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Biomedical subjects

James R Baker

Publications and source records attributed to James R Baker.

At least 37 records · Page 2Linked to original sources
PubMed2005

Targeting and inhibition of cell growth by an engineered dendritic nanodevice.

The cellular uptake and cytotoxicity of an engineered multifunctional dendritic nanodevice containing folic acid (FA) as the targeting molecule, methotrexate (MTX) as the chemotherapeutic drug, and fluorescein (FI) as the detecting agent were studied in vitro. FI and FA were conjugated to the generation 5 poly(amidoamine) (G5) dendrimer carrier through a thiourea and amide linkage and MTX was conjugated through an ester linkage to the carrier to generate the trifunctional dendritic device, G5-FI-FA-MTX. This trifunctional dendrimer-drug conjugate bound to FA receptor-expressing KB cells in a dose-dependent and saturable manner. Confocal microscopic analysis demonstrated cellular internalization of the conjugate. G5-FI-FA-MTX induced a time- and dose-dependent inhibition of cell growth in KB cells. The targeted dendrimer conjugates G5-FI-FA-MTX and G5-FA-MTX inhibited cell growth in KB cells, whereas the nontargeted G5-MTX failed to induce growth inhibition. These studies show the potential of G5-FI-FA-MTX or G5-FA-MTX for targeting and growth suppression of tumor cells that overexpress FA-receptors.

Antineoplastic Agents↗
PubMed2005

Targeting cancer cells with DNA-assembled dendrimers: a mix and match strategy for cancer.

The unique biology of cancer requires the development of a multifunctional drug delivery system which can be efficiently manufactured to target subtle molecular alterations that distinguish a cancer cells from the many types of healthy cells found in the body. We sought to produce dendrimers conjugated to different bio-functional moieties [fluorescein (FITC) and folic acid (FA)], then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that over-express the high affinity folate receptor. This study demonstrates a unique molecular platform based on the DNA-directed assembly of dendritic polymers for the delivery of different agents to cancer cells. While only nanometers in diameter (the size of proteins), this DNA-linked dendrimer nanocluster platform is considered to allows for the delivery of drugs, genetic materials, and imaging agents to cancer cells, offering the potential for developing combinatorial therapeutics.

Animals↗
PubMed2005

HPLC separation of different generations of poly(amidoamine) dendrimers modified with various terminal groups.

Polyamidoamine (PAMAM) dendrimers of different generations with various terminal groups were analyzed, for the first time, using a combination of high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) techniques. Separation of amine-terminated dendrimers from generation 1 through generation 9 (G1NH(2)-G9NH(2)) was achieved using reversed-phase HPLC with elution time increasing gradually as the density of terminal amine groups increases as a function of generation. Furthermore, separation of dendrimers with terminal amino, acetamide, hydroxyl, and carboxylate groups was obtained. It has also been shown that HPLC can be used to separate dendrimers based on some structural defects inherent during the syntheses of PAMAM dendrimers. MALDI-TOF mass spectra of G1NH(2) identify the major imperfections present during typical synthesis processes. The absolute molar masses (M(n)) and molar mass distributions of the dendrimers were measured using the SEC system equipped with multiangle laser light scattering and refractive-index detectors. Findings from this study suggest HPLC can be a vital tool for characterization and preparative separation of PAMAM dendrimers.

Amides↗
PubMed2005

Capillary electrophoresis of polycationic poly(amidoamine) dendrimers.

Generation 2 to generation 5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications.

Aminopyridines↗
PubMed2005

Analysis of poly(amidoamine)-succinamic acid dendrimers by slab-gel electrophoresis and capillary zone electrophoresis.

Ethylenediamine (EDA)-core poly(amidoamine) (PAMAM) succinamic acid dendrimers (Ex.SAH, where x refers to the generation) were synthesized and analyzed by polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), potentiometric acid-base titration, and capillary zone electrophoresis (CZE). Various generations (E1.SAH-E7.SAH) PAMAMs and a succinamic acid terminated core-shell tecto(dendrimer) (E5(E3.SAH)(n)) were first analyzed by PAGE. PAGE results show that the relative mobilities of generation 2 to generation 7 dendrimers decreased with the increasing number of generations. The molecular mass of a generation 5 core generation 3 shell tecto(dendrimer) (denoted as E5(E3.SAH)(n)) was determined to be between the Mw of E6.SAH and E7.SAH. CZE analysis allowed the evaluation of electrophoretic properties of given-generation dendrimers. The electrophoretic mobilities of individual generations PAMAM polyanions are similar, indicating that the separation mainly depends on their approximately identical charge/mass ratio. The E5(E3.SAH)(n) tectodendrimer had a lower electrophoretic mobility, which was consistent with its lower charge/mass ratio. The combination of PAGE and CZE analysis provides an alternative and effective way to characterize this group of PAMAM-succinamic acid dendrimers.

Dendrimers↗
PubMed2005

Synthesis and functional evaluation of DNA-assembled polyamidoamine dendrimer clusters for cancer cell-specific targeting.

We sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5'-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics.

Carrier Proteins↗
PubMed2005

Silver/dendrimer nanocomposites as biomarkers: fabrication, characterization, in vitro toxicity, and intracellular detection.

We have synthesized water-soluble, biocompatible, fluorescent, and stable silver/dendrimer nanocomposites that exhibit a potential for in vitro cell labeling. Amino-, hydroxyl-, and carboxyl-terminated ethylenediamine core generation 5 poly(amidoamine) dendrimers were utilized to prepare aqueous silver(I)-dendrimer complexes (with the molar ratio of 25 Ag+ per dendrimer) at the biologic pH of 7.4. Conversion of silver(I)-dendrimer complexes into dendrimer nanocomposites was achieved by irradiating the solutions with UV light to reduce the bound Ag+ cations to zerovalent Ag0 atoms, which were simultaneously trapped in the dendrimer network, resulting in the formation of {(Ag0)25-PAMAM_E5.NH2}, {(Ag0)25-PAMAM_E5.NGly}, and {(Ag0)25-PAMAM_E5.NSAH} dendrimer nanocomposites (DNC), respectively. The silver-DNCs were characterized by means of UV-vis, fluorescence spectroscopy, dynamic light-scattering, zeta potential measurements, high-resolution transmission electron microscopy, X-ray energy dispersive spectroscopy, and selected area electron diffraction. The cytotoxicity of dendrimers and related silver nanocomposites was evaluated using an XTT colorimetric assay of cellular viability. The cellular uptake of nanoparticles was examined by transmission electron and confocal microscopy. Results indicate that {(Ag0)25-PAMAM_E5.NH2}, {(Ag0-)25-PAMAM_E5.NGly}, and {(Ag0)25-PAMAM_E5.NSAH} form primarily single particles with diameters between 3 and 7 nm. The dendrimer nanocomposites are fluorescent, and their surface charge, cellular internalization, toxicity, and cell labeling capabilities are determined by the surface functionalities of dendrimer templates. The {(Ag0)25-PAMAM_E5.NH2} and {(Ag0)25-PAMAM_E5.NSAH} nanocomposites exhibit potential application as cell biomarkers.

Animals↗
PubMed2004

Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells.

Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto's thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-gamma and IL-1beta cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-gamma/IL-1beta pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.

Apoptosis↗
PubMed2004

Trapping cavitation bubbles with a self-focused laser beam.

We observed that laser-induced cavitation bubbles in water can be trapped in a self-focused laser beam. Both optical imaging and acoustic detection have been utilized to confirm bubble trapping. Transverse and longitudinal trapping forces were measured to be as large as 87 and 11 pN, respectively. This result is contrary to conventional wisdom, since the mechanism of trapping in conventional optical tweezers implies that a low-index particle (a bubble being the limiting case) should be antitrapped.

Equipment Design↗
PubMed2004

Rapid access to azepine-fused oxetanols from alkoxy-substituted maleimides.

[reaction: see text] UV irradiation of alkoxy-substituted N-alkenylmaleimides induces a sequence involving a [5 + 2] cycloaddition followed by a Norrish-Yang cyclization. The resulting highly strained alkylidene oxetanol-fused azepines are formed in good yield and with high diastereoselectivities

Journal Article↗
PubMed2004

IFNgamma sensitization to TRAIL-induced apoptosis in human thyroid carcinoma cells by upregulating Bak expression.

TRAIL preferentially induces apoptosis in tumor cells and virus-infected cells. Unlike other tumor necrosis factor family members, TRAIL does not kill cells from most normal tissues and has thus been proposed as a promising new cancer treatment. Our study demonstrated that IFNgamma combined with TRAIL can trigger apoptosis in vitro in several resistant thyroid tumor cell lines, such as thyroid anaplastic carcinoma cells (ARO cells), while either agent alone exerts only a minimal effect. We further tested this effect on a mouse thyroid tumor model, when in vivo tumor growth was also significantly inhibited by this combination. The mechanism of how IFNgamma sensitized thyroid carcinoma cells to TRAIL-induced apoptosis was investigated by screening global gene alterations in ARO cells treated with IFNgamma. Microarray data revealed that a proapoptotic gene, Bak, is markedly upregulated by IFNgamma, and this was confirmed by RNase protection assay. Western blot analysis also showed a significant increase in Bak at the protein level. Upregulation of Bak and sensitization for apoptosis by IFNgamma was blocked by overexpression of antisense Bak in ARO cells. Furthermore, overexpression of Bak sensitized ARO cell to TRAIL-induced apoptosis without the need for IFNgamma pretreatment. This suggests that Bak is a regulatory molecule involved in IFNgamma-facilitated TRAIL-mediated apoptosis in thyroid cancer cells.

Animals↗
PubMed2004

Direct observation of lipid bilayer disruption by poly(amidoamine) dendrimers.

Atomic force microscopy (AFM) is employed to observe the effect of poly(amidoamine) (PAMAM) dendrimers on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers. Aqueous solutions of generation 7 PAMAM dendrimers cause the formation of holes 15-40 nm in diameter in previously intact bilayers. This effect is observed for two different branch end-groups--amine and carboxyl. In contrast, carboxyl-terminated core-shell tectodendrimer clusters do not create holes in the lipid membrane but instead show a strong affinity to adsorb to the edges of existing bilayer defects. A possible mechanism for the formation of holes in the lipid bilayer is proposed. The dendrimers remove lipid molecules from the substrate and form aggregates consisting of a dendrimer surrounded by lipid molecules. Dynamic light scattering (DLS) measurements as well as 31P NMR data support this explanation. The fact that tectodendrimers behave differently suggests that their cluster-like architecture plays an important role in their interaction with the lipid bilayer.

Dimyristoylphosphatidylcholine↗
PubMed2004

Quality assessment of selected commercially available whitefly and aphid biological control agents in the United States.

This study assessed the quality of three commercially available natural enemies used for pest management in greenhouses: the whitefly parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae), the aphid parasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae), and the aphid predatory midge Aphidoletes aphidimlyza (Rondani) (Diptera: Cecidomyiidae). Shipment packaging was consistent for all natural enemies. However, there was high variability in delivery punctuality, product cost, and product information provided by each of the six selected companies. Product quantity, percentage of emergence upon arrival, percentage of total emergence, percentage of females, and percentage of flying insects were assessed using International Organization for Biological Control (IOBC) recommended procedures. The parameters with greatest variability between companies were percentage of emergence upon arrival (0.9-10.5%) and percentage of flying insects (35.4-85.0%) for E. formnosa; product quantity (623.3-833.8 aphid mummies), percentage of emergence upon arrival (6.1-41.2%) and percentage of females (51.1-54.8%) for A. colemani; and percentage of emergence upon arrival (0.0-7.7%) and percentage of females (54.6-76.2%) for A. aphlidimyza. Results are discussed in terms of the value to consumers and compared with IOBC standards.

Animals↗
PubMed2004

A novel MEA/AFM platform for measurement of real-time, nanometric morphological alterations of electrically stimulated neuroblastoma cells.

Studies of electrically induced morphological changes in neurons have either been limited by the resolution of light microscopy or the cell fixation required for electron microscopy. Atomic force microscopy (AFM), however, mechanically maps cell topography, offering exquisite resolution of evolving processes in three dimensions. In this paper, we present a microelectrode array (MEA) based platform for the real-time detection of subtle, electrically induced variations in neuronal morphology, with AFM. This platform required the customized design and production of a silicon-based MEA, integration with a commercial AFM, and the development of biological techniques for culture of neuroblastoma (SH-SY5Y) cells onto the device. Biphasic pulse trains (1 Hz) of electric current were delivered to a microelectrode interfaced with a neuroblastoma cell, and the AFM continuously recorded a cross-sectional height profile. Proof-of-principle experiments demonstrate that electric stimulation may induce fluctuations ranging in the 100-300-nm range, 75-fold greater than the systemic resolution, but smaller than the resolution of light microscopy modalities. In addition, the real-time capabilities of AFM captured a collapse (30%-40%) of a neurite cross section, seconds after electric stimulation. Ultimately, this platform can be used to nanocharacterize cell responses to electric stimulation and other biochemical cues, for use in neuronal patterning and regeneration studies.

Cell Culture Techniques↗
PubMed2004

Interleukin-1beta and tumor necrosis factor (TNF)-alpha sensitize human thyroid epithelial cells to TNF-related apoptosis-inducing ligand-induced apoptosis through increases in procaspase-7 and bid, and the down-regulation of p44/42 mitogen-activated protein kinase activity.

Primary thyroid cells are resistant to TNF-related apoptosis-inducing ligand (TRAIL). Previously we showed that the combination of IL-1beta and TNFalpha facilitated TRAIL-mediated apoptosis in these cells and enhanced cell surface expression of TRAIL receptors. The aim of this study was to further characterize the mechanism by which these cytokines sensitized primary thyroid cells to TRAIL-mediated apoptosis. IL-1beta and TNFalpha increased the concentrations of procaspase-7 and Bid. In contrast, the p44/42 MAPK (Erk) pathway was active in thyroid cells and this activity was significantly decreased after exposure to IL-1beta/TNFalpha. A MAPK kinase inhibitor (U0126) could enhance the cytokine-induced sensitization of thyroid cells to TRAIL, reinforcing the inhibitory role of Erk on TRAIL signaling. In conclusion, IL-1beta/TNFalpha treatment sensitizes human thyroid cells to TRAIL-mediated apoptosis through increased surface expression of TRAIL receptors, increased expression of procaspase-7 and Bid, and the inhibition of p44/42 MAPK (Erk) pathway.

Apoptosis↗
PubMed2004

Detection and analysis of tumor fluorescence using a two-photon optical fiber probe.

The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing as little as 0.3% GFP-expressing cells. TPOFF results were similar to both flow-cytometric analysis of tumor cells after isolation and suspension, and fluorescence determined by microscope images of cryosectioned tumors. TPOFF was then used to measure GFP fluorescence from tumors in live mice. The fiber probe detected fluorescently-labeled Herceptin antibody targeted to HER2-expressing tumors in severe combined immunodeficient mice. Dendrimer nanoparticles targeted by folic acid and having 6-TAMRA as a fluorescent probe were also used to label KB cell tumors in vivo. The fiber probe documented a fourfold increase in tumor fluorescence in animals that received the targeted dendrimer. These results suggest TPOFF can be used as a minimally invasive system for identifying tumor markers and monitoring drug therapy.

Animals↗