ABSTRACT
A PCR method was developed by which to rapidly and accurately determine the rrn arrangement of Salmonella enterica serovars. Primers were designed to the genomic regions flanking each of the seven rrn operons. PCR analysis using combinations of these primers will distinguish each of the possible arrangements of the rrn skeleton.
Subject(s)
Chromosome Mapping/methods , Genes, rRNA , Polymerase Chain Reaction/methods , RNA, Ribosomal/genetics , Salmonella enterica/genetics , DNA Primers , Genes, Bacterial , Salmonella enterica/classification , Time FactorsABSTRACT
E. coli and Salmonella typhimurium are widely used bacterial hosts for genetic manipulation of DNA from prokaryotes and eukaryotes. Introduction of foreign DNA by electroporation or transduction into E. coli and Salmonella is limited by host restriction of incoming DNA by the recipient cells. Here, we describe a simple method that temporarily inactivates host restriction, allowing high-frequency DNA transfer. This technique might be readily applied to a wide range of bacteria to increase DNA transfer between strains and species.
Subject(s)
DNA, Recombinant/genetics , Gene Transfer Techniques , Bacteriophage P22/genetics , Bacteriophage lambda/genetics , Biotechnology , Conjugation, Genetic , Deoxyribonucleases, Type I Site-Specific/antagonists & inhibitors , Electroporation , Escherichia coli/enzymology , Escherichia coli/genetics , Hot Temperature , Salmonella enteritidis/enzymology , Salmonella enteritidis/genetics , Salmonella typhimurium/enzymology , Salmonella typhimurium/genetics , Species SpecificityABSTRACT
Ingestion of oleander plant, containing the cardiac glycoside oleandrin, has been reported to induce fatal poisonings. Derivatives of oleandrin are structurally similar to digoxin. We investigated the cross-reactivities of oleandrin and its aglycone metabolite, oleandrigenin, in several commercially available digoxin immunoassays; assessed their ability to inhibit Na,K-ATPase catalytic activity; and measured their binding to proteins in serum. As assayed with ACS:180, Stratus, RIA, On-Line, and TDx digoxin assays, oleandrin at 100 micromol/L in digoxin-free serum gave apparent digoxin values of 0, 0.83, 2.24, 2.37, and 5.34 nmol/L, respectively, whereas oleandrigenin at that concentration gave results of 0, 0.52, 0.77, 4.94, and 1.40 nmol/L. Study of Na,K-ATPase inhibition showed IC50 values (micromol/L) of 0.22 for ouabain, 0.62 for oleandrin, 1.23 for oleandrigenin, and 2.69 for digoxin. At 25 degrees C, 96% of oleandrin and 48% of oleandrigenin were bound to serum proteins. Because detection of oleandrin and oleandrigenin by digoxin immunoassays is variable between assays as well as between congeners, assessment of cross-reactivity is warranted for each assay. The inhibition of Na,K-ATPase by oleandrin and oleandrigenin confirms that they likely exert their toxic effects through inhibition of sodium pump activity. In cases of digitalis-like poisoning with suspicion of oleander ingestion, a combination of digoxin immunoassays may be useful to effectively rule out the presence of oleander.
Subject(s)
Cardenolides/blood , Digoxin/blood , Enzyme Inhibitors/blood , Immunoassay , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Blood Proteins/metabolism , Cardenolides/pharmacology , Cardiac Glycosides/blood , Humans , Immunoassay/methods , Immunoassay/statistics & numerical data , Sensitivity and SpecificityABSTRACT
A 64-year-old man developed severe hyperbilirubinemia of predominantly conjugated fraction in 1978, eight years after a myocardial infarction and development of congestive heart failure. In 1975, he was admitted elsewhere for symptoms suggestive of chronic hepatitis, but liver biopsy revealed replacement of hepatocytes by red blood cells which was interpreted as a result of left-sided cardiac failure. In 1978, liver biopsy showed congestive liver disease with cardiac sclerosis. Despite initial improvement, his condition deteriorated, he became encephalopathic, and died in a coma. This case is reported to illustrate that chronic congestive heart failure can present with severe jaundice and terminate in hepatic coma.
