No abstract

The bacterium Agrobacterium tumefaciens transforms eukaryotic hosts by transferring DNA to the recipient cell where it is integrated and expressed. Bacterial factors involved in this interkingdom gene transfer have been described, but less is known about host-cell factors. Using the yeast Saccharomyces cerevisiae as a model host, we devised a genetic screen to identify yeast mutants with altered transformation sensitivities. Twenty-four adenine auxotrophs were identified that exhibited supersensitivity to A. tumefaciens-mediated transformation when deprived of adenine. We extended these results to plants by showing that purine synthesis inhibitors cause supersensitivity to A. tumefaciens transformation in three plant species. The magnitude of this effect is large and does not depend on prior genetic manipulations of host cells. These data indicate the utility of yeast as a model for the transformation process and identify purine biosynthesis as a key determinant of transformation efficiency. These findings should increase the utility of A. tumefaciens in genetic engineering.A grobacterium tumefaciens, a Gram-negative soil bacterium, genetically transforms plants by transferring DNA to the host cell where it is integrated into the host chromosome and expressed. Exogenous DNA sequences introduced into transferred DNA (T-DNA) vectors can be delivered to plants, making A. tumefaciens a cornerstone of plant genetic engineering. Under controlled conditions, A. tumefaciens can also transform mammalian cells and a variety of fungi, including the yeast Saccharomyces cerevisiae (1-6).Understanding the cellular factors influencing transformation will provide broader insights into the mechanisms underlying interkingdom DNA transfer and should increase the utility of A. tumefaciens in genetic engineering. Bacterial factors that control virulence gene induction as well as processing and delivery of the T-DNA have been studied extensively (7,8). Recently, a few host-cell factors have been identified that participate in A. tumefaciens-mediated transformation. Studies in Arabidopsis thaliana have implicated histone H2A in chromosomal integration of the T-DNA (9). Studies in S. cerevisiae have implicated a nuclear pore protein in T-DNA nuclear import (10) and nonhomologous end-joining proteins in T-DNA chromosomal integration (11). To date, however, the facile yeast system has not been used to perform a large-scale screen to identify host factors that influence transformation sensitivity. Consequently, we devised a genetic screen to isolate yeast mutants with altered sensitivity to A. tumefaciens-mediated transformation. This approach revealed an unexpected link between transformation efficiency and de novo biosynthesis of adenine, an essential purine precursor of DNA, RNA, and ATP. Materials and MethodsStrains and Plasmids. The supervirulent A. tumefaciens strain EHA105 harboring pKP506 served as the bacterial donor strain in yeast-transformation experiments (1). The pKP506 plasmid contains the yeast TRP1 marker and the ARS1 rep...

In light of recent warnings by the United States (US) Surgeon General and Centers for Disease Control (CDC) guidelines for recommending more prudent use of opioid narcotics, the search for a non-opioid alternative for aborting acute migraines is particularly relevant. The CDC also estimates the prevalence of opioid dependence may be as high as 26% among patients prescribed opioids for chronic pain, not due to cancer, in the primary care setting. Given such staggering data, it is imperative that we, as caretakers, not foster opioid dependence but rather continue to investigate non-opioid therapies for the management of acute migraines in the emergent care settings. Our literature review demonstrates that metoclopramide should be used more frequently as first-line therapy for an acute migraine over opioids. The use of opioids specifically has been discouraged as migraine treatment by the American Headache Society citing “insufficient evidence” as the main reason. Metoclopramide, specifically using the 10 mg dose, has been cited as “highly likely to be effective” by the same guidelines. Another major issue with opioids is the growing potential for abuse, thus minimizing the use of these drugs for only special circumstances would be beneficial overall.

Background: Renal biopsies are uncommonly performed in horses and little is known about their diagnostic utility and associated complication rate.Objective: To describe the techniques, the complication rate, risk factors, and histopathology results; as well as evaluate the safety and diagnostic utility of renal biopsy in the horse.Animals: One hundred and forty-six horses from which 151 renal biopsies were obtained. Animals ranged in age from 48 hours to 30 years.Methods: Multicenter retrospective study, with participation of 14 institutions .Results: Renal biopsy in horses was associated with a similar rate of complications (11.3%) to that occurring in humans and companion animals. Complications were generally associated with hemorrhage or signs of colic, and required treatment in 3% of cases. Fatality rate was low (1/151; 0.7%). Biopsy specimens yielded sufficient tissue for a histopathologic diagnosis in most cases (94%) but diagnoses had only fair (72%) agreement with postmortem findings. Risk factors for complications included biopsy specimens of the left kidney (P 5 .030), a diagnosis of neoplasia (P 5 .004), and low urine specific gravity (P 5 .030). No association with complications was found for age, sex, breed, institution, presenting complaint, other initial clinicopathologic data, biopsy instrument, needle size, or use of ultrasonographic guidance.Conclusions and Clinical Importance: Renal biopsy in horses has low morbidity and results in a morphological histopathologic diagnosis in 94% of cases. However, this procedure might result in serious complications and should only be used when information obtained would be likely to impact decisions regarding patient management and prognosis.