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PubMed2006

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics I. Differential centrifugation fractionation B16F10 cells and use to study the intracellular fate of HPMA copolymer - doxorubicin.

Polymer therapeutics are being designed for lysosomotropic, endosomotropic and transcellular drug delivery. Their appropriate intracellular routing is thus crucial for successful use. For example, polymer-anticancer drug conjugates susceptible to lysosomal enzyme degradation will never deliver their drug payload unless they encounter the appropriate activating enzymes. Many studies use confocal microscopy to monitor intracellular fate, but there is a pressing need for more quantitative methods able to define intracellular compartmentation over time. Only then will it be possible to optimise the next generation of polymer therapeutics for specific applications. The aim of this study was to establish a subcellular fractionation method for B16F10 murine melanoma cells and subsequently to use it to define the intracellular trafficking of N-(2-hydroxyproplylmethacrylamide) (HPMA) copolymer-bound doxorubicin (PK1). Free doxorubicin was used as a reference. The cell cracker method was used to achieve cell breakage and optimised to reproducibly achieve approximately 90% breakage efficiency. This ensured that subsequent subcellular fractionation experiments were representative for the whole cell population. To characterise the subcellular fractions obtained by differential centrifugation, DNA (nuclei), succinate dehydrogenase (mitochondria), N-acetyl-beta-glucosaminidase (lysosomes), alkaline phosphatase (plasma membrane) and lactate dehydrogenase (cytosol) were selected as markers and their assay was carefully validated. The relative specific activity (RSA) of the fractions obtained from B16F10 cells were: nuclei (2.2), mitochondria (4.1), lysosomes (3.7) and cytosol (2.5). When used to study the intracellular distribution at non-toxic concentrations of PK1 and doxorubicin, time-dependent accumulation of PK1 in lysosomes was evident and the expected nuclear localisation of free doxorubicin was seen. Live cell fluorescence microscopy and confocal co-localisation studies gave qualitative corroboration of these results, but by using this method, we were unable to accurately define organelle localisation. In conclusion, the B16F10 subcellular fractionation method developed here provides a useful tool to allow comparison of the intracellular trafficking of other polymer conjugates.

Alkaline Phosphatase↗
PubMed2001

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles.

In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.

Animals↗
PubMed1976

Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus.

Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of gamma-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in the gamma-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in gamma-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed gamma-glutamyl transpeptidase activity localized in the capillary walls.

Acid Phosphatase↗
PubMed1984

Aqueous density fractionation of mineralizing tissues: an efficient method applied to the preparation of enamel fractions suitable for crystal and protein studies.

An aqueous density fractionation for calcifying tissues was tested for its ability to prepare fractions corresponding to precise mineralization stages, suitable for further protein and crystal studies. Two fractions of immature enamel corresponding to different densities were prepared, using cooled cesium salt saturated solutions, and compared for crystal size and amelogenin molecular weight distribution. For the first time, a steep increase in crystal width was directly correlated to protein degradation in keeping with increasing mineralization. Thus, this paper describes a method for obtaining an accurate fractionation of a calcifying tissue according to its true density heterogeneity. The recovered fractions are shown to be suitable for both crystal and protein studies.

Amelogenin↗
PubMed2003

Size-based analysis of incinerator fly ash using gravitational SPLITT fractionation, sedimentation field-flow fractionation, and inductively coupled plasma-atomic emission spectroscopy.

Fly ash has been regarded as hazardous because of its high adsorption of toxic organic and/or inorganic pollutants. Fly ash is also known to have broad distributions of different chemical and physical properties, such as size and density. In this study, fly ash emitted from a solid waste incinerator was pre-fractionated into six sub-populations by use of gravitational SPLITT fractionation (GSF). The GSF fractions were then analyzed by sedimentation field-flow fractionation (SdFFF) and ICP-AES. SdFFF analysis showed the fly ash has a broad size distribution ranging from a few nanometers up to about 50 microm. SdFFF results were confirmed by electron microscopy. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of the GSF fractions showed the fly-ash particles contain a variety of inorganic elements including Ca, Si, Mg, Fe, and Pb. The most abundant in fly ash was Ca, followed by Si then Mg. No correlations were found between trace element concentration and particle size.

Carbon↗
PubMed1992

Release of different fractions of epidermal growth factor from human platelets in vitro: preferential release of 140 kDa fraction.

Platelet-rich plasma in acidic-citrate-dextrose anticoagulant was kept for 5 days in an oxygen-permeable bag at 22 degrees C in an incubator/rotator. Platelet count remained stable throughout the experiment. On days 0, 3 and 5, aliquots were removed; platelets were isolated by centrifugation at 22 degrees C, 1500 g for 20 min, reconstituted to the original volume with PBS buffer, and the contents of alpha-granules were released by repeated freezing and thawing. Epidermal growth factor (EGF) and beta-thromboglobulin (beta-TG) in the platelet-poor plasma and platelet lysates were determined by radioimmunoassays. Results indicated that in platelet-free plasma, both total EGF and beta-TG increased 3-5-fold after 5 days; this amount represented 10-20% of the factors stored in the platelets. Correspondingly, the EGF and beta-TG contents of the platelet lysates exhibited accompanying decreases. HPLC fractionation showed that the main EGF fraction which progressively decreased in the lysates and increased in plasma had a molecular mass of 140 kDa. The contents of the 67 kDa and 6 kDa fractions did not change substantially. We conclude that under these conditions, the 140 kDa fraction was released preferentially. In view of these and previous experiments, it seems likely that different organs contribute to plasma EGF fractions.

Adolescent↗
PubMed2006

New fractionation scheme for lipid classes based on "in-cell fractionation" using sequential pressurized liquid extraction.

A new preparation scheme is proposed to fractionate neutral lipids (acylglycerines, sterol esters, long-chain free fatty acids) from polar phospholipids in biological matrices. This fractionation is mandatory in the microbial community, for the control of bioremediation processes, in the study of phytoplankton growth in lakes and rivers, and in the quality control of processed food. Basically, a two-step pressurized liquid extraction (PLE) scheme is combined with an "in-cell-fractionation" using silica-based sorbents placed at the outlet of the PLE cartridge. The optimized extraction scheme consists of n-hexane/acetone (9:1, v/v) extraction at 50 degrees C (2 cycles, 10 min each) to obtain neutral lipids followed by chloroform/methanol (1:4, v/v) extraction at 110 degrees C (2 cycles, 10 min each). Thermally pre-treated silicic acid and cyanoproyl-modified silica turned out to be appropriate sorbents to ensure clear-cut boundaries between neutral lipids and phospholipids. The proposed protocol is superior to commonly used approaches consisting of an exhaustive lipid extraction followed by off-line lipid fractionation using solid-phase extraction (SPE) regarding fractionation efficiency, time and solvent consumption. In this paper, it is also shown that the transmethylation using trimethylchlorosilane/methanol (1:9, v/v) provides a complete reaction to give methyl esters without artefact formation across the array of different lipid classes even with polyunsaturated fatty acid moieties.

Chemical Fractionation↗
PubMed1999

Enzyme distributions in subcellular fractions of BHK cells infected with Semliki forest virus: evidence for a major fraction of sphingomyelin synthase in the trans-golgi network.

BHK cells either untreated or infected with Semliki Forest virus have been fractionated on sucrose density gradients. Virus infection caused an increase in density of a membrane fraction enriched in sphingomyelin (SM), cholesterol, SM synthase and sialyltransferase activity. This increase in density was related to incorporation of viral proteins into this fraction, which is likely to contain trans-Golgi network (TGN) membranes. In contrast, glucosylceramide synthase and galactosyltransferase activities (markers for cis/medial and trans-Golgi respectively) underwent no density shift and alkaline phosphodiesterase, a plasma membrane marker, was only slightly density-shifted in infected cells. When cells were incubated with NBD-ceramide to enable them to synthesise NBD-SM and then washed with albumin to remove surface label, fluorescence in untreated cells was concentrated in a single juxtanuclear spot but in infected cells this region of bright fluorescence was larger and extended around the nucleus. After fractionation of these cells, NBD-SM (but only a small proportion of the NBD-ceramide) was found to be shifted into the higher density fraction in infected cells. This work provides further evidence that SM synthase is not mainly localised in the early Golgi cisternae as previously thought, but is associated more with a cholesterol-rich compartment which could be the TGN.

Alphavirus Infections↗
PubMed2001

Amifostine before fractionated irradiation protects bone growth in rats better than fractionation alone.

The aim of this study was to determine the independent and combined effects of 100 mg/kg and 200 mg/kg doses of the radioprotectant amifostine and radiotherapy dose fractionation in preserving the integrity of or minimizing damage to the physis during high-dose radiation exposure in an animal model. Thirty-six weanling four-week-old male Sprague-Dawley rats were randomized into six study groups of six animals each. The distal femur and proximal tibia in the right leg of each animal was exposed to X-irradiation, with the contralateral left leg serving as the nonirradiated control. Three groups received a single 25 Gy radiotherapy dose: one group alone, a second group preceded by 100 mg/kg amifostine, and a third preceded by 200 mg/kg amifostine. Three groups received a total of 25 Gy in three equal fractions: one group alone, a second group preceded by 100 mg/kg amifostine, and a third preceded by 200 mg/kg amifostine. Fractionation of the 25 Gy radiation dose reduced the mean percent overall limb growth loss to 44.8%, a statistically significant reduction compared to a mean 58.8% reduced growth with the single 25 Gy dose. Addition of amifostine at 100 and 200 mg/kg before each of the three fractions of radiotherapy further decreased the mean percent overall limb growth loss to 35.2% and 28.5%, respectively, both statistically significant reductions beyond that achieved by fractionation alone.

Amifostine↗
PubMed1965

The distribution of ribosomal ribonucleic acids among subcellular fractions from bacteria and the adverse effect of the membrane fraction on the stability of ribosomes.

1. The distributions of nucleic acids and protein among fractions obtained by differential centrifugation from species of Pseudomonas, Aerobacter, Escherichia, Proteus and Bacillus have been studied. 2. The DNA in a cell wall-membrane fraction obtained by low-speed centrifugation from the Gram-negative species could be removed by homogenizing and subsequent washing. About 7-14% of the total RNA remained firmly attached and resembled ribosomal RNA in base composition. A similar fraction from the Gram-positive B. subtilis contained about one-half of the total bacterial DNA and only 60% of this could be removed by homogenizing and subsequent washing. 3. A deposit obtained by high-speed centrifugation could be separated into a heavy ribosome layer and a light turbid layer. In E. coli B the latter contained about equal concentrations of RNA and DNA and accounted for about one-half of the total bacterial NADP-activated 6-phosphogluconate dehydrogenase. 4. The washed cell wall-membrane fraction from most species accelerated the degradation of ribosomes. In Pr. vulgaris the activity of this fraction was exceptionally high and resulted in the progressive degradation of ribosomes during their isolation from this species. 5. A possible connexion between ribosome degradation and the synthesis of flagella is discussed in the light of these results.

Bacillus subtilis↗
PubMed1986

Intractable unphysiologically low adenylate energy charge values in synaptosome fractions: an explanatory hypothesis based on the fraction's heterogeneity.

Synaptosomes prepared and incubated in a variety of ways from rat cerebra exhibited intractable, unphysiologically low adenylate energy charge values (approximately 0.37-0.60), low total adenine nucleotide contents (approximately 8-10 nmol/mg protein), and much higher adenylate kinase apparent Keq values (approximately 3-8) as compared to intact brain tissue (values of approximately 0.90, 25 nmol/mg, and 0.74, respectively). Synaptosomes prepared from mouse, dog, and chicken cerebra had values essentially identical to those from rat. When incubated under oxygen in a physiological salt solution containing glucose, synaptosomes metabolized more glucose to lactic acid than to CO2, and the addition of 100 microM veratridine caused a two- to threefold stimulation of O2 uptake, lactate accumulation, and CO2 output. It is known that synaptosome fractions contain a substantial number (at least 30-45% by volume) of cytoplasm-containing particles devoid of mitochondria (henceforth termed "cytosolic particles"), and that approximately 80% of brain hexokinase is bound to the outer mitochondrial membrane. For the cytosolic particles, lacking oxidative phosphorylation, to maintain their "in vivo" ATP turnover would require about a 19-fold increase in the glycolytic rate, which is not possible due to limiting amounts of hexokinase, and thus these particles are postulated to be responsible for the high level of aerobic lactate accumulation and the intractable low energy charge values found in synaptosome fractions. The mitochondria-containing particles are postulated to have a normal energy charge, a submaximal glycolytic rate, and minimal lactate production, on the basis of the capacity of veratridine to stimulate synaptosomal O2 uptake and CO2 and lactate output. Calculations based on this "two populations of particles" hypothesis indicate that for synaptosome fractions in general, (1) the cytosolic particles contain approximately 35-64% of the total adenine nucleotides and maintain an energy charge of approximately 0.12; (2) the cytosolic particles and mitochondria-containing particles have adenylate kinase apparent Keq values of approximately 0.21-1.66 and 0.74, respectively, revealing that the higher apparent Keq values of the synaptosome fractions probably are not real departures from equilibrium: and (3) approximately 31-45% of synaptosome fraction protein is contained in debris, which, when taken into account, yields total adenine nucleotide contents in the cytosolic particles and mitochondria-containing particles of approximately 15-24 and approximately 11-19 nmol/mg of particle protein, respectively.

Adenosine Diphosphate↗
PubMed2005

Size fractionation of marine sediments by pinched inlet gravitational split-flow thin fractionation and the study of size dependent PCDD/Fs concentrations from different bay areas.

Pinched inlet gravitational split-flow thin fractionation (PI-GSF) has been applied to the continuous size fractionation of marine sediments in order to study the difference in sediment size distribution and the concentration of PCDD/Fs contained in different particle sizes. A PI-GSF channel, known to improve the separation efficiency by reducing the sample inlet thickness, was utilized to fractionate sediments collected from three different bay areas (Geoje, Ulsan, and Pohang) in Korea into 5 different sub-populations (<2.0, 2.0-5.0, 5.0-10, 10-20, 20-63 microm in diameter). The sorted sediment fractions from PI-GSF were examined using electron microscopy to obtain size distribution and the results showed a variation in particle size distribution between bay areas. When the collected particle fractions were examined for size dependent levels of PCDD/Fs, the concentrations of total PCDD/Fs were shown to be much greater for samples collected close to heavy industry complexes than sediments from bay areas without major industry.

Journal Article↗
PubMed1985

Small sucrose density gradients: reproducible and automatic fractionation using a commercial fraction collector (Pharmacia).

A simple, inexpensive modification of a programmable commercial fraction collector (Frac 100; Pharmacia, Sweden) was described. This modification allowed reproducible automatic fractionation of small sucrose gradients by rinsing the hanging drop at the delivery head of the fraction collector into the sampling tube, using signals from the fraction collector to control this specialized function. The described principle may also be useful whenever fraction size is near to drop volume and high resolution and reproducibility are desired.

Animals↗
PubMed1983

Fractionation by micrococcal nuclease digestion of Drosophila embryo chromatin: isolation of a fraction enriched in two major nonhistone proteins.

Limited digestion of Drosophila melanogaster embryo nuclei with mitochondrial nuclease, followed by selective solubilization in 0.1 M NaCl, yields a soluble nucleoprotein fraction (S3) enriched in two dominant protein bands of apparent molecular weight of 44000 and 48000. The analysis of the nucleosome monomer and multimer peaks, separated on sucrose gradients after slight digestion with micrococcal nuclease, shows that these proteins are associated with chromatin subunits, and that they are principally found in the subnucleosome region of fraction S3. This doublet is tentatively identified as a member of the noncanonical HMG of Drosophila. The thermal denaturation and the circular dichroism spectra of the fraction soluble in 0.1 M NaCl (S3) and insoluble fraction (P3) show that both fractions are also structurally different.

Animals↗
PubMed1984

The influence of fraction size in radiotherapy on the late normal tissue reaction--I: Comparison of the effects of daily and once-a-week fractionation on human skin.

A comparison of the normal tissue reactions to daily and once-a-week fractionation was performed on postoperatively irradiated bilateral parasternal fields in patients with breast cancer. Both schedules resulted in identical acute skin erythema and pigmentation. However, the development of telangiectasia was significantly more pronounced after once-a-week fractionation. These findings imply that the Cumulative Radiation Effect (CRE) formula has to be modified. The slope of the iso-effect dose versus number of fractions was 0.24 for acute skin reactions and estimated to be about 0.34 for late skin reactions at a dose level equivalent to 46 Gy in 2 Gy fractions with conventional fractionation. The beta/alpha ratio was 0.12 Gy-1 for acute and about 0.31 Gy-1 for late effects, for 200 kV X rays.

Breast Neoplasms↗
PubMed1994

Flow cytometric analysis of DNA ploidy, percent S-phase fraction, and total proliferative fraction as prognostic indicators of local control and survival following radiation therapy for prostate carcinoma.

PURPOSE: Treatment recommendations for localized prostate cancer may be improved by the identification of tumor factors prognostic for local control and survival. In this retrospective study, flow cytometric deoxyribonucleic acid (DNA) ploidy analysis and cell cycle analysis were performed on paraffin-embedded biopsy material to determine if additional prognostic factors could be identified in patients treated with radiation therapy. METHODS AND MATERIALS: Seventy patients with T1-4NxM0 tumors were identified in whom the primary treatment had been radical radiation therapy with no prior or concurrent endocrine therapy and in whom sufficient prostatic tissue was available for flow cytometric analysis. There were 40 diploid, 26 aneuploid, and 4 multiploid cases. Aneuploid and multiploid cases were combined for analysis. Cell cycle data were obtained on all diploid and 10 aneuploid cases. RESULTS: The histologic differentiation of the tumor (well or moderate vs. poor) was an independent predictor of overall survival and disease-free survival (p = 0.05 and 0.01, respectively). Local control was worse in the poorly differentiated patients, although this was not statistically significant in a multivariate analysis (p = 0.08). Neither T-stage, deoxyribonucleic acid ploidy (diploid vs. nondiploid), percent S-phase fraction, nor total proliferative fraction (S-phase fraction + G2M) significantly predicted for any of these endpoints. Within the diploid and well or moderately differentiated subgroup (n = 25), S-phase (< 4.2 vs. > or = 4.2) was a significant predictor of local control (100% vs. 51%, p = 0.03). A comparable distinction could be made using total proliferative fraction (< 10% vs. > or = 10%) with local control rates of 100% vs. 56% (p = 0.05). Among the poorly differentiated tumors, no similarly favorable subgroup was identified. CONCLUSIONS: This retrospective and multivariate analysis identifies both histology and percent S-phase or total proliferative fraction as predictors of local control following irradiation, and confirms that histology, but not DNA ploidy, is significant for overall survival. If these previously unreported findings are confirmed by prospective studies, S-phase should be added to histology as a parameter in the evaluation of clinical trials.

Cell Division↗
PubMed2006

A partially coupled, fraction-by-fraction modelling approach to the subsurface migration of gasoline spills.

The subsurface spreading behaviour of gasoline, as well as several other common soil- and groundwater pollutants (e.g. diesel, creosote), is complicated by the fact that it is a mixture of hundreds of different constituents, behaving differently with respect to e.g. dissolution, volatilisation, adsorption and biodegradation. Especially for scenarios where the non-aqueous phase liquid (NAPL) phase is highly mobile, such as for sudden spills in connection with accidents, it is necessary to simultaneously analyse the migration of the NAPL and its individual components in order to assess risks and environmental impacts. Although a few fully coupled, multi-phase, multi-constituent models exist, such models are highly complex and may be time consuming to use. A new, somewhat simplified methodology for modelling the subsurface migration of gasoline while taking its multi-constituent nature into account is therefore introduced here. Constituents with similar properties are grouped together into eight fractions. The migration of each fraction in the aqueous and gaseous phases as well as adsorption is modelled separately using a single-constituent multi-phase flow model, while the movement of the free-phase gasoline is essentially the same for all fractions. The modelling is done stepwise to allow updating of the free-phase gasoline composition at certain time intervals. The output is the concentration of the eight different fractions in the aqueous, gaseous, free gasoline and solid phases with time. The approach is evaluated by comparing it to a fully coupled multi-phase, multi-constituent numerical simulator in the modelling of a typical accident-type spill scenario, based on a tanker accident in northern Sweden. Here the PCFF method produces results similar to those of the more sophisticated, fully coupled model. The benefit of the method is that it is easy to use and can be applied to any single-constituent multi-phase numerical simulator, which in turn may have different strengths in incorporating various processes. The results demonstrate that the different fractions have significantly different migration behaviours and although the methodology involves some simplifications, it is a considerable improvement compared to modelling the gasoline constituents completely individually or as one single mixture.

Computer Simulation↗
PubMed1980

Allogeneic hemopoietic stem cell transplantation using mouse spleen cells fractionated by lectins: in vitro study of cell fractions.

Mouse spleen cells sequentially agglutinated by soybean agglutinin (SBA) and peanut agglutinin (PNA) previously had been shown to be sufficiently depleted of graft-versus-host activity to allow reconstitution of lethally irradiated allogeneic recipient mice. We have now tested the extent of T-cell depletion in this cell fraction by various in vitro assays, including cytotoxicity testing with anti-Thy-1 antiserum, mitogenic response to the T-cell mitogens concanavalin A and phytohemagglutinin and allogeneic responsiveness in the mixed lymphocytes culture assay. By these criteria the SBA+, PNA+ spleen fraction, used previously in the in vivo experiments, was found to possess about 1% T-cell contamination. The slight contamination with T cells previously found in the singly agglutinated SBA fraction can be removed by a second fractionation with SBA, thus eliminating the possibility that a minor T-cell subpopulation bears receptors for SBA. Finally, we demonstrated that the twice-agglutinated fraction, by SBA and PNA or by SBA alone, contains a significant number of prothymocytes, thereby indicating that mouse prothymocytes bear receptors for both SBA and PNA. The implication of these findings to bone marrow transplantation in humans is discussed.

Animals↗