scholarly journals Preparation of Rat Gingival Mitochondria with an Improved Isolation Method

2010 ◽  
Vol 2010 ◽  
pp. 1-6
Author(s):  
Noriaki Kaneko ◽  
Tetsuya Rikimaru ◽  
Tetsuyuki Fujimura ◽  
Shigeyasu Mori ◽  
Saburo Hidaka ◽  
...  
Keyword(s):  
Optimal Condition ◽  
Respiratory Control ◽  
Adenosine Diphosphate ◽  
Respiratory Control Ratio ◽  
Differential Centrifugation ◽  
Isolation Method ◽  
High Quality

In order to establish a method of obtaining rat gingival mitochondria (Mt), Mt fractions were prepared in various combinations of homogenizing time with collagenase concentration. Rat gingival tissues were excised, minced, treated with collagenase, homogenized, and subjected to differential centrifugation rates. Both the respiratory control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio of the Mt fraction prepared in a combination of 40, 50, or 60 sec homogenization with collagenase in a concentration range of 0.115%–0.130% (w/v) were measured. The values for the RCR and ADP/O ratio of the Mt fraction obtained in an optimal condition was and , respectively. These results suggest that Mt of fairly high quality can be obtained through this refined combination of the homogenizing time and collagenase concentration.

OXIDATIVE PHOSPHORYLATION BY MUSCLE MITOCHONDRIA OF DYSTROPHIC MICE

1967 ◽  
Vol 45 (8) ◽  
pp. 1271-1278 ◽  
Author(s):  
Klaus Wrogemann ◽  
M. C. Blanchaer
Keyword(s):  
Skeletal Muscle ◽  
Oxidative Phosphorylation ◽  
Respiratory Control ◽  
Laboratory Strain ◽  
Adenosine Diphosphate ◽  
Isolation Procedure ◽  
Respiratory Control Ratio ◽  
Differential Centrifugation ◽  
Dystrophic Mice ◽  
Phosphorylation Rate

Oxidative phosphorylation was studied in mitochondria isolated from the skeletal muscle of control and dystrophic mice of the Jackson Laboratory strain 129/Re, aged 32–104 days. The isolation procedure included a preliminary incubation of the muscle minced in a medium containing a proteinase (Nagarse) followed by gentle homogenization and differential centrifugation. Polarographic estimations in the presence and absence of adenosine diphosphate (ADP) indicated that the rate of oxygen uptake, ADP/0 ratio, respiratory control ratio, and phosphorylation rate were not significantly different in the mitochondria isolated from control and dystrophic mice. Bovine serum albumin increased the ADP/0 and respiratory control ratios, but the values for the control and dystrophic preparations again did not differ significantly in the presence of albumin.

scholarly journals The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration

1982 ◽  
Vol 202 (3) ◽  
pp. 731-737 ◽  
Author(s):  
N G Dippenaar ◽  
M D Brand
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Mitochondrial Protein ◽  
Respiratory Control ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Respiratory Control Ratio ◽  
Differential Centrifugation ◽  
Mesenteric Lymph ◽  
Efflux Pathway

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2-3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.

scholarly journals Development of new total RNA isolation method for tissues with rich phenolic compounds

2020 ◽  
Vol 45 (4) ◽  
pp. 343-350
Author(s):  
Zafer Seçgin ◽  
Gökhan Gökdemir ◽  
Elif Seda Atabay ◽  
Aslıhan Kurt Kızıldoğan ◽  
Musa Kavas
Keyword(s):  
Phenolic Compounds ◽  
Rna Sequencing ◽  
Rna Isolation ◽  
Wall Structure ◽  
Isolation Method ◽  
High Quality ◽  
Ctab Method ◽  
Total Rna Isolation

AbstractBackgroundRNAs to be used in transcriptome analysis must be of high quality and pure in order to ensure maximum representation of the expressed genes. RNA isolation is difficult in hazelnut tissues containing large amounts of secondary metabolite, phenolic compounds and the cell wall structure. Commonly used protocols for RNA isolation are those that require a lot of labor and time and also do not allow sufficient RNA isolation when applied to tissues rich in phenolic compounds. This study was aimed to develop an efficient method for isolation of total RNAs from bud of hazelnut to be used in RNA sequencing.Materials and methodsAn optimized new method was successfully applied on three different hazelnuts genotypes (Çakıldak, Palaz, Tombul) and about 25 times higher amount of total RNAs per mg fresh tissues were obtained compared to classical CTAB method. Different methods have been tried for the isolation of RNA from hazelnut tissues and the determination of the quality of the obtained RNAs.ResultsThe quality and quantity of isolalated total RNAs were determined by spectrophotometer, electrophoresis and PCR. This success has been caught without any compromise of purity since A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were >2.0 in all purified RNAs.ConclusionThe total RNAs isolated with new protocol was found to be suitable for RNA sequencing and other molecular applications.

scholarly journals Effect of hyperbaric oxygen therapy on liver function during intermittent ischemia

2013 ◽  
Vol 28 (suppl 1) ◽  
pp. 61-65 ◽  
Author(s):  
Leticia Botigeli Baldim ◽  
Ricardo Nejo Jr ◽  
Maria Eliza Jordani Souza ◽  
Maria Cecília Jordani Gomes ◽  
Maria Aparecida Neves Cardoso Picinato ◽  
...  
Keyword(s):  
Liver Function ◽  
Hyperbaric Oxygen ◽  
Mitochondrial Function ◽  
Hyperbaric Oxygen Therapy ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Oxygen Therapy ◽  
Respiratory Control ◽  
Respiratory Control Ratio ◽  
Hepatic Pedicle

PURPOSE: To analyze the effects of hyperbaric oxygen therapy on liver function in rats previously subjected to ischemia and reperfusion. METHODS: A randomly distribution of 23 Wistar rats was conducted into three groups: SHAM, animals subjected to surgical stress without restricting blood flow by clamping the hepatic pedicle, IR, rats underwent hepatic vascular occlusion intermittently for two complete cycles of 15 minutes of ischemia followed by 5 min of reperfusion, IR / HBO, rats underwent hepatic pedicle clamping and thereafter exposed to hyperbaric oxygen pressure of 2 absolute atmospheres for 60 minutes. We evaluated liver function through mitochondrial function, determined by the stages 3 and 4 of respiration, respiratory control ratio (RCR) and mitochondrial permeability transition (Swelling). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also quantified . We analyzed the results using the Mann-Whitney test and were considered significant all results with p <0.05. RESULTS: There were significant differences between the results of stage 3 in SHAM vs IR group ; of the stage 4 in the groups IR vs SHAM and SHAM vs IR /HBO; of the Respiratory Control Ratio (RCR) in the group IR vs IR / HBO ; of alanine aminotransferase in the groups IR vs SHAM , SHAM vs IR/HBO and IR vs IR / HBO; aspartate aminotransferase in the groups SHAM vs IR and SHAM vs IR / HBO. CONCLUSION: The whole analysis of the mitochondiral function indicators permits us to conclude that the hyperbaric oxygen therapy acted as a protective agent of the mitochondrial function, minimizing the ischemia-reperfusion injury of the hepatic parenchyma.

scholarly journals Low Vacuum Filtration Method as an Alternative Extracellular Vesicle Concentration Method: Comparison with Ultracentrifugation and Differential Centrifugation

2020 ◽  
Author(s):  
Anna Drożdż ◽  
Agnieszka Kamińska ◽  
Magdalena Surman ◽  
Agnieszka Gonet-Surówka ◽  
Robert Jach ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Culture Media ◽  
Membrane Surface ◽  
Method Comparison ◽  
Environmental Scanning Electron Microscopy ◽  
Drug Carriers ◽  
Differential Centrifugation ◽  
Isolation Method ◽  
Filtration Method ◽  
Vacuum Filtration

Recent years brought great focus in the field of development of extracellular vesicles (EVs) based drug-delivery systems. Considering possible applications of EVs as a drug carriers the isolation process is a crucial step. To solve problems related with EV isolation, we created and validated a new EVs isolation method &ndash; Low Vacuum Filtration (LVF) and compared it with two commonly applied procedures - differential centrifugation (DC) and ultracentrifugation (UC). EVs isolated from endothelial cells culture media have been characterized by a) transmission electron microscopy (TEM) b) nanoparticle tracking analysis (NTA), c) western blot and d) Fourier-Transform Infrared Spectroscopy (FTIR). Additionally, the membrane surface have been imaged with Environmental Scanning Electron Microscopy (ESEM). We showed that LVF is reproducible and efficient method for EVs isolation form conditioned media. Additionally, we observed correlation between ATR-FTIR spectra quality and the EVs and proteins concentration. ESEM imaging confirmed that actual pore diameter are close to the values calculated theoretically. LVF method is an easy, fast and inexpensive EVs isolation method which allows for isolation of both ectosomes and exosomes from high volume sources with good repeatability. We think that it could be an efficient alternative for commonly applied methods.

scholarly journals Effect of Captopril on Serum Lipid Levels and Cardiac Mitochondrial Oxygen Consumption in Experimentally-Induced Hypercholesterolemia in Rabbits

2011 ◽  
pp. S177-S184 ◽  
Author(s):  
Z. KOJIC ◽  
K. GOPCEVIC ◽  
D. MARINKOVIC ◽  
G. TASIC
Keyword(s):  
Oxygen Consumption ◽  
Serum Lipid ◽  
Respiratory Control ◽  
Adenosine Diphosphate ◽  
Lipid Levels ◽  
Mitochondrial Oxygen Consumption ◽  
Respiration Rates ◽  
Serum Lipid Levels ◽  
Hypercholesterolemic Diet ◽  
Oxygen Ratio

Angiotensin converting enzyme inhibitors are widely used in therapy of cardiovascular diseases. However, the consensus on effects of these inhibitors in control of myocardial oxygen consumption during the process of experimental hypercholesterolemia and under the condition of endothelial dysfunction has not been reached. Here we examined effects of captopril, an angiotensin converting enzyme inhibitor, on serum lipid levels and oxygen consumption rate in mitochondria isolated from heart of rabbits treated by hypercholesterolemic diet. During the twelve-week period, the Chinchilla male rabbits were daily treated by saline (controls); 1 % cholesterol diet; 5 mg/kg/day captopril or 1 % cholesterol + 5 mg/kg/day captopril. Total- and high-density lipoprotein cholesterol and triglyceride in serum were measured spectrophotometricly. The left ventricle mitochondrial fraction was isolated and myocardial oxygen consumption was measured by Biological Oxygen Monitor. Mitochondria isolated from hearts of rabbits exposed to hypercholesterolemic diet showed significantly reduced respiration rates (state 3 and state 4) with altering adenosine diphosphate/oxygen ratio, whereas the respiratory control ratio was not affected when compared to controls. Mitochondria from cholesterol/captopril–treated animals showed significantly reduced respiration rates without altering adenosine diphosphate/oxygen ratio index or respiratory control ratio. Although captopril did not exert the favorable effect on serum lipid levels in cholesterol-treated animals, it restored the mitochondrial oxygen consumption. Further studies should be performed to define the underlying physiological and/or pathophysiological mechanisms and clinical implications.

Low Temperature Deposited High Quality ITO Films Prepared by E-Beam Evaporation

1990 ◽  
Vol 187 ◽  
Author(s):  
C. S. Chang ◽  
J. C. Wang ◽  
L. C. Kuo
Keyword(s):  
Electron Beam ◽  
Low Temperature ◽  
Substrate Temperature ◽  
Optimal Condition ◽  
Deposition Rate ◽  
Oxygen Pressure ◽  
Electron Beam Evaporation ◽  
Film Properties ◽  
High Quality ◽  
Ito Films

AbstractAn electron beam evaporation method has been used to prepare tin doped indium oxide (ITO) films with 95 wt.% In2O3 and 5 wt.% SnO2 in an oxygen atmosphere. It was found that the deposition rate and oxygen pressure strongly influence the film properties when the substrate temperature was lower than 200°C. In an optimal condition, highly transparent (transmittance ˜ 90% at wavelength 570 nm) and conductive (resistivity – 3×10−4Ω-cm) films of thickness around 2000 Å at substrate temperature as low as 180°C can be obtained.

Characterization of mitochondria isolated from the freezing-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis): substrate preferences, salt effects, and pH effects on warm- and cold-acclimated animals

1985 ◽  
Vol 63 (2) ◽  
pp. 373-379 ◽  
Author(s):  
James S. Ballantyne ◽  
Kenneth B. Storey
Keyword(s):  
Salt Concentration ◽  
Respiratory Control ◽  
Ph Effects ◽  
Respiratory Control Ratio ◽  
Eurosta Solidaginis ◽  
Optimal Range ◽  
Salt Effects ◽  
Substrate Preferences ◽  
Optimal Ph

The mitochondria of the freezing-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) have been isolated and characterized. Proline is the preferred substrate of mitochondria from both warm- and cold-acclimated animals based on state 3 rates. Lipid is used as a substrate by warm- and cold-acclimated mitochondria assayed at 20 °C, but not by the mitochondria from cold-acclimated animals assayed at 1 °C. Cold-acclimated mitochondria assayed at 1 °C have a higher and broader optimal range of salt concentration for the oxidation of proline based on the respiratory control ratio (RCR) than those from warm-acclimated animals oxidizing the same substrate at 20 °C. The optimal pH for warm-acclimated mitochondria oxidizing proline at 20 °C is low (6.2) based on the RCR, but rises to pH 7.0 in cold-acclimated animals at 1 °C. It is suggested that the broad optimal salt concentration in the cold-acclimated animals and the very low optimal pH in warm-acclimated animals are adaptations for survival in this freezing-tolerant larva.

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