E.Lucio Benedetti

Presence of a lattice structure in membrane fragments rich in nicotinic receptor protein from the electric organ of Torpedo marmorata.

Recently Cohen et al. [l] have developed a fractionation method of Torpedo electric tissue which yields membrane fragments particularly rich in cholinergic (nicotinic) receptor protein. Observation of this purified fraction by both freeze-etching and negative staining reveals a lattice organization of particles. Similar particles are seen in negatively stained preparations of highly purified receptor protein from Electrophoms and most probably correspond to the cholinergic receptor protein.

Cultured calf lens epithelium. I. Methods of cultivation and characteristics of the cultures

Abstract Methods for culturing lens epithelial cells are described. The younger cultures contain mostly epithelial-like cells whereas in the long term cultures the cuboidal cells elongate and form parallel arrays. Other ultrastructural changes during aging of the cells are also observable. During the first weeks of culturing the pattern of proteins synthesized gradually changes. In particular the synthesis of the cell-specific proteins, the crystallins, ceases almost completely, with the exception of one β-crystallin component. The cell-elongation associated with the aging of the cultures is not accompanied by the appearance of the putative characteristics of differentiated lens cells, e.g. γ-crystallin synthesis or alterations in the lactate dehydrogenase pattern.

Selective reassociation of the crystallins

Abstract Under proper conditions a mixture of all the unfolded polypeptide chains which in the native state compose the soluble crystallins reassociate to the “native polymers” α-, β H -, β L - and γ-crystallin. On the other hand, after dissociation and reassociation α- and β-crystallin polypeptides tend to form rod-like hybrids. The presence of both types of subunits in these complexes has been proven by immunoelectrophoresis and polyacrylamide gel electrophoresis in 6 m -urea.

Cultured calf lens epithelium. II. The effect of dexamethasone

Abstract Lens epithelial cells in long-term culture elongate and orientate parallel to each other when a low concentration of dexamethasone is added to the medium. These changes in morphology are not observed when other hormones are applied. Cycloheximide and colchicine both prevent the elongation induced by dexamethasone. Any significant difference in the pattern of proteins synthesized by epithelial-like and elongated cells could not be demonstrated. The present results may suggest that the elongation process is not intimately linked to specific biochemical changes such as γ-crystallin synthesis occurring during lens fibre differentiation in vivo.

Sequence analysis of peptide fragments from the intrinsic membrane protein of calf lens fibers MP26 and its natural maturation product MP22

Calf lens fiber plasma membranes, containing only the intrinsic membrane protein MP26 and its maturation product MP22 were treated with proteolytic enzymes such as trypsin, protease V8 from S. aureus or with chemical agents as CNBr in formic acid. The cleavage products, purified by electrophoresis, were analysed for their amino acid composition and N‐terminal sequences. Proteolysis gave rise to peptides which were mainly shortened at the C‐terminal end of the molecules. While the V8 protease produced a fragment with a similar N‐terminal sequence as the maturation product MP22, trypsin yielded another cleavage product. Chemical hydrolysis yielded large fragments (11–15 kDa) with hydrophobic N‐terminal sequences. Our results suggest that MP26 is characterised by an N‐terminal signal sequence and possesses other hydrophobic domains which could function as untranslocated insertion sequences.

Laparoscopy in Ureteral Engineering: A Feasibility Study

OBJECTIVE We recently bioengineered a ureter substitute from a seeded scaffold implanted by open surgery in the omentum. In view of the development of laparoscopy in the treatment of benign conditions of the ureter, obtaining a ureter substitute by minimally invasive techniques would be a desirable objective. However, conflicting results about the biological impact of carbon dioxide insufflation on the microcirculation of intra-abdominal organs prompted us to investigate first whether the results obtained by open surgery, in terms of vascular supply and maturation, could be reproduced laparoscopically. MATERIALS AND METHODS Bladder full-thickness tissue was harvested laparoscopically from three pigs for urothelial and smooth muscle cell primary cultures subsequently used to seed a small intestinal submucosa (SIS) matrix. After 2 wk, the in vitro seeded constructs were shaped around silicone drains and transferred laparoscopically into the abdomen for omental maturation. Three weeks later, the constructs were harvested for histological, immunohistochemical, and electron microscopic analysis. RESULTS The laparoscopic procedures were performed successfully in all animals. After omental maturation, the constructs were vascularized and comprised of a well-differentiated multilayered urothelium with umbrella cells, over connective tissue and smooth muscle cells, with no evidence of fibrosis or inflammation. Electron microscopic analysis showed characteristics of a terminally differentiated urothelium. CONCLUSION As shown by conventional microscopy, immunochemistry, and electron microscopy, carbon dioxide insufflation does not impact cell growth and differentiation. These findings validate the laparoscopic approach for omental maturation of ureter substitutes.

Fatty acid acylation of lens fiber plasma membrane proteins: MP26 and α-crystallin are palmitoylated

We describe in this report the fatty acylation of some of the main polypeptides from the eye lens fibers. MP26, the major lens fiber plasma membrane protein, and probably MP22, its natural degradation product, are palmitoylated in a post‐translational process. This is also the case for α‐crystallin, a major cytoplasmic structural protein shown to interact directly with the plasma membrane. Furthermore, a 65 kDa non‐identified polypeptide and a high molecular weight component are also modified in the same way.

INTERMEDIATE FILAMENT-LIKE PROTEINS INLEISHMANIA

The cytoskeleton of Leishmania, and other Trypanosomatidae organisms, consists of a flagellum and a highly stable net of subpellicular microtubules that are cross-linked to each other and to the plasma membrane (Hou et al., 1992). The occurrence of additional cytoskeletal constituents such as microfilaments or intermediate filaments has not been reported, although an actin gene from Leishmania has been cloned and sequenced (Arruda and Matsudaira, 1994). None the less, there must exist cytoskeletal components within the cytoplasm, capable of positioning and directing the movement of organelles during cell division. Intermediate filaments are found both in the nucleus and cytoplasm of eukaryotic cells. Whereas lamins appear ubiquitous, cytoplasmic intermediate filaments are absent from some vertebrate (Fuchs, 1991) and invertebrate cells (Bartnik et al., 1986). Moreover, they have not been identified in unicellular eukaryotes (Klymkowsky, 1995). In this work we have used polyclonal antibodies against the intermediate filament proteins vimentin and desmin as a preliminary attempt to identify such structures in Leishmania.