ABSTRACT
Organisms belonging to the genus Staphylococcus were isolated on mannitol salt agar from the feces of wild caught Cope's gray treefrogs (Hyla chrysoscelis) from east-central Kansas. All 222 presumptive isolates were confirmed as coagulase-negative staphylococci with Staphylococcus sciuri and Staphylococcus xylosus being most prevalent. Antibiotic susceptibility patterns to five different antibiotics were determined and the results indicated 99% of all isolates were resistant to penicillin G and 59% of the isolates were resistant to oxacillin, a clinical substitute for methicillin. Due to the significance of methicillin resistance in the genus Staphylococcus, 10 randomly chosen oxacillin resistant organisms were analyzed for the presence of the mecA gene, which is known to code for methicillin resistance. The gene was detected in four of the 10 organisms examined. These data indicate that gray treefrogs are harboring inordinately large numbers of methicillin resistant staphylococci as part of their normal flora and that the mechanism of methicillin resistance may be independent of mecA.
Subject(s)
Anura/microbiology , Staphylococcus/drug effects , Animals , Coagulase/analysis , Disease Reservoirs/veterinary , Drug Resistance , Feces/microbiology , Genes, Bacterial , Methicillin Resistance , Oxacillin/pharmacology , Penicillins/pharmacology , Species Specificity , Staphylococcus/enzymology , Staphylococcus/isolation & purification , United StatesABSTRACT
Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.
Subject(s)
Biotin/chemistry , Immunoglobulin Fragments/chemistry , Immunoglobulin Light Chains/chemistry , Immunoglobulins/chemistry , Animals , Antibodies, Monoclonal/chemistry , Azides/chemistry , Cell Separation/methods , Flow Cytometry/methods , HIV Antibodies/chemistry , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin M/chemistry , Mice , Photochemistry , Protein Denaturation , Tumor Cells, CulturedABSTRACT
The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Affinity Labels/metabolism , Benzophenones/metabolism , Plant Proteins , Ribulose-Bisphosphate Carboxylase/metabolism , Adenosine Diphosphate/metabolism , Amino Acid Sequence , Binding Sites , Electrophoresis, Polyacrylamide Gel , Kinetics , Molecular Sequence Data , Peptide Fragments/isolation & purification , Photolysis , Plants, Toxic , Ribulose-Bisphosphate Carboxylase/isolation & purification , Nicotiana/enzymologyABSTRACT
1. Resting metabolic rates at 25 degrees C were determined for juvenile midland painted turtles that had recently been fed or fasted for 1, 2, 4, 6, 10, 14 or 19 days. 2. Recently fed turtles had an oxygen consumption rate of 211 microliter O2/g/hr. This decreased by 32% on the first day of the fast and by 69% by the 19th day. 3. Mass of the turtles (4.91-14.30 g) did not affect the rate of oxygen consumption (VO2).
