Comparative gene mapping of three cancer related genes in the pig

Comparative mapping of genes to the porcine genome will provide a palette of possible candidate genes, which may be associated with particular polygenic traits of interest. Three interesting genes examined for this swine-human comparative gene mapping study are the Breast Cancer Tumor Susceptibility (Type I) gene (BRCA1), the Fibroblast Growth Factor (Type VII)(FGF7) gene, and the Deleted in Pancreatic Cancer (Type IV) (DPC4) gene. While unique in some of their relative functions, each of these genes have been associated with several types of human cancer. The purpose of this thesis research was to use comparative mapping techniques to find the relative genetic and/or physical location of three Type I loci, or coding genes, in the porcine genome. In doing so, the goal of furthering the porcine genome map has progressed. Likewise, the comparative map between swine and humans has been enhanced. In addition to other currently used models for human disease, the pig model may be employed in the future based on results such as those observed in this study

Epinephrine Impairs Lipid Resuscitation from Bupivacaine Overdose

Background Lipid emulsion infusion reverses local anesthetic-induced cardiac toxicity, but the effect of adding epinephrine has not been studied. We compared escalating doses of epinephrine on recovery with lipid infusion in a rat model of bupivacaine overdose. Methods Rats anesthetized with isoflurane received an IV bolus of 20 mg/kg bupivacaine, producing asystole (zero time) in all animals. Ventilation (100% oxygen) and chest compressions were started immediately, and at 3 min the rats received one of six IV treatments (n = 5 for all groups): 5 ml/kg saline followed by infusion for 2 min at 1.0 ml x kg x min, and a second 5 ml/kg bolus at 5 min; or the same bolus and infusion treatment using 30% lipid emulsion plus a single injection of epinephrine at one of five doses: 0 (lipid control), 1, 2.5, 10, or 25 mcg/kg. An electrocardiogram and arterial pressure were monitored continuously, and arterial blood gas was measured at 7.5 and 15 min. Results Epinephrine improved initial return of spontaneous circulation (rate-pressure product > 30% baseline) but only 3 of 5 rats at 10 mcg/kg and 1 of 5 rats at 25 mcg/kg sustained return of spontaneous circulation by 15 min. Lipid alone resulted in slower but more sustained recovery. Epinephrine doses above a threshold near 10 mcg/kg increased lactate, worsened acidosis, and resulted in poor recovery at 15 min, as compared with lipid controls. There was tight correlation of epinephrine dose to serum lactate at 15 min. Conclusions Epinephrine over a threshold dose near 10 mcg/kg impairs lipid resuscitation from bupivacaine overdose, possibly by inducing hyperlactatemia

Cytochrome Oxidase Deficiency Protects Escherichia coli from Cell Death but Not from Filamentation Due to Thymine Deficiency or DNA Polymerase Inactivation

Temperature-sensitive DNA polymerase mutants (dnaE) are protected from cell death on incubation at nonpermissive temperature by mutation in the cydA gene controlling cytochrome bd oxidase. Protection is observed in complex (Luria-Bertani [LB]) medium but not on minimal medium. The cydA mutation protects a thymine-deficient strain from death in the absence of thymine on LB but not on minimal medium. Both dnaE and Δthy mutants filament under nonpermissive conditions. Filamentation per se is not the cause of cell death, because the dnaE cydA double mutant forms long filaments after 24 h of incubation in LB medium at nonpermissive temperature. These filaments have multiply dispersed nucleoids and produce colonies on return to permissive conditions. The protective effect of a deficiency of cydA at high temperature is itself suppressed by overexpression of cytochrome bo3, indicating that the phenomenon is related to energy metabolism rather than to a specific effect of the cydA protein. We propose that filamentation and cell death resulting from thymine deprivation or slowing of DNA synthesis are not sequential events but occur in response to the same or a similar signal which is modulated in complex medium by cytochrome bd oxidase. The events which follow inhibition of replication fork progression due to either polymerase inactivation, thymine deprivation, or hydroxyurea inhibition differ in detail from those following actual DNA damage

Cell Death in Escherichia coli dnaE(Ts) Mutants Incubated at a Nonpermissive Temperature Is Prevented by Mutation in the cydA Gene

Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40°C in Luria-Bertani medium. Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density. Cells survive at 40°C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids. Deletion of recA or sulA has no effect. We isolated suppressors which survived for long periods at 40°C but did not form colonies. The suppressors protected against hydroxyurea-induced killing. Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene. The DNA content of dnaE mutants increased about eightfold in 4 h at 40°C, as did the DNA content of the suppressed strains. The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs. Transformation activity also increased. 4′,6′-Diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40°C, indicating that there was segregation of the newly formed DNA. We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation. These observations support the view that E. coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis

Resuscitation with Lipid <i>versus</i> Epinephrine in a Rat Model of Bupivacaine Overdose

Background Lipid emulsion infusion reverses cardiovascular compromise due to local anesthetic overdose in laboratory and clinical settings. The authors compared resuscitation with lipid, epinephrine, and saline control in a rat model of bupivacaine-induced cardiac toxicity to determine whether lipid provides a benefit over epinephrine. Methods Bupivacaine, 20 mg/kg, was infused in rats anesthetized with isoflurane, producing asystole in all subjects. Ventilation with 100% oxygen and chest compressions were begun immediately, along with intravenous treatment with 30% lipid emulsion or saline (5-ml/kg bolus plus continuous infusion at 0.5 ml . kg . min) or epinephrine (30 microg/kg). Chest compressions were continued and boluses were repeated at 2.5 and 5 min until the native rate-pressure product was greater than 20% baseline. Electrocardiogram and arterial pressure were monitored continuously and at 10 min, arterial blood gas, central venous oxygen saturation, and blood lactate were measured. Effect size (Cohen d) was determined for comparisons at 10 min. Results Lipid infusion resulted in higher rate-pressure product (P &lt; 0.001, d = 3.84), pH (P &lt; 0.01, d = 3.78), arterial oxygen tension (P &lt; 0.05, d = 2.8), and central venous oxygen saturation (P &lt; 0.001, d = 4.9) at 10 min than did epinephrine. Epinephrine treatment caused higher lactate (P &lt; 0.01, d = 1.48), persistent ventricular ectopy in all subjects, pulmonary edema in four of five rats, hypoxemia, and a mixed metabolic and respiratory acidosis by 10 min. Conclusions Hemodynamic and metabolic metrics during resuscitation with lipid surpassed those with epinephrine, which were no better than those seen in the saline control group. Further studies are required to optimize the clinical management of systemic local anesthetic toxicity