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Biomedical subjects

James J La Clair

Publications and source records attributed to James J La Clair.

16 recordsLinked to original sources
PubMed2006

A systems perspective to digital structures in molecular analysis.

Within the last decade, the advance of miniaturization has opened the window to new systems that permit digital and molecular science to intersect, suggesting a new role for organic chemistry. Currently, fusion of molecules and electronic digits, as well as molecular-based digital structures, have expanded the conventional interpretation of the digit. This emergence has already generated new technological platforms with unique applications for molecular analysis and computation. We provide a brief overview of the conventional understanding of digital devices, examine the concept of molecular-based digits, and suggest new architectures by examining studies conducted on the compact discs. This analysis presents a perspective for the unique interaction of molecules and digits and the development of digital-based platforms for molecular analysis.

Journal Article↗
PubMed2006

Geometry in digital molecular arrays.

The development of digital molecular devices arises through the appropriate geometric positioning of a molecular assay. A detailed evaluation of the digital media reveals the critical aspects of geometric positioning in terms of developing an analytically-robust system for molecular analysis. This study reveals an explicit digital compact disc based assay for molecular affinity events.

Molecular Structure↗
PubMed2006

Phorboxazole analogues induce association of cdk4 with extranuclear cytokeratin intermediate filaments.

The cellular localization profile and molecular association of the phorboxazoles were examined with a streamlined target elucidation system using synthetic fluorescent probes. Cellular image analyses identified the binding of phorboxazole analogues to cytosolic components. Proteomic analysis directed at fluorescently labeled cytosolic fractions indicated that the primary targets observed microscopically were cytokeratins, as verified by determination of low nanomolar binding to cloned and expressed proteins. Phorboxazole probes localized the essential cell cycle promoter cdk4 upon cytokeratin networks.

Cell Cycle↗
PubMed2005

Design and total synthesis of a fluorescent phorboxazole A analog for cellular studies.

To enable studies to elucidate the intracellular processing and targeting of the potent cytostatic/apoptotic anticancer natural products phorboxazoles A and B, a fluorescent derivative has been developed. This involved the total syntheses of the terminal alkyne 33-O-Me-45,46-dehydrobromophorboxazole A (MDHBPA) and a terminal vinyl iodide derivative of the blue fluorescent dye N,N,-dimethyl-7-aminocoumarin (DMC). Sonogashira coupling of these partners provided enyne DMC-MDHBPA in high yield.

Aminocoumarins↗
PubMed2005

In vivo reporter labeling of proteins via metabolic delivery of coenzyme A analogues.

We demonstrate site-specific reporter labeling of proteins within live cells. By accessing the coenzyme A (CoA) metabolic pathway with a cell-permeable pantetheine analogue, we deliver CoA-bound reporter molecules for post-translational protein modification in vivo. These methods may be applied to natural product pathway manipulation as well as applications in conventional molecular and cellular biology.

Carrier Proteins↗
PubMed2005

Fluorescent profiling of natural product producers.

The identification of natural product producer organisms remains a problem for both isolation and natural product classification. A concise screen is developed through fluorescent modification of a set of natural products that offer a common activity. Through real-time multicolor microscopy, the processing, storage, and effects of a natural product are rapidly screened at the level of the strain and individual organism.

Animals↗
PubMed2005

A three-component photoreversible tag for thiols.

[structure: see text] A one-pot coupling of a 1,3-diketone, an aldehyde, and an alkanethiol has been developed to produce a protected sulfide. Through use of an o-nitrophenylbenzaldehyde, this method provides a one-step route to a photochemically reversible thiol-protecting group. The kinetics of photolysis were established using (1)H NMR analysis, which allows for the rate to be based on the entire reaction scheme.

Journal Article↗
PubMed2004

Modular synthesis of pantetheine and phosphopantetheine.

[structure: see text] D-Pantetheine and D-phosphopantetheine, precursors to coenzyme A, have been synthesized though a linear sequence from three modules (M1-M3) in 9 and 10 steps, respectively. These routes provide access to analogues of coenzyme A containing modified cystamines, beta-alanines, and pantoic acid residues. All three modules were joined using conventional methods of peptide synthesis. The chiral component, M3, was derived from D-pantolactone.

Molecular Structure↗
PubMed2004

Manipulation of carrier proteins in antibiotic biosynthesis.

Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here we describe a method to covalently label carrier proteins from PK and NRP synthases using the enzymatic transfer of a modified coenzyme A analog by a 4'-phosphopantetheinyltransferase. Using this method, carrier proteins can be loaded with single fluorescent or affinity reporters, providing novel entry for protein visualization, Western blot identification, and affinity purification. Application of these methods provides an ideal tool to track and quantify metabolically engineered pathways. Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.

Anti-Bacterial Agents↗
PubMed2003

Molecular screening on a compact disc.

A method is described to screen the recognition between small molecule ligands and biomolecules using a conventional compact disc (CD) player. A procedure was developed to attach ligands to the reading face of a CD by activating the terminus of polycarbonate, a common polymer composite within the reading face of a CD. Terminal residues of the polycarbonate surface 1 were phosphorylated by reaction with ethyl-(N,N)-diisopropylamine-buffered dichloro-(N,N)-diisopropylaminophosphate to yield surface-bound chlorophosphate 2. Ligands containing a primary alcohol were condensed with 2 providing a polycarbonate capped with phosphodiester linked ligands 3-6. Displays were generated on the surface of a CD by printing tracks of ligands 3-6 on the disc with an inkjet printer. Using this method, discs were created with entire assemblies of ligand molecules distributed into separate blocks. A molecular array was developed by assembling collections of these blocks to correlate with the CDROM-XA formatted data stored within the digital layer of the disc. Regions of the disc containing a given ligand or set of ligands was marked by its spatial position using the tracking and header information. Recognition between surface expressed ligands and biomolecules was screened by an error determination routine.

Journal Article↗
PubMedDate not supplied

Chemical defense in ascidians of the didemnidae family.

Fluorescent analogues (DB1 and TA1) of the secondary metabolites didemnin B (DB) and tamandarin A (TA) were synthesized to investigate the potential chemical defense mechanisms of tunicates in the family Didemnidae. These compounds were found to alter predator-prey relations. Five species of freshwater fish and one marine fish, the damselfish Amphiprion ocellaris, were acclimated to a diet of mosquito larvae. Fish showed an immediate, negative reaction to mosquito larvae treated with >/=5 ng of DB1 or TA1, with consumption of larvae resulting in regurgitation. Both freshwater and marine fish learned to avoid tainted prey by associating species of larvae with "distaste". Distaste for a given organism also arose when depsipeptides DB1 or TA1 were transferred to the fish from the surrounding medium. Fluorescence microscopy in fish indicated that a similar processing and localization followed ingestion and absorption of DB1 or TA1. Fluorescent labeling of DB or TA provided an ideal tool to conduct short-term studies of predator-prey relationships between fish and marine invertebrate larvae.

Animals↗
PubMedDate not supplied

A site-specific bifunctional protein labeling system for affinity and fluorescent analysis.

Most covalent protein labeling schemes require a choice between visual and affinity properties, requiring the use of multiple fusion systems where both attributes are needed. While not disruptive at the single experiment level, this detail becomes critical when addressing high-throughput experimentation. Here we develop a uniform site-specific protein tag for use in both fluorescent and affinity screening. Covalent protein tagging with a stilbene reporter via promiscuous phosphopantetheinyltransferase (PPTase) modification enables a switchable, antibody-elicited fluorescent response in solution or on affinity resin. For demonstration purposes, VibB, a natural fusion protein harboring a carrier protein domain, was labeled with a stilbene tag through PPTase modification with a stilbene-labeled coenzyme A analogue. Analysis of the resulting stilbene-tagged VibB was accomplished by fluorescent and Western blot analysis with anti-stilbene monoclonal antibody EP2-19G2. The illustration of this method for general application to fusion protein analysis offers a dual role in assisting both solution-based fluorescent analysis and surface-based affinity detection and purification.

Affinity Labels↗