Biomedical subjects

J Paul Robinson

Publications and source records attributed to J Paul Robinson.

8 recordsLinked to original sources

PubMed2003

Micro-assembly of functionalized particulate monolayer on C18-derivatized SiO2 surfaces.

This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions. For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.

Adsorption↗

PubMed2003

Composite surface for blocking bacterial adsorption on protein biochips.

The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO(2) surfaces of a biochip that had been modified with a C(18) coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C(18)-derivatized SiO(2) surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.

Adsorption↗

PubMed2003

DPI induces mitochondrial superoxide-mediated apoptosis.

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.

Animals↗

PubMed2003

Investigations of phagosomes, mitochondria, and acidic granules in human neutrophils using fluorescent probes.

The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX-Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes. Added R123 and MTG selectively accumulated in mitochondria. Added R123, MTG, and DHR increased the fluorescence of CMX-Ros and LysoTracker Red. This is the first FCM and CLSM demonstration of ROS formation in phagosomes. A distinct subpopulation of neutrophil granules, termed oxidative response granules, also was identified. Neutrophil mitochondrial membrane potential may be evaluated by incubating the cells with R123 and MTG, but results with CMX-Ros should be interpreted with caution. HE and DHR seem to measure a common pathway in the oxidative burst. The simultaneous application of several probes for investigations of organelles carries the risk of probe interference.

Acids↗

PubMed2003

Simultaneous mechanical loading and confocal reflection microscopy for three-dimensional microbiomechanical analysis of biomaterials and tissue constructs.

At present, mechanisms by which specific structural and mechanical properties of the three-dimensional extracellular matrix microenvironment influence cell behavior are not known. Lack of such knowledge precludes formulation of engineered scaffolds or tissue constructs that would deliver specific growth-inductive signals required for improved tissue restoration. This article describes a new mechanical loading-imaging technique that allows investigations of structural-mechanical properties of biomaterials as well as the structural-mechanical basis of cell-scaffold interactions at a microscopic level and in three dimensions. The technique is based upon the integration of a modified, miniature mechanical loading instrument with a confocal microscope. Confocal microscopy is conducted in a reflection and/or fluorescence mode for selective visualization of load-induced changes to the scaffold and any resident cells, while maintaining each specimen in a "live," fully hydrated state. This innovative technique offers several advantages over current biomechanics methodologies, including simultaneous visualization of scaffold and/or cell microstructure in three dimensions during mechanical loading; quantification of macroscopic mechanical parameters including true stress and strain; and the ability to perform multiple analyses on the same specimen. This technique was used to determine the structural-mechanical properties of three very different biological materials: a reconstituted collagen matrix, a tissue-derived biomaterial, and a tissue construct representing cells and matrix.

3T3 Cells↗

PubMed2002

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production.

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.

Apoptosis↗

PubMed2002

Detection of reactive oxygen species by flow cytometry after spinal cord injury.

The monitoring of reactive oxygen species (ROS) levels in injured nervous tissue is critical for both studying the mechanism of secondary damage and evaluating the effectiveness of antioxidants. Flow cytometry is an excellent method to detect ROS in cultured cells and naturally suspended individual cells. However, its use in nervous tissue is limited due to the difficulties in obtaining single cells in suspension. We have developed a new method which minimizes the error during conventional dissociation. Specifically, we introduced a fixation step (with formaldehyde) between the dye loading and dissociation. As a result, the post-injury ROS signals detected by flow cytometry increase significantly when using hydroethidine as superoxide indicator. The injury-induced elevation of ROS obtained from this new method was also in better agreement with the two other standard ROS detection methods, fluorescence microscopy and lipid peroxidation assay. Furthermore, more pronounced decrease of ROS was found in this improved method in response to treatment with a superoxide scavenger, manganese(III)tetrakis(4-benzoic acid)porphyrin. Based on these observations, we suggest that the data obtained from the cells by this new method are more accurate than those from the classic cell dissociation method that dissociates cells directly from fresh tissues.

Animals↗

PubMed2002

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure.

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.

Animals↗