scholarly journals In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates.

1976 ◽  
Vol 68 (2) ◽  
pp. 319-338 ◽  
Author(s):  
P J Oates ◽  
O Touster
Keyword(s):  
Electron Microscopy ◽  
Blood Cells ◽  
Fusion Reaction ◽  
Light And Electron Microscopy ◽  
Yeast Cells ◽  
Eosin Y ◽  
Vacuole Fusion ◽  
Lysosomal System

Fusion of phagolysosomes (PLs) has been demonstrated to occur in vitro. Two separate cell homogenates of the ameba Acanthamoeba sp. (Neff) were prepared, each rich in PLs labeled with distinctive particulate markers. Portions of each were incubated together in vitro and fusion occurred as evidenced by the appearance of PLs containing both types of markers. Fusion was confirmed by electron microscopy, including serial sectioning. The membranes of fused vacuoles excluded the dye eosin Y. Surviving cells in the homogenates were not responsible for the observed fusion. Fusion was obtained using either synthetic markers (polystyrene and polyvinyltoluene latex) or biological markers (autoclaved yeast cells and glutaraldehyde-fixed goat red blood cells), or a combination of both. The specificity of PL fusion in vivo appeared to be maintained in vitro. As determined by light and electron microscopy, the fusion reaction was dependent on time and temperature, and on the initial presence of membrane around both marker particles. A minimum of 10% of the vacuoles fused by 10 min of incubation at 30 degrees C, and no rupture of the vacuoles was detected during this time. After 10 min of incubation, vacuole rupture began and fusion ceased. At a constant initial vacuole concentration, the extent of PL fusion in vitro was quantitatively reproducible. This appears to be a promising system for further investigation of membrane fusion in the lysosomal system.

scholarly journals Optimization of Innovative Three-Dimensionally-Structured Hybrid Vesicles to Improve the Cutaneous Delivery of Clotrimazole for the Treatment of Topical Candidiasis

Pharmaceutics ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 263 ◽  
Author(s):  
Maria Letizia Manca ◽  
Iris Usach ◽  
José Esteban Peris ◽  
Antonella Ibba ◽  
Germano Orrù ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Chemical Properties ◽  
Chemical Characterization ◽  
Yeast Cells ◽  
Unilamellar Vesicles ◽  
Transmission Electron ◽  
Physico Chemical

New three-dimensionally-structured hybrid phospholipid vesicles, able to load clotrimazole in a high amount (10 mg/mL), were obtained for the first time in this work by significantly reducing the amount of water (≤10%), which was replaced with a mixture of glycerol and ethanol (≈90%). A pre-formulation study was carried out to evaluate the effect of both the composition of the hydrating medium and the concentration of the phospholipid on the physico-chemical properties of hybrid vesicles. Four different three-dimensionally-structured hybrid vesicles were selected as ideal systems for the topical application of clotrimazole. An extensive physico-chemical characterization performed using transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), 31P-NMR, and small-angle X-ray scattering (SAXS) displayed the formation of small, multi-, and unilamellar vesicles very close to each other, and was capable of forming a three-dimensional network, which stabilized the dispersion. Additionally, the dilution of the dispersion with water reduced the interactions between vesicles, leading to the formation of single unilamellar vesicles. The evaluation of the in vitro percutaneous delivery of clotrimazole showed an improved drug deposition in the skin strata provided by the three-dimensionally-structured vesicles with respect to the commercial cream (Canesten®) used as a reference. Hybrid vesicles were highly biocompatible and showed a significant antifungal activity in vitro, greater than the commercial cream Canesten®. The antimycotic efficacy of formulations was confirmed by the reduced proliferation of the yeast cells at the site of infection in vivo. In light of these results, clotrimazole-loaded, three-dimensionally-structured hybrid vesicles appear to be one of the most innovative and promising formulations for the treatment of candidiasis infections.

scholarly journals FORMATION OF BONE TISSUE IN CULTURE FROM ISOLATED BONE CELLS

1974 ◽  
Vol 61 (2) ◽  
pp. 427-439 ◽  
Author(s):  
Itzhak Binderman ◽  
Dan Duksin ◽  
Arieh Harell ◽  
Ephraim Katzir (Katchalski) ◽  
Leo Sachs
Keyword(s):  
Electron Microscopy ◽  
Bone Tissue ◽  
Bone Cells ◽  
Light And Electron Microscopy ◽  
Bone Collagen ◽  
The Matrix ◽  
Three Stages ◽  
And Function

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.

Ultraviolet microbeam irradiation of microtubules in vitro. The action spectrum for local depolymerization of marginal band microtubules in vitro matches that for reducing birefringence of chromosomal spindle fibres in vivo

1988 ◽  
Vol 91 (4) ◽  
pp. 469-478
Author(s):  
K. Hughes ◽  
A. Forer ◽  
P. Wilson ◽  
C. Leggiadro
Keyword(s):  
Electron Microscopy ◽  
Blood Cells ◽  
Monochromatic Light ◽  
Action Spectrum ◽  
Living Cells ◽  
Ultraviolet Microbeam ◽  
Marginal Band ◽  
Spindle Fibres

Marginal bands were isolated from newt red blood cells and, using monochromatic light from an ultraviolet microbeam, the marginal band microtubules were irradiated in vitro to produce areas of reduced birefringence (ARBs). The ARBs neither moved nor changed shape after they were formed, though the marginal bands sometimes changed shape during the irradiation. Marginal band ARBs were regions in which the microtubules were locally depolymerized, as determined by electron microscopy and immunofluorescence. The action spectrum for producing ARBs on marginal band microtubules in vitro matches very closely the action spectrum for producing ARBs on crane-fly spermatocyte chromosomal spindle fibres in vivo, which indicates that ARBs in vivo are produced by the ultraviolet light acting directly on the microtubules (as opposed to an intermediate component), and confirms, without complications inherent in the fixation of living cells, that ARBs on spindle fibres in vivo are regions in which microtubules are locally depolymerized.

scholarly journals THE ABSORPTION OF FAT BY INTESTINE OF GOLDEN HAMSTER IN VITRO

1963 ◽  
Vol 17 (3) ◽  
pp. 597-607 ◽  
Author(s):  
Elliott W. Strauss
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Bile Salts ◽  
Intracellular Transport ◽  
Light And Electron Microscopy ◽  
Lipid Absorption ◽  
Apical Cytoplasm ◽  
Everted Sacs

Everted sacs of intestine from golden hamsters were incubated at 37°C for at least 1 hour in vitro with emulsified lipid after removal of both pancreatic lipase and bile salts. The fine structure of intestinal epithelium is well preserved under these conditions. Absorption of fat by the intestinal mucosa in vitro closely resembles lipid absorption in vivo, as observed by both light and electron microscopy. The physiological significance of these observations is discussed. Tubular elements of the agranular endoplasmic reticulum are often strikingly abundant in the apical cytoplasm of intestinal absorptive cells. These have a role in the intracellular transport of fat since they frequently contain droplets of lipid derived from the incubation medium. The rate of fat accumulation in the epithelium appears to be proportional to the concentration in the medium.

scholarly journals Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae.

1996 ◽  
Vol 132 (5) ◽  
pp. 787-794 ◽  
Author(s):  
Z Xu ◽  
W Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Fusion Reaction ◽  
S Phase ◽  
Low Molecular Weight ◽  
Vacuole Fusion ◽  
Vacuole Inheritance ◽  
Segregation Structure

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a "segregation structure") into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one "low molecular weight activity" (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.

A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers

Parasitology ◽  
1981 ◽  
Vol 82 (3) ◽  
pp. 345-355 ◽  
Author(s):  
D. T. Hart ◽  
K. Vickerman ◽  
G. H. Coombs
Keyword(s):  
Electron Microscopy ◽  
Buoyant Density ◽  
Light And Electron Microscopy ◽  
Host Cells ◽  
Leishmania Mexicana ◽  
Cell Debris ◽  
Simple Method ◽  
Large Numbers

SUMMARYA rapid method for the bulk isolation of purified Leishmania mexicana mexicana amastigotes from parasite-induced lesions in experimentally infected mice is described. The procedure includes purification steps based on differences in net cell charge, lysis susceptibility and buoyant density between parasite and host cells. Yields of up to 2 × 1010 untransformed amastigotes with minimal contamination with host cells and cell debris can be obtained. At least 90 % of the purified amastigotes are viable as judged by light and electron microscopy, the staining of their lysosomes with acridine orange, their ability to transform to promastigotes and their infectivity to macrophages in vivo and in vitro.

Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 14-15
Author(s):  
Conly L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Quantitative Analysis ◽  
Light Microscopy ◽  
Living Cell ◽  
Light And Electron Microscopy ◽  
Time Behavior ◽  
Dynamic Interactions ◽  
Positive Identification ◽  
Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

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