Biomedical subjects

F Pavalko

Publications and source records attributed to F Pavalko.

3 recordsLinked to original sources

PubMed2001

Peroxynitrite causes endothelial cell monolayer barrier dysfunction.

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated barrier dysfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD, Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,.NO rapidly combines with superoxide (O) to form the powerful oxidant peroxynitrite (ONOO(-)), which we hypothesized would cause PAEC monolayer barrier dysfunction. To test this hypothesis, we treated PAEC with ONOO(-) (500 microM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 microM). SIN-1-mediated ONOO(-) formation was confirmed by monitoring the oxidation of dihydrorhodamine 123 to rhodamine. Both ONOO(-) and SIN-1 increased albumin clearance (P < 0.05) in the absence of cytotoxicity and altered the architecture of the cytoskeletal proteins actin and beta-catenin as detected by immunofluorescent confocal imaging. ONOO(-)-induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO(-) and SIN-1 nitrated tyrosine residues, including those on beta-catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO(-) into PAEC monolayers via liposomes also resulted in barrier dysfunction. These results indicate that ONOO(-) directly alters endothelial cytoskeletal proteins, leading to barrier dysfunction.

Actins↗

PubMed1999

Structure-function studies of the eighth hydrophobic domain of a serotonin receptor.

The most prominent structural feature of the G protein-coupled receptor superfamily is their seven hydrophobic domains, which are postulated to form membrane-spanning alpha helices. Some members of the G protein-coupled receptor family, specifically several serotonin (5-HT) receptors, possess eight hydrophobic domains. The importance of this extra hydrophobic domain, located at the N terminus of the receptor, is unknown. This question was addressed by deleting the extra hydrophobic region from the 5-HT2C receptor and comparing its function and topology with those of the wild-type receptor. Immunofluorescence microscopy was used to determine the location of the N terminus of the epitope-tagged wild-type and mutant receptors. The N terminus of both receptors was extracellular, suggesting that the extra hydrophobic domain does not change the topology of this receptor and is unlikely to be a membrane-spanning alpha helix. Radioligand-binding studies in transfected cells and expression studies in Xenopus oocytes demonstrated that seven hydrophobic domains were sufficient for normal function in these assays. Interestingly, the mutant receptor, now containing seven hydrophobic domains, is expressed at higher levels in transfected cells than the wild-type receptor containing eight hydrophobic domains, suggesting that the extra hydrophobic domain does impact the activity of this receptor by regulating its expression.

Amino Acid Sequence↗

PubMed1990

Actin-membrane interaction in focal adhesions.

Focal adhesions are regions of the plasma membrane where cells in tissue culture adhere strongly to the underlying extracellular matrix, and which at their cytoplasmic face serve to anchor bundles of actin microfilaments. They provide an experimental model for studying the links between the cytoskeleton and the extracellular matrix. Members of the integrin family of extracellular matrix receptors are prominent components, spanning the membrane in focal adhesions, but there is evidence that other membrane components are also needed for these structures to form. A number of proteins are concentrated at the cytoplasmic face of focal adhesions. Recent efforts have sought to determine the links between actin and the integrin cytoplasmic domains. Using in vitro binding assays, two potential bridges between actin and integrin have been identified. One involves talin, which has recently been shown to bind actin directly. The other involves the actin-binding protein, alpha-actinin, which has been found to interact with several integrins. The physiological significance of these two potential bridges between actin and integrin remains to be determined in vivo.

Actinin↗