scholarly journals ENERGY-LINKED ULTRASTRUCTURAL TRANSFORMATIONS IN ISOLATED LIVER MITOCHONDRIA AND MITOPLASTS

1972 ◽  
Vol 53 (2) ◽  
pp. 450-465 ◽  
Author(s):  
Charles R. Hackenbrock
Keyword(s):  
Osmium Tetroxide ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Rapid Freezing ◽  
Chemical Fixation ◽  
Spherical Form ◽  
Osmolar Concentration ◽  
Special Case ◽  
Isolated Liver ◽  
Ultrastructural Transformation

An investigation was carried out in which microsamples of isolated rat liver mitochondria and freshly prepared mitoplasts in defined energy states were freeze-cleaved. Parallel microsamples were fixed with osmium tetroxide and with glutaraldehyde followed by osmium tetroxide as previously used in this laboratory for the preservation of energy-linked mitochondrial configurations. The details of the orthodox configuration of energized mitochondria and the condensed configuration of de-energized mitochondria, as revealed previously by chemical fixation, are confirmed in this report for nonfixed, freeze-cleaved mitochondria. The precise agreement in preservation of configuration obtained by the physical fixation of rapid freezing and by chemical fixation establishes unequivocally that mitochondria undergo energy-linked ultrastructural transformation between the condensed and the orthodox configurations which are thus natural structural states related to the metabolic activity of the mitochondrion. Configurations observed by freeze-cleaving and by chemical fixation reveal that mitoplasts also undergo a specific and dramatic ultrastructural transformation with the induction of oxidative phosphorylation. The transformation appears to be isovolumetric and therefore is thought to be mediated through energized conformational activity in the surface electron-transport membrane of the mitoplast. Passively swollen, spherical, osmotically active mitoplasts could not be fixed rapidly enough by chemical fixatives as normally used without altering the spherical form. In this special case preservation of configurational form required rapid freezing or chemical fixatives of low osmolar concentration.

scholarly journals ION-INDUCED ULTRASTRUCTURAL TRANSFORMATIONS IN ISOLATED MITOCHONDRIA

1969 ◽  
Vol 42 (1) ◽  
pp. 221-234 ◽  
Author(s):  
Charles R. Hackenbrock ◽  
Arnold I. Caplan
Keyword(s):  
Marked Decrease ◽  
Active Form ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Dense Matrix ◽  
Low Levels ◽  
Osmolar Concentration ◽  
Oriented Membranes ◽  
Ultrastructural Transformation ◽  
Distinct Membrane

The energized uptake of low levels of Ca2+ in the presence and absence of phosphate by isolated rat liver mitochondria, and the perturbation effected by this activity on ultrastructural and metabolic parameters of mitochondria have been investigated. In the presence of phosphate, low levels of Ca2+ are taken up by mitochondria and result in various degrees of ultrastructural expansion of the inner mitochondrial compartment. This indicates that low levels of Ca2+ in the presence of phosphate, are accumulated in an osmotically active form into the water phase of the inner compartment. The first clearly observable quantitative increase in the volume of the inner compartment occurs after the accumulation of 100 nmoles Ca2+/mg protein. An accumulation of 150–200 nmoles Ca2+/mg protein, which is equivalent to the osmolar concentration of endogenous K+, is required to effect a doubling of the volume of the inner compartment. This degree of osmotic perturbation occurs as mitochondria transform from a condensed to an orthodox conformation. The osmotically induced orthodox conformation differs from the mechanochemically induced orthodox conformation previously described, in that its development is concomitant with a marked decrease in acceptor control and oxidative phosphorylation efficiency and it fails to transform to a condensed conformation in response to addition of ADP. In the absence of added phosphate, a maximum of 190 nmoles Ca2+/mg protein was found to be taken up by mitochondria (state 6). Ca2+ is apparently bound under state 6 conditions since the uptake does not effect an ultrastructural expansion of the inner compartment. Phosphate added after state 6 Ca2+ binding, however, results in an immediate ultrastructural expansion of the inner compartment. The addition of phosphate to mitochondria in the absence of exogenous Ca2- fails to effect an osmotic ultrastructural transformation. Under state 6 conditions, the binding of between 40 and 190 nmoles Ca2+/mg protein results in the formation of dense matrix inclusions which appear to be composed of tightly packed, concentrically oriented membranes. Under conditions in which the bound Ca2+ is subsequently released, there is a concomitant loss in the density of these matrix inclusions, leaving behind morphologically distinct membrane whorls in the mitochondrial matrix.

scholarly journals Evidence for β-adrenergic activation of Na+-dependent efflux of Ca2+ from isolated liver mitochondria

1982 ◽  
Vol 204 (1) ◽  
pp. 369-371 ◽  
Author(s):  
Timothy P. Goldstone ◽  
Martin Crompton
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Adrenergic Agonist ◽  
Isolated Rat Liver ◽  
Dependent Activity ◽  
Adrenergic Activation ◽  
Isolated Liver ◽  
Isolated Rat ◽  
Dependent Mechanism

The existence of a Na+-dependent mechanism for Ca2+ efflux from isolated rat liver mitochondria was confirmed. The activity of this system is decreased by 60% in mitochondria isolated from perfused livers. The Na+-dependent activity is fully restored by infusion of either 1μm-adrenaline or 1μm-isoprenaline, but the α-adrenergic agonist phenylephrine is ineffective.

scholarly journals Effects of glucagon and N6O2′-dibutyryladenosine 3′:5′-cyclic monophosphate on calcium transport in isolated rat liver mitochondria

1978 ◽  
Vol 176 (1) ◽  
pp. 295-304 ◽  
Author(s):  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Retention Time ◽  
Calcium Transport ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Dibutyryl Cyclic Amp ◽  
Oxygen Utilization ◽  
Isolated Rat Liver ◽  
Isolated Mitochondria ◽  
Isolated Liver ◽  
Stimulation Of

1. The administration of glucagon to fed rats by intraperitoneal injection, or the perfusion of livers from fed rats with glucagon by the method of Mortimore [Mortimore (1963) Am.J. Physiol. 204, 699–704] was associated with increases of 15- and 5-fold respectively, in the time for which a given load of exogenous Ca2+ is retained by mitochondria subsequently isolated from the liver. This effect of glucagon was (a) also induced by N6O2′-dibutyryl cyclic AMP, (b) completely blocked by cycloheximide, (c) relatively slow in onset (15–60 min) and (d) associated with a stimulation of about 20% in the rates of ADP-stimulated oxygen utilization and Ca2+ transport measured in the presence of succinate. 2. Perfusion of livers with glucagon resulted in the isolation of mitochandria which showed a 50% increase, no significant change and a 40% increase in the concentrations of endogenous Ca, Mg and Pi respectively, when compared with mitochondria isolated from control perfused livers. 3. The administration of insulin or adrenaline to fed rats induced increases of 10- and 8-fold respectively, in the time for which Ca2+ is retained by isolated liver mitochondria. Perfusion of livers with insulin had no effect on mitochondrial Ca2+ retention time. 4. The perfusion of livers from starved rats with glucagon, or the administration of either glucagon or insulin to starved rats, increased by about 2.5- and 15-fold respectively, the time for which isolated mitochondria retain Ca2+. 5. Mechanisms which may be responsible for the observed alterations in Ca2+-retention time are discussed.

scholarly journals PREPARATION OF ISOLATED RAT LIVER MITOCHONDRIA FOR ELECTRON MICROSCOPY

1970 ◽  
Vol 44 (2) ◽  
pp. 278-289 ◽  
Author(s):  
W. H. Butler ◽  
J. D. Judah
Keyword(s):  
Electron Microscopy ◽  
Comparative Study ◽  
Potassium Permanganate ◽  
Rat Liver ◽  
Osmium Tetroxide ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Rat Liver ◽  
Isolated Rat

A comparative study of the fixation of isolated rat liver mitochondria was undertaken. If the criterion is adopted that after processing, the mitochondria should resemble as closely as possible rat liver mitochondria in situ, the procedure found to produce such preservation was that of fixation in suspension in veronal-buffered 2% potassium permanganate. Fixation in osmium tetroxide produced variable results, while mitochondria fixed in glutaraldehyde were contracted. We suggest that in cases where fixation procedures modify the morphological appearance of mitochondria, the significance of such changes must be treated with caution.

Mitochondrial swelling during cold exposure of the rat and hamster

1962 ◽  
Vol 202 (6) ◽  
pp. 1037-1040 ◽  
Author(s):  
Joseph B. Boatman ◽  
Marie M. Boucek ◽  
Marvin J. Rabinovitz
Keyword(s):  
Cold Exposure ◽  
Room Temperature ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Thyroid Uptake ◽  
Severe Reduction ◽  
Absorbance Changes ◽  
Recording Spectrophotometer ◽  
Rat Thyroid ◽  
Isolated Liver

Swelling of isolated liver mitochondria of hamsters and rats was measured in a Beckman DK ratio-recording spectrophotometer after 0, 1, 2, 5, 10, 15, and 20 days of cold exposure at 5 C. Swelling was determined by absorbance changes over 10-min intervals at 520 mµ at 37 C in 0.3 m sucrose with and without 1.5 x 10–7 m l-thyroxin added. Thyroid uptake and plasma content of 0.5 µc I131 was measured in additional animals at 2, 6, 18, 48, and 144 hr of cold exposure. Swelling rates of rat liver mitochondria increased with time, while hamster liver mitochondria showed no change during 20 days of exposure to cold. Added thyroxin increased swelling rates equally of both rat and hamster liver mitochondria, reducing the early refractory period of the swelling curve. Rat thyroid uptakes of I131 were higher at room temperature than the hamster. Cold exposure resulted in a severe reduction of hamster thyroid uptake of I131 while reduction of rat thyroid uptake was minor. Differences in mitochondrial response to swelling media between the cold-exposed hamster and rat paralleled the different functional levels of the thyroid during cold exposure.

Effects of 5-5'-Diphenyl-2-thiohydantoin (DPTH) and Thyroxine on the Ultrastructure and Chemical Composition of Rat Liver

1971 ◽  
Vol 29 ◽  
pp. 570-571
Author(s):  
E. A. Elfont ◽  
R. B. Tobin ◽  
D. G. Colton ◽  
M. A. Mehlman
Keyword(s):  
Chemical Composition ◽  
Rat Liver ◽  
Future Study ◽  
Mode Of Action ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Effective Inhibitor ◽  
Biochemical Effects ◽  
Glycerophosphate Dehydrogenase ◽  
Stimulation Of

Summary5,-5'-diphenyl-2-thiohydantoin (DPTH) is an effective inhibitor of thyroxine (T4) stimulation of α-glycerophosphate dehydrogenase in rat liver mitochondria. Because this finding indicated a possible tool for future study of the mode of action of thyroxine, the ultrastructural and biochemical effects of DPTH and/or thyroxine on rat liver mere investigated.Rats were fed either standard or DPTH (0.06%) diet for 30 days before T4 (250 ug/kg/day) was injected. Injection of T4 occurred daily for 10 days prior to sacrifice. After removal of the liver and kidneys, part of the tissue was frozen at -50°C for later biocheailcal analyses, while the rest was prefixed in buffered 3.5X glutaraldehyde (390 mOs) and post-fixed in buffered 1Z OsO4 (376 mOs). Tissues were embedded in Araldlte 502 and the sections examined in a Zeiss EM 9S.Hepatocytes from hyperthyroid rats (Fig. 2) demonstrated enlarged and more numerous mitochondria than those of controls (Fig. 1). Glycogen was almost totally absent from the cytoplasm of the T4-treated rats.

Observations of thick and thin sections in a field-emission SEM

1994 ◽  
Vol 52 ◽  
pp. 1018-1019
Author(s):  
W. P. Wergin ◽  
S. Roy ◽  
E. F. Erbe ◽  
C. A. Murphy ◽  
C. D. Pooley
Keyword(s):  
Electron Microscope ◽  
Field Emission ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope ◽  
Scanning Electron

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.

Immunocytochemistry after fast freeze fixation-freeze substitution: Advantages and limits

1990 ◽  
Vol 48 (3) ◽  
pp. 42-43
Author(s):  
Marie-Thérèse Nicolas
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Acrylic Resin ◽  
Expected Improvement ◽  
List Type ◽  
Chemical Fixation ◽  
Freeze Fixation ◽  
Liquid Chemical ◽  
Luciferase Enzyme ◽  
Lower Background

An alternative to aqueous chemical fixation consists in immobilizing physically the specimen by freezing it as fast as possible without using any cryoprotectant. This Fast Freeze Fixation (FFF) followed by Freeze Substitution (FS) avoids osmotic artefacts due to the slow penetration of liquid chemical fixative. Associated with Immuno-Gold labeling (IGS), FFF-FS allows a more precise localization of antigens.Using the bioluminescent bacteria Vibrio harveyi, a comparison of IGS with an antibody directed against its luciferase (enzyme of the luminescent reaction) has been done after liquid chemical fixation versus FFFFS. This later technique, beside an expected improvement of the ultrastructure always shows a better preservation of antigenicity and a lower background. In the case of FFF-FS technique (Figure 3):–labeling in acrylic resin (LRWhite) is 2 to 4 fold more intense than in epoxy resin (Epon),–but the ultrastructure is always better in Epon.–but the ultrastructure is always better in Epon.–The addition of fixatives in the substitution medium, results in a decrease of labeling which is more important in the case of a mixture of fixatives than with osmium tetroxide alone; with one exception: the substitution with glutaraldehyde which produces a dramatic increase in the density of the labeling but also, at the same time, a swelling of the cells of about 30%.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

Two isoprenylated flavonoids from Dorstenia psilurus activate AMPK, stimulate glucose uptake, inhibit glucose production and lower glycemia

2019 ◽  
Vol 476 (24) ◽  
pp. 3687-3704 ◽  
Author(s):  
Aphrodite T. Choumessi ◽  
Manuel Johanns ◽  
Claire Beaufay ◽  
Marie-France Herent ◽  
Vincent Stroobant ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Human Cancer ◽  
Glucose Production ◽  
Liver Mitochondria ◽  
New Drugs ◽  
Structural Isomer ◽  
Rat Liver Mitochondria ◽  
Ionization Mass ◽  
Ampk Activation ◽  
Starting Point

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze–thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKβ1−/− mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

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