Search results for "Immunogold labelling"

Basic Electron Microscopy

2006

Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization.

1999

p21(Cip1), first described as an inhibitor of cyclin-dependent kinases, has recently been shown to have a function in the formation of cyclin D-Cdk4 complexes and in their nuclear translocation. The dual behavior of p21(Cip1) may be due to its association with other proteins. Different evidence presented here indicate an in vitro and in vivo interaction of p21(Cip1) with calmodulin: 1) purified p21(Cip1) is able to bind to calmodulin-Sepharose in a Ca(2+)-dependent manner, and this binding is inhibited by the calmodulin-binding domain of calmodulin-dependent kinase II; 2) both molecules coimmunoprecipitate when extracted from cellular lysates; and 3) colocalization of calmodulin and p21(Cip…

3D-Ultrastructure, Functions and Stress Responses of Gastropod (Biomphalaria glabrata) Rhogocytes

2014

Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastropods and other molluscs, with great variation in their number, shape and size. They are enveloped by a lamina of extracellular matrix. Their most characteristic feature is the "slit apparatus", local invaginations of the plasma membrane bridged by cytoplasmic bars, forming slits of ca. 20 nm width. A slit diaphragm creates a molecular sieve with permeation holes of 20×20 nm. In blue-blooded gastropods, rhogocytes synthesize and secrete the respiratory protein hemocyanin, and it has been proposed-though not proven-that in the rare red-blooded snail species they might synthesize and secrete the he…

Novel molluskan biomineralization proteins retrieved from proteomics: a case study with upsalin.

2012

12 pages; International audience; The formation of the molluskan shell is regulated by an array of extracellular proteins secreted by the calcifying epithelial cells of the mantle. These proteins remain occluded within the recently formed biominerals. To date, many shell proteins have been retrieved, but only a few of them, such as nacreins, have clearly identified functions. In this particular case, by combining molecular biology and biochemical approaches, we performed the molecular characterization of a novel protein that we named Upsalin, associated with the nacreous shell of the freshwater mussel Unio pictorum. The full sequence of the upsalin transcript was obtained by RT-PCR and 5'/3…

Application of X-ray microanalysis to study of the expression of endothelial adhesion molecules on human umbilical vein endothelial cells in vitro

1994

A semi-quantitative procedure is described, which allows the evaluation of expression levels of endothelial adhesion molecules on cultured human umbilical vein endothelial cells (HUVEC) using energy dispersive X-ray microanalysis (EDX). As a model two adhesion molecules, E-selection (CD62E; ELAM-1/endothelial leukocyte adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1; CD54), were localized by the use of the silver-enhancement colloidal gold method after stimulation of HUVEC with endotoxin lipopolysaccharide (LPS), tumour necrosis factor (TNF) or a phorbol ester (PMA). The analysis was performed in a scanning electron microscope (SEM) at an accelerating voltage of 15 kV wit…

Protein mapping of calcium carbonate biominerals by immunogold

2007

The construction of metazoan calcium carbonate skeletons is finely regulated by a proteinaceous extracellular matrix, which remains embedded within the exoskeleton. In spite of numerous biochemical studies, the precise localization of skeletal proteins has remained for a long time as an elusive goal. In this paper, we describe a technique for visualizing shell matrix proteins on the surface of calcium carbonate crystals or within the biominerals. The technique is as follows: freshly broken pieces of biominerals or NaOCl then EDTA-etched polished surfaces are incubated with an antibody elicited against one matrix protein, then with a secondary gold-coupled antibody. After silver enhancement,…

Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis.

2005

We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, …

A novel pyruvate kinase (PK-S) from boar spermatozoa is localized at the fibrous sheath and the acrosome

2007

Boar spermatozoa contain a novel pyruvate kinase (PK-S) that is tightly bound at the acrosome of the sperm head and at the fibrous sheath in the principal piece of the flagellum, while the midpiece contains a soluble pyruvate kinase (PK). PK-S could not be solubilized by detergents, but by trypsin with no loss of activity. Purified PK-S as well as PK-S still bound to cell structures and soluble sperm PK have all kinetics similar to those of rabbit muscle PK-M1. The PK-S subunit had a relative molecular mass of 64 ± 1 × 103(n= 3), i.e. slightly higher than that of PK-M1, and carried an N-terminal extension (NH2-TSEAM-COOH) that is lacking in native PK-M1. Evidence is provided that PK-S is en…

A monoclonal antibody against an adult-specific cuticular protein of Tenebrio molitor (Insecta, Coleoptera)

1989

International audience; To study the sequential expression of the epidermal program in the mealworm Tenebrio molitor, monoclonal antibodies were prepared against the water-soluble proteins from preecdysial adult cuticle. Among the 16 clones obtained, one of them (named K2F6) recognized a 20-kDa antigen, found only in adult extracts but not in the larval or pupal ones, as revealed by immunoblot analysis. Our results strongly suggest an epidermal origin for this protein. The monoclonal antibody K2F6 fails to react with water-soluble proteins from fat body and hemolymph taken during the deposition of the 20-kDa antigen. Electron microscopic immunogold localization of this antigen showed that i…

Gastrin-cholecystokinin-like immunoreactivity in the central nervous system of the milkweed bug, Spilostethus pandurus. Ultrastructural aspects of th…

1995

Summary All the ganglia belonging to the central nervous system of adults of the milkweed bug Spilostethus pandurus (Hemiptera) were screened immunohistochemically for vertebrate gastrin-cholecystokinin (CCK-8(s))-like peptides. Several large reactive perikarya are present in the median part of the protocerebrum, their processes extending to the dorsal ‘aorta’. These cell bodies are also paraldehyde fuchsin-positive, ie they are A-type cells. In the lateral part of the protocerebrum, in the deutocerebrum and tritocerebrum, and in the suboesophageal, prothoracic and abdominal ganglia, a few immunoreactive cell bodies send axonal processes into their respective neuropiles. The A-type cells re…