scholarly journals Subfractionation of hepatic endosomes in Nycodenz gradients and by free-flow electrophoresis. Separation of ligand-transporting and receptor-enriched membranes

1985 ◽  
Vol 232 (1) ◽  
pp. 25-32 ◽  
Author(s):  
W H Evans ◽  
N Flint
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Binding Sites ◽  
Average Diameter ◽  
Free Flow ◽  
Density Range ◽  
Short Intervals ◽  
Electrophoresis Separation ◽  
Isopycnic Centrifugation

The complexity of rat liver endosome fractions containing internalized radioiodinated asialotransferrin, asialo-(alkaline phosphatase), insulin and prolactin was investigated by using free-flow electrophoresis and isopycnic centrifugation in Nycodenz gradients. Two subfractions were separated by free-flow electrophoresis. Both subfractions contained receptors for asialoglycoprotein and insulin. Glycosyltransferase activities were associated with the more electronegative vesicles, whereas 5′-nucleotidase and alkaline phosphodiesterase activities were associated with the less electronegative vesicles. Three subfractions were separated on Nycodenz gradients. Two subfractions, previously shown to become acidified in vitro, contained the ligands. At short intervals after uptake (1-2 min), ligands were mainly in subfraction DN-2 (density 1.115 g/cm3), but movement into subfraction DN-1 (density 1.090 g/cm3) had occurred 10-15 min after internalization. Low amounts of glycosyltransferase activities were associated with subfraction DN-2, and 5′-nucleotidase and alkaline phosphodiesterase activities were mainly located in subfraction DN-1. The binding sites for asialoglycoproteins and insulin were distributed towards the higher density range in the Nycodenz gradients, thus indicating a segregation of receptor-enriched vesicles and those vesicles containing the various ligands 10-15 min after internalization. Electron microscopy of the subfractions separated on Nycodenz gradients indicated that whereas the ligand-transporting fractions consisted mainly of empty vesicles (average diameter 100-150 nm), the receptor-enriched component was more granular and smaller (average diameter 70-95 nm). The properties of the endosome subfraction are used to assign their origin to the regions of the endocytic compartment where ligand-receptor dissociation and separation occur.

scholarly journals Site-specific inhibition of myosin-mediated motility in vitro by monoclonal antibodies.

1985 ◽  
Vol 100 (4) ◽  
pp. 1024-1030 ◽  
Author(s):  
P F Flicker ◽  
G Peltz ◽  
M P Sheetz ◽  
P Parham ◽  
J A Spudich
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Functional Assay ◽  
Specific Inhibition ◽  
Myosin Molecule ◽  
Site Specific ◽  
Dictyostelium Myosin ◽  
Cell Biol

Monoclonal antibodies directed against seven different sites on Dictyostelium myosin (Peltz, G., J. A. Spudich, and P. Parham, 1985, J. Cell Biol., 100: 1016-1023) were tested for their ability to inhibit movement of myosin in vitro, using the Nitella-based myosin-mediated bead movement assay (Sheetz, M. P., R. Chasan, and J. A. Spudich, 1984, J. Cell Biol., 99: 1867-1871). To complement this functional assay, we located the binding sites of these antibodies by electron microscopy, using the rotary shadowing technique. One antibody bound to the 18,000-dalton light chain and inhibited movement completely. All of the remaining antibodies bound to various positions along the rod portion of the myosin molecule, which is approximately 1,800 A long. Antibodies that bound to the rod about 470, 680, and 1400 A from the head-tail junction did not alter myosin movement. One antibody appeared to bind very close to the head-tail junction and to inhibit movement 50%. Surprisingly, three antibodies that bound about 1,200 A from the head-tail junction inhibited movement completely. This inhibition did not depend on using intact IgG, since Fab' fragments had the same effect.

scholarly journals Effect of crosslinking with riboflavin and ultraviolet a (UVA) on the scleral tissue structure

2017 ◽  
Vol 10 (2) ◽  
pp. 6-12
Author(s):  
Mukharram M Bikbov ◽  
Valentina K Surkova ◽  
Emin L Usubov ◽  
Nikolaj A Nikitin ◽  
Mikhail Nikolaevich Astrelin
Keyword(s):  
Electron Microscopy ◽  
Aqueous Solution ◽  
Average Diameter ◽  
Tissue Structure ◽  
In Vitro Experiments ◽  
Pathological Changes ◽  
Ultraviolet A ◽  
Intermediate Space ◽  
Special Software

Purpose. To evaluate the effect of the scleral crosslinking with riboflavin and ultraviolet A (UVA) on the scleral tissue structure in vitro experiments. Material and methods. The study was conducted on 7 porcine cadaver eyes. Two parallel scleral strips were excised from each eyeball, one of which was subjected to crosslinking procedure (instillation of 0.1% aqueous solution of riboflavin mononucleotide for 20 minutes, ultraviolet irradiation of 3 mW /cm2 for 30 minutes), second was used as control. Scleral structure was evaluated by light (Van Gieson’s stain) and electron microscopy. Morphometric analysis of the microphotographs was performed using special software. Results. As a result of crosslinking, the average packing density of collagen fibers increased by 8.2%, the area of the intermediate space decreased by 5.2%, the average diameter of collagen fibrils increased by 12%. There were no pathological changes in scleral structures. Conclusion. Obtained results confirm the efficacy of scleral crosslinking with riboflavin/UVA in the formation of additional crosslinks, and the procedure safety for the scleral tissue.

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

2020 ◽  
Vol 122 (1) ◽  
pp. 116-129
Author(s):  
Susana G. Guerreiro ◽  
Ronald E. Unger ◽  
Nuno M. F. S. A. Cerqueira ◽  
Anne Sartoris ◽  
Maria J. Martins ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Migration ◽  
Binding Sites

Preparation and Characterization of Mno.5Zno.5Fe2O4 @Au Composite Nanoparticles and its Anti-Tumor Effect on Glioma Cells

2011 ◽  
Vol 138-139 ◽  
pp. 907-913
Author(s):  
Yun Tao Li ◽  
Jing Liu ◽  
Li Wang ◽  
Jia Zhang ◽  
Zi Yu Wang ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Electron Microscopy ◽  
Au Nanoparticles ◽  
Glioma Cells ◽  
Trisodium Citrate ◽  
Average Diameter ◽  
Composite Nanoparticles ◽  
Au Nanoparticle ◽  
Core Shell

To explore the preparation method and characters of a new gold nanoshells on maganese-zinc ferrite (Mno.5Zno.5Fe2O4@Au) composite nanoparticles. Mno.5Zno.5Fe2O4@Au nanoparticles with core/shell structure were synthesized by reduction of Au3+ with trisodium citrate in the presence of Mno.5Zno.5Fe2O4 magnetic nanoparticles (MZF-NPs) prepared by improved co-preciption with the character of superparamagnetism and detected by transmission electron microscopy (TEM), scanning electron microscopy (SEM), x-ray diffraction (XRD), energy dispersive spectrometry (EDS) and Marven laser particle size analyzer.Thermodynamic test was used to observe temperature change of various doses of Mno.5Zno.5Fe2O4@Au nanoparticles. The cytotoxicity of the Mno.5Zno.5Fe2O4@Au composite nanoparticles in vitro was tested by the MTT assay. The therapeutic effect of Mno.5Zno.5Fe2O4@Au composite nanoparticles combined with magnetic fluid hyperthermia (MFH) on human glioma cells were evaluated in vitro by an MTT assay.The results indicated that the Mno.5Zno.5Fe2O4@Au composite nanoparticles were prepared successfully. The core/shell particles were spherical with exact average diameter of them was 66.9nm.EDS showed each Mno.5Zno.5Fe2O4@Au nanoparticle contained Mn, Zn, Fe, O and Au elements, and this proved Au had successfully attached to Mn0.5Zn0.5Fe2O4.The result of thermodynamic test showed that Mno.5Zno.5Fe2O4@Au composite nanoparticles could serve as a heating source under alternating magnetic field (AMF) exposure leading to reach their steady temperature (40-45°C). Moreover, Mno.5Zno.5Fe2O4@Au composite nanoparticles didn’t show cytotoxicity in vitro. The therapeutic result reveals that Mno.5Zno.5Fe2O4@Au composite nanoparticles can significantly inhibit the growth of glioma cells.The conclusion was that the self-prepared Mno.5Zno.5Fe2O4@Au composite nanoparticles had strong magnetic responsiveness and good power absorption capabilities in the high frequency AMF,then they could suggested to be useful for glioma hyperthemia. Mno.5Zno.5Fe2O4 @Au composite nanoparticles can not only be directed to tumor region in a given magnetic field more exactly but also produce marked thermotherapy.

Sem Studies of Co-Cr-Mo Surgical Implant Alloy Corrosion Behavior

1982 ◽  
Vol 40 ◽  
pp. 520-521
Author(s):  
Ann Chidester Van Orden ◽  
John L. Chidester ◽  
Anna C. Fraker ◽  
Pei Sung
Keyword(s):  
Electron Microscopy ◽  
Corrosion Behavior ◽  
Anodic Polarization ◽  
Saline Solution ◽  
Saturated Calomel Electrode ◽  
Physiological Saline ◽  
X Ray ◽  
Physiological Saline Solution ◽  
Scanning Electron

The influence of small variations in the composition on the corrosion behavior of Co-Cr-Mo alloys has been studied using scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDX), and electrochemical measurements. SEM and EDX data were correlated with data from in vitro corrosion measurements involving repassivation and also potentiostatic anodic polarization measurements. Specimens studied included the four alloys shown in Table 1. Corrosion tests were conducted in Hanks' physiological saline solution which has a pH of 7.4 and was held at a temperature of 37°C. Specimens were mechanically polished to a surface finish with 0.05 µm A1203, then exposed to the solution and anodically polarized at a rate of 0.006 v/min. All voltages were measured vs. the saturated calomel electrode (s.c.e.).. Specimens had breakdown potentials near 0.47V vs. s.c.e.

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

1977 ◽  
Vol 35 ◽  
pp. 460-461
Author(s):  
Tai-Te Chao ◽  
John Sullivan ◽  
Awtar Krishan
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Tubulin Polymerization ◽  
Vinca Alkaloids ◽  
Antimitotic Activity ◽  
Transmission Electron ◽  
Flow Microfluorometry ◽  
Brain Tubulin

Maytansine, a novel ansa macrolide (1), has potent anti-tumor and antimitotic activity (2, 3). It blocks cell cycle traverse in mitosis with resultant accumulation of metaphase cells (4). Inhibition of brain tubulin polymerization in vitro by maytansine has also been reported (3). The C-mitotic effect of this drug is similar to that of the well known Vinca- alkaloids, vinblastine and vincristine. This study was carried out to examine the effects of maytansine on the cell cycle traverse and the fine struc- I ture of human lymphoblasts.Log-phase cultures of CCRF-CEM human lymphoblasts were exposed to maytansine concentrations from 10-6 M to 10-10 M for 18 hrs. Aliquots of cells were removed for cell cycle analysis by flow microfluorometry (FMF) (5) and also processed for transmission electron microscopy (TEM). FMF analysis of cells treated with 10-8 M maytansine showed a reduction in the number of G1 cells and a corresponding build-up of cells with G2/M DNA content.

Sites of Insulin Action Using Ferritin Labeling

1971 ◽  
Vol 29 ◽  
pp. 540-541
Author(s):  
Burton B. Silver ◽  
Ronald S. Nelson
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Anterior Pituitary ◽  
Insulin Action ◽  
Diabetic Rats ◽  
Insulin Antibody ◽  
Fat Pad ◽  
Target Organs ◽  
Uranium Salts ◽  
Sugar Levels

Some investigators feel that insulin does not enter cells but exerts its influence in some manner on the cell surface. Ferritin labeling of insulin and insulin antibody was used to determine if binding sites of insulin to specific target organs could be seen with electron microscopy.Alloxanized rats were considered diabetic if blood sugar levels were in excess of 300 mg %. Test reagents included ferritin, ferritin labeled insulin, and ferritin labeled insulin antibody. Target organs examined were were diaphragm, kidney, gastrocnemius, fat pad, liver and anterior pituitary. Reagents were administered through the left common carotid. Survival time was at least one hour in test animals. Tissue incubation studies were also done in normal as well as diabetic rats. Specimens were fixed in gluteraldehyde and osmium followed by staining with lead and uranium salts. Some tissues were not stained.

Some Structural Features of Metaphase Chromosomes of Mice and Men

1967 ◽  
Vol 25 ◽  
pp. 190-191
Author(s):  
Godfrey C. Hoskins ◽  
Betty B. Hoskins
Keyword(s):  
Electron Microscopy ◽  
Electron Micrographs ◽  
Structural Features ◽  
Metaphase Chromosomes ◽  
The Future ◽  
Of Mice And Men ◽  
Mouse Cells ◽  
Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

Electron Microscopy of Fibroblasts from Myotonic Muscular Dystrophy Patients

1981 ◽  
Vol 39 ◽  
pp. 498-499
Author(s):  
S. E. Miller ◽  
G. B. Hartwig ◽  
R. A. Nielsen ◽  
A. P. Frost ◽  
A. D. Roses
Keyword(s):  
Electron Microscopy ◽  
Muscular Dystrophy ◽  
Genetic Diseases ◽  
Skin Fibroblasts ◽  
Inborn Errors ◽  
Cytoplasmic Inclusions ◽  
Cultured Skin ◽  
Myotonic Muscular Dystrophy ◽  
Glycosaminoglycan Metabolism

Many genetic diseases can be demonstrated in skin cells cultured in vitro from patients with inborn errors of metabolism. Since myotonic muscular dystrophy (MMD) affects many organs other than muscle, it seems likely that this defect also might be expressed in fibroblasts. Detection of an alteration in cultured skin fibroblasts from patients would provide a valuable tool in the study of the disease as it would present a readily accessible and controllable system for examination. Furthermore, fibroblast expression would allow diagnosis of fetal and presumptomatic cases. An unusual staining pattern of MMD cultured skin fibroblasts as seen by light microscopy, namely, an increase in alcianophilia and metachromasia, has been reported; both these techniques suggest an altered glycosaminoglycan metabolism An altered growth pattern has also been described. One reference on cultured skin fibroblasts from a different dystrophy (Duchenne Muscular Dystrophy) reports increased cytoplasmic inclusions seen by electron microscopy. Also, ultrastructural alterations have been reported in muscle and thalamus biopsies from MMD patients, but no electron microscopical data is available on MMD cultured skin fibroblasts.

Alterations of ultrastructure and elemental composition in cultured human epidermal keratinocytes after treatment with nitrofurazone and silver sulfadiazine

1987 ◽  
Vol 45 ◽  
pp. 676-677
Author(s):  
A. R. Crooker ◽  
M. C. Myers ◽  
T. L. Beard ◽  
E. S. Graham
Keyword(s):  
Electron Microscopy ◽  
Elemental Composition ◽  
Pyrolytic Carbon ◽  
Human Keratinocytes ◽  
Silver Sulfadiazine ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Culture Systems ◽  
Cytotoxicity Endpoints

Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.

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