Biomedical subjects

Martin J Brown

Publications and source records attributed to Martin J Brown.

3 recordsLinked to original sources

PubMed2004

Assessment of lymphocyte-mediated cytotoxicity using flow cytometry.

Cytotoxic lymphocytes, including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill target cells by releasing granules containing perforin and granzymes, and/or via Fas-Fas ligand interactions. Both pathways lead to prompt activation within target cells of caspase cascades responsible for apoptosis induction and cell death. We have utilized cell-permeable fluorogenic caspase substrates and multiparameter flow cytometry to detect caspase activation in target cells, and applied these tools to quantify and visualize cytotoxic lymphocyte activities. This novel assay, referred to as the flow cytometric cytotoxicity (FCC) assay, is a nonradioactive single-cell-based assay that provides a more rapid, biologically informative, and sensitive approach to measure cytotoxic lymphocyte activity when compared to other assays such as the 51chromium (51Cr) release assay. In addition, the FCC assay can be used to study CTL-mediated killing of primary target cells of different cell lineages that are frequently not amenable to study by the 51Cr release assay. Furthermore, the FCC assay enables evaluation of the phenotype and fate of both target and effector cells, and as such, provides a useful new approach to illuminate the biology of cytotoxic lymphocytes.

Apoptosis↗

PubMed2003

Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization.

Lymphocyte microvilli mediate initial rolling-adhesion along endothelium but are lost during transmigration from circulation to tissue. However, the mechanism for resorption of lymphocyte microvilli remains unexplored. We show that chemokine stimulation of human peripheral blood T (PBT) cells is sufficient to induce rapid resorption of microvilli. Microvilli in other cells are regulated by ezrin/radixin/moesin (ERM) proteins, which link the plasma membrane to the cortical F-actin cytoskeleton; maintenance of these linkages requires ERM activation, reflected by phosphorylation at a specific carboxy-terminal threonine residue. Carboxyphosphorylated-ERM (cpERM) proteins in resting PBT cells show a punctate peripheral distribution consistent with localization to microvilli. cpERM dephosphorylation begins within seconds of stimulation by chemokines (stromal derived factor 1 alpha [SDF-1 alpha] or secondary lymphoid tissue cytokine), and ERM proteins lose their punctate distribution with kinetics paralleling the loss of microvilli. The cpERM proteins are preferentially associated with the cytoskeleton at rest and this association is lost with chemokine-induced dephosphorylation. Transfection studies show that a dominant-negative ERM construct destroys microvilli, whereas a construct mimicking cpERM facilitates formation of microvilli, retards chemokine-induced loss of microvilli, and markedly impairs chemokine-induced polarization. Thus, chemokine induces rapid dephosphorylation and inactivation of cpERM, which may in turn facilitate 2 aspects of cytoskeletal reorganization involved in lymphocyte recruitment: loss of microvilli and polarization.

Blood Proteins↗

PubMed2000

Forest Canopy Stratification-Is It Useful?

It has long been recognized that the forest canopy has a complex structure that is significant for environmental interactions, regeneration, growth, and biotic habitat. Not only is the structure variously complex, but also there are many ways to conceptualize that complexity. Yet the persistent theme when considering the structure of canopies continues to be that of stratification: whether structural units are arranged in layers above the ground. We examined the use of the terms "stratification," "layering," and others in connection with canopy structure and found they had various meanings (often only implied) that were difficult to reconcile and to measure. We applied the definitions to the structure of a single, well-studied canopy located in Virginia, U.S.A., and found they failed to define consistently and clearly the presence, number, or location of strata. Additionally, we found the concept had limitations related to scale dependence, point of reference, and spatial averaging. Thus, asserting that a forest is stratified or naming the number of layers generally provided no guide to its structure. We propose alternative ways of conceptualizing and studying the forest canopy that avoid most of the problems associated with stratification. Among these are direct measurement and mapping of structural and environmental variables that have clear potential connections with canopy functions and viewing the distribution of structures or environmental conditions within the canopy as ecological gradients.

canopy↗