Biomedical subjects

M C Alliegro

Publications and source records attributed to M C Alliegro.

22 records · Page 2Linked to original sources

PubMed1988

Storage and mobilization of extracellular matrix proteins during sea urchin development.

After fertilization, sea urchin embryos surround themselves with an extracellular matrix, or hyaline layer, to which cells adhere during early development. Hyalin, the major protein component of the hyaline layer has been isolated and partially characterized in several laboratories. Although other proteins are present in the hyaline layer, little is known about their origin, distribution, or functions. The present report characterizes a set of hyaline layer proteins that are secreted after fertilization from a class of vesicles that are distinct from cortical granules. The group of proteins in these vesicles were identified by a monoclonal antibody (8d11) which recognizes a carbohydrate epitope common to each of these molecules. 8d11 polypeptides range in molecular weight from 105 to 225 kDa. Oogonia and oocytes in early stages of vitellogenesis do not express the antigen. The proteins are first observed by immunofluorescence during oogenesis as a peripheral band in mid-vitellogenic oocytes. Following germinal vesicle breakdown 8d11 moves to be distributed evenly throughout the cytoplasm. The proteins are transported to the egg surface by a cytochalasin-sensitive mechanism after fertilization, and secreted predominately within the first 30 min of development. 8d11 proteins are depleted in areas of cell contact during early embryogenesis, and become concentrated on the apical surface of ectoderm cells where they are assembled into high-molecular-weight aggregates. Three of the molecules in this group may be proteins previously described as "apical lamina" proteins. These observations provide evidence of a third pathway (cortical granules and basal lamina granules being the other two) for synthesis, storage, and exocytosis of matrix proteins that are release after fertilization.

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PubMed1988

Echinonectin: a new embryonic substrate adhesion protein.

An extracellular matrix molecule has been purified from sea urchin (Lytechinus variegatus) embryos. Based on its functional properties and on its origin, this glycoprotein has been given the name "echinonectin." Echinonectin is a 230-kD dimer with a unique bow tie shape when viewed by electron microscopy. The molecule is 12 nm long, 8 nm wide at the ends, and narrows to approximately 4 nm at the middle. It is composed of two 116-kD U-shaped subunits that are attached to each other by disulfide bonds at their respective apices. Polyclonal antibodies were used to localize echinonectin in paraffin-embedded, sectioned specimens by indirect immunofluorescence. The protein is stored in vesicles or granules in unfertilized eggs, is released after fertilization, and later becomes localized on the apical surface of ectoderm cells in the embryo. When used as a substrate in a quantitative in vitro assay, echinonectin is highly effective as an adhesive substrate for dissociated embryonic cells. Because of the quantity, pattern of appearance, distribution, and adhesive characteristics of this protein, we suggest that echinonectin serves as a substrate adhesion molecule during sea urchin development.

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PubMed1985

Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs.

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.

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PubMed1983

Polyspermy inhibition in the oyster, Crassostrea virginica.

Inhibition of polyspermy is a critical response during fertilization which ensures that only one sperm nucleus will fuse with the female pronucleus to restore the diploid state. Oyster (Crassostrea virginica) eggs prevent polyspermy by a process occurring at the cell surface. However, 5 min after fertilization, there are still functional sperm receptor sites available for penetration by supernumerary sperm. Reinsemination experiments indicate that there is no decrease in the number of penetration sites during this interval. Yet, the number of sperm entering eggs is restricted to one per fertilized egg at a sperm:egg ratio of 1000:1. At a sperm:egg ratio of 10(5):1, an average of only six sperm were able to penetrate each egg. Gamete binding experiments indicate that there is a gradual decrease in the number of sperm bound to eggs starting at approximately 75 sec and continuing until all sperm are detached. Since eggs are fertilized within seconds of insemination and this process takes at least 12 min, it is considered an unlikely mechanism for the polyspermy block. There are no ultrastructural correlates to the polyspermy block nor to the unbinding of sperm, such as secretion of cortical granules or fertilization envelope formation. Based on reinsemination experiments, kinetic data, and ultrastructural observations, we suggest a physiological block to polyspermy which prevents fusion of gamete plasma membranes, and is mediated by an inhibitory effect directly upon the sperm.

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