scholarly journals Pressure-induced depolymerization of spindle microtubules. III. Differential stability in HeLa cells.

1976 ◽  
Vol 69 (2) ◽  
pp. 443-454 ◽  
Author(s):  
E D Salmon ◽  
D Goode ◽  
T K Maugel ◽  
D B Bonar
Keyword(s):  
Hydrostatic Pressure ◽  
Light Microscopy ◽  
Hela Cells ◽  
Atmospheric Pressure ◽  
Differential Effect ◽  
Polarization Microscopy ◽  
Spindle Fiber ◽  
Ultrastructural Studies ◽  
Differential Stability ◽  
Spindle Microtubules

Evidence from light microscopy (principally polarization microscopy) has demonstrated that hydrostatic pressure can reversibly inhibit mitosis by rapidly depolymerizing the spindle fiber microtubules. We have confirmed this finding in ultrastructural studies of mitotic HeLa cells incubated at 37 degrees C and pressurized at 680 atm (10,000 psi). Althouth there are many spindle microtubules in the cells at atmospheric pressure, electron micographs of cells pressurized for 10 min (and fixed while under pressure in a Landau-Thibodeau chamber) show few microtubules. Pressure has a differential effect on the various types of spindle microtubules. Astral and interpolar MTs appear to be completely depolymerized in pressurized cells, but occasional groups of kinetochore fiber microtubules are seen. Surprisingly, the length and density of microtubules of the stem bodies and midbody of telophase cells appear unchanged by pressurization. In cells fixed 10 min after pressure was released, microtubules were again abundant, the density often appearing to be higher than in control cells. Reorganization seems incomplete, however, since many of the microtubules are randomly oriented. Unexpectedly, kinetochores appeared diffuse and were difficult to identify in sections of pressurized cells. Even after 10 min of recovery at atmospheric pressure, their structure was less distinct than in unpressurized cells.

scholarly journals NITROUS OXIDE: EFFECTS ON THE MITOTIC APPARATUS AND CHROMOSOME MOVEMENT IN HELA CELLS

1973 ◽  
Vol 58 (1) ◽  
pp. 96-106 ◽  
Author(s):  
B. R. Brinkley ◽  
Potu N. Rao
Keyword(s):  
Nitrous Oxide ◽  
Hela Cells ◽  
Metaphase Plate ◽  
Microtubule Assembly ◽  
Chromosome Movement ◽  
Spindle Microtubule ◽  
Mitotic Apparatus ◽  
Ultrastructural Studies ◽  
Multipolar Spindles ◽  
Spindle Microtubules

When HeLa cells were grown in the presence of nitrous oxide (N2O) under pressure (80 lb/in2) mitosis was inhibited and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy. When the cells were returned to a 37°C incubator, mitosis was resumed and the cells entered G1 synchronously. Ultrastructural studies of N2O-blocked cells revealed a bipolar spindle with centriole pairs at each pole. Both chromosomal and interpolar (pole-to-pole) microtubules were also present. Thus, N2O, unlike most c-mitotic agents, appeared to have little or no effect upon spindle microtubule assembly. However, the failure of chromo somes to become properly aligned onto the metaphase plate indicated an impairment in normal prometaphase movement. The alignment of spindle microtubules was frequently atypical with some chromosomal microtubules extending from kinetochores to the poles, while others extended out at acute angles from the spindle axis. These ultrastructural studies indicated that N2O blocked cells at a stage in mitosis more advanced than that produced by Colcemid or other c-mitotic agents. Like Colcemid, however, prolonged arrest in mitosis with N2O led to an increased incidence of multipolar spindles.

Electron Microscopy of Mycoplasma Pneumoniae

1971 ◽  
Vol 29 ◽  
pp. 258-259 ◽  
Author(s):  
E. S. Boatman ◽  
G. E. Kenny
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Growth Phase ◽  
Hela Cells ◽  
Sulfonic Acid ◽  
Gamma Globulin ◽  
Horse Serum ◽  
Morphological Aspects ◽  
The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

1995 ◽  
Vol 53 ◽  
pp. 902-903
Author(s):  
J. R. Kuhn ◽  
M. Poenie
Keyword(s):  
Mitotic Spindle ◽  
Actin Filaments ◽  
Cell Shape ◽  
Dynamic Structure ◽  
Living Cells ◽  
Cytoskeletal Proteins ◽  
Polarization Microscopy ◽  
Video Enhancement ◽  
Ultrastructural Studies ◽  
Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

scholarly journals Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduvidae)

1989 ◽  
Vol 84 (2) ◽  
pp. 171-188 ◽  
Author(s):  
Margherita A. Barracco ◽  
Clarice T. Loch
Keyword(s):  
Endoplasmic Reticulum ◽  
Light Microscopy ◽  
Rough Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Panstrongylus Megistus ◽  
Ultrastructural Studies ◽  
Hemocyte Count

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrogylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, espcieally rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER).They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrondensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrondense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasme is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/cubic millimeter of hemolymph in the 4th intar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.

Regulation of Mitosis

1969 ◽  
Vol 5 (3) ◽  
pp. 745-755
Author(s):  
W. T. JACKSON
Keyword(s):  
Mitotic Spindle ◽  
Time Lapse ◽  
Polarization Microscopy ◽  
Animal Cells ◽  
Higher Plant ◽  
Competitive Antagonism ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Giant Nuclei

Earlier studies on the effects of the herbicide isopropyl N-phenylcarbamate (IPC) on mitosis revealed blocked metaphases, multinucleate cells, giant nuclei and an increase in number of partly contracted chromosomes. It was assumed that IPC, like colchicine, was causing these effects by disruption of the spindle apparatus by destroying the spindle microtubules. The animal hormone melatonin causes an increase in birefringence of the mitotic spindle in animal cells, presumably by increasing the number of microtubules. We have studied the effects of IPC, melatonin, and combinations of the two on mitosis in dividing endosperm cells of the African blood lily (Haemanthus katherinae Baker) in vivo by phase-contrast and polarization microscopy. Both qualitative and quantitative data are presented. Interpretation of these results has been aided materially by a time-lapse cinemicrographic analysis of dividing cells subjected to 1 and 10 p.p.m. IPC (unpublished) and by an accompanying fine-structural analysis of untreated and IPC-treated cells. Mitosis was disrupted by 0.01-10 p.p.m. IPC, the severity of the effect depending on both concentration and stage of mitosis of the cell at the time of treatment. Concentrations of IPC that caused cessation of chromosome movement also caused loss of birefringence of the mitotic spindle. Melatonin increased birefringence of the mitotic spindle in these plant cells and partly nullified the adverse effects of IPC. The results of this study demonstrate that the herbicide IPC, under our conditions, causes disruption of mitosis and loss of birefringence of the spindle. And it has been established that an animal hormone is capable of increasing the birefringence, and presumably the number of microtubules, of the mitotic spindle in dividing endosperm cells of a higher plant. Although melatonin is capable of partly nullifying the effects of IPC, a competitive antagonism is not postulated.

Improvement in the efficiency of oxidative phosphorylation in the freshwater eel acclimated to 10.1 MPa hydrostatic pressure

2000 ◽  
Vol 203 (19) ◽  
pp. 3019-3023
Author(s):  
M. Theron ◽  
F. Guerrero ◽  
P. Sebert
Keyword(s):  
Hydrostatic Pressure ◽  
Oxidative Phosphorylation ◽  
Atmospheric Pressure ◽  
High Hydrostatic Pressure ◽  
Anguilla Anguilla ◽  
Respiratory Chain Complexes ◽  
Complex Iv ◽  
Control Value ◽  
Chain Complexes ◽  
Freshwater Eel

Previous studies have suggested that the efficiency of oxidative phosphorylation in the freshwater eel (Anguilla anguilla) is increased after acclimation to high hydrostatic pressure. Analysis at atmospheric pressure of the respiratory chain complexes showed that, after 21 days at 10.1 MPa, the activity of complex II was decreased to approximately 50 % (P<0.01) of the control value and that cytochrome c oxidase (complex IV) activity was significantly increased to 149 % of the control value (P<0.05). ADP/O ratios calculated from mitochondrial respiration measurements were significantly increased after acclimation to high hydrostatic pressure (2.87 versus 2.52, P<0.001) when measured in the presence of pyruvate plus malate at atmospheric pressure. These results clearly show an increased oxidative phosphorylation efficiency in response to high-pressure acclimation.

Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor

1999 ◽  
Vol 112 (22) ◽  
pp. 3899-3909 ◽  
Author(s):  
G. Skretting ◽  
M.L. Torgersen ◽  
B. van Deurs ◽  
K. Sandvig
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
Hela Cells ◽  
Diphtheria Toxin ◽  
Surface Receptors ◽  
Caveolin 1 ◽  
Ultrastructural Studies ◽  
Diphtheria Toxin Receptor ◽  
Early Endosomes ◽  
Toxin Receptor

We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.

Cryopreservation of HeLa Cells at a High Hydrostatic Pressure of 1.0–1.5 kbar

BIOPHYSICS ◽  
2021 ◽  
Vol 66 (1) ◽  
pp. 98-106
Author(s):  
S. V. Ugraitskaya ◽  
N. V. Shishova ◽  
E. R. Valeeva ◽  
S. A. Kaurova ◽  
N. E. Shvirst ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Hela Cells ◽  
High Hydrostatic Pressure

Tensile fracture and fractographic analysis of 1045 spheroidized steel under hydrostatic pressure

1990 ◽  
Vol 5 (1) ◽  
pp. 83-91 ◽  
Author(s):  
A. S. Kao ◽  
H. A. Kuhn ◽  
O. Richmond ◽  
W. A. Spitzig
Keyword(s):  
High Pressure ◽  
Hydrostatic Pressure ◽  
Fracture Surface ◽  
Atmospheric Pressure ◽  
Fracture Mechanism ◽  
Shear Crack ◽  
Applied Pressure ◽  
Void Formation ◽  
Tensile Fracture ◽  
Central Crack

Void formation in tensile test under hydrostatic pressure is characterized through quantitative metallography, and the fracture mechanism under pressure is analyzed by fractography. Transition of the fracture surface from the cup-and-cone under atmospheric pressure to a slant structure under high pressure is explained on the basis of the void development leading to fracture and the concomitant change in fracture mechanism. The concept of “shear blocks” is introduced to illustrate the features observed on the fracture surface of specimens tested under high pressure. It is postulated that shear blocks evolve to connect the central crack regions with the shear crack initiated on neck surface due to the severe necking deformation under applied pressure.

scholarly journals DC Electrical Ageing of XLPE under Hydrostatic Pressure

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Fadila Benlizidia Lalam ◽  
Faouzi Djemmal
Keyword(s):  
Hydrostatic Pressure ◽  
Direct Current ◽  
Atmospheric Pressure ◽  
Pressure Effect ◽  
Power Model ◽  
Lifetime Data ◽  
Confidence Bounds ◽  
Polyethylene Films ◽  
Thermally Activated ◽  
Thermally Activated Process

The experimental electrical ageing, of cross-linked polyethylene films 100 μm thick, was investigated under high hydrostatic pressure of 300 bar and at atmospheric pressure. The tests are conducted on direct current (dc) for up to 1000 h ageing and at temperature of 70°C. The use of the Weibull statistic, with the estimation of confidence bounds at 90%, has shown that the hydrostatic pressure has a real effect on the lifetime. These lifetime data are qualitatively analyzed with the inverse power model. It was found that thermally activated process is able to describe the pressure effect on the electrical ageing of XLPE.

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