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PubMed1998

Oral anti-inflammatory and anti-ulcerogenic activities of a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia (Turneraceae).

Anti-inflammatory studies were conducted on rats or mice using a crude hydroalcoholic extract of the aerial parts of Turnera ulmifolia and it's partitioned fractions, i.e. the aqueous, ethyl acetate and ethanolic fractions. The hydroalcoholic extract and it's fractions (aqueous and ethanolic) inhibited carrageenan-induced edema. However, only the ethanolic fraction was used in the other experiments due to it's yield. The extract also inhibited the cotton pellet granuloma and the increase of vascular permeability induced by histamine, 5-hydroxytryptamine and prostaglandin E2, but not that produced by bradykinin. The extract or the fraction did not present analgesic activity in the writhing test using acetic acid and did not reduce croton oil-induced ear edema in mice. When the ethanolic fraction and LPS were administered i.p. to Balb/C mice 72 h before blood or peritoneal fluid collection, no changes were observed in the white or total blood cell counts in the peripheral blood. On the other hand, changes were observed in both total and differential cell counts in the peritoneal exudate since all doses of the fraction reduced the number of total leukocytes (mainly lymphocytes) obtained from the peritoneal exudate. In contrast to nonsteroidal anti-inflammatory drugs, the administration of the hydroalcoholic extract or the ethanolic fraction alone did not potentiate gastric mucosal lesions induced by aspirin. The extract and the fraction inhibited the appearance of gastric lesions induced by indomethacin, ethanol and pylorus ligature, but not those induced by stress. As also observed with carbenoxolone, the ethanolic fraction increased the wall mucus in hypothermical-restraint stress-induced gastric lesions. The anti-ulcerogenic effect of the extract and of the ethanolic fraction may be related to an increase of mucosal defensive factors, such as prostaglandin and mucus. The anti-inflammatory actions of the extract and the fraction may be due to an inhibitory effect on histamine and cyclooxygenase II, but not on cyclooxygenase I, because the extract and it's fraction present both anti-inflammatory and anti-ulcerogenic effects. The major substances present in the ethanolic fraction are flavonoids which will be isolated and identified.

Administration, Oral↗
PubMed2005

Phytochemicals and antioxidant activity of milled fractions of different wheat varieties.

The health-promoting effects of whole-grain consumption have been attributed in part to their unique phytochemical contents and profiles that complement those found in fruits and vegetables. Wheat is an important component of the human diet; however, little is known about the phytochemical profiles and total antioxidant activities of milled fractions of different wheat varieties. The objectives of this study were to investigate the distribution of phytochemicals (total phenolics, flavonoids, ferulic acid, and carotenoids) and to determine hydrophilic and lipophilic antioxidant activity in milled fractions (endosperm and bran/germ) of three different wheat varieties, two of which were grown in two environments. Grain samples of each of the wheat varieties were milled into endosperm and bran/germ fractions. Each fraction was extracted and analyzed for total phenolics, ferulic acid, flavonoids, carotenoid contents, and hydrophilic and lipophilic antioxidant activities. Total phenolic content of bran/germ fractions (2867-3120 micromol of gallic acid equiv/100 g) was 15-18-fold higher (p < 0.01) than that of respective endosperm fractions. Ferulic acid content ranged from 1005 to 1130 micromol/100 g in bran/germ fractions and from 15 to 21 micromol/100 g in the endosperm fractions. The bran/germ fraction flavonoid content was 740-940 micromol of catechin equiv/100 g. On average, bran/germ fractions of wheat had 4-fold more lutein, 12-fold more zeaxanthin, and 2-fold more beta-cryptoxanthin than the endosperm fractions. Hydrophilic antioxidant activity of bran/germ samples (7.1-16.4 micromol of vitamin C equiv/g) was 13-27-fold higher than that of the respective endosperm samples. Similarly, lipophilic antioxidant activity was 28-89-fold higher in the bran/germ fractions (1785-4669 nmol of vitamin E equiv/g). Hydrophilic antioxidant activity contribution to the total antioxidant activity (hydrophilic + lipophilic) was >80%. In whole-wheat flour, the bran/germ fraction contributed 83% of the total phenolic content, 79% of the total flavonoid content, 51% of the total lutein, 78% of the total zeaxanthin, 42% of the total beta-cryptoxanthin, 85% of the total hydrophilic antioxidant activity, and 94% of the total lipophilic antioxidant activity. Our results showed that different milled fractions of wheat have different profiles of both hydrophilic and lipophilic phytochemicals. These findings provide information necessary for evaluating contributions to good health and disease prevention from whole-wheat consumption.

Antioxidants↗
PubMed1966

Interactions of calf-thymus histone fractions in aqueous solution with 8-anilinonaphthalene-1-sulphonic acid.

1. The interactions of histone fractions with 8-anilinonaphthalene-1-sulphonic acid were investigated by fluorimetry and spectrofluorimetry and the results were interpreted with the aid of equilibrium-dialysis techniques. 2. Characteristic differences were found between the various histone fractions, and with fractions F3 and F2a the binding was found to be salt-dependent. 3. Evidence was obtained indicating a slow change of the physical state of fractions F3 and F2a in the presence of salt, and the binding by these two fractions in the presence of salt was greater by an order of magnitude than by fractions F1 and F2b. 4. Conditions favouring binding were also those favouring histone aggregation; SO(4) (2-) ions activated binding at a lower concentration than Cl(-) ions; urea, guanidinium ions and high concentrations of I(-) ions were inhibitory to binding. 5. After histones had been kept in the presence of salt for a long time the reversal of interaction on decreasing the salt concentration was incomplete. 6. The inhibition of binding by fraction F2a in the presence of urea or fraction F2b depended on the time sequence of addition of the reagents. 7. Artificial nucleoproteins made by precipitating DNA with the histone fractions in neutral 0.14m-sodium chloride showed the same order of interaction as was found for the fractions in solution. 8. Comparison of the binding by fraction F2a with that by bovine plasma albumin showed that in both cases there were a large number of weakly binding sites but that fraction F2a lacked the small number of strongly binding sites found in albumin. No slow change of binding in the presence of salt was found for albumin. 9. Binding by fraction F2b increased the affinity of the protein for further molecules of the adsorbate. 10. The results are discussed in relation to the close relationship between binding and aggregation and the possible role of non-polar interactions as determined by the balance between polar and non-polar amino acids in the histone fractions.

Aniline Compounds↗
PubMed1957

Changes in droplet fractions from rat kidney cells after intraperitoneal injection of egg white.

1. Kidney homogenates from rats injected with egg white and from control rats were fractionated simultaneously into six fractions and the content of acid phosphatase, ribonuclease, desoxyribonuclease, cathepsin, and beta-glucuronidase in corresponding fractions from treated and untreated animals was compared. These observations were correlated with the amount of dark brown bottom sediments in fractions NDrI, DrII, and DrIII, and with the number of droplets in fraction NDrI. 2. It was found that after injection of egg white the amount of small droplets decreased as indicated by the decrease of the dark brown bottom layer in the sediment of fraction DrIII and by the concomitant decrease of hydrolytic enzymes in the same fraction, and that the number of large droplets increased as indicated by the increase of brown sediment in fraction NDrI and the increase in the number of droplets counted in a bacterial counting chamber in the same fraction. It was concluded that the treatment with egg white induced the transformation of small droplets into large droplets. 3. The decrease of hydrolytic enzymes in the fractions containing the small droplets was accompanied by a marked increase of these enzymes in the supernatant fluid. The enzyme content of fraction NDrI was not increased after treatment, although it contained greatly increased numbers of large droplets. Counting of the droplets in this fraction showed decreased enzymatic activity of the average large droplet after treatment with egg white. It was suggested that during the transformation of small into large droplets, a portion of the hydrolytic enzymes was released into the surrounding cytoplasm, and that this was partly responsible for the increased enzyme content of the supernatant fluid after fractionation of the kidney homogenate. In contrast to the four other hydrolytic enzymes, beta-glucuronidase was not increased in the supernatant fluid. 4. Eighteen hours after intraperitoneal injection of egg white, the specific enzymatic activities of kidney homogenates showed a 25 to 35 per cent increase for cathepsin, ribonuclease, and desoxyribonuclease, no change for acid phosphatase and beta-glucuronidase, and approximately a 7 per cent decrease for cytochrome oxidase. The increase of cathepsin, ribonuclease, and desoxyribonuclease in the total homogenate was interpreted as an indication of the formation of new enzymes, and it was suggested that this partly accounted for the increase of these enzymes in the supernatant fluid. 5. The activation of the enzymes by osmotic effects was investigated in vitro by incubation of droplet fractions in the presence of different concentrations of sucrose.

Acid Phosphatase↗
PubMed1982

Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-dependent initiation factor.

Mouse and human cell extracts (S100) can support an accurate and efficient transcription initiation on homologous ribosomal RNA gene (rDNA) templates. The cell extracts were fractionated with the aid of a phosphocellulose column into four fractions (termed A, B, C and D), including one containing a major part of the RNA polymerase I activity. Various reconstitution experiments indicate that fraction D is an absolute requirement for the correct and efficient transcription initiation by RNA polymerase I on both mouse and human genes. Fraction B effectively suppresses random initiation on these templates. Fraction A appears to further enhance the transcription which takes place with fractions C and D. Although fractions A, B and C are interchangeable between mouse and human extracts, fraction D is not; i.e. initiation of transcription required the presence of a homologous fraction D for both templates. The factor(s) in fraction D, however, is not literally species-specific, since mouse D fraction is capable of supporting accurate transcription initiation on a rat rDNA template in the presence of all the other fractions from human cell extract under the conditions where human D fraction is unable to support it. We conclude from these experiments that a species-dependent factor in fraction D plays an important role in the initiation of rDNA transcription in each animal species.

Animals↗
PubMed1996

Comparison of cell profiles in separately evaluated fractions of bronchoalveolar lavage (BAL) fluid in children.

BACKGROUND: Bronchoalveolar lavage (BAL) has been widely accepted as a routine procedure in the differential diagnosis of respiratory diseases in adults. However, there is only limited information about the value of BAL in children and there is no standardisation of the procedures. METHODS: The difference in cellular profiles of sequential BAL fractions from children was analysed to assess the effect of evaluating bronchial and alveolar fractions separately. Twenty five children (mean age 13.4 years) were examined by fibreoptic bronchoscopy under general anaesthesia, combined with local application of lignocaine. The investigation was part of the clinical evaluation of an infective pulmonary problem. Bronchoalveolar lavage was performed from the right middle lobe using four 20 ml aliquots of normal saline and the four fractions of recovered fluid were evaluated separately. RESULTS: The total cell count was lowest in the first aspirate (1.8 x 10(6)) and increased gradually from fraction 1 to fraction 4. Similarly, the viability of the cells was lowest in fraction 1 and gradually improved from fraction 1 to fraction 4. In the differential count the number of neutrophils decreased from fraction 1 (8.3%) to fraction 4, with fractions 2 (5.4%), 3 (3.4%), and 4 (2.4%) being significantly different from fraction 1. There were no differences in the numbers of eosinophils, lymphocytes, or macrophages between individual fractions. CONCLUSIONS: These results show that the cell profile of the first "bronchial" fraction is different from subsequent samples. It should be evaluated separately while the second and third aliquots may be pooled.

Adolescent↗
PubMed1989

Investigation on interaction of fractionated 3H-heparin with plasma proteins by gel filtration chromatography.

Interaction of fractionated 3H-heparin with plasma proteins was investigated by gel filtration chromatography. The peak of the elution profile for fractionated 3H-heparin in rat plasma was observed at the higher molecular weight fraction than that for fractionated 3H-heparin in buffer without any protein, suggesting the existence of fractionated 3H-heparin binding protein(s) in plasma. Then the interaction of fractionated 3H-heparin with such plasma proteins as albumin, antithrombin III, thrombin and alpha-globulin was investigated. The elution profile for fractionated 3H-heparin in albumin solution, antithrombin III solution and the solution containing antithrombin III and thrombin were different from that for fractionated 3H-heparin in plasma. The elution profile for fractionated 3H-heparin in alpha-globulin solution, of a concentration close to that in plasma, was comparable to that of fractionated 3H-heparin in plasma, though two peaks were found. The major peak corresponded to that of fractionated 3H-heparin in plasma, and the minor peak eluted at a higher molecular weight fraction. When alpha-globulin concentration was decreased, the major peak shifted to the lower molecular weight fraction, and the minor peak was diminished and then disappeared. Thus it was suggested that alpha-globulin is dominant for plasma protein binding of fractionated 3H-heparin.

Alpha-Globulins↗
PubMed1985

Repair in mouse lung of multifraction X rays and neutrons: extension to 40 fractions.

We have extended our previous multiple irradiations of mouse lung from 20 to 40 fractions of both X-ray and neutron radiation in order to test whether the repair parameters previously derived will hold for lower doses per fraction, down to 1.1 Gy of X rays and 0.18 Gy of 3 MeV neutrons per fraction. Repair parameters were calculated from measurements of breathing rate and lethality at monthly intervals up to 17 months after irradiation with 1, 10, 20 or 40 equal fractions. Sparing of neutron damage was negligible when the neutron dose was divided into multiple fractions, but progressively greater repair of lung damage was seen after increasing numbers of X-ray fractions. A significant increase in the iso-effect dose for 40 fractions of X rays was found compared with 20 fractions, even when two fractions per day were given at intervals of about 6 hours, as was the case in the 40 fraction experiment. The data were well fitted by the linear quadratic formula for response vs. dose per fraction and the ratio alpha/beta yielded values of approximately 3 Gy after X rays and 30 to 40 Gy after neutron irradiation; these values are not different from alpha/beta ratios found for up to 20 fractions. The single dose RBE was less than 2, increasing to about 6 at the lowest dose per fraction measured, in agreement with previous results. The ratio of the alpha component for neutrons to that for X rays was about 8, which is therefore the limiting RBE predicted for infinitely small doses per fraction.

Animals↗
PubMed1995

Derivation of the optimum dose per fraction from the linear quadratic model.

The linear quadratic equation for fractionated radiotherapy has already been adapted to include a time factor for tumour repopulation: loge cell kill (E) is given as a function of dose per fraction (d), number of fractions (n), overall treatment time (T) and the clonogen doubling time (Tp). By incorporating a normal tissue isoeffect and replacing the relationship between T and n by a function f, the equation for E can be rewritten as a more complex function of d. In this form, E and d are continuous variables so that the dose per fraction (d') required to produce maximum values of E for isoeffective late normal tissue effects can be found by differential calculus. The derived equation takes the form (beta kTp-alpha Tp)d2 + 1.386fd + 0.693fk = 0 and when solved for d provides a direct estimation of the optimum dose per fraction. Where normal tissue sparing is possible and the tumour dose z is related to the normal tissue dose d, the optimum dose per fraction z' can be found by solving the equation (beta kTp-alpha gTp)z2 + 1.386fgz + 0.693fk = 0 The results show that a critical minimum dose per fraction is required to counteract rapid tumour clonogen repopulation in both conventional and accelerated radiotherapy. The calculus method is reasonably accurate for larger fraction numbers, when clonogen doubling times are 3.5 days or longer and for conventional radiotherapy given 5 days per week. The model is even more accurate for accelerated hyperfractionated radiotherapy providing that there is complete repair between successive fractions. Where greater normal tissue sparing is possible, as with focal teletherapy methods and brachytherapy, higher tumour doses per fraction can be used to increase further the tumour cell kill without exceeding normal tissue tolerance. These predicted doses per fraction are consistent with clinical experience when the given constraints in terms of frequency of treatment are considered. The model described can be used for tumours in which repopulation occurs at a constant rate throughout treatment. For tumours in which accelerated repopulation occurs, the optimum dose per fraction can be separately calculated for the initial phase of slow repopulation (for which very small doses per fraction are optimal) and also for the second phase of rapid repopulation (for which either accelerated hyperfractionated treatments or hypofractionated focal methods of treatment would be appropriate). The limitations of the model are fully discussed including the need for accurate radiobiological predictive assays. In the future such assays of pre-treatment doubling times and tumour cell radiosensitivities could be used to determine reasonable ranges for the optimum dose per fraction in experimental tumours and subsequently in clinical trails. This approach could produce major improvements in the therapeutic potential of radiotherapy.

Cell Division↗
PubMedDate not supplied

Interactions of organic compounds with wastewater dissolved organic matter: role of hydrophobic fractions.

The role of structural fractions of dissolved organic matter (DOM) from wastewater in the sorption process of hydrophobic organic compounds is still not clear. In this study, DOM from two wastewater treatment plants (Lachish and Netanya, Israel) was fractionated to hydrophobic acid (HoA) and hydrophobic neutral (HoN) fractions. The fractions were characterized and their sorptive capabilities for s-triazine herbicides and polycyclic aromatic hydrocarbons (PAHs) were studied. For all sorbates, the binding to the HoN fractions was much higher than to HoA fractions. The HoA fractions were more polar than the HoN fractions, containing a higher level of carboxylic functionalities. However the higher binding coefficients of atrazine (2-chloro-4-ethylamine-6-isopropylamino-s-triazine) and ametryn [2-(ethylamino)-4-isopropylamino-6-methyl-thio-s-triazine] obtained for the HoN fractions suggest that their sorption is governed by hydrophobic-like interactions rather than H bonding. The values of binding coefficients of PAHs measured for the HoN fractions were within the range reported for humic acids and much higher than other fractions, suggesting that this fraction plays an important role in the overall sorption of these compounds by DOM. Higher sorption coefficients were measured for the Netanya DOM sample containing higher level of hydrophobic fractions (HoA + HoN) than the Lachish DOM, suggesting that the sorption of hydrophobic organic compounds by DOM is governed by the level of these structural substances. The evaluation of mobility of organic pollutants by wastewater irrigation requires not only assessment of the total carbon concentration but also, more importantly, the content of the hydrophobic fractions.

Adsorption↗
PubMed1990

Antimicrobial supplementation of growing pigs: the effect of porcine sera fractions on in vitro muscle cell proliferation.

Sera obtained from pigs before and after subtherapeutic levels of ASP250 supplementation (pre and post serum pools) have been subjected to comparative fractionation by using gel filtration and affinity chromatography on immobilized Cibacron Blue F3G-A. Comparable serum fractions obtained from pre- and post-ASP250 blood sera were assayed in muscle cell culture bioassays designed to measure their effect on proliferation. Pre- and post-ASP250 sera were subjected to gel filtration and divided into the following fractions: fraction 1, Kav less than .17; fraction 2, Kav = .17 to .41; fraction 3, Kav = .41 to .59. Post-ASP250 fractions 2 and 3 increased proliferation rate in cultured muscle cells to a greater extent than comparable pre-ASP250 fractions (P less than .001). Chromatography of fraction 3 on immobilized Cibacron Blue F3G-A showed that both pre- and post-ASP250 fraction 3 contained a putative inhibitor of myogenic cell proliferation as well as mitogenic factors. However, negative growth factor activity was greater in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). Additionally, positive growth factor activity was lower in pre-ASP250 fraction 3 than in post-ASP250 fraction 3 (P less than .05). These data suggest that levels and(or) activities of both positive and negative muscle growth factors in serum may be altered by the addition of antimicrobials to the diets of growing pigs.

Animals↗
PubMed2003

Fractions of the semen of red deer (Cervus elaphus)--their occurrence and characteristics in different periods of season.

During a complete season 145 ejaculates were collected from 4 red deer stags (Cervus elaphus) using a modified artificial vagina. Differences found in characteristics of ejaculates allowed to identify four different types of ejaculates: pre-mating, mating, transitory, and post-mating ejaculate. These types of ejaculates occurred exclusively during corresponding periods of the season in the sequence mentioned above. Three fractions of ejaculates were identified: grey, white, and yellow. Pre- and post-mating ejaculates were homogenous and grey. The mating- and transitory ejaculate consisted of two fractions; the mating ejaculate consisted exclusively of white and yellow fractions and the transitory one contained exclusively the white and grey fraction. Respective fractions of ejaculates differed also in consistency. Pre- and post-mating ejaculates as well as the grey fraction of the transitory ejaculate were watery, the white fraction of the mating and transitory ejaculate were milky and the yellow fraction was honey-like. All respective fractions of ejaculates contained spermatozoa, except the yellow fraction. Volume, sperm concentration and pH of the respective fractions of ejaculates were examined. The yellow fraction had the largest volume and contributed most to differences between the ejaculates as concerns the volume. Sperm concentration was highest in the white fraction of the mating and the transitory ejaculate. The objective of this report was to confirm the previous findings about occurrence of distinguishable fractions and to describe their characteristics in respect to different periods of the season.

Animals↗
PubMedDate not supplied

Influence of ethanol and benzene on cytochrome P-450 fractions in rat liver microsomes.

The repartition and characteristics of liver microsomal cytochrome P-450 fractions from benzene- or ethanol-treated rats were compared to those observed either in untreated animals, or in rats treated by classic inducers, namely phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone. DEAE-cellulose chromatography allowed the separation of four main cytochrome P-450 fractions called A (nonabsorbed), Ba, Bb, and Bc (successively eluted by a NaCl gradient). In control rats, and in ethanol- and benzene-treated animals, fractions A were predominant; phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, and benzene induced Bb fractions. Enzymatic and immunological methods allowed a characterization of those cytochrome P-450 fractions. Fractions A are similar in all cases, and mainly active towards aniline. This aniline hydroxylase activity is especially increased by ethanol. As a rule, fractions Ba and Bc behave similarly and exhibit rather low monooxygenase activities. On the contrary, fractions Bb differ from each other as a function of the inducer. Phenobarbital-induced Bb fraction is different from all other Bb fractions and especially active towards benzphetamine. 3-Methylcholanthrene- and beta-naphthoflavone-induced Bb fractions are identical, but they are different from all other Bb fractions, and especially active towards 7-ethoxycoumarin. Fraction Bb induced by benzene is different from those induced by classic inducers, but may be identical to the Bb fraction of control animals. As a whole, benzene and ethanol appear to display inducing properties different from those of phenobarbital or polycyclic aromatic hydrocarbon-like inducers.

Animals↗
PubMed1983

Styrene oxide metabolism in rhesus monkey liver: enzyme activities in subcellular fractions and in isolated hepatocytes.

The activity and the kinetic behavior of expoxide hydrolase were studied in various subcellular fractions of rhesus monkey liver isolated by differential centrifugation. The purity of the fractions was estimated by morphometric electron microscopy. Hydrolase activity was measured by a specific radiometric assay with [7-3H]styrene oxide as substrate. All isolated subcellular fractions catalyzed the hydration of styrene oxide at a significant rate. With a saturating concentration of substrate (1 mM), the enzymatic activity (nanomole of product per minute per milligram of protein; mean +/- S.E.) turned out to be 1.51 +/- 0.45 (nuclear fraction), 3.50 +/- 1.11 (mitochondrial fraction), 14.8 +/- 2.26 (microsomal fraction) and 1.69 +/- 0.37 (soluble fraction). The hydrolase obeyed Michaelis-Menten kinetics in each fraction. Vmax (nanomole per minute per milligram; mean +/- S.E.) was 1.64 +/- 0.65 (nuclear fraction) 3.87 +/- 1.71 (mitochondrial fraction), 19.8 +/- 5.4 (microsomal fraction) and 2.72 +/- 1.36 (soluble fraction). The Km (millimole; mean +/- S.E.) values in the fractions were 0.09 +/- 0.02, 0.07 +/- 0.01, 0.23 +/- 0.15 and 0.64 +/- 0.40, respectively. The metabolism of styrene oxide was also studied in isolated hepatocytes from rhesus monkey. These cells hydrated the substrate easily whereas the conjugation of styrene oxide with glutathione was not measurable. Our results show that epoxide hydrolase is present in all subcellular fractions of the rhesus monkey liver. Styrene oxide is preferentially metabolized by hydration to styrene glycol in the isolated hepatocytes of this species and no conjugation with glutathione was found under the incubation conditions used.

Animals↗
PubMed2005

New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues.

To clarify the mechanism of the anticarcinogenic effect of conjugated linoleic acid (CLA), its intracellular distribution needs to be determined. Subcellular fractionation using centrifugation techniques is a method that is frequently used for isolation of cell organelles from different tissues. But as the size and density of the organelles differ, the method needs to be optimised for every type of tissue. The novelty of this study is the application of a subcellular fractionation method to human healthy and cancerous renal and testicular tissue. Separation of total tissue homogenate into nuclei, cytosol, and a mixture of mitochondria and plasma membranes was achieved by differential centrifugation. As mitochondria and plasma membranes seemed to be too similar in size and weight to be separated by differential centrifugation, discontinuous density-gradient centrifugation was carried out successfully. The purity of the subcellular fractions was checked by measuring the activity of marker enzymes. All fractions were highly enriched in their corresponding marker enzyme. However, the nuclear fractions of kidney and renal cell carcinoma were slightly contaminated with mitochondria and plasma membrane fractions of all tissues with lysosomes. The fraction designated the cytosolic fraction contained not only cytosol, but also microsomes and lysosomes. The CLA contents of the subcellular fractions were in the range 0.13-0.37% of total fatty acids and were lowest in the plasma membrane fractions of all types of tissue studied. C16:0, C18:0, C18:1 c9, C18:2 n-6, and C20:4 n-6 were found to be the major fatty acids in all the subcellular fractions studied. However, marked variations in fatty acid content between subcellular fractions and between types of tissue were detectable. Because of these differences between tissues, no general statement on characteristic fatty acid profiles of single subcellular fractions is possible.

Adipose Tissue↗
PubMed1975

Subcellular fractionation of pig platelets.

Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes or alpha-granules, mitochondria and dense granules. With this technique of platelet homogenization, 80% of the serotonin and 93% of the beta-N-acetylglucosaminidase were found to be particulate. In the gradient, mitochondria were sharply banded in a fraction (density 1.16--1.17) having a specific activity 10--100 times higher than the other fractions of the gradient. Serotonin-containing granules were found in a pellet of density greater than 1.27 and contained 60% of the serotonin and adenine nucleotides of the granule fraction. The lysosome markers that were monitored, acid phosphatase and beta-N-acetylglucosaminidase, exhibited different distribution patterns. Acid phosphatase showed the highest specific activity in the microsomal fraction with only 2.8% in the granule fraction, and this latter amount also appeared to be associated with membranes upon further fractionation. Beta-N-acetylglucosaminidase was present in both the granule fraction and in the microsomal fraction with nearly the same specific activity. However, that present in the granule fraction was clearly associated with granules that distributed over a wide range of densities on a sucrose gradient. The calcium distribution was followed to attempt to determine its subcellular location; 19% was found in the same subfraction as the serotonin-containing granules, but at least 50% of the particulate calcium was associated with granules distinctly separate from the storage granules.

Acid Phosphatase↗
PubMed1988

Phosphatidylinositol kinase, phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions.

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [gamma-32P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150-200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4-11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40-70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP2 in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

Immunofractionation of high density lipoprotein subclasses 2 and 3. Similarities and differences of fractions isolated from male and female populations.

High density lipoprotein (HDL) subclasses 2 and 3 isolated from male and female populations were further subfractionated by immunoaffinity techniques. Each subclass gave rise to 2 fractions: one contained apolipoprotein (apo) A-I but no apo A-II (LpAI); the other contained apo A-I and apo A-II (LpAI,AII). The bulk fraction (HDL-3(LpAI,AII)) comprised over 70% of total HDL and was present in similar concentrations in both populations. There were, however, significant male-female differences in plasma levels of the minor HDL-3 fraction i.e. HDL-3(LpAI). Females had significantly higher plasma concentrations of both fractions within HDL-2. These fractions also exhibited strong, positive correlations with total HDL cholesterol concentrations, both in males as well as females. It suggests that metabolic activities giving rise to both HDL-2(LpAI) and HDL-2(LpAI,AII) determine plasma HDL cholesterol concentrations. Several similarities were noted between the male and female populations. Triglyceridaemia was negatively correlated with HDL-2 derived fractions and positively correlated with the bulk fraction HDL-3(LpAI,AII). Compositional data showed that the fraction (LpAI) had a lower esterified cholesterol to total cholesterol ratio than the fraction (LpAI,AII), the differences being more apparent at the HDL-3 level. Additionally, analysis of the surface components of HDL-3 fractions suggested that (LpAI,AII) had a greater potential than (LpAI) for absorbing lipoprotein surface material. Finally, the relative concentrations of the individual components of fractions within the same population and defined by the same apolipoprotein criterion showed highly significantly, positive correlations. Such correlations were not apparent for apolipoprotein dissimilar fractions. These observations could reflect a metabolic link between apolipoprotein similar fractions.

Adult↗