scholarly journals Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA.

1975 ◽  
Vol 65 (2) ◽  
pp. 309-323 ◽  
Author(s):  
R S Tuan ◽  
K P Chang
Keyword(s):  
Plasma Membranes ◽  
Structural Integrity ◽  
Light And Electron Microscopy ◽  
Heat Denaturation ◽  
Marker Enzyme ◽  
Genome Complexity ◽  
Numerical Abundance ◽  
Cellular Components ◽  
Isopycnic Centrifugation

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote-containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.

scholarly journals Characterization of a rapid cellular-fractionation technique for hepatocytes. Application in the measurement of mitochondrial membrane potential in situ

1984 ◽  
Vol 219 (2) ◽  
pp. 375-382 ◽  
Author(s):  
S B Shears ◽  
C J Kirk
Keyword(s):  
Incubation Medium ◽  
Plasma Membranes ◽  
Electrical Potential ◽  
Cell Fractionation ◽  
Marker Enzyme ◽  
Cell Nuclei ◽  
Electrical Potentials

A rapid cellular-fractionation technique [Hoek, Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.

scholarly journals The characterization of rat kidney plasma membranes prepared by zonal centrifugation

1972 ◽  
Vol 129 (4) ◽  
pp. 919-928 ◽  
Author(s):  
R. G. Price ◽  
D. G. Taylor ◽  
D. Robinson
Keyword(s):  
Alkaline Phosphatase ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Marker Enzymes ◽  
Subcellular Organelles ◽  
High Degree ◽  
Isopycnic Centrifugation ◽  
Zonal Rotor

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO3 by isopycnic centrifugation in a linear sucrose gradient in an ‘A’-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5′-nucleotidase, l-leucine β-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine β-naphthylamidase, l-leucine p-nitroanilidase and 5′-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.

scholarly journals Isolation and characterization of subcellular membranes of Entamoeba invadens.

1976 ◽  
Vol 71 (2) ◽  
pp. 357-369 ◽  
Author(s):  
H H van Vliet ◽  
F Spies ◽  
W A Linnemans ◽  
A Klepke ◽  
J A Op den Kamp ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Plasma Membranes ◽  
Simple Technique ◽  
Sucrose Solution ◽  
Phospholipid Composition ◽  
High Ratio ◽  
Isolation And Characterization ◽  
Entamoeba Invadens ◽  
Isopycnic Centrifugation

A method is described for the isolation of subcellular membranes of Entamoeba invadens. Plasma membranes were obtained by rate centrifugation followed by isopycnic centrifugation on a sucrose gradient. Intact phagolysosomes floated in a 10% sucrose solution providing a simple technique for isolation. Phagolysosomal membranes were collected by isopycnic centrifugation, after lysis of the phagolysosomes. Microsomes were obtained by differential centrifugation. Membrane fractions were examined by electron microscopy, and the contamination of each fraction was determined with marker enzymes. Mg2+-ATPase is associated with the plasma membrane. Acid phosphatase (beta-glycerophosphate) was associated mainly with phagolysosmal membranes. Plasma membranes also contained acid phosphatase activity which hydrolyzes p-nitrophenylphosphate but not beta-glycerophosphate. The localization of the two phosphatases was confirmed cytochemically. Isolated plasma membranes were contaminated with phagolysosomal membranes (15%) and with microsomes (25%). No more than 5% of the phagolysosomal membrane fraction consisted of plasma membranes. Contamination of the microsomes by plasma and phagolysosomal membranes was 10% and 7%, respectively. Plasma membranes and phagolysosomal membranes had a high ratio of cholesterol to phospholipid (0.93 and 1.05 mumol/mumol, respectively). Microsomes were relatively poor in cholesterol (0.39 mumol/mumol). Microsomes, plasma, and phagolysosomal membranes contained increasing amounts of spingolipids (12%, 17%, and 28%). Phagolysosomal membranes had a high percentage of phosphatidylserine but little phosphatidylcholine. Microsomes were rich in phosphatidylcholine (45%). Differences in phospholipid composition between plasma and phagolysosomal membranes are discussed in view of the phagocytic process.

scholarly journals Water- and solute-accessible spaces of purified peroxisomes. Evidence that peroxisomes are permeable to NAD+

1983 ◽  
Vol 210 (3) ◽  
pp. 685-693 ◽  
Author(s):  
P Van Veldhoven ◽  
L J Debeer ◽  
G P Mannaerts
Keyword(s):  
Structural Integrity ◽  
Organic Layer ◽  
Peroxisome Proliferator ◽  
Marker Enzyme ◽  
Beta Oxidation ◽  
Percoll Gradients ◽  
Liver Homogenates ◽  
High Degree ◽  
Water Space ◽  
Isopycnic Centrifugation

Peroxisomes were purified from liver homogenates from rats, treated with the peroxisome proliferator clofibrate, by a combination of differential centrifugation and isopycnic centrifugation in iso-osmotic self-generating Percoll gradients. Structural integrity of the peroxisomes appeared to be preserved as evidenced by a high degree of catalase latency, the absence of catalase release during purification and the exclusion of inulin (mol.wt. +/- 5000). Spaces for water and solutes were measured after incubation of the peroxisomes in iso-osmotic sucrose with radioactive water or solutes and separation of the organelles from their media by centrifugation through an organic layer. Extraperoxisomal water was corrected for by the use of radioactive dextran or inulin. The sucrose, glucose, urea, methanol and acetate-accessible spaces were identical, suggesting that these spaces represent the volume in which molecules that can cross the membrane distribute. This volume equalled 50-65% of the water space. Urate and NAD+, a cofactor of peroxisomal beta-oxidation of fatty acids, also distributed in this volume, but were also partly bound. Urate and NAD+ binding was not abolished by sonication, which released the bulk of matrix catalase activity, but NAD+ binding was seriously diminished. The peroxisomal water and sucrose spaces were estimated to be 107 microliters and 55 microliters per g of liver tissue from a clofibrate-treated rat. From quantitative morphometric data [Anthony, Schmucker, Mooney & Jones (1978) J. Lipid Res. 19, 154-165] and our marker enzyme analyses, as well as from our experimentally determined water spaces of mitochondrial and microsomal fractions, it could be calculated that the volume contamination by lysosomes, mitochondria and microsomes did not exceed 1, 8 and 6% respectively. Our data indicate that apparently intact peroxisomes are permeable to a number of small molecules, including NAD+. Whether the NAD+-binding sites in sonicated peroxisomes mirror the likely existence of a membrane carrier requires further investigation.

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).

Neuropharmacological Characterization of Dioclea altissima Seed Lectin (DAL) in Mice: Evidence of Anxiolytic-like Effect Mediated by Serotonergic, GABAergic Receptors and NO Pathway

2020 ◽  
Vol 26 (31) ◽  
pp. 3895-3904
Author(s):  
João R.C. Araújo ◽  
Adriana R. Campos ◽  
Marina de Barros M.V. Damasceno ◽  
Sacha A.A.R. Santos ◽  
Maria K.A. Ferreira ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Elevated Plus Maze ◽  
Biological Activities ◽  
Plant Lectins ◽  
Plus Maze ◽  
Marble Burying ◽  
Gabaergic Receptors ◽  
The Central Nervous System ◽  
Hole Board

Background: Plant lectins have shown promising biological activities in the central nervous system (CNS). Objective: This study evaluated the effect of DAL, a lectin isolated from the seeds of the Dioclea altissima species, having binding affinity to D-glucose or D-mannose residues, on mice behavior. Methods: Mice (n=6/group) were treated (i.p.) with DAL (0.25, 0.5 or 1 mg/kg) or vehicle and subjected to several tests (open field/OFT, marble-burying/MBT, hole-board/HBT, elevated plus maze/PMT, tail suspension/ TST, forced swimming/FST or rotarod/RRT). Pizotifen, cyproheptadine, flumazenil, L-NAME, 7-NI, Larginine or yohimbine were administered 15 min before DAL (0.5 mg/kg) and the animals were evaluated on PMT. It was also verified whether the DAL effect depended on its structural integrity and ability to interact with carbohydrates. Results: The results showed there were no neurobehavioral changes in the mice at the RRT, FST and locomotion in the OFT. DAL (0.25, 0.5 or 1 mg/kg) increased the behavior of grooming and rearing in the OFT, head dips in the HBT, pedalling in the TST and decreased the number of marbles hidden in the MBT. In the PMT, DAL (0.25, 0.5 and 1 mg/kg) and Diazepam increased the frequency of entries in the open arms and the time of permanence in the open arms without affecting the locomotor activity. The effect of DAL was dependent on carbohydrate interaction and protein structure integrity and it prevented by pizotifen, cyproheptadine, flumazenil, L-NAME and 7-NI, but not by L-arginine or yohimbine. Conclusion: DAL was found to have an anxiolytic-like effect mediated by the 5-HT and GABAergic receptors and NO pathway.

scholarly journals Characterization of cobalamin receptor sites in brush-border plasma membranes of the tapeworm Spirometra mansonoides.

1983 ◽  
Vol 258 (7) ◽  
pp. 4261-4265
Author(s):  
P A Friedman ◽  
P P Weinstein ◽  
J F Mueller ◽  
R H Allen
Keyword(s):  
Brush Border ◽  
Plasma Membranes ◽  
Receptor Sites
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