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Biomedical subjects

Timothy J Drake

Publications and source records attributed to Timothy J Drake.

8 recordsLinked to original sources
PubMed2005

Simultaneous monitoring of the expression of multiple genes inside of single breast carcinoma cells.

Monitoring gene expression is at the center of research for a wide variety of medical, biological, and biotechnological applications. Currently no method exists for true multiple gene expression monitoring inside of a single living cell that allows for the gene expression profile of the cell to be directly compared with another single living cell. By microinjecting multiple molecular beacons with different fluorophores inside of single breast carcinoma cells and monitoring with advanced fluorescent microscopy, the expression of multiple genes can be simultaneously monitored inside single living cells. Using ratiometric analysis as a basis for the measurements allows the different gene expression levels to be compared from cell to cell. Not only does this allow differentiation of individual mRNA expression levels between multiple single cells but it also allows for mRNA expression trend analysis at the single cell level.

Actins↗
PubMed2005

Molecular beacons for bioanalytical applications.

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties. They act like switches and are normally in a closed state, when the fluorophore and the quencher are brought together to turn "off" the fluorescence. When prompted to undergo conformational changes that open the hairpin structure, the fluorophore and the quencher are separated, and fluorescence is turned "on." This Education will outline the principles of MBs and discuss recent bioanalytical applications of these probes for in vitro RNA and DNA monitoring, biosensors and biochips, real-time monitoring of genes and gene expression in living systems, as well as the next generation of MBs for studies on proteins, the MB aptamers. These important applications have shown that MBs hold great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.

Biosensing Techniques↗
PubMed2005

Single cell glutamate analysis in Aplysia sensory neurons.

Glutamate is the major excitatory neurotransmitter in the central nervous system. However, techniques and assays available for the determination and detection of glutamate are limited. Here we have applied an effective glutamate assay toward the high-throughput analysis of single neurons. Initial physiological studies and recent immunohistochemical data strongly suggested that mechanosensory neurons could use L-glutamate as a co-transmitter (in addition to sensorin) in the sensory-motor synapse of Aplysia californica. We have evaluated the levels of glutamate in these cells and compared them to other non-sensory Aplysia neurons. Since this is the first report of this assay in single cellular analysis, a series of chemical and cellular controls were also done. Based on our results, we were able to determine the concentration levels inside single Aplysia sensory neurons to be 29 mM, with significant heterogeneity between individual cells. In comparison to the pleural mechanosensory neurons, non-sensory abdominal neurons contained approximately 3 mM glutamate. These elevated levels in the sensory neurons confirm the earlier findings [Dale N, Kandel ER. L-glutamate may be the fast excitatory transmitter of Aplysia sensory neurons. Proc Natl Acad Sci USA 1993;90:7163-7.], suggesting that glutamate plays a role as neurotransmitter in these cells.

Animals↗
PubMed2005

Investigation of molecular beacon aptamer-based bioassay for platelet-derived growth factor detection.

This report describes studies on the use of a molecular-beacon aptamer (MBA) as a synthetic high-affinity DNA probe that exhibits fluorescence resonance energy transfer (FRET) in response to a specific protein biomarker, platelet-derived growth factor (PDGF). As a step toward the application of the MBA in a fluorescence-based assay for biological specimens, we examined the influence of certain physical and chemical parameters of incubation that would affect DNA conformation and DNA-backbone modification, and thus improve nuclease resistance. This bioassay is compatible with pH, temperature, and monovalent cation levels typically encountered in biological samples, and phosphorothioate backbone-modified MBA is able to exhibit specific FRET. With minimal sample processing and without assay optimization, the MBA is able to detect as little as 10 ng PDGF per mug of serum proteins from cell-culture media. We also show that different sets of known fluorophore-quencher pairs can be successfully used in the MBA for sensitive detection of the PDGF target. It should, therefore, be possible to develop multiplex bioassays that monitor either quenching or enhancement for the simultaneous detection of several biomarkers by using MBAs created from high-affinity DNA ligands for the desired protein targets. Interestingly, we observed that, with a DNA ligand with multiple binding sites for a standard multimeric protein target, the FRET bioassay could be accomplished by using a mixture of two individually labeled DNAs-one carrying the fluorophore and the other with the matching quencher. This observation has significant implications in the future design of more selective DNA-based FRET bioassays that use more than one ligand for the same protein target.

Biological Assay↗
PubMed2005

Stochasticity of manganese superoxide dismutase mRNA expression in breast carcinoma cells by molecular beacon imaging.

Visual and quantitative monitoring of cell-to-cell variation in the expression of manganese superoxide dismutase (MnSOD) mRNA by using novel ratiometric imaging with molecular beacons (MB) reveals a distinct change in patterns following induction of human breast-carcinoma cells with lipopolysaccharide. Interestingly, the pattern of cell-to-cell variation in a cell line stably transfected with a plasmid bearing a cDNA clone of MnSOD and overproducing the enzyme is significantly different from the pattern associated with MnSOD induction. The levels and the patterns of cell-population heterogeneity for beta-actin mRNA expression do not show distinct changes either following induction or in stably transfected cells. These results are significant in light of the reported relationship between this enzyme and malignant phenotype of breast-carcinoma cells. Use of MBs in ratiometric image analyses for cytoplasmic mRNAs represents a novel means of directly examining the stochasticity of transcription of MnSOD and other genes implicated in cellular phenotype regulation.

Base Sequence↗
PubMed2004

Functionalized nanoparticles for liquid atmospheric pressure matrix-assisted laser desorption/ionization peptide analysis.

Nanoparticles for the extraction of peptides and subsequent analysis using atmospheric pressure matrix-assisted laser desorption/ionization (APMALDI) have been evaluated. The atmospheric pressure source allows for particles to be directly introduced in the liquid matrix, minimizing sample loss and analysis time. Described in this work are two sample preparation procedures for liquid APMALDI analysis: a C18 functionalized silica nanoparticle for hydrophobic extractions, and an aptamer functionalized magnetite core nanoparticle for rapid, affinity extractions. The C18 particles provide a non-selective support for rapid profiling applications, while the aptamer particles are directed towards reducing the complexity in biological samples. The aptamer functionalized particles provide a more selective analyte-nanoparticle interaction whereby the tertiary structure of the analyte becomes more critical to the extraction. In both cases, the liquid APMALDI matrix provides a support for ionization, and acts as the releasing agent for the analyte-particle interaction. Additionally, analyte enrichment was possible due to the large surface-to-volume ratio of the particles. The experiments conducted with functionalized nanoparticles, in an atmospheric pressure liquid matrix, present a basis for further methodologies and utilities of silica nanoparticles to be developed.

Adsorption↗
PubMed2004

Molecular beacons.

This opinion covers the field of molecular beacons (MBs), in which nucleic acids are molecularly engineered to have unique functions for the investigation of biomolecules. Molecular beacons have been used in a variety of formats, and this review discusses four: first, in vitro RNA and DNA monitoring; second, biosensors and biochips based on MBs; third, real-time monitoring of genes and gene expression in living systems; and finally, the next generation of molecular beacons that will be highly useful for studies with proteins, molecular beacon aptamers. These unique applications have shown that MBs holds great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.

Biosensing Techniques↗