Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Exposure to nuclear magnetic resonance imaging procedure attenuates morphine-induced analgesia in mice

Adult male mice exposed to a Nuclear Magnetic Resonance Imaging (NMRI) procedure during the mid-dark period and injected with morphine (10 mg/kg) failed to exhibit the normal nocturnally enhanced morphine analgesia response to a thermal stimulus that was displayed by mice exposed to a sham imaging procedure and treated with morphine (p less than .01). When tested during the mid-light period, animals exposed to the NMRI procedure and given morphine displayed attenuated analgesia levels relative to sham exposed mice (p less than .01) treated with morphine. However, the morphine induced analgesia was not totally abolished since the imaged mice still exhibited analgesia relative to saline treated mice (p less than .01). These results suggest that the magnetic and/or radio-frequency fields associated with the NMRI procedure alter both day- and night-time responses to morphine. These results may reflect magnetic field induced alterations in neuronal calcium binding and/or alterations in nocturnal pineal gland activity.     

Characterization of polymorphic microsatellite markers for the Carnaby's cockatoo (Calyptorhynchus latirostris) and related black cockatoo species

Microsatellite loci were isolated from Carnaby's black cockatoo (Calyptorhynchus latirostris: Aves), a highly valued, endangered, and endemic species of bird from Western Australia. This study describes three dinucleotide and one tetranucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with between two and nine alleles and moderate levels of variability. Two additional dinucleotide markers which were monomorphic in the Carnaby's cockatoo were able to amplify and were polymorphic in two other species of black cockatoo, greatly increasing the utility of these markers.     

Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding.

Monosaccharide binding competition, lectin affinity chromatography, and glycosylation inhibitors have been used to determine if glycosylation plays a role in thrombin-receptor interactions. Mannose appeared to specifically inhibit thrombin binding to mouse embryo (ME) and hamster fibroblasts. Concanavalin A bound to antibody-purified receptor fractions, and was used as an affinity ligand to purify receptor fractions that retained thrombin binding activity. Cells treated with tunicamycin (6.25 ng/ml) for 24 h lost approximately 35% of their high-affinity thrombin binding sites, yet binding of receptor monoclonal antibody TR-9 was not affected, indicating that the receptor was present in the membrane, but unable to bind thrombin. Thus thrombin receptor glycosylation may be directly involved in thrombin binding.     

Magnetic circular dichroism studies on the heme and tryptophan components of cytochrome c peroxidase

We have measured the magnetic circular dichroism of cytochrome c peroxidase and some of its derivatives from 250-350 nm. Comparison of the changes observed on conversion to the catalytic intermediate (cytochrome c peroxidase-I) with spectra obtained from horseradish peroxidase and its derivatives and model compounds of protoheme leads us to the conclusion that the observed changes in the magnetic circular dichroism spectra reflect conversion of the heme to the ferryl state. No evidence was found for modification of tryptophan in cytochrome c peroxidase-I.     

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.     

Cutaneous trichosporosis

Of 269 patients with cutaneous trichosporosis a majority of 170 (63.2%) showed the infection as intertrigo in the genitocrural and perianal areas. The predominant symptoms were itching and burning. Trichosporosis has been found to be less common in the other sites. Trichosporosis due to Trichosporon beigelii should be kept in mind as one of the differential diagnosis in cases of genitocrural intertrigo and other cutaneous infections by fungi and bacteria in the tropics.     

Use of Metabolic Inhibitors to Estimate Protozooplankton Grazing and Bacterial Production in a Monomictic Eutrophic Lake with an Anaerobic Hypolimnion

Inhibitors of eucaryotes (cycloheximide and amphotericin B) and procaryotes (penicillin and chloramphenicol) were used to estimate bacterivory and bacterial production in a eutrophic lake. Bacterial production appeared to be slightly greater than protozoan grazing in the aerobic waters of Lake Oglethorpe. Use of penicillin and cycloheximide yielded inconsistent results in anaerobic water and in aerobic water when bacterial production was low. Production measured by inhibiting eucaryotes with cycloheximide did not always agree with [³H]thymidine estimates or differential filtration methods. Laboratory experiments showed that several common freshwater protozoans continued to swim and ingest bacterium-size latex beads in the presence of the eucaryote inhibitor. Penicillin also affected grazing rates of some ciliates. We recommend that caution and a corroborating method be used when estimating ecologically important parameters with specific inhibitors.