Biomedical subjects

M M Allietta

Publications and source records attributed to M M Allietta.

6 recordsLinked to original sources

PubMed1997

Changes in vesicle morphology induced by lateral phase separation modulate phospholipase A2 activity.

The action of phospholipase A2 (PLA2) toward zwitterionic bilayers is modulated by lateral phase separation of reaction products and substrate. The experiments here address the mechanism of this modulation. PLA2 is particularly active toward lipid dispersions containing reaction products and substrates in which lateral phase separation has occurred. Here, we study PLA2 activity in two related model systems: first in a system in which lateral phase separation can be produced a priori, and second in a system in which the action of PLA2 produces sufficient reaction product in situ such that lateral phase separation occurs. The dispersions in which lateral phase separation occurs a priori form either disk micelles or disk vesicles, not canonical vesicles. When lateral phase separation occurs due to in situ PLA2 activity, there is an abrupt change in vesicle structure and a simultaneous profound increase in catalytic rate. This observation is surprising in light of several reports that vesicles remain intact even when the entire outer monolayer has been hydrolyzed. Membrane curvature and the associated structural defects and dynamic fluctuations in membrane structure have been proposed to modulate PLA2 activity. The mechanism by which lateral phase separation modulates PLA2 activity has been unclear. The data presented here indicate that lateral phase separation affects PLA2 activity by altering membrane curvature and/or inducing defects in the membrane structure.

Kinetics↗

PubMed1996

Epidermal growth factor and platelet-derived growth factor-BB induce a stable increase in the activity of low density lipoprotein receptor-related protein in vascular smooth muscle cells by altering receptor distribution and recycling.

Low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor, expressed by vascular smooth muscle cells (VSMCs) in normal arteries and in atherosclerotic lesions. In this investigation, we demonstrate a novel mechanism for the regulation of LRP activity in cultured rat aortic VSMCs. Cells that were treated with platelet-derived growth factor-BB (PDGF-BB) or epidermal growth factor (EGF) for 24 h bound increased amounts of the LRP ligand, activated alpha2-macroglobulin (alpha2M), at 4 degrees C. The Bmax for activated alpha2M was increased from 56 +/- 5 to 178 +/- 24 and 143 +/- 11 fmol/mg cell protein by PDGF-BB and EGF, respectively, while the KD was unchanged. Northern and Western blot analyses demonstrated that neither PDGF-BB nor EGF increase LRP mRNA or protein levels. Instead, LRP was redistributed to the cell surface and remained localized primarily in coated pits, as determined by surface protein biotinylation, affinity labeling, and immunoelectron microscopy studies. The increase in cell-surface LRP was partially explained by a 50% decrease in receptor endocytosis rate; however, at 37 degrees C, PDGF-BB- and EGF-treated VSMCs still bound/internalized increased amounts of activated alpha2M and subsequently released increased amounts of trichloroacetic acid-soluble radioactivity. The cytokine-induced shifts in LRP subcellular distribution were stable when VSMCs were challenged with a saturating concentration of ligand and then incubated, in the absence of cytokine, for 2.5 h at 37 degrees C. Regulation of LRP distribution and activity may be an important aspect of the VSMC response to the atherogenic cytokines, PDGF-BB and EGF.

Animals↗

PubMed1995

Cell wall anchoring to cytoplasmic membrane of Candida albicans.

Cell wall ultrastructure of the opportunistic pathogenic yeast Candida albicans was investigated by stereoscopic freeze-etching technique. Three wall layers were distinguishable by this technique. No clear periplasmic space was evident. Bilayer membrane invaginations were extensive. The outermost regions of the membrane invaginations were lined with thin, spine-like fibrils, which extended into the cell wall. We suggest that the fibrils along the invaginations are involved in anchoring the cell wall to the membrane.

Candida albicans↗

PubMed1995

A cytokeratin 8-like protein with plasminogen-binding activity is present on the external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines.

Plasminogen binding to cell surfaces may be important for tumor invasion and other processes that involve cellular migration. In this investigation, the principal plasminogen-binding protein was identified in the plasma membrane fraction of rat hepatocytes. The protein had an apparent mass of 59 kDa, was insoluble in a spectrum of detergents, and was identical to cytokeratin 8 (CK 8) as determined by sequence analysis of nine amino acids at the N terminus of two cyanogen bromide fragments. The 59 kDa protein bound CK 8-specific antibody in western blot analyses. These studies demonstrate that CK 8 or a CK 8-like protein binds plasminogen. Given this newly determined and potentially important CK 8 function, immunofluorescence and immunoelectron microscopy studies were performed to determine whether CK 8 may be present on the external surfaces of unpermeabilized, viable hepatocytes. All of the cells in each preparation were immunopositive with two separate CK 8-specific antibodies. A punctate pattern of immunofluorescence was detected on the cell surface with approximately even intensity from cell to cell. By immunoelectron microscopy, CK 8 was preferentially associated with microvilli. In order to determine whether other epithelial cells express cell-surface CK 8, immunofluorescence and immunoelectron microscopy studies were performed with HepG2 hepatocellular carcinoma cells and with BT20 and MCF-7 breast carcinoma cells. The pattern of antigen expression was equivalent with each cell type and comparable to that observed with hepatocytes. These studies support the hypothesis that CK 8 is associated with the external cell surface where it may express important proteinase receptor function.

Amino Acid Sequence↗

PubMed1988

Electron microscopic identification of exposed plasmin epitopes in alpha 2-macroglobulin-plasmin complex using monoclonal antibody-colloidal gold adducts.

Monoclonal antibodies 10-F-1, directed against the K4 region of plasminogen, and 10-V-1, directed against the K1-3 region of plasminogen, were adducted to colloidal gold. These antibody-gold adducts bound specifically to alpha 2-macroglobulin (alpha 2M)-plasmin. Greater than 90% of the apparent binding was eliminated when alpha 2M-methylamine was substituted for the alpha 2M-plasmin. The plasmin epitope recognized by 10-F-1 was identified at the extreme pole of the alpha 2M-plasmin complex, suggesting that plasmin protrudes from the end of the hollow cylinder formed by alpha 2M. The complexes formed between alpha 2M-plasmin and 10-V-1 were indistinguishable from those formed with 10-F-1. This suggests that exposure of plasmin surface structure in alpha 2M-plasmin, while substantial, may be limited to the single region of the inhibitor. Evidence for ternary complexes containing one alpha 2M and two plasmin molecules was obtained in the form of antibody-gold bound at both poles of alpha 2M-plasmin. The fraction of alpha 2M-plasmin that associated with more than one antibody was small. The data presented here are considered in relation to current models of alpha 2M structure and function.

Antibodies, Monoclonal↗

PubMed1979

Two species of lysosomal organelles in cultured human fibroblasts.

Cultured diploid human skin fibroblasts were fractionated by a procedure that maximizes recovery of particles containing acid hydrolases. The cells were detached by controlled trypsinization, disrupted by N2 cavitation at low pressure and fractionated at 18,000 x g on a self-generating gradient of colloidal silica. This procedure separated two species of particles that could be consisered lysosomal. The denser one (peak density 1.11) was apparently free of other contaminants, but the more buoyant one (peak density 1.085) sedimented with or close to the peaks of other organelles, including mitochondria, Golgi, endoplasmic reticulum and plasma membranes. The two populations of particles contained acid hydrolases (phosphatase, six glycosidases and four cathepsins) in roughly equal proportions, displayed latency, had similar turnover of 35S-mucopolysaccharide in normal as well as in iduronidase-deficient cells, and were recipients of alpha-L-iduronidase, previously shown to be acquired by receptor-mediated endocytosis. Acid phosphatase staining of the intact fibroblasts showed residual bodies scattered throughout the cytoplasm and, near the nucleus, a prominent network of tubules and associated dilatations and knob-like enlargements. In both thin and thick sections, these appeared continuous, as if forming a three-dimensional network similar to the network described by Novikoff (1976) as GERL. Ultrastructural studies of the isolated fractions showed the denser lysosomal peak to be composed of small round or oblong acid phosphatase-positive bodies. The more buoyant peak contained the nonlysosomal organelles predicted from the biochemical markers, small acid phosphatase-positive bodies and large multivesiculated structures in which acid phosphatase was localized in a matrix surrounding apparently empty vesicles. These large structures may represent fragments of GERL. We suggest that the dense and buoyant lysosomal organelles originate primarily from residual bodies and the GERL network, respectively.

Cathepsins↗