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Vitro Techniques (vitro + techniques)

Distribution by Scientific Domains

Medical Sciences	50%
Life Sciences	50%

Selected Abstracts

Characterization of L-plastin interaction with beta integrin and its regulation by micro-calpain,

CYTOSKELETON, Issue 5 2010
E. Le Goff
Abstract Recent evidences suggest that plastin/fimbrin is more than a simple actin cross-linking molecule. In this context and based on the fact that other members of the same family interact with transmembrane proteins, such as integrins, we have investigated a possible interaction between L-plastin and integrins. By combining coimmunoprecipitation of endogenous proteins and in vitro techniques based on solid phase and solution assays, we demonstrate that L-plastin is an additional binding partner for the ,-chain of integrin and confirmed that both proteins display some colocalization. We then show that L-plastin binds to the cytoplasmic domain of ,1 integrin and to ,1 and ,2 peptides. Using recombinant L-plastin domains, we demonstrate that the integrin-binding sites are not located in NH2 terminal part of L-plastin but rather in the two actin-binding domains. Using pull-down, cross-linking experiments, and enzyme-linked immunosorbent assay, we show that the L-plastin/integrin complex is regulated by ,-calpain cleavage and is not directly dissociated by calcium. Indeed, despite the ability of calpain to cleave both proteins, only the cleavage of , integrin hindered the formation of the L-plastin/integrin complex. We discuss these results in the light of the three-dimensional structure of the actin-binding domains of L-plastin. © 2010 Wiley-Liss, Inc. [source]

Aryl hydrocarbon bioaccessibility to small mammals from arctic plants using in vitro techniques

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2007
Sarah A. Armstrong
Abstract Through their diet, herbivores inhabiting contaminated sites may be chronically exposed to a variety of aryl hydrocarbons (e.g., dioxins and polycyclic aromatic hydrocarbons [PAHs]). However, little is known about how differences in morphology and physiology among plant species alter the environmental accumulation of aryl hydrocarbons or their release and subsequent activity in the gastrointestinal tract of herbivores after ingestion. In the present study, the activity of aryl hydrocarbons during digestion was examined using six Arctic plant species growing in impacted and reference sites near Inuvik, Northwest Territories, Canada. The plant species studied were black spruce (Picea mariana), labrador tea (Ledum groenlandicum), bog birch (Betula glandulosa), green alder (Alnus crispa), water sedge (Carex aquatilis), and little-tree willow (Salix arbusculoides). Plants were digested using a simulator of the upper digestive tract, and aryl hydrocarbon release was evaluated using an aryl hydrocarbon-receptor assay. Bioaccessible aryl hydrocarbon activity varied among the plant species tested. The species with the greatest activity was green alder, and the species with the least activity was black spruce. Further investigation revealed that digested plant extracts may antagonize the aryl hydrocarbon receptor and prevent bioactivation of the aryl compound benzo[a]pyrene. Thus, PAH risk from the ingestion of vegetation varies among plant species and may depend on antagonists present in the vegetation. [source]

Intravenous immunoglobulin and salicylate differentially modulate pathogenic processes leading to vascular damage in a model of Kawasaki disease

ARTHRITIS & RHEUMATISM, Issue 7 2009
Andrew C. Lau
Objective Kawasaki disease (KD) is a multisystem vasculitis affecting children and is characterized by immune activation in the acute stage of disease. Systemic inflammation eventually subsides, although coronary arteritis persists, resulting in aneurysm formation. KD is the leading cause of acquired heart disease among children in North America. Accepted treatment guidelines include high-dose intravenous immunoglobulin (IVIG) and aspirin in the acute phase. Although this therapy is effective, the cellular and molecular mechanisms involved are not clear. The aim of this study was to examine the effect of IVIG and salicylate at each stage of disease development. Methods Using a murine model of KD, we established and validated several in vitro techniques to reflect 3 key steps involved in disease pathogenesis, as follows: thymidine incorporation to evaluate T cell activation, enzyme-linked immunosorbent assay to measure tumor necrosis factor , (TNF,) production, and real-time polymerase chain reaction to examine TNF,-mediated expression of matrix metalloproteinase 9 (MMP-9). Results At therapeutic concentrations, IVIG, but not salicylate, effectively reduced the immune response leading to TNF, expression. Unexpectedly, pharmacologic doses of salicylate were not able to inhibit TNF, production and in fact enhanced its production. Neither drug directly regulated MMP-9 expression but did so only indirectly via modulating TNF,. TNF, activity was a prerequisite for local expression of MMP-9 at the coronary artery. Conclusion Therapeutic concentrations of IVIG and salicylate differentially modulate the expression of TNF, and its downstream effects. Further dissection of the biologic effects of aspirin in acute KD is necessary for the rational design of therapy. [source]

Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells

CLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 6 2008
Masahiko Ayaki MD
Abstract Purpose:, The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Methods:, Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. Results:, HCEC survival was 20,30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. Conclusions:, The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative. [source]