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PubMed1977

Chromatin fractionation related to cell type and chromosome condensation but perhaps not to transcriptional activity.

A chromatin fractionation procedure has been developed that involves the shearing of swollen chromatin and the separation of chromatin fragments by sucrose gradient centrifugation. When chromatin from rapidly growing HeLa cells was fractionated, four partially resolved peaks were obtained. Partial characterization of the chromatin fractions indicated that each contained similar lengths of DNA formed in a complex with histones in nucleosomes. The most likely difference between the fractions is the degree of intra-or interstrand association of the fibers of nucleosomes. Chromatins from chicken erythrocytes and mitotic chromosomes were analyzed by the procedure. These chromatins yielded gradient profiles that were distinctly different from each other and from interphase chromatin. These results suggested that the fractionation procedure reflected at least some of the differences in the structure of chromatin known to exist in vivo. The association of pulse-labeled RNA with a particular chromatin fraction is frequently used to support claims of successful separation of transcriptionally active chromatin from inactive chromatin. Since our data show that the slowly sedimenting fractions preferentially bind RNA whose synthesis was clearly not in progress at the start of the fractionation, this criterion is suspect. The presence of equal amounts of satellite DNA in all fractions of mouse L-cell chromatin indicated that the method did not fractionate on the basis of the in vivo transcriptional activity.

Animals↗
PubMed1975

Effects of thrombin on washed, human platelets: changes in the subcellular fractions.

Pressure homogenization and subcellular fractionation has been performed on washed, human platelets and platelets treated with thrombin to undergo the so-called release reaction. Electron microscopy revealed that the particulate zones obtained from the control sample corresponded to membrane vesicles (B), small storage granules (D) as well as mitochondria and larger storage granules (E). Only a few storage granules could be observed in the particulate zones isolated from thrombin-treated platelets. Visual comparison of the sucrose gradient patterns revealed that one granule fraction (D) had disappeared from the thrombin-treated sample. Sodium dodecysulfate-polyacrylamide gel electrophoresis showed a major protein band (mol. wt 145 500 plus or minus 1000) in the extracellular phase (supernatant after removal of the platelets) of the thrombin-treated sample and in the granule fractions (D and E) of the control (mol. wt 147 000 plus or minus 1000). Incubation of whole, washed platelets with thrombin for 5 min at 37 degrees C followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis of the isolated membrane fraction revealed no reproducible differences in the protein band pattern compared to membranes isolated from control platelets. However, after treatment with thrombin for 30 min, a protein band (mol. wt 183 000 plus or minus 3500) had disappeared. The distribution of protein and beta-N-acetylglucosaminidase activity among the subcellular fractions were measured. Both were mainly recovered in the soluble fraction (greater than 77%). The granule fractions, D and E of the control contained 3.0% plus or minus 0.8% and 6.4% plus or minus 1.3% of the total amount of beta-N-acetylglucosaminidase in the gradient. Fraction E of the thrombin-treated cells contained 3.3% plus or minus 1.0% of total while fraction D was lacking.

Animals↗
PubMed2005

Fractionation of surface sediment fines based on a coupled sieve-SPLITT (split flow thin cell) method.

In traditional sediment grain-size separation using sieve technique, the bulk of the organic matter passes through the smallest mesh size (generally 38 microm) and is not further fractionated. In this study, a common sieve separation has therefore been coupled with an extra high capacity split flow thin cell fractionation (EHC-SPLITT) instrument to separate the bulk surface sediment not only into size-based sieve fractions (> 100, 63-100, 38-63 and < 38 microm) but particularly to further fractionate hydrodynamically the fine fraction (< 38 microm) using the EHC-SPLITT. Compared to the few previous studies using a smaller high capacity (HC) SPLITT cell, the EHC-SPLITT evaluated in detail here has several advantages (e.g., 23 times higher throughput and allowance for large particle diameters). First, the EHC-SPLITT was calibrated with particle standards. Then, its ability to fractionate fine surface sediments hydrodynamically was demonstrated with material from biogeochemically distinct regimes using two cutoff velocities (1 and 6 m d(-1)). The results from particle standards indicated a good agreement between theory and experiment and a satisfactory mass recovery for the sieve-SPLITT method (80-97%) was observed for sediment samples. The mass distributions revealed that particles < 38 microm were predominant (70-90%), indicating the large need for a technique such as the EHC-SPLITT to further fractionate the fine particles. There were clearly different compositions in the EHC-SPLITT-mediated sub-fractions of the sediment fines as indicated by analyses of organic and inorganic parameters (POC, Si, Fe and Al). The EHC-SPLITT technique has the potential to provide information of great utility to studies of benthic boundary layer transport and off-shelf export and how such processes fractionate geochemical signals.

Calibration↗
PubMed1982

Localization of dolichol in the lysosomal fraction of rat liver.

The distribution of dolichol and/or dolichol esters in subcellular fractions prepared from a rat liver homogenate has been investigated. After saponification of the various fractions dolichol was isolated and quantitated by high performance liquid chromatography in three systems. The degree of purity of the subcellular preparations was examined by marker enzymes and by electron microscopy. Using differential centrifugation it was found that the level of dolichol was highest in the mitochondria-lysosome fraction. Upon further resolution of this fraction by sucrose density centrifugation it was found that the majority of the dolichol was associated with the lysosome-rich fraction. In contrast, the mitochondrial fraction had only a low level of dolichol. This novel observation was confirmed by the finding that dolichol was greatly enriched in a highly purified lysosome fraction preparations isolated by Metrizamide density centrifugation. The enrichment of dolichol in this purified preparation paralleled the observed enrichment of the lysosomal enzyme activity in this fraction. All of these data suggest that the majority of cellular dolichol and/or dolichol esters is localized in the lysosome fraction. The significance of this finding in relation to the metabolism of dolichol is discussed.

Acetylglucosaminidase↗
PubMed1983

Membrane lipids of hepatic tissue. I. Neutral lipids from subcellular fractions of liver and hepatoma 7288CTC.

Enriched subcellular fractions of nuclei, mitochondria, endoplasmic reticulum (ER), plasma membrane, and cytosol were prepared from liver and hepatoma 7288CTC taken from male inbred BUF rats. Purity was established by marker enzyme activities and distribution of DNA, RNA, sialic acids, total phospholipids, and cholesterol. The subcellular fractions of hepatoma differed from those of liver: 5'-Nucleotidase activity was elevated in ER and mitochondria, cytosol RNA was increased, and cholesterol was elevated in all hepatoma subcellular fractions. Neutral lipid classes of hepatoma subcellular fractions differed quantitatively from those of liver: Hepatoma nuclei and mitochondria contained elevated levels of free fatty acids (FFA) and triglycerides (TG). Generally, the fatty acid profiles of FFA, TG, and sterol esters from hepatoma subcellular fractions were more uniform and showed less organelle specificity than did liver. Hepatoma FFA and TG contained lower percentages of palmitate and higher percentages of stearate in all subcellular fractions than did liver. The sterol esters from most hepatoma subcellular fractions compared to those from liver were characterized by much higher levels of long-chain fatty acids of 20 carbon atoms or longer. The oleate-to-vaccenate ratio in FFA of liver subcellular fractions exhibited some specificity, but not that of hepatoma subcellular fractions. The oleate-to-vaccenate ratio in the acyl chains of liver and hepatoma TG did not reveal organelle specificity.

Animals↗
PubMed1982

The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies.

Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.

Cell Fractionation↗
PubMed1990

Identification and isolation of an axonal plasma membrane enriched fraction from cerebellar granule cell neurites.

A procedure is described to isolate a fraction enriched in cerebellar granule cell neuritic membranes. Morphological markers that are specific for either the granule cell perikarya or neuritic membranes have been identified. Concanavalin A (Con A) has been shown to bind predominantly to the granule cell neurites whereas, the enzymes acetylcholinesterase (AChE) and 2',3',cyclic nucleotide-3'-phosphohydrolase (CNPase) are localized predominantly in the neuronal cell bodies. The membrane fraction enriched in Con A binding has been used to generate a monoclonal antibody which morphologically recognized the cerebellar granule cell neuritic membrane. Following fractionation of the granule cells, each marker was used to identify the cellular origin of the fractions. The neuritic markers Con A and the neuritic membrane antibody MR2 bound predominantly to membranes found in the 29.1% and 31.5% region of the sucrose gradient. The perikaryal markers, CNPase and AChE activity were most enriched in membrane fractions found at a sucrose concentration of 23% and 21%, respectively. Morphological examination of the neuritic enriched fraction shows that it contains predominantly membranous material with few subcellular organelles. The protein profiles of the cerebellar granule cell fractions are unique when compared with the protein profiles of other neuronal and non-neuronal fractions. The membrane fraction isolated from the cerebellar granule cells should prove useful in furthering our understanding of the axonal influence on glial development.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1998

Neurotoxic effects of fractionated diesel exhausts following microinjections in rat hippocampus and striatum.

Exhaust emissions from a heavy-duty diesel vehicle, separated into particulate and semivolatile phases and thereafter fractionated according to polarity, were studied in the adult rat brain after intracranial microinjections using cresyl violet staining and immunohistochemistry. Intrastriatal as well as intrahippocampal injections of particulate fractions III [containing mononitro-polycyclic aromatic hydrocarbon (PAH)], IV (dinitro-PAH and quinones) and V (polar material) and of semivolatile fractions IV and V, in amounts corresponding to a driven length of 19.5 m, caused major lesions with tissue loss and disappearance of immunoreactivity for glial fibrillary acidic protein, tyrosine hydroxylase, and acetylcholine esterase. Particulate fractions I ("light" aliphatic hydrocarbons) and II ("heavy" aliphatic hydrocarbons and PAH) and semivolatile fraction III produced smaller lesions; semivolatile fractions I and II led to lesions equivalent to those of the vehicle dimethyl sulfoxide alone. Microinjected doses of particulate fractions III or IV corresponding to driven lengths of 2.0 and 9.8 m produced a variable lesion. Thus, fractions containing nitro-derivatives of PAH, quinones, and polar material caused the greatest damage after intracranial injections. It is concluded that intracerebral microinjections of fractionated motor vehicle exhausts provide a method for systematic testing of direct neurotoxicity.

Animals↗
PubMed1986

Subcellular fractionation and distribution of cholinergic binding sites in fetal human brain.

Conventional subcellular fractionation techniques have been applied to human fetal brain (13-15 weeks gestation) and the fractions have been characterized by assaying for marker enzymes, cholinergic binding sites and electron microscopy. Fractionation of the homogenate resulted in a nuclear pellet (P1), a crude mitochondrial pellet (P2) and a supernatant (S2). Further resolution of the P2 fraction by density gradient centrifugation resulted in two bands at the gradient interfaces and a pellet. The P2 and subsequently the P2B fraction contained intact plasma membrane profiles as judged by the predominance of adenylate cyclase activity and the presence of occluded lactate dehydrogenase which constituted over 70% of the total activity in these fractions. Morphological examination of the gradient fractions revealed that the P2B fraction contains membrane bound structures which resemble synaptosomes prepared from neonatal rat brain. These structures have a granular matrix in which mitochondria and frequently, neurofilaments were observed. Very few synaptic vesicles were present and there was no evidence for post synaptic attachments. The cholinergic markers choline acetyltransferase, acetylcholinesterase and receptor sites defined by quinuclidinyl benzilate and alpha-bungarotoxin binding were enriched in fractions P2 and P2B which contained the bulk of nerve ending particles. This enriched preparation of fetal synaptosomes may be valuable for functional studies on pre-synaptic terminals in developing brain.

Brain↗
PubMed2003

[Time factor and repopulation during fractionated radiotherapy. Comparison between two xenografted human squamous cell carcinoma].

BACKGROUND: A series of experiments were performed to determine the local tumour control of two human squamous cell carcinoma lines in nude mice. An accelerated-fractionated radiation therapy regime is compared to a conventional-fractionated therapy regime. MATERIAL AND METHODS: KB is a well established human nasopharyngeal squamous cell carcinoma line (ATCC CCL 17). In nude mice KB grows as an low differentiated carcinoma. PEC MB is an undifferentiated squamous cell carcinoma of the maxillary sinus, which was successfully established in nude mice by our group 1993. Both tumors were serially passaged in nude mice. Local irradiation was given without anaesthesia under ambient conditions to air breathing animals using 18 MeV electrons of an linear accelerator (Mevatron 77, Siemens, Munich). Each dose level group consists of six to eight animals. The radiation treatments were given in ten equals fractions using graded dose levels of 2, 3, 4.5, 6 and 8 Gy. The interfraction time interval was 6 hours in the accelerated-fractionated group and 24 hours in the conventional-fractionated group (Figure 1). In the conventional-fractionated group a therapy break was given after 5 fractions for 72 h. The endpoint of the experiments was the dose, which was necessary to control 50% of the tumors (TCD(50)). The TCD(50) values were calculated after 60 days (Tables 1a and 1b). RESULTS: The experiments show, that with increasing overall treatment time of 8 3/4 days using the same number of fractions under ambient conditions the tumor control dose of the tumor KB increases from 36.3 Gy (95% CI 30.9...42.7) to 44.3 Gy (38.3...51.2) (Figure 2a). For the tumor PEC MB the tumor control dose increases from 39.5 Gy (33.4...46.7) to 45.5 Gy (37.0...56.0 (Figure 2b). CONCLUSION: This observed increase of the dose necessary to control the squamous cell carcinoma KB and PEC MB can be caused by repopulation of clonogenic tumors cells, however, other mechanism such as an increasing fraction of hypoxic tumor cells can not be ruled out.

Animals↗
PubMed1978

Protein and enzyme distribution in microsomal and myelin fractions from rat and Jimpy mouse brain.

The protein, glycoprotein and enzyme composition of myelin and myelin-related fraction (SN 4) from rat forebrain was compared with that of microsomal fractions. Acetylcholinesterase was largely confined to the microsomal fractions, wheras 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP) showed a high specific activity in myelin and SN 4 fractions. Nevertheless, the total specific activities of CNP present in microsomal membranes and in a water-soluble form were not negligible, and suggest that this enzyme has a wide distribution among subcellular particles. A high molecular weight protein was identified in myelin and all the other fractions studied. This protein (X), which co-migrates with the major myelin glycoprotein, was present in myelin and in fractions lacking typical myelin components as well as in fractions from a myelin deficient mutant, the Jimpy mouse. The results suggest that the X protein is probably a contaminant in isolated myelin, although the occurrence of this protein as an intrinsic component of several different membranes cannot be ruled out. Despite substantial overlap in density upon zonal centrifugation between SN 4 and microsomal fractions, the enzyme patterns of the fractions were different.

Acetylcholinesterase↗
PubMed1987

Antibodies to the neuronal cytoskeleton are elicited by Alzheimer paired helical filament fractions.

Antibodies were raised to paired helical filament (PHF) enriched fractions obtained from brains of individuals with Alzheimer disease by extraction with ionic detergent followed by sucrose gradient centrifugation. Electron microscopic examination showed that the fractions were enriched in Alzheimer PHF but contained also lipofuscin, amyloid, granular material and membranous elements. Analysis of these fractions with SDS-PAGE stained with Coomassie blue showed only a faint band at approximately 60 kDa while most of the material was excluded from the stacking gel. BALB/c mice were injected weekly with 100 or 200 micrograms of these fractions or corresponding fractions from age-matched control brains. The 3 mice injected with Alzheimer brain, but not the 5 mice injected with control brain fractions, produced antibodies that reacted with central and peripheral nervous system axons, Alzheimer neurofibrillary tangles in intact tissue as well as with isolated, SDS-treated paired helical filaments. In gel strips antibodies from all 3 mice injected with Alzheimer brain fractions reacted with the 200-kDa and 168-kDa but not the 68-kDa neurofilament subunits. The 3 antisera reacted also with some forms of the microtubule-associated protein tau. Adsorptions with the insoluble fraction from Alzheimer but not from control brains blocked staining of axons and NFT by all 3 antisera. Adsorption with highly purified neurofilament proteins or with a preparation containing the 200-kDa and 168-kDa neurofilament subunits blocked axon and NFT immunostaining only in one antiserum. Adsorptions with microtubule protein, heat-stable microtubule-associated protein, or a preparation of tau did not completely block immunostaining by any of the 3 antisera. These results demonstrate that fractions enriched with Alzheimer paired helical filaments contain insoluble neurofilament, tau and other yet unidentified antigens.

Aged↗
PubMed1994

Distribution and characteristics of Ca+2-phosphatidylserine-dependent protein kinase C in subcellular fractions and lamellar bodies of adult rabbit lung.

Pulmonary surfactant prevents lung collapse at minimal alveolar diameter. Since surfactant acts extracellularly, secretion is vitally important in regulating the alveolar surfactant levels. Studies with phorbol esters which stimulate protein kinase C (PKC) activity suggest PKC is involved in regulating surfactant secretion. This study was done to characterize PKC activity in adult rabbit lung fractions. Lungs were removed, homogenized and subcellular fractions prepared by centrifugation on a discontinuous sucrose gradient. Calcium-phosphatidylserine-dependent PKC activity was assayed in fractions in the presence of 4 microM phorbol 12-myristate 13-acetate, 1mM EDTA, 8 mole% phosphatidylserine and 1mM Ca2+ by measuring the transfer of 32P from [gamma-32P]ATP to protein. Concurrent assays were done without Ca+2 or PS. Ca+2-PS dependent PKC activity was defined as the difference between the two. Select fractions were incubated with PKC inhibitors sangivamycin, acridine orange or 9-aminoacridine and activity measured. The results showed the majority of the PKC activity was in the cytosolic fraction (87%, specific activity, 142 pmoles/min/mg) but the lamellar bodies also appeared to contain a small amount of PKC activity (approximately 4.0%, 151 pmoles/min/mg). PKC inhibitors were used to examine the characteristics of the enzyme in the microsomal and lamellar body fractions. Sangivamycin was the most potent inhibitor. Some differences in the inhibition characteristics between the lamellar body and microsomal fractions were observed. However using an add-back approach with the lamellar body fraction, indicated that the small quantity of activity in this fraction be attributed to contamination by microsomes. These results indicate that PKC is active in adult rabbit lung subcellular compartments but is probably not associated with the intracellular surfactant storage organelles.

Animals↗
PubMed1988

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions.

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.

Animals↗
PubMed1987

Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme.

Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.

Animals↗
PubMed1986

Preparation and characterization of submitochondrial fractions from adrenal cells.

A method for preparing submitochondrial fractions from adrenocortical cells was developed by adapting a procedure that has been successful with yeast mitochondria. The method is based upon osmotic swelling, sonication and centrifugation in sucrose. The preparation yields highly purified fractions of outer membrane, intermembrane space, inner membrane and a less purified fraction of matrix. Recoveries are good so that 10(7) cells yield approximately 170 micrograms of inner membrane protein and 12 micrograms of outer membrane protein. Electron microscopy shows that the outer membrane fraction consists of vesicles (0.2-0.6 micron diameter) while inner membrane appears as densely packed sheets of membranous material. Two-dimensional polyacrylamide gels (isoelectric focusing followed by electrophoresis) of all the fractions give highly reproducible patterns of protein spots with Coomassie staining. Steroidogenic proteins were found only in inner membrane fractions which were shown to contain cytochrome P-450 C27 side-chain cleavage and P-450 11 beta-hydroxylase together with adrenodoxin and adrenodoxin reductase. Inner membrane catalyzes side-chain cleavage of cholesterol (conversions to pregnenolone) and 11 beta-hydroxylation (DOC----corticosterone) when substrate and NADPH are added. The preparation yields highly purified submitochondrial fractions from Y-1 mouse adrenal tumor cells and from porcine and bovine adrenocortical mitochondria. The method has the virtue of yielding highly purified intermembrane fluid which is not true of other methods for fractionation of adrenal mitochondria. The procedure also yields cleaner preparations of the two membranes than two other published methods currently used to prepare submitochondrial fractions from adrenocortical cells.

Adrenal Glands↗
PubMedDate not supplied

Characterization of two subcellular fractions isolated from myelinated axons.

Myelin and a heavy membrane fraction (1.0/1.2 fraction) were isolated from rabbit white matter by a slight modification of the procedure for bovine CNS. The specific activities of acetylcholinesterase and Na+, K+-ATPase were higher in the 1.0/1.2 fraction than in myelin. In contrast, the cerebroside content and 2'3'-cyclic nucleotide 3'-phosphohydrolase activity in the 1.0/1.2 fraction were 4.5 and 3.4 times lower than in myelin. Total lipids accounted for only 30% of the 1.0/1.2 fracton's dry weight; for myelin, they represented 70%. Polacrylamide gel electrophoresis showed the presence of many high molecular weight proteins and glycoproteins in the 1.0/1.2 fraction but myelin components were practically missing. Cytochrome c oxidase and NADPH-cytochrome c reductase activities suggested about 15% contamination in the 1.0/1.2 fraction but less than 5% for myelin. In electron micrographs of the 1.0/1.2 fraction, there were many membraneous profiles that varied in size, some mitochondrial fragments, and only a few lamellar whorls of compact myelin. The results suggest that the 1.0/1.2 fraction is different from other myelin-related fractions and is probably enriched in axolemma.

Animals↗
PubMed2006

Antioxidant and radioprotective effect of the active fraction of Pilea microphylla (L.) ethanolic extract.

The ethanolic extract of Pilea microphylla (L.) was defatted, successively fractionated with acetone and the residue so obtained was found to be most potent when subjected to detailed free radical scavenging and in vivo radioprotection studies. The most active fraction reacts with free radicals, such as DPPH (50 microM), ABTS(.)(-) (100 microM) and (.)OH (generated by Fenton reaction) with IC(50) value of 23.15 microg/ml, 3.0 microg/ml and 310 microg/ml, respectively. The most active fraction inhibited iron-induced lipid peroxidation in phosphatidyl choline liposomes with an IC(50) of 13.74 microg/ml. The kinetics of scavenging of DPPH and ABTS(.)(-) radicals were followed at different concentrations of the fraction by employing stopped-flow studies. The observed first order decay rate constants at 200 microg/ml and 50 microg/ml of fraction with DPPH (50 microM) and ABTS(.)(-) (50 microM) were found to be 0.4s(-1) and 2.1s(-1), respectively. The fraction when screened for in vivo radioprotection in Swiss albino mice showed 80% protection at a dose of 900 mg/kg and with a DRF of about 1.12. The fraction was also found to protect livers of irradiated mice from depletion of endogenous antioxidant enzymes like glutathione, GST, SOD, catalase and thiols. The fraction also protected the villi height, increased the number of crypt cells while offering general protection to the intestine from acute radiation effects. The fraction also protected the hematopoietic system as assessed by endogenous spleen colony assay, contributing to the overall radioprotective ability.

Animals↗