Rapid multichannel microfluorometry for metabolic and spectral studies in single living cells

A multichannel microspectrofluorometer has been developed for operation on two modes, a ‘morphological’ mode for the assay of intracellular fluorochromes (e.g. NAD(P)H) in correlation with topography, and a ‘spectral’ mode for wavelength analysis of natural cell fluorescence. This instrument is based on an electron bombardment silicon camera tube (EBS) operated in conjunction with a multiscaling computer. The total NAD(P)H emission from 2 × 30 μm cell strips can be analysed in real time (32.8 msec frame scan) with a signal-to-noise ratio over 100:1. The metabolic changes in cytoplasmic regions are compared with those in regions comprising cytoplasm + nucleus, where the major contribution may be nuclear (cf earlier studies). The observation of a ‘multilocalized’ and asynchronous metabolic response is facilitated with substrates such as glucose-1-phosphate, associated with a longer lag period before the initiation of fluorescence changes. The latter largely occur in the 440–480 nm region. Fluorescence spectra recorded from intracellular regions are nearly super-posable to the spectrum obtained from NAD(P)H crystals.     

Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods

A modified fluorescein diacetate (FDA) assay has been compared with standard NCCLS broth macrodilution and broth microdilution methods for the detection of antifungal activity. The FDA assay was performed in a medium containing bacteriological peptone, NaCl, yeast extract and glucose (0.2%, 0.1%, 0.1% and 1% w/v, respectively) and buffered with 10 mM BES buffer. The MICs of amphotericin B, fluconazole, miconazole and flucytosine (representing three major classes of antifungal agents) obtained by the three methods were compared. The results obtained with the FDA assays correlated well with the NCCLS macrodilution method for MICs of amphotericin B, miconazole and fluconazole, but not for flucytosine. However, the MIC values of flucytosine obtained with the FDA assay were well within the quality control range for the two reference strains recommended by the NCCLS. The FDA assay described is an attractive alternative to the NCCLS methods for screening for antifungal agents, with the added advantage of objectivity of fluorescence measurement.     

Searching for protein signatures using a multilevel alphabet

Short motifs are known to play diverse roles in proteins, such as in mediating the interactions with other molecules, binding to membranes, or conducting a specific biological function. Standard approaches currently employed to detect short motifs in proteins search for enrichment of amino acid motifs considering mostly the sequence information. Here, we presented a new approach to search for common motifs (protein signatures) which share both physicochemical and structural properties, looking simultaneously at different features. Our method takes as an input an amino acid sequence and translates it to a new alphabet that reflects its intrinsic structural and chemical properties. Using the MEME search algorithm, we identified the proteins signatures within subsets of protein which encompass common sequence and structural information. We demonstrated that we can detect enriched structural motifs, such as the amphipathic helix, from large datasets of linear sequences, as well as predicting common structural properties (such as disorder, surface accessibility, or secondary structures) of known functional‐motifs. Finally, we applied the method to the yeast protein interactome and identified novel putative interacting motifs. We propose that our approach can be applied for de novo protein function prediction given either sequence or structural information. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Microbial and host cells acquire enhanced oxidant-scavenging abilities by binding polyphenols

The dilemma whether supplementations of dietary antioxidants might prevent the adverse consequences of oxidative stress, the inadequacy of the analytical methods employed to quantify oxidant scavenging ability (OSA) levels in whole blood and the distribution and fate of polyphenols and their metabolites in various body compartments following oral consumption are discussed. While none-metabolized polyphenols might exert their antioxidant effects mainly in the oral cavity, metabolized polyphenols might be beneficial in the gastrointestinal tract to counteract the toxicity of oxidants and also of the sequelae of inflammatory processes. Although only micromolar amounts of polyphenols and their metabolites eventually reach the blood circulation, these may nevertheless still be highly effective as scavengers of reactive oxygen and nitrogen species because of their ability to synergize with plasma low molecular-weight antioxidants and with albumin. Polyphenols can avidly bind to surfaces of microorganisms and of blood cells to markedly enhance their OSA, therefore the routine quantifications of antioxidant levels conducted in clinical settings should always use catalase-rich whole blood but not as customary, plasma alone. In addition to their antioxidant and metal chelating properties, polyphenols may also act as signaling agents capable of affecting metabolic, inflammatory, autoimmune, carcinogenic and aging processes.     

Novel positron emission tomography tracer distinguishes normal from cancerous cells

Development of tumor-specific probes for imaging by positron emission tomography has broad implications in clinical oncology, such as diagnosis, staging, and monitoring therapeutic responses in patients, as well as in biomedical research. Thymidylate synthase (TSase)-based de novo biosynthesis of DNA is an important target for drug development. Increased DNA replication in proliferating cancerous cells requires TSase activity, which catalyzes the reductive methylation of dUMP to dTMP using (R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. In principle, radiolabeled MTHF can be used as a substrate for this reaction to identify rapidly dividing cells. In this proof-of-principle study, actively growing (log phase) breast cancer (MCF7, MDA-MB-231, and hTERT-HME1), normal breast (human mammary epithelial and MCF10A), colon cancer (HT-29), and normal colon (FHC) cells were incubated with [(14)C]MTHF in culture medium from 30 min to 2 h, and uptake of radiotracer was measured. Cancerous cell lines incorporated significantly more radioactivity than their normal counterparts. The uptake of radioactively labeled MTHF depended upon a combination of cell doubling time, folate receptor status, S phase percentage, and TSase expression in the cells. These findings suggest that the recently synthesized [(11)C]MTHF may serve as a new positron emission tomography tracer for cancer imaging.     

Preparation of monoclonal antibodies able to discriminate between testosterone and 5α-dihydrotestosterone

A procedure for production of monoclonal antibodies to testosterone is described. The method involves immunization of rats with a bovine serum albumin conjugate of testosterone 3-(0-carboxymethyl)oxime followed by polyethylene glycol induced hybridization of the immune lymphocytes with mouse myeloma cells. The resulting hybridomas were cloned and the antibodies produced by each clone were characterized. All the antibodies obtained showed high affinity for testosterone, (K_a = 10¹⁰ l/mol), but clones differed widely in the degree of cross-reaction of the antibodies with other steroids, such as 5α-dihydrotestosterone (range 2–100%) and androstenedione (< 0.1–4%). Large quantities of the selected specific antibodies can be obtained by mass growth of the hybridoma line in culture or as tumors in irradiated or nude mice. Monoclonal antibody preparations may improve standardization of immunoassay methods.