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PubMed2005

The use of a sequential leaching procedure for heavy metal fractionation in green liquor dregs from a causticizing process at a pulp mill.

A five-stage sequential leaching procedure was used to fractionate heavy metals (Cd, Cu, Pb, Cr, Zn, Fe, Mn, Ni, Co, As, V, Ba and Ti) in green liquor dregs into the following fractions: (1) water-soluble fraction (H2O), (2) exchangeable fraction (CH3COOH), (3) easily reduced fraction (HONH3Cl), (4) oxidizable fraction (H2O2 + CH3COONH4), and (5) residual fraction (HF + HNO3 + HCl). The green liquor dregs were derived from a causticizing process at a pulp mill at Kemi, Northern Finland. According to the leaching studies, the leachability of heavy metals in the water-soluble fraction varied between 0.5 and 2 mg kg(-1) expressed on a dry weight (d.w.) basis, indicating relatively low bioavailability of the metals. However, the concentration of Mn (2065 mg kg(-1); d.w.) showed a strong and of Zn (17.6 mg kg(-1); d.w.), Ni (39.7 mg kg(-1); d.w.) and Ba (32.0 mg kg(-1); d.w.) slightly tendency to be extracted in the exchangeable fraction. In addition, Zn, Mn, Ni, Co, V and Ba showed clear leachability in the easily reduced fraction, as well as Cd, Cu, Cr, Zn, Mn, As and Ba in the oxidizable fraction. For Cd, Cu, Cr, Zn, Mn, Ni, Co, Ba and Ti, the sum of leachable heavy metal concentrations in fractions 1-5 agreed relatively well with the "total" heavy metal concentrations. Recoveries of the sum of fractions 1-5 were 84-56% of those obtained by the US EPA method 3052 (i.e. concentrations obtained after microwave oven digestion with a mixture of HF and HNO3).

Chemical Fractionation↗
PubMed1992

The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells.

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5'-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.

Adenocarcinoma↗
PubMed2002

Lens membrane fraction associated intermediate filaments of different aged rats.

PURPOSE: To describe the intermediate filament proteins vimentin, filensin and phakinin associated with different fractions isolated from neonatal, 10 day old and 20 day old rat lenses. METHODS: Fractions were isolated by differential and density gradient centrifugation of lens homogenates from neonatal, 10 day old and 20 day old rats. Aliquots of the 8 M urea soluble proteins of each fraction were separated by SDS PAGE, transferred to PVDF membranes, the membranes were probed with antibodies to vimentin, filensin or phakinin, and analyzed by computer. RESULTS: Over the 20 day growth period, the water soluble fraction increased and the most abundant membrane fraction was characterized by a significant increase in its urea insoluble protein and a significant decrease in its urea soluble protein. There were no significant quantitative changes in any of the other fractions. The concentration of each intermediate filament protein was greatest in the cytoskeletal fraction and over the 20 day period, the amount of vimentin associated with this fraction dramatically decreased, and the amounts of filensin and phakinin dramatically increased. Among the membrane fractions, the greatest concentration of each intermediate filament protein was found in the non sedimenting membrane fraction (NSMF) which was the least abundant fraction recovered. Filensin and phakinin associated with the other three major membrane fractions increased over the 20 day growth period, but the level of vimentin did not significantly change. CONCLUSIONS: The NSMF may represent a domain of the lens plasma membrane particularly important in interaction between plasma membrane and cytoskeleton and as the membrane-cytoskeleton protein architecture of rat lens changes over the first 20 days of life, the changes are readily detected in the different membrane fractions.

Animals↗
PubMed2003

Single- versus multiple-fraction radiotherapy in patients with painful bone metastases: cost-utility analysis based on a randomized trial.

BACKGROUND: Radiotherapy is an effective palliative treatment for cancer patients with painful bone metastases. Although single- and multiple-fraction radiotherapy are thought to provide equal palliation, which treatment schedule provides better value for the money is unknown. We compared quality-adjusted life expectancy (the overall valuation of the health of the patients) and societal costs for patients receiving either single- or multiple-fraction radiotherapy. METHODS: A societal cost-utility analysis was performed on a Dutch randomized, controlled trial of 1157 patients with painful bone metastases that compared pain responses and quality of life from a single-fraction treatment schedule of 8 Gy with a treatment schedule of six fractions of 4 Gy each. The societal values of life expectancies were assessed with the EuroQol classification system (EQ-5D) questionnaire. A subset of 166 patients also answered additional questionnaires to estimate nonradiotherapy and nonmedical costs. Statistical tests were two-sided. RESULTS: Comparing the single- and multiple-fraction radiotherapy schedules, no differences were found in life expectancy (43.0 versus 40.4 weeks, P =.20) or quality-adjusted life expectancy (17.7 versus 16.0 weeks, P =.21). The estimated cost of radiotherapy, including retreatments and nonmedical costs, was statistically significantly lower for the single-fraction schedule than for the multiple-fraction schedule ($2438 versus $3311, difference = $873, 95% confidence interval [CI] on the difference = $449 to $1297; P<.001). The estimated difference in total societal costs was larger, also in favor of the single-fraction schedule, but it was not statistically significant ($4700 versus $6453, difference = $1753, 95% CI on the difference = -$99 to $3604; P =.06). For willingness-to-pay between $5000 and $40 000 per quality-adjusted life year, the single-fraction schedule was statistically significantly more cost-effective than the multiple-fraction schedule (P< or =.05). CONCLUSIONS: Compared with multiple-fraction radiotherapy, single-fraction radiotherapy provides equal palliation and quality of life and has lower medical and societal costs, at least in The Netherlands. Therefore, single-fraction radiotherapy should be considered as the palliative treatment of choice for cancer patients with painful bone metastases.

Adult↗
PubMed1978

Biochemical and morphological characterization of subcellular fractions isolated from rabbit colon muscle.

From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondrial fragments and microsomal vesicles at 35--45% sucrose and a small protein fraction at 45--55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content was equally distributed between the microsomal fractions 35% and 35--45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome c oxidase was found to be concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35--45%. The NADH-oxidase activity was highest in the 35% fraction and the 5'-nucleotidase activity in the 40,000 X g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [3H]-ouabain and the incorporation of [3H]-leucine was most pronounced in the 35% fraction. In a K+-free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. From these results we concluded that the fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35--45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.

Adenosine Triphosphatases↗
PubMed1985

Morphological and functional characterization of interstitial cells from mouse testes fractionated on Percoll density gradients.

Cell fractions were obtained by separation on Percoll density gradients after dissociation of mature mouse testes by mechanical or collagenase dispersion, and the ultrastructure, hCG receptor properties, and hCG-stimulated testosterone (T) production of these fractions were compared. Gradients were fractionated according to specific gravity, and all cell types were quantitated using morphometric techniques. Three peaks of specific [125I]iodo-hCG binding corresponding to densities of 1.0667 g/cm3 (fractions 2-3), 1.045 g/cm3 (fractions 6-7), and 1.0365-1.0215 g/cm3 (fraction 9) were obtained after collagenase dispersion, but the second peak of binding (fractions 6-7) was not observed after mechanical dispersion. Morphometric studies were performed by light microscopy on the cells present in the fractions corresponding to the first and second hCG binding peaks obtained by either mechanical or collagenase dispersion; the third representing germ cells and membranous debris was not studied further. Regardless of the method of preparation, morphologically intact Leydig cells represented 60-80% and 7-10% of the cells in fractions 2-3 and 6-7 that were associated with the first and second peaks of hCG binding Leydig cells obtained from fractions 6-7 contained greater numbers of lipid inclusions than Leydig cells from fractions 2-3. Mechanically dispersed Leydig cells exhibited similar numbers of hCG receptors in the dense and light Leydig cells, but hCG stimulated T production per Leydig cell was significantly greater in the dense Leydig cells containing few lipid inclusions. T production by dense and light collagenase-dispersed Leydig cells was not significantly different. The second hCG binding peak in collagenase-dispersed cell fractions 6-7 was associated with an increase in the number of an indeterminate connective cell type released from the testes by collagenase treatment, whose ultrastructure and limited hCG-binding capacity suggested that they may represent Leydig cell precursors. It is concluded that the identification and quantitation of different cell types in isolated testicular cell fractions is, therefore, of fundamental importance in the interpretation of receptor and secretory capacities of enriched preparations of Leydig cells.

Animals↗
PubMed1992

Macromolecule-macromolecule interaction in drug distribution. II. Effect of alpha-globulin on saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes in primary culture.

The uptake of fractionated [3H]heparin was investigated in rat parenchymal hepatocytes in primary culture. The initial uptake of fractionated [3H]heparin was found to be saturable with the maximum uptake velocity (Vmax) of 10.1 +/- 1.46 pmol/min/mg protein and the Michaelis constant (Km) of 284 +/- 47.9 nM. The effect of alpha-globulin, the major protein binding to fractionated [3H]heparin, on the saturable uptake profile of fractionated [3H]heparin was also investigated. The uptake clearance was reduced, depending on the concentration of fractionated [3H]heparin, by the addition of 1 mg/ml alpha-globulin. We assumed that fractionated 3H-heparin bound to alpha-globulin was not available for uptake and that the reduction in the uptake clearance was solely attributable to the saturable binding of fractionated [3H]heparin to alpha-globulin. The uptake clearance versus concentration profile was analyzed to obtain the dissociation constant (Kd) of 31.8 nM and the capacity (n) of 0.047 for the binding of fractionated [3H]heparin to alpha-globulin. The saturable binding of fractionated [3H]heparin to alpha-globulin was supported by in vitro binding experiments using gel chromatography, in which bound fractionated [3H]heparin decreased with the concentration of fractionated [3H]heparin in the presence of alpha-globulin. In conclusion, the present study demonstrated the saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes and the saturable binding of fractionated [3H]heparin to alpha-globulin. The saturable uptake may suggest the involvement of a specific transport system such as receptor-mediated endocytosis.

Alpha-Globulins↗
PubMed2004

Dose fractionation in stereotactic radiotherapy for parasellar meningiomas: radiobiological considerations of efficacy and optic nerve tolerance.

OBJECT: Benign meningiomas have been shown to be equally well controlled with single-dose radiosurgery (15 Gy) and fractionated doses of 54 Gy in 30 fractions after adequate follow up. For a subset of patients with meningioma, the optic apparatus is dose limiting when considering single-dose stereotactic radiosurgery, with tolerance estimated to be 8 to 10 Gy. Recently, hypofractionated regimens have been used to treat benign meningiomas with a small number of fractions. An analysis of the expected efficacy of hypofractionation compared with the estimated optic tolerance to fractionated radiotherapy was undertaken. METHODS: Using the assumption that 15 Gy in one fraction and 54 Gy in 30 fractions are isoeffective for control of benign meningioma, an alpha/beta for meningioma is calculated to be 3.28 Gy. Invoking a 10% error for these doses (15 Gy+/-10% is equivalent to 54 Gy+/-10%) results in upper and lower limits of the estimate for alpha/beta of 3.85 Gy and 2.7 Gy. Using these estimates, isoeffect curves for control of meningioma were constructed for fraction numbers of one to 45. Best estimates of optic nerve/chiasm tolerance to single doses of radiation are 8 to 10 Gy, with the reported incidence of optic neuropathy increasing significantly at higher doses. This is consistent with the optic ret model, which also predicts for optic tolerance following fractionated radiotherapy. Comparison of optic tolerance and estimates of efficacious doses at fraction numbers between one and 30 were made. Statistical estimates of patient numbers and duration of follow up required to rule out optic neuropathy following radiotherapy were made. Single doses of radiation required to treat benign meningioma optimally (13.5-16.5 Gy) clearly exceed the estimated and reported clinical tolerance of the optic nerves and chiasm. The application of equivalent biological doses in a small number of fractions continues to exceed optic tolerance until at least 25 fractions are applied. CONCLUSIONS: The use of small numbers of fractions to treat patients with meningioma when portions of optic nerve or chiasm receive full dose may result in undertreatment of the tumor and/or exceeding optic nerve tolerance. In such cases standard fractionation is recommended. Ruling out a low, yet unacceptable, risk of optic neuropathy may require the close study of many patients with long-term follow-up evaluation.

Dose Fractionation, Radiation↗
PubMed1981

Lipid composition of axolemma-enriched fractions from human brains.

The lipid composition was determined for axolemma-enriched fractions and myelin which were isolated via a preparation of purified myelinated axons. The myelin had a lipid composition which was compatible with that previously reported for myelin isolated by alternative procedures. The most dense axolemma-enriched fraction contained 25.3% cholesterol, 25.8% galactolipid (21.3% cerebrosides and 4.8% sulfatides), and 48.9% phospholipid. The major phospholipids were the ethanolamine phospholipid (19.8% of total lipid weight; 49.0% in the plasmalogen form) and choline phospholipids (18.7% of total lipid weight; 16.0% in the plasmalogen form) with lesser amounts of sphingomyelin, phosphatidylserine, and phosphatidylinositol also present; the ganglioside content was 13.9 micrograms of acetylneuraminic acid per mg protein. The less dense axolemma-enriched fraction had a lipid composition which was intermediate between that of myelin and the more dense axolemma-enriched fraction. On the average, less than 2.3% of the total protein in the axolemma-enriched fraction was myelin basic protein. Both axolemma-enriched fractions stained uniformly with Luxol fast blue and demonstrated specific saxitoxin-binding which was enriched 2- to 7-fold over that of the whole white matter homogenate from which the fractions were isolated. The choline and ethanolamine phospholipids in that most dense axolemma-enriched fractions contained a greater percentage of unsaturated fatty acids compared with the comparable phospholipids in myelin. The content of unsaturated fatty acids in these phospholipids of the axolemma-enriched fraction was not as great as that of human CNS synaptic plasma membranes. However, the chain length distribution of these phospholipid fatty acids was similar in myelin, synaptic plasma membrane, and the axolemma-enriched fraction. The distribution of aldehydes derived from the ethanolamine phospholipids of the more dense axolemma-enriched fraction closely resemble the distribution of the comparable aldehydes in the myelin fraction. The possible origin and function of the lipids in the axolemma-enriched fractions are discussed.

Aged↗
PubMed1988

Partial characterization of a fraction from human follicular fluid that initiates the human sperm acrosome reaction in vitro.

A human follicular fluid (HFF) fraction prepared by Sephadex G-75 column chromatography has been previously shown by this laboratory to initiate the human sperm acrosome reaction (AR) in vitro. In the present report, the apparent molecular weight (MW) of this AR activity determined by a longer G-75 column than was used in the previous work was 50,000 +/- 5,106. The G-75 Sephadex void volume fractions of some but not all HFF samples were also found to contain some AR-initiating activity. The occasional void volume activity was less potent than that of the 50,000 MW fraction and was not studied further. Further characterization of the 50,000 MW fraction was carried out. A time-course study demonstrated that maximum AR were obtained within 5 min following the addition of the 50,000 MW fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining revealed that the 50,000 MW fraction was still a relatively crude preparation. Treatment of the 50,000 MW fraction with chloroform:methanol did not extract the AR-initiating activity into the lipid phase. The AR-initiating activity of the untreated 50,000 MW fraction was precipitated when it was boiled, but the activity was partially resistant to boiling after overnight incubation. Treatment of the 50,000 MW fraction with pronase E or with several glycosaminoglycan hydrolases did not destroy the activity. Pronase treatment resulted in a higher amount of boiling-resistant AR-initiating activity. The AR-initiating activity of the untreated 50,000 MW fraction was partially dialyzable, but the activity of an undialyzed fraction did not pass through an ultrafiltration membrane with a 10,000 MW cut-off. However, treatment of the 50,000 MW fraction with protease, peptide:N-glycosidase F, and to a lesser extent chondroitinase ABC yielded an active lower MW activity which could pass through such an ultrafiltration membrane. The lower MW activity released by peptide:N-glycosidase F eluted in the included volume (5,000-1,000) of a Sephadex G-25 column. Neutral hexose but not protein or peptide was detected in the G-25 peak of AR-initiating activity. These results suggest that the AR-initiating activity present in the 50,000 MW fraction of HFF: 1) is present either as two different AR factors (a high-MW factor and a low-MW, noncovalently bound factor) or as a single factor responsible for both the nondialyzable and dialyzable AR-initiating activities (the latter being enzymatically released from the former), and 2) may be at least partially associated with N-linked oligosaccharides of a glycoprotein or proteoglycan.

Acrosome↗
PubMed2000

Contribution of planar (0-1 ortho) and nonplanar (2-4 ortho) fractions of Aroclor 1260 to the induction of altered hepatic foci in female Sprague-Dawley rats.

The hepatic tumor promoting activity of the planar 0-1 ortho ( approximately 9.7% w/w) and the nonplanar 2-4 ortho ( approximately 90.3% w/w) fraction of the commercial PCB mixture Aroclor 1260 was studied using a medium-term two-stage initiation/promotion bioassay in female Sprague-Dawley rats. Fractionation was carried out on an activated charcoal column. The composition of the effluent from the column was tested by GC-ECD. The absence of planar compounds in the 2-4 ortho fraction was confirmed by GC-MS analysis. The dioxin-like toxic potency of the fractions was determined with the DR-CALUX assay. The animal experiment was started with the initiation procedure (diethylnitrosamine injection, 30 mg/kg body wt ip, 24 h after (2)/(3) hepatectomy), followed 6 weeks later by the promotion treatment, which consisted of a weekly subcutaneous injection during 20 weeks. Exposure groups (n = 10) received the following treatments (dose/kg body wt/week): Aroclor 1260 (10 mg), 0-1 ortho fraction (0.97 mg), 2-4 ortho fraction (1, 3, or 9 mg), a reconstituted 0-4 ortho fraction (9.97 mg), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153; 1 or 9 mg), 2,3,7,8-TCDD (1 microg; positive control) or corn oil (1 ml; vehicle control). One group did not receive a promotion treatment. All exposure groups exhibited a significantly increased volume fraction of the liver occupied by hepatic foci positive for the placental form of glutathione-S-transferase-p compared to the corn oil control, except for the groups treated with 0-1 ortho fraction and 1 mg PCB 153/kg body wt/week. Approximately 80% of the total tumor promoting capacity of the reconstituted 0-4 ortho fraction could be explained by the 2-4 ortho PCB fraction while the 0-1 ortho fraction had only a negligible contribution. These results suggest that the majority of the tumor promotion potential of PCB mixtures resides in the non-dioxin-like fraction, which is not taken into account in the toxic equivalency factor (TEF) approach for risk assessment of PCBs. This may result in an underestimation of the tumor promotion potential of environmental PCB mixtures.

Animals↗
PubMed1999

Oxygen tension in transplanted mouse osteosarcomas during fractionated high-LET- and low-LET radiotherapy--predictive aspects for choosing beam quality?

PURPOSE: The lower OER of high-LET radiations, compared to conventional (low-LET) radiations, has often been put forward as an argument for using high-LET radiotherapy in the management of hypoxic tumours. Among the different neutron beams used in therapy, the reactor fission neutrons have the lowest OER. The aim of the present study is to follow the variations of tumour oxygenation status during fractionated irradiation with different radiation qualities. Little information is available so far after fractionated high-LET irradiation. In addition, the RBE of reactor fission neutrons for effects on tumours and on normal tissues are compared. MATERIAL AND METHODS: Murine OTS 64-osteosarcomas were transplanted in 102 balb-C mice and irradiated by 36 Gy of photons in fractions of 3 Gy five times a week (group P-36/3) or by 12 Gy of reactor fission neutrons in fractions of 2 Gy two times a week (group N-12/2). Irradiations started at a tumor volume of 500 to 600 mm3. A third group received no radiotherapy, but all investigations (group CG). Tumor volume and tumor oxygenation were measured once a week under therapy and during three weeks after therapy. For in vivo-evaluation of oxygen status a computerized polarographic needle electrode system (KIMOC pO2 histograph, Eppendorf) was used. The median pO2 and the hypoxic fraction (pO2 values < 5 mm Hg) of single tumors and of total groups were calculated from pooled histograms and from row data as well. RESULTS: In correlation with the increase of tumor volume, from day 1 to day 42 of follow-up the median pO2 decreased from 20 mm to 8 mm Hg and the hypoxic fraction increased from 7% to 31%. After fractionated photon therapy a growth delay of three weeks was observed. Six weeks after beginning of the irradiation the median tumor volume had been doubled again. After fission neutron therapy growth delay continued until the end of the follow-up period. In both of the irradiated groups a significant decrease of median pO2 values and an increase of the hypoxic fraction were observed under radiotherapy. Hypoxia was more intensive after neutrons with a decrease of the median pO2 from 20 mm Hg to 1 mm Hg vs. 10 mm Hg after photon therapy and with an increase of the hypoxic fraction from 7% to 78% vs. 36% respectively. Two weeks after the end of therapy the median pO2 and the hypoxic fraction of both treated groups reached the levels prior to irradiation indicating a complete reoxygenation. CONCLUSION: During fractionated irradiation of murine osteosarcomas with photons and reactor fission neutrons, a marked hypoxia was observed for both radiation qualities, but hypoxia was more intense during fractionated neutron irradiation. After irradiation, a complete reoxygenation occurred in both groups independently of the degree of hypoxia observed during the treatment. The RBE of reactor fission neutrons, after fractionated irradiation, was much higher for effects on murine osteosarcomas compared to their RBE observed for normal tissues in previous experiments. Present data are in agreement with our clinical observations on more than 300 patients treated with reactor fission neutrons for advanced and hypoxic tumours with various histologies.

Animals↗
PubMed1992

Adsorption from salivary fractions at solid/liquid and air/liquid interfaces.

Ellipsometry and the drop-volume technique were used to study the interfacial behaviour of fractions obtained from unstimulated whole saliva. Fractionation was by gel filtration on a Superdex 200 Hiload column equilibrated with 10 mM potassium phosphate buffer, pH 6.8, containing 0.15 M NaCl. The fractions were reconstituted to have the same absorbance at 215 nm (estimated molecular-weight range, F1 greater than 760-460 K, F2 205-39 K, F3 14-4.5 K, F4 4.5-2.5 K, F5 1.5-0.85 K, F6 0.85 less than or equal to 0.5 K). The fractions were analysed for amino acid composition and studied by hydrophobic interaction chromatography on a Phenyl-Superose column. Fraction 3 contained the largest amounts of proline, followed by fractions 4 and 2. Fraction 3 showed the highest relative hydrophobicity. Ellipsometric measurements on negatively charged silica surfaces and methylated hydrophobic surfaces revealed that larger amounts of material adsorbed on hydrophobic than on hydrophilic surfaces. On hydrophilic surfaces the largest amounts were adsorbed from the high molecular-weight fraction 1. Fractions 4 and 6 did not give any adsorption at all on these surfaces. Fraction 3 gave the largest amounts adsorbed on the hydrophobic surfaces. Drop-volume measurements showed distinct differences in the ability of the salivary fractions to lower the surface tension. Fractions 2 and 3 showed the greatest reduction in surface tension. It was concluded that the adsorption behaviour of salivary proteins showed a wide variation among the different fractions and that it is influenced by the physicochemical characteristics of the interfaces present in the mouth.

Adsorption↗
PubMed1976

Effects of estrogen on gene expression in the chick oviduct. Isolation and fractionation of chromatin non-histone proteins.

Non-histones isolated from hen oviduct chromatin have been fractionated by a variety of methods. Chromatin was dissociated in 2 M NaC1, 5 M Urea, 0.1% beta-mercaptoethanol and 0.01 M Tris - HC1, pH 8.3, and the DNA removed by ultracentrifugation. After desalting by gel filtration the chromatin proteins were separated into three distinct fractions by stepwise elution with 0.10 M NaC1, 0.25 M Na C1 and 15% guanidine - HC1 from Bio-Rex 70 columns. Fractions I and II contain only non-histones and Fraction III contains histones plus a small amount of non-histones. Further fractionation of the non-histones was achieved by ammonium sulfate precipitation and DEAE-cellulose chromatography for Fraction I and phosphocellulose chromatography and gel filtration on Bio-Gel A-15 m for Fraction II. The histone and non-histones present in Fraction III were separated by gel filtration on Bio-Gel A-0.5 m. All fractionation methods have been used preparatively with reasonable recoveries of protein (greater than or equal to 60%). The fractions have been characterized by acrylamide gel electrophoresis. The integrity of the histones was maintained during the fractionation procedure indicating that proteolytic degradation was unlikely to have occurred. There was no selective loss of chromatin proteins during the ultracentrifugation and desalting steps and the non-histones were separated into distinct fractions with enrichment of some species not apparent prior to fractionation of the chromatin proteins.

Animals↗
PubMed1980

Electrophoretic fractionation of serum gamma-glutamyl transpeptidase in human hepatic cancer.

Serum gamma-glutamyl transpeptidase from patients with various hepatobiliary diseases was fractionated by polyacrylamide gradient gel slab electrophoresis to study the specific patterns of gamma-glutamyl transpeptidase fractions in hepatic cancer. On zymograms of normal serum gamma-glutamyl transpeptidase, a total of 10 fractions was observed. Additionally, fractions I', I" and II' were recognized in sera from hepatocellular carcinoma patients. Among these, fraction I', which migrated slightly, but significantly, slower than fraction I was the most specific; it was found in 55% of the hepatocellular carcinoma patients. Fractions I" and II' were also relatively specific, each was observed in about 29% of these patients. Fractions V to IX were observed in few hepatocellular carcinoma cases. Fraction I' is thought to be a hepatoma-related fraction, highly specific for the serum of hepatocellular carcinoma patients. Fractions I" and II' were also thought to be hepatoma-related fractions of gamma-glutamyl transpeptidase. We suggest that fractions I', I" and II' may be useful in the diagnosis of hepatocellular carcinoma.

Carcinoma, Hepatocellular↗
PubMed1985

Influence of cell concentration on the contribution of unfrozen fraction and salt concentration to the survival of slowly frozen human erythrocytes.

We have shown previously that the survival of human red blood cells during slow freezing at 2% hematocrit is dependent more on the magnitude of the unfrozen fraction than on the salt concentration in that unfrozen fraction. In parallel, first Nei and more recently Pegg and colleagues have shown that survival is affected by the hematocrit of the suspension. Freezing at hematocrits above 30% becomes increasingly damaging. The present studies were designed to see whether there is a link between the two phenomena. Cells were suspended at nominal hematocrits of 0.4, 2, 8, 40, or 60% in five test solutions of glycerol-NaCl. The test solutions were of such composition that when frozen to a specified temperature, the magnitude of the unfrozen fraction differed but the NaCl concentration (ms) remained constant. At low hematocrits (0.4 to 8%), red cell survival was dependent predominantly on the unfrozen fraction and was relatively independent of the salt concentration in that fraction. This we term the "rheological" effect because injury appears to be related to interaction with the ice walls and perhaps is due to shearing forces or cell deformation. But at high hematocrits (40 or 60%), cell survival became dependent on both the unfrozen fraction and the salt concentration in that fraction. When freezing occurs at high hematocrits, increasing numbers of cells are presumably brought into contact with their neighbors. Furthermore, they are increasingly shrunken cells, for the progressive removal of liquid water, which is responsible for the crowding, also causes a rise in ms and the consequent osmotic shrinkage of cells. Our data suggest that at unfrozen fractions above those producing injurious rheological forces, the tight packing of less shrunken cells (i.e., high hematocrit, low ms) and the extensive shrinking of loosely packed cells (high ms, low hematocrit) are both quite innocuous. Injury becomes substantial only when extensively shrunken cells are brought into close contact (i.e., high ms, high hematocrit). At high hematocrit the cells occupy a substantial fraction of the unfrozen space, and the water that they lose during slow freezing adds substantially to the volume of extracellular ice. Accordingly, we defined other measures of unfrozen fraction that include these perturbations. However, we found that the conclusions on the relation between survival, unfrozen fraction, and hematocrit were not affected by the method of expressing the unfrozen fraction. Freezing at high hematocrit to high ms and low values of unfrozen fraction is one way to produce contact between shrunken cells at low temperatures.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Preservation↗
PubMed1984

Separation of macrophages on discontinuous bovine serum albumin (BSA) density gradients: cytotoxic effects of fractionated cells from normal donors and cancer patients.

Human monocytes from normal donors as well as breast or colon cancer patients were fractionated on five-step discontinuous bovine serum albumin (BSA) density gradients, and the monocytes from each fraction were allowed to mature into macrophages during a 5 day incubation period. The macrophages were then examined both for their ability to kill tumor cells after activation with lipopolysaccharide (LPS) and the quantity of prostaglandin E2 (PGE2) synthesized. When the macrophages obtained from normal donors were fractionated, fractions 2 and 4 which comprised 58% of the total cell population, were cytotoxic for tumor cells. In contrast, when the macrophages from breast cancer patients were fractionated, only the high density cells found in fraction 4 were cytotoxic. It is also conceivable that since fraction 4 comprises only 26% of the total macrophages recovered, this may be the reason unfractionated macrophages from breast cancer patients are unable to kill tumor cells. When the colon cancer patients' macrophages were fractionated on BSA density gradients, fractions 1, 2 and 4, which comprised 79% of the total cell population, were cytotoxic for tumor cells. Prostaglandin E2 synthesis was also analyzed and it was found that fraction 3 consistently synthesized increased quantities of PGE2 when compared with the other three fractions. Furthermore, since fraction 3 was non-cytotoxic for tumor cells, it is conceivable that the increased synthesis of PGE2 by fraction 3 rendered these macrophages non-cytotoxic.

Animals↗
PubMed1994

Single dose versus fractionated total body irradiation before bone marrow transplantation: radiobiological and clinical considerations.

PURPOSE: This present review is intended to evaluate the specific influence of fractionation of total body irradiation on the outcome of a subsequent bone marrow transplantation. METHODS AND MATERIALS: Available experimental and clinical data on the influence of fractionation on leukemia cell killing, immunosuppression, and sparing of normal tissues were analyzed. RESULTS: Review of available data shows: (a) The role of fractionation on leukemia cell killing may vary with the leukemia type. For acute nonlymphoblastic leukemia, a few experimental and several clinical studies show no or little fractionation effect; a 12-13 Gy fractionated scheme could, therefore, be more efficient than a conventional 10 Gy single dose total body irradiation. For chronic myelogenous leukemia, some sensitivity to fractionation is suggested, so that an increase in total or fractional dose may be necessary in fractionated schemes to equate the efficacy of a 10 Gy single dose. For acute lymphoblastic leukemia, a high fractionation sensitivity was observed for some leukemic cell lines in vitro, without undisputable clinical confirmation for the moment. (b) Numerous experimental studies have demonstrated that the immunosuppressive effect of total body irradiation, a major determinant of engraftment, is highly fractionation sensitive. In humans, high rates of graft failures have been reported when T-cell depletion of the graft was associated to fractionated total body irradiation schedules. (c) A large amount of radiobiological and clinical data have demonstrated that late radiation-induced injuries to normal tissues and organs are highly fractionation sensitive. However, in a context of total body irradiation for bone marrow transplantation, the number of other determinants of normal tissue damage makes it difficult to demonstrate a clear-cut advantage of fractionated over single dose scheme, with a possible exception for children. CONCLUSIONS: In 1994, available data suggest that very cautious attempts could be made to adapt total body irradiation schedules to the potential normal tissue toxicity, T-cell depletion, and to the type of leukemia.

Bone Marrow↗