Sexual assault evidence samples require the use of a specific process known as a differential digestion to separate sperm from nonsperm cells prior to DNA extraction. An automated differential digestion process was developed using a selective degradation technique, which uses DNase I to digest the remaining nonsperm DNA in the sperm fraction. The use of DNase on pristine samples, as well as aged and degraded samples, was assessed to ensure that the quantity and quality of the sperm DNA were not compromised or adversely affected. Samples processed using the selective degradation technique yielded comparable DNA yield and DNA typing data to the conventional differential digestion process. The automated process utilized 96-well plates for high throughput and incorporated microscope slide preparations for confirmation of sperm. It reduced processing time by about sixfold and was paramount in the elimination of the Oakland Police Department Criminalistics Laboratory's sexual assault kit backlog.
In the present study, we have examined the role of reduced glutathione (GSH) in the modulation of Ca2+ ionophore A-23187-induced gastric mucosal cellular disruption. Experiments were conducted using cells isolated from rabbit oxyntic mucosa. Cell injury was assessed by release of the lysosomal enzyme acid phosphatase, by trypan blue dye uptake, and by examination of lipid peroxidation. Cellular GSH was assessed spectrophotometrically by measuring total soluble reduced thiol content. Ionophore A-23187 (3-25 microM) induced a concentration-dependent injury to mucosal cells and a concentration-dependent reduction in cellular GSH content. Removal of Ca2+ from the incubation medium abolished both the disruptive action and the GSH-lowering effect of A-23187. Gastric cellular GSH content was reduced significantly by preincubation of cells with an agent that depletes reduced sulfhydryls, diethyl maleate (DEM; 1 mM). Furthermore, DEM significantly augmented cellular disruption in response to A-23187. Similarly, incubation of cells with L-cysteine (1 mM), a stimulant of glutathione synthesis, increased mucosal cellular GSH content and decreased the cellular disruptive effect of A-23187. Exogenous addition of GSH to the cell suspension significantly reduced Ca2+ ionophore-induced cellular disruption and hastened the recovery of cellular free Ca2+ concentration ([Ca2+]) to baseline values. Similarly, exogenous GSH reduced cellular disruption produced by cyclopiazonic acid and thapsigargin, agents that increase intracellular [Ca2+] ([Ca2+]i) by inhibiting Ca2+ sequestration. These data suggest that a sustained increase in [Ca2+]i contributes to the pathogenesis of gastric mucosal cellular injury. This effect appears to be mediated by a reduction in the cellular content of GSH.
High-resolution melt (HRM) analysis of the VNTR region of the human D1S80 locus, a 16-bp repeat minisatellite from approximately 400 to over 700 bp in length, was investigated. A Qiagen Rotor-Gene Q using the Type-it PCR HRM kit was used to acquire HRM curves for 14 single, and 16 biallelic, dsDNA samples. The HRM analysis was applicable over a range of DNA concentrations; however the characteristics of the melt curve did depend on the forward and reverse primer ratio. Despite the large amplicon size and the similarities of the repeat sequences, it was possible to discriminate different genotypes. Heterozygotes were clearly different from the homozygous variants and even small differences in the repeat sequence could be differentiated. However, the melt analysis requires a high-resolution system with temperature resolution of 0.02°C or better in order to sort out differences in these large amplicons of near identical GC content (in this case 56%). HRM analysis of amplicons with large repeat sequences can be used as a means of comparing DNA fragments. Examination of multiple sequences can be used to differentiate DNA samples and demonstrate the potential of HRM analysis as a rapid and inexpensive prescreening technique in forensic applications.
A sustained increase in cytosolic Ca2+ can damage gastric mucosal cells. The present study has examined the role of Ca2+ in thromboxane B2 (TXB2)-mediated damage of rabbit isolated gastric mucosal cells. Cells were isolated from rabbit oxyntic mucosa by collagenase-EDTA digestion. Cell metabolic activity and cell damage were estimated by alamar blue dye absorbance and trypan blue uptake, respectively. Cellular Ca2+ was monitored by indo-1 dye fluorescence. Addition of TXB2 (10(-6) and 10(-8) M) to the cell suspension resulted in a decrease in metabolic activity, and this effect was reduced when Ca2+ was removed from the incubation medium. TXB2 addition to the incubation medium resulted in an increase in cytosolic Ca2+ and incubation of cells with the intracellular Ca2+ chelator, BAPTA-AM (20 microM), reduced cell injury in response to TXB2. Incubation of cells with the Ca2+ ionophore A23187 (1-25 microM) resulted in a dose-dependent increase in trypan blue uptake and a reduction in cell metabolism. Cell injury in response to A23187 were exacerbated by addition of TXB2 (10(-8) M) to the cell suspension. TXB2 treatment reduced cellular content of reduced glutathione (GSH), while exogenous GSH addition (10 mM) reduced TXB2-mediated cell injury. These data demonstrate that TXB2 can directly injure gastric mucosal cells. Gastric mucosal cellular damage in response to TXB2 is mediated in part by a disruption of Ca2+ homeostasis as well as a reduction in cellular GSH content.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.