Antonio Sesso

Permeabilities and stabilities of large dihexadecylphosphate and dioctadecyldimethylammonium chloride vesicles

Abstract Sodium dihexadecylphosphate (DHP) vesicles, with a mean external diameter of 0.27 μm, were obtained by chloroform vaporization followed by gel filtration. The relative volume of the internal aqueous compartment, estimated measuring entrapment of [ 14 C] sucrose, was 13 liters/mol. DHP and dioctadecyldimethyl ammonium chloride (DODAC) large vesicles were stable up to 20 days at room temperature. In contrast, small sonicated DHP and DODAC vesicles were stable for some hours after preparation. Both DHP and DODAC large vesicles were impermeable toward most solutes tested. Large DHP vesicles responded as ideal osmometers toward gradients of sucrose, NaCl, or NaOH. The behavior of large vesicles of synthetic amphiphiles and of phospholipids was found to be analogous in the gel state.

Characterization of dioctadecyldimethylammonium chloride vesicles prepared by membrane extrusion and dichloromethane injection

Abstract Vesicles of dioctadecyltrimethylammonium chloride were prepared by sonication, membrane extrusion and dichloromethane injection. The diameter of the vesicles were determined using quasi-elastic light scattering and freeze fracture electron microscopy. The incorporation of water soluble probes in the vesicles allowed the determination of a molar volume of 6 to 8 L/mole for the CH2Cl2 injected vesicles and 1.0 L/mole for the membrane extruded vesicles, in very good agreement with the data calculated from the vesicle diameters. The permeability of the injected CH2Cl2 vesicles is dependent on temperature and probe structure. Membrane extruded vesicles are more permeable than injected vesicles, both below and above the phase transition temperature.

Morphometric studies on venom secretory cells from Bothrops jararacussu (Jararacuçu) before and after venom extraction

A comparative morphometrical analysis was carried out on secretory cells from Bothrops jararacussu venom glands, before manual extraction of the venom (milking) and 4 and 8 days after milking. At the 8th day after milking, the cytoplasmic volume increased by 160%. The rough endoplasmic reticulum (RER) volume density increase, up to the 8th day after milking, is mainly due to widening of the intra-scisternal space. The total volume and membrane surface of the RER. Golgi apparatus and subcomponents, secretory vesicles and mitochondria, increased during the experimental period while the volume and surface densities of these organelles, with the exception of the RER, did not vary. The numerical density of Golgi-associated microvesicles per Golgi volume unit also increased. The greatest relative increments in these parameters occurred within the first 4 days. These results are compatible with an increased rate of membrane synthesis and transport in the milked glands and suggest that the membrane biogenesis, degradation and circulation that takes place in the first week after milking is achieved through coordinated cellular mechanisms that maintain the rate between total membrane surface and total cytoplasmic volume unaltered.

Salivary duct carcinoma: ultrastructural and histogenetic considerations

Two cases of salivary duct carcinoma were examined by light and electron microscopy. Histologically, the tumor presented cribriform and papillary patterns together with comedonecrosis. Electron microscopy revealed duct cells with microvilli, interdigitations, and apical vesicles. No myoepithelial cells were observed. These findings suggest a ductal origin other than from the intercalated duct or its precursor element. The importance of separating the salivary gland adenocarcinomas is discussed.

Mitochondrial Swelling and Incipient Outer Membrane Rupture in Preapoptotic and Apoptotic Cells

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix. Anat Rec, 2012.

Ultrastructural morphometric study on developing acinar cells of the rat pancreas and parotid gland.

The fraction of cellular volume occupied by the cytoplasm has been assessed by point-counting volumetry in 0.5 micrometer araldite sections. Measurement of the transection radius of the nuclei of acinar cells allowed the assessment of the mean nuclear volume according to the method of Bach. With these data, the cytoplasm volume was calculated in cubic micrometers. Ultrastructural morphometric data were obtained by means of superimposing on the electron photomicrographies (x 21,000 or x 28,500) a test system of 84 evenly spaced segments according to Weibel et al. The analysis of the values obtained for the pancreas and parotid gland allowed for the supposition that the process of maturation in the rat, either for the pancreas or the parotid gland, occurs simultaneously with the mitotic process, this last being held as the predominating one in the first 3--4 postnatal weeks. The cytodifferentiation process becomes more marked in the last phases studied and on the 40th day it already shows parameters held as similar to those of the adult animal.

A kinetic and structural study of two-step aggregation and fusion of neutral phospholipid vesicles promoted by serum albumin at low pH

The addition of bovine serum albumin (BSA) to 25 +/- 5 nm diameter single bilayer phosphatidylcholine (PC) vesicles (SBV) (pH 3.5) gives rise to readily visible transient turbidity. Studies of this system, employing a series of techniques, including time-dependent turbidity changes, membrane filtration, centrifugation, Sepharose chromatography and freeze fracture electron microscopy demonstrated that the process involves aggregation and fusion of the vesicles. At least three distinct time-dependent steps have been characterized: (1) the rapid initial formation (in approx. 5 min) of large aggregates (responsible for the visible turbidity) composed of SBV interconnected by BSA in its F form. The formation of these aggregates may be reversed by raising the pH or adding excess BSA to the system at this stage; (2) spontaneous collapse of these large aggregates, in an irreversible step, to form a heterogeneous population of vesicles; (3) fusion produces as the final product of the process, a relatively homogeneous population of larger (50 +/- 10 nm diamter) vesicles. This system serves as a convenient and simple model system for the detailed study of protein-mediated aggregation and fusion of membranes at the molecular level.

Freeze-fracture and thin-section study of condensing vacuoles in rat pancreatic acinar cells

Early and late developmental forms of condensing vacuoles are prominent in the relatively low rate secreting acinar cells of suckling rat pancreas. These vacuoles, when studied in freeze-fracture replicas and ultrathin sections under standard processing conditions, showed a biphasic evolution. During the first stage the condensing vacuoles (referred to as CV1) enlarge, accumulating contents of rather low electron density. Fracture faces with irregular patterns, possibly the result of fusion (pinching off) of microvesicles with (from) the condensing vacuoles, were occasionally encountered. The infrequency of such images indicates that fusion-fission during the growth stage must be a very rapid event. One common type of surface irregularity is gibbosities (or convexities) in the P fracture face with complementary images in the E fracture face. The significance of these irregularities, which are in apparent discordance with the theory of microvesicular transport, is unclear. By the end of the growing period the condensing vacuoles are large and smooth-surfaced (referred to as CV2) with contents of intermediate electron density (between that of the initial growing stage and that of the mature zymogen granule). The number of intercalated particles on both the large irregularly surfaced CV1) and large smooth-surfaced condensing vacuoles (CV2) membranes is high and comparable to that of the Golgi saccule and endoplasmic reticulum membranes. During the second stage, the smooth-surfaced condensing vacuoles undergo volume reduction associated with progressive increase in the electron density of their contents, thus becoming zymogen granules. Concomitant with size reduction, the number of intercalated particles in the membranes with CV2 diminishes markedly. The process of membrane retrieval appears to be accomplished selectively by pinching off coated microvesicles heavily studded with intercalated particles.

Inverted ductal papilloma of minor salivary gland origin: morphological aspects and cytokeratin expression

Ultrastructural features and cytokeratin expression of inverted ductal papillomas of minor salivary gland origin were studied. Under the electron microscope, an increased number of desmosomes and mucus-like granules in some cells were the most striking features. Immuno-histochemical study revealed that tumor cells displayed strongly positive reactions with cytokeratins 13 and 14, and less strong reactions with cytokeratins 7, 8, 18 and 5D3. These results support the hypothesis that an inverted ductal papilloma can be derived from the proximal portion of a salivary gland excretory duct.

Deposition of BaSO4 in the tight junctions of amphibian epithelia causes their opening ; apical Ca2+ reverses this effect

Selective deposition of BaSO4 in the tight junctions (TJs) of frog skins led to profound and reversible functional alterations of these structures, as revealed by changes of tissue conductance (G), clamping current (I), and fluxes of extracellular markers (sulfate (JSO4) and sucrose (JSUC)). Experiments were performed with nominally Ca2+ -free simple salt solutions on the apical side (usually KCl) and Na2SO4-Ringer on the inner side of skins. The deposition of BaSO4 in the TJs was obtained by diffusion and/or migration through the paracellular path of Ba2+ from the apical solution and SO42−from the inner solution. A brief presence (2 to 6 min) of apical Ba2+ (Ba2+ pulse) is followed (i.e., when Ba2+ is removed from the apical fluid) by a large increase of G, I, JSO4 and JSUC, above pre-Ba2+ levels. These attain a steady state within 15 to 30 min (overshoot phase), characterizing a conspicuous increase of the paracellular permeability. During the overshoot phase, a second Ba2+ pulse blocks the paracellular route while apical Ba2+ is present, leading to a new and larger overshoot when the Ba2+ pulse is terminated. Addition of apical Ca2+ triggers the resealing of the TJs, resulting in a full recovery of G, I, JSO4 and JSUC. This Ca2+ -induced recovery persists when apical Ca2+ is removed. The presence of a normal Ca2+ concentration in the inner bathing Ringer does not induce the recovery process. Tissues remain viable after being submitted to the Ba2+ treatment and the subsequent overshoot. Experiments performed in the urinary bladder of Rana catesbeiana and skins and urinary bladders of Bufo marinus indicate that Ba2+ effect can also be elicited in these tissues. The above results seem to report general properties of the TJs. Incidentally, they warn about the use of Ba2+ as an ion channel blocker in epithelial membranes in association with SO42−-containing solutions on the contralateral side.