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Biomedical subjects

B Kachar

Publications and source records attributed to B Kachar.

104 records · Page 6Linked to original sources

Freeze-fracture cytochemistry: replicas of critical point-dried cells and tissues after fracture-label.

Applications of the new fracture-labeling techniques for the observation of cytochemical labels on platinum-carbon replicas are described. Frozen cells, embedded in a cross-linked protein matrix, and frozen tissues are fractured with a scalpel under liquid nitrogen, thawed, labeled, dehydrated by the critical point drying method, and replicated. This method allows direct, high-resolution, two-dimensional chemical and immunological characterization of the cellular membranes in situ, as well as detection of sites within cross-fractured cytoplasm and extracellular matrix.

Animals↗

Freeze-fracture study of rat ventral prostate: the columnar epithelial cell.

The organization of the secretory epithelium of the rat ventral prostate as seen by freeze-fracture is analyzed. Ultrastructural interpretation of the tall columnar epithelial cell is facilitated by the axial regionalization of the cytoplasm. The endoplasmic reticulum, which exists in all the regions of the cell, appears to have a role in the maintenance of cellular organization. Its cisternae delimit the spaces for the nucleus, the Golgi, and the apical secretory apparatus. Elements involved in the formation of secretory products for export are located within the apical region of the cell and are regionally separated from the Golgi complex. Structural and functional specializations of the plasma membrane in the apical, lateral, and basal areas are illustrated. Artifacts related to specimen preparation procedures are reported. Displacement of membrane particles, which we observed in the fracture faces of plasma and intracellular membranes, appears related to fixation at low temperature. Fixation at 37 degrees C, which does not induce membrane-particle displacement, results in proliferation of tight-junctional elements.

Animals↗

Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells.

The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membrane is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin. The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.

Animals↗

Freeze-fracture study of rat ventral prostate: secretory mechanisms in the epithelial cell.

Membrane events during apocrine and merocrine secretion of rat ventral prostate epithelial cells were analyzed by freeze-fracture. Early morphological manifestation of both secretory modes involves focal clearance of microvilli characterized by their progressive inclination and attenuation over the apical membrane. Merocrine secretion includes invasion of the apical microfilament web by the secretory vesicle and clearance of membrane particles at the site of its interaction with the apical plasmalemma. During apocrine secretion, a portion of the plasma membrane cleared of microvilli projects into the lumen. Growth of this projection is accompanied by a progressive, but partial, clearing of membrane particles and results in the formation of a large bleb containing dilated endoplasmic reticulum cisternae. Completion of the process involves "degeneration" of the bleb and its release by constriction of a neck, and possibly, fusion of vesicular or tubular structures. Swelling and blebbing of microvilli are shown to be preparation artifacts.

Animals↗

Particle displacement in epithelial cell membranes of rat prostate and pancreas induced by routine low temperature fixation.

When carried out at 4 degrees C in contrast to 37 degrees C, glutaraldehyde fixation for routine freeze-fracture specimen preparation results in displacement of membrane particles of epithelial cells of rat ventral prostate. Particle-free areas are observed in endoplasmic reticulum (ER), nuclear envelope, and Golgi membranes; particle aggregation is observed in lateral and basal portions of the plasma membrane. Multilamellar arrangement of lipids can be observed in some of the large particle-free areas. Thin section observation indicates that in the ER, particle-free areas coexist with segregation of cytoplasmic ground substance from the membrane surfaces. Particle-free patches are also observed in the ER membranes of pancreatic tissues fixed at 4 degrees C.

Animals↗

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.

Animals↗

Morphometric evaluation of the number of exocrine pancreatic cells during early postnatal growth in the rat.

The number of the various cell categories of the exocrine pancreas of the rat was evaluated by morphometric methods in paraffin sections of glands from rats aged 2, 5, 15, 20 and 33 days. The evolution of these cell types could be properly expressed by equations of the type y = aoek.x, where y = cell number, and x = age in days. The time necessary for each cell type to duplicate was thus obtained. The percentages (y') of acinar, intercalated duct and connective tissue cells also proved to be age-dependent and could be expressed by second-degree equations.

Age Factors↗

Imaging faces of shadowed magnetite (Fe(3)O(4)) crystals from magnetotactic bacteria with energy-filtering transmission electron microscopy.

We used energy-filtering transmission electron microscopy to image magnetite crystals isolated from uncultured magnetotactic bacteria. These magnetite crystals were shadowed in high vacuum with platinum at 45 degrees. The shadowed crystals were observed in a Zeiss (Thornwood, NY) CEM902 transmission electron microscope. Imaging shadowed crystals with inelastically scattered electrons provided information of the decoration pattern of small platinum particles over crystal surfaces, and thus information on surface characteristics of crystals. Results were comparable to those obtained from scanning electron microscopy using a field emitter gun. Electron energy loss spectra of the crystals as well as of the supporting film were recorded to evaluate variations of image contrast with energy losses. Results indicated that the contrast is attenuated with inelastic imaging and that the effect of contrast tuning caused a contrast inversion at a given point between 100 and 150 eV. We believe this approach can be useful for studying multilayered materials by transmission electron microscopy.

Bacteria↗

Electrokinetic shape changes of cochlear outer hair cells.

Rapid mechanical changes have been associated with electrical activity in a variety of non-muscle excitable cells. Recently, mechanical changes have been reported in cochlear hair cells. Here we describe electrically evoked mechanical changes in isolated cochlear outer hair cells (OHCs) with characteristics which suggest that direct electrokinetic phenomena are implicated in the response. OHCs make up one of two mechanosensitive hair cell populations in the mammalian cochlea; their role may be to modulate the micromechanical properties of the hearing organ through mechanical feedback mechanisms. In the experiments described here, we applied sinusoidally modulated electrical potentials across isolated OHCs; this produced oscillatory elongation and shortening of the cells and oscillatory displacements of intracellular organelles. The movements were a function of the direction and strength of the electrical field, were inversely related to the ionic concentration of the medium, and occurred in the presence of metabolic uncouplers. The cylindrical shape of the OHCs and the presence of a system of membranes within the cytoplasm--laminated cisternae--may provide the anatomical substrate for electrokinetic phenomena such as electro-osmosis.

Animals↗

Surface domains in the pathogenic protozoan Tritrichomonas foetus.

The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus. The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.

Animals↗

Coordinated flagellar and ciliary beating in the protozoon Tritrichomonas foetus.

Tritrichomonas foetus is a flagellated protozoon found in urogenital tract of cattle. Its free movement in liquid medium is powered by the coordinated movement of three flagella projecting towards the anterior region of the cell, and one recurrent flagellum that forms a junction with the cell body and ends as a free projection in the posterior region of the cell. We have used video microscopy and digital image processing to analyze the relationships between the movements of these flagella. The anterior flagella beat in a ciliary type pattern displaying effective and recovery strokes, while the recurrent flagellum beats in a typical flagellar wave form. One of the three anterior flagella has a distinctive pattern of beating. It beats straight in its forward direction as opposed to the ample beats performed by the others. Frequency measurements obtained from cells swimming in a viscous medium shows that the beating frequency of the recurrent flagellum is approximate twice the frequency for the three anterior flagella. We also observed that the costa and the axostyle do not show any active motion. On the contrary, they form a cytoskeletal base for the anchoring and orientation of the flagella.

Animals↗