeply penetrating boluses increased by an average of 2.5% in the low-exposure group (P greater than 0.05) and 28.0% in the high-exposure group (P less than 0.0001). CD measured with shallowly penetrating boluses decreased by 4.3% (P less than 0.0001) in the low-exposure group and increased by an average of 18.3% in the high-exposure group (P less than 0.05). Papain exposure caused EAD to increase in some lungs and decrease in others. For deep bolus penetrations, EAD changed by an average of -0.8% in the low-exposure group (P greater than 0.05) and +21.1% in the high-exposure group (P greater than 0.05). Both EAD and CD appeared to be sensitive to lung injury. However, changes in EAD were less consistent than those in CD, possibly due to changes caused by lung injury in the regional distribution of inspired aerosol. 相似文献
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Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed. 相似文献
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Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1–84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1–84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP−/− and PTHR1−/− mice. However, the mammary buds in PTHrP (1–84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models. 相似文献