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PubMed1989

Intracellular distribution of copper in the liver of copper-loaded sheep--a subcellular fractionation study.

Eighteen ewes in two groups were dosed orally with CuSO4 to induce chronic Cu toxicity. Copper dosing was stopped at the first rise of acid AP activity in the serum in group 1 sheep and on the first day of haemolysis in group 2 sheep. Liver samples were obtained 1 week prior to the start of Cu dosing, at the first rise of acid phosphatase (AP) activity in serum and on the first day of haemolysis. These liver samples were homogenized and were separated into nuclear (N), heavy mitochondrial (MH), light mitochondrial (ML), microsomal (MI) and cytosolic (CY) fractions by centrifugation. The Cu concentration and specific activities of AP were determined in the liver, LH and subcellular fractions. The composition of the fractions was studied by light and electron microscopy. In the predosing biopsies, the concentration and percentage of Cu and the total specific activity of AP were highest in the ML fractions. With increasing Cu loading, the concentration of Cu in all fractions increased; the percentage of Cu increased in the N and MH fractions, decreased in the ML and MI fractions and was maintained at a constant level in the CY fractions. The total specific activities of AP in LH, N, MH, MI and CY fractions were increased and the activity was highest in the MH fraction. The results indicate that the increase in the concentration of Cu in liver cells was predominantly in lysosomes and cytosol. Furthermore, it is suggested that the necrosis of isolated hepatocytes observed in chronic Cu-poisoned sheep may be due to a saturation of the uptake of Cu into the lysosomal system of the cell, leading to the accumulation of toxic levels of Cu in the cytosol.

Acid Phosphatase↗
PubMed1985

Separation and analysis of two plasma membrane fractions from bovine thyroid which differ in TSH binding and TSH activation of adenylate cyclase.

A tissue disruption technique leading to the separation of thyroid epithelial cell components from interfollicular material has been used to study the distribution and the properties of membrane adenylate cyclase originating from intraglandular thyroid and non-thyroid cells. Bovine thyroid fragments were forced through a metallic sieve. The material which filtrates was composed of open cells and cell debris (fraction A); the material remaining on the sieve contained the basal lamina and the interfollicular material as shown by photon and electron microscopic observations (fraction B). Homogenates (HA and HB) were prepared from fractions A and B and centrifuged on a 41% sucrose layer to prepare membrane fractions: MA and MB, which were tested for the presence of adenylate cyclase, TSH-responsive adenylate cyclase and 125I-labelled TSH binding activity. HA and HB contained respectively 70% and 30% of the total thyroid adenylate cyclase activity. MA and MB were similarly enriched in 5'-nucleotidase and adenylate cyclase: 8- to 10-fold as compared to the corresponding homogenates. MA and MB exhibited a marked difference in the response to TSH: TSH either alone or in the presence of Gpp(NH)p stimulated the adenylate cyclase of MA and did not have any effect on MB. Fractionation of MA by isopycnic centrifugation on Percoll gradients yielded a membrane peak exhibiting a TSH-responsive adenylate cyclase activity and a 125I-labelled TSH binding activity displaceable by an excess of unlabelled TSH. A membrane peak at the same density was obtained from MB but its adenylate cyclase did not respond to TSH and there was no specific binding of labelled TSH. Our data indicate that an important fraction of membrane adenylate cyclase of the thyroid does not seem to be coupled with TSH receptor; the major part of this fraction (MB) likely originates from intraglandular non-thyroid epithelial cells. The separation of this membrane fraction from the thyroid cell plasma membrane fraction (MA) allows to increase the response of this latter fraction to TSH.

Adenylyl Cyclases↗
PubMed2005

Factors influencing the use of single vs multiple fractions of palliative radiotherapy for bone metastases: a 5-year review.

AIMS: Evidence from a number of randomised trials and meta-analyses supports the use of single-fraction radiotherapy for the palliation of painful bone metastases. This study explores patient and treatment factors that influence the choice of single compared with multiple-fraction radiotherapy for the treatment of bone metastases in clinical practice. MATERIALS AND METHODS: The Princess Margaret Hospital Palliative Radiation Oncology Program Database served as the basis for our report. All courses of treatment delivered for bone metastases were extracted. Courses were classified into single or multiple fractions. Clinical characteristics were compared between the two groups. RESULTS: Between 1998 and 2002, 882 courses of radiotherapy were delivered for the treatment of bone metastases, of which 283 (32%) were a single fraction. The proportion of single-fraction treatments was 37% in 1998, 30% in 1999 and 43% in 2000, but dropped to 26% and 28% in 2001 and 2002, respectively (P = 0.02). Patients treated with single fractions were significantly older (68 +/- 12 years vs 64 +/- 12 years), and had more weight loss and poor performance status. Single fractions included 20% of treatments in palliative irradiation of the spine, 36% in the pelvis and long bones, and 59% in the chest wall (P < 0.001). There was no significant difference in patients' gender, primary cancers, number of metastatic sites, treating physicians, enrollment in a clinical trial and general radiotherapy waiting time in our department. Multivariate analysis indicated age (P = 0.001), performance status (P < 0.001), anatomical site (P < 0.001) and year of radiotherapy (P = 0.006) as significant. CONCLUSION: One-third of palliative radiotherapy courses for bone metastases in our programme were given as single fractions. Performance status, age and anatomical site were significant factors affecting single compared with multiple fractionation. The variation in the use of single fractions over time may reflect the dynamic process of interpretation and application of evidence from clinical trials to practice.

Aged↗
PubMed1997

Red wine and fractionated phenolic compounds prepared from red wine inhibit low density lipoprotein oxidation in vitro.

The oxidative modification of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. This study examined the effect of red wine, ethanol and red wine stripped of phenols on copper-mediated and azo-initiated LDL oxidation. Red wine containing phenolic compounds (0.025-20 mg/l gallic acid equivalents) increased the lag time of conjugated diene formation, inhibited the generation of thiobarbituric acid reactive substances (TBARS) and decreased the relative electrophoretic mobility of LDL in a concentration-dependent manner. These changes were not apparent in LDL incubated with ethanol or red wine stripped of phenols. In other experiments, red wine (75 mg/l gallic acid equivalents) was incubated with plasma at 37 degrees C for 3 h. The LDL isolated from this plasma displayed a 60% increase in lag time following copper-mediated oxidation. Uptake of this LDL by cultured J774 macrophages was three-fold lower than control LDL. Red wine was fractionated into phenolic acids (fraction 1), catechins and monomeric anthocyanidins (fraction 2), flavonols (fraction 3) and polymeric anthocyanidins (fraction 4). All red wine fractions prolonged the time before LDL oxidation. Fraction 2 displayed a significantly greater antioxidant activity than fractions 3 and 4 (but not fraction 1) in at least one pro-oxidant model. In conclusion we have shown that antioxidant compounds in red wine can associate with LDL particles following an incubation in whole plasma, can exert an antioxidant effect and, in so doing, can inhibit the uptake of the lipoprotein by macrophages. This antioxidant effect of red wine was apparent in most of the phenolic fractions separated from wine, particularly catechins, monomeric anthocyanidins and phenolic acids.

Amidines↗
PubMed2002

Factors affecting risk of symptomatic temporal lobe necrosis: significance of fractional dose and treatment time.

PURPOSE: To study the factors affecting the risk of symptomatic temporal lobe necrosis after different fractionation schedules. METHODS AND MATERIALS: One thousand thirty-two patients with T1-2 nasopharyngeal carcinoma treated with radical radiotherapy in Hong Kong during 1990-1995 were studied. They were treated at four different centers with similar techniques but different fractionation schedules: 984 patients were given 1 fraction daily throughout (q.d.), and 48 patients were irradiated twice daily (b.i.d.) for part of the course. The median total dose was 62.5 Gy (range 50.4-71.2), dose per fraction was 2.5 Gy (range 1.6-4.2), and overall treatment time (OTT) was 44 days (range 29-70). In addition, 500 patients received supplementary doses for parapharyngeal extension, 113 received booster doses by brachytherapy, and 114 received sequential chemotherapy using cisplatin-based regimes. RESULTS: Altogether, 24 patients developed symptomatic temporal lobe necrosis: 18 from the q.d. group and 6 from the b.i.d. group. The 5-year actuarial incidence ranged from 0% (after 66 Gy in 33 fractions within 44 days) to 14% (after 71.2 Gy in 40 fractions within 35 days). Multivariate analyses showed that the risk was significantly affected by the fractional effect of the product of total dose and dose per fraction (hazard ratio [HR] = 1.04, 95% confidence interval [CI] 1.02-1.05), OTT (HR 0.88, 95% CI 0.80-0.97), and b.i.d. scheduling (HR 13, 95% CI 3-54). Repeating the analyses for patients treated with the q.d. schedules confirmed the independent significance of OTT in addition to the product of total dose and dose per fraction. CONCLUSION: The tentative results suggest that in addition to fractional dose, the OTT also had significant impact on the risk of temporal lobe necrosis, and b.i.d. scheduling increased the hazard further.

Adolescent↗
PubMed1998

Immunocontraceptive activity guided fractionation and characterization of active constituents of neem (Azadirachta indica) seed extracts.

A novel approach for immunocontraception by intervention of local cell mediated immunity in the reproductive system by using single intrauterine application of neem oil has been described earlier. The reversible block in fertility was reported to last for 107-180 days in female Wistar rats (Upadhyay et al., 1990. Antifertility effects of neem oil by single intrauterine administration: A novel method of contraception. Proceedings Of The Royal Society Of London B 242, 175-180) and 7-11 months in monkeys (Upadhyay et al., 1994. Long term contraceptive effects of intrauterine neem treatment (IUNT) in bonnet monkeys: An alternative to intrauterine contraceptive devices. Contraception 49, 161-167). The present study, describes the identification and characterization of the biologically active fraction from neem seeds (Azadirachta indica A. Juss. Family Meliaceae), responsible for the above activity in adult female Wistar rats. Initial studies with the mechanically extracted oil and solvent extracts of neem seeds have revealed that the antifertility activity was present in constituents of low to intermediate polarity. A hexane extract of neem seeds was reported to be biologically active (Garg et al., 1994. Comparison of extraction procedures on the immunocontraceptive activity of neem seed extracts. Journal of Ethnopharmacology 22, 87-92). Subsequently, hexane extract was sequentially fractionated through the last active fraction using various separation techniques and tested for antifertility activity at each step. Preparative HPLC was used for isolating individual components of the active fraction in quantities, sufficient for characterization. An analytical HPLC method was developed for standardization of the fraction. The active fraction was identified to be a mixture of six components, which comprises of saturated, mono and di-unsaturated free fatty acids and their methyl esters. Dose response study was performed with the last active fractions. The antifertility activity with the active fraction was reversible in nature and it was completely active until 5% concentration. There was no systemic toxic effect following the administration of the active fraction. This study, for the first time, proposes an active fraction from neem seeds, responsible for long term and reversible blocking of fertility after a single intrauterine administration with high efficacy.

Animals↗
PubMed2001

Single fraction external beam radiation therapy in the treatment of localized metastatic bone pain. A review.

Bone metastases are a frequent complication of cancer, and frequently cause pain. Indications for radiotherapy for bone metastases include pain, risk for pathologic fracture, and neurological complications arising from spinal cord compression, nerve root pain or cranial nerve involvement. There are numerous fractionation patterns of external beam radiation therapy for painful bone metastasis, both fractionated schedules and single fraction regimens. All prospective randomized trials that evaluated differences in the outcomes associated with various fractionated regimens versus single fraction regimens unequivocally showed that single fraction regimens (mostly 8 Gy) are at least equal with various fractionated regimens. The single fraction regimens have an additional advantage of being more convenient to both patients and hospitals. However, there are still numerous questions that are left unanswered in these trials, such as the "optimal" single fraction that should be used, the possibility for retreatment, and prognostic factors that may help identify those patients more likely to respond to a single fraction radiation therapy in the treatment of painful bone metastasis.

Bone Neoplasms↗
PubMed2003

Distribution of zeptomole-abundant doxorubicin metabolites in subcellular fractions by capillary electrophoresis with laser-induced fluorescence detection.

Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at < 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies.

Animals↗
PubMed1976

Sequence composition of the template-active fraction of rat liver chromatin.

Rat liver chromatin has been separated into nuclease-sensitive and -resistant fractions after mild digestion with DNAase II. The nuclease-sensitive material is further fractionated into Mg2+ -soluble and -insoluble chromatin fractions. The kinetics of production of these chromatin fractions have been investigated. After a brief enzyme treatment (5 min at 10 enzyme units/A260 unit of chromatin at pH 6.6), 11% of the input chromatin DNA is found in the Mg2+ -soluble fraction. This DNA has a weight-average single-strand length of about 400 nucleotides and, as determined by renaturation kinetics, comprises a subset of nonrepetitive DNA sequences and a subset of families of middle repetitive sequences. This demonstrates the nonrandom distribution of repetitive and single copy sequences in the Mg2+ -soluble fraction of chromatin. Previous studies have shown that the Mg2+ -soluble fraction is enriched in nonrepeated sequences which are transcribed in vivo (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F., and Bonner, J. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). We now report that the Mg2+ -soluble fraction of liver chromatin contains a low proportion of sequences in common with the Mg2+ -soluble fraction of brain chromatin. Thus, fractionation does not depend on some general property of chromatin but is specific with regard to the template activity of the tissue from which the chromatin was obtained.

Animals↗
PubMed1995

Distribution of calretinin, calbindin D28k, and parvalbumin in subcellular fractions of rat cerebellum: effects of calcium.

The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane-associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.

Animals↗
PubMed1974

Isolation and characterization of plasma membrane fractions from garfish Lepisosteus osseus olfactory nerve.

Garfish Lepisosteus osseus olfactory nerve, because of its large size and the unusually high concentration of axonal membrane, is an excellent source of axonal membrane. A procedure is described for the isolation of two types of plasma membranes from the nerve which are obtained in yields of about 20 mg (fraction I) and 1.5 mg (fraction II) per g of wet nerve. Both membrane fractions consist mostly of rounded membrane vesicles, with a unit membrane thickness of approximately 7.5 nm. The two membrane fractions are different in their lipid to protein ratios, Na-K ATPase activities, polypeptide patterns on sodium dodecyl sulfate (SDS) gel electrophoresis, and fatty acid compositions. They have similar phospholipid composition. On the basis of the relative concentration of axonal and Schwann cell plasma membranes in the nerve, the Na-K ATPase activities of the two membrane fractions and a comparison of the properties of the membrane fractions to those of squid and lobster nerve membrane preparations, fraction I seems to be the axonal membrane and fraction II the Schwann cell plasma membrane. Fraction I has a low protein to lipid ratio. Its polypeptide pattern on SDS gel appears to be much more complex as compared to that of fraction II membrane.

Adenosine Triphosphatases↗
PubMed1975

Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.

Alkaline Phosphatase↗
PubMed2005

Effect of patient variation on standard- and hypo-fractionated radiotherapy of prostate cancer.

Recent publications suggested that the alpha/beta ratio in the well-known linear quadratic (LQ) model could be as low as 1.5 Gy for prostate cancer, indicating that prostate cancer control might be very sensitive to changes in the dose fractionation scheme. This also suggests that the standard-fractionation scheme based on large alpha/beta ratios may not be optimal for the radio-therapeutic management of prostate cancer. Hypo-fractionated radiotherapy for prostate cancer has received more attention recently as an alternative treatment strategy, which may lead to reduced treatment time and cost. However, hypo-fractionated radiotherapy may be more sensitive to patient variation in terms of disease control than standard-fractionated radiotherapy. The variation of LQ parameters alpha and beta for a patient population may compromise the outcome of the treatment. This effect can be studied by the introduction of the sigmaalpha and sigmabeta parameters, which are the standard deviations of Gaussian distributions around alpha0 and beta0. The purpose of this study is to examine the effect of patient variation in alpha and beta on tumour control probability for standard- and hypo-fractionated radiotherapy of prostate cancer. The tumour control probability based on the LQ model is calculated using parameters alpha, beta, sigmaalpha and sigmabeta. Our results show that sigmaalpha is an important parameter for radiotherapy fractionation, independent of the alpha/beta ratio. A large sigmaalpha will result in a significant increase in the radiation dose required to achieve the same 95% TCP. Compared with the standard-fractionated scheme, sigmaalpha has a smaller effect on hypo-fractionated treatment at lower alpha/beta ratios. On the other hand, for lower alpha/beta ratios, the beta term also plays a more important role in cell-killing and therefore the patient variation parameter sigmabeta must be considered when designing a new dose fractionation scheme.

Cell Survival↗
PubMed2006

Fractionation of grape seed extract and identification of gallic acid as one of the major active constituents causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells.

The anti-cancer efficacy of grape seed extract (GSE) against prostate cancer (PCA) via its anti-proliferative, pro-apoptotic and anti-angiogenic activities in both cell culture and animal models have recently been described by us. GSE is a complex mixture containing gallic acid (GA), catechin (C), epicatechin (EC) and several oligomers (procyanidins) of C and/or EC, some of which are esterified to GA. To determine which components are most active against PCA, an ethyl acetate extract of GSE was separated by reverse-phase high-performance liquid chromatography (HPLC) into three fractions. Fraction 1 was far more effective than others in causing growth inhibition and apoptotic death of human PCA DU145 cells. Of the components in this fraction, GA showed a very strong dose- and time-dependent growth inhibition and apoptotic death of DU145 cells, but C and procyanidins B1 (EC-C dimer), B2 (EC-EC dimer) and B3 (C-C dimer) were nearly ineffective. Mechanistic studies demonstrated a strong caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavages by GA in DU145 cells. Procyanidin oligomers eluting in HPLC Fractions 2 and 3 were obtained in larger quantities by separating GSE into eight fractions (I-VIII) on a gel filtration column. All fractions were analyzed by HPLC-UV and negative-ion electrospray mass spectrometry. Fractions I-III contained the active compound GA and inactive components C, EC, B1 and B2. Fraction IV contained other dimers and a dimer-GA ester and was also less active than GSE in DU145 cells. Fractions V-VIII, however, caused significant growth inhibition and apoptosis with the highest activity present in the later fractions that contained procyanidin trimers and GA esters of dimers and trimers. Together, these observations identify GA as one of the major active constituents in GSE. Several procyanidins, however, and especially the gallate esters of dimers and trimers also may be efficacious against PCA and merit further investigation.

Adenocarcinoma↗
PubMed2006

Dosimetric and radiobiological impact of dose fractionation on respiratory motion induced IMRT delivery errors: a volumetric dose measurement study.

Respiratory motion can introduce substantial dose errors during IMRT delivery. These errors are difficult to predict because of the nonsynchronous interplay between radiation beams and tissues. The present study investigates the impact of dose fractionation on respiratory motion induced dosimetric errors during IMRT delivery and their radiobiological implications by using measured 3D dose. We focused on IMRT delivery with dynamic multileaf collimation (DMLC-IMRT). IMRT plans using several beam arrangements were optimized for and delivered to a polystyrene phantom containing a simulated target and critical organs. The phantom was set in linear sinusoidal motion at a frequency of 15 cycles/min (0.25 Hz). The amplitude of the motion was +/- 0.75 cm in the longitudinal direction and +/- 0.25 cm in the lateral direction. Absolute doses were measured with a 0.125 cc ionization chamber while dose distributions were measured with transverse films spaced 6 mm apart. Measurements were performed for varying number of fractions with motion, with respiratory-gated motion, and without motion. A tumor control probability (TCP) model for an inhomogeneously irradiated tumor was used to calculate and compare TCPs for the measurements and the treatment plans. Equivalent uniform doses (EUD) were also computed. For individual fields, point measurements using an ionization chamber showed substantial dose deviations (-11.7% to 47.8%) for the moving phantom as compared to the stationary phantom. However, much smaller deviations (-1.7% to 3.5%) were observed for the composite dose of all fields. The dose distributions and DVHs of stationary and gated deliveries were in good agreement with those of treatment plans, while those of the nongated moving phantom showed substantial differences. Compared to the stationary phantom, the largest differences observed for the minimum and maximum target doses were -18.8% and +19.7%, respectively. Due to their random nature, these dose errors tended to average out over fractionated treatments. The results of five-fraction measurements showed significantly improved agreement between the moving and stationary phantom. The changes in TCP were less than 4.3% for a single fraction, and less than 2.3% for two or more fractions. Variation of average EUD per fraction was small (< 3.1 cGy for a fraction size of 200 cGy), even when the DVHs were noticeably different from that of the stationary tumor. In conclusion, IMRT treatment of sites affected by respiratory motion can introduce significant dose errors in individual field doses; however, these errors tend to cancel out between fields and average out over dose fractionation. 3D dose distributions, DVHs, TCPs, and EUDs for stationary and moving cases showed good agreement after two or more fractions, suggesting that tumors affected by respiration motion may be treated using IMRT without significant dosimetric and biological consequences.

Artifacts↗
PubMed1981

Comparative studies of two membrane fractions isolated from chemotrophically and phototrophically grown cells of Rhodopseudomonas capsulata.

Light and heavy membrane fractions have been isolated by equilibrium sucrose density centrifugation from Rhodopseudomonas capsulata 938 GCM grown aerobically in the dark (chemotrophically) and anaerobically in the light (phototrophically). The densities of the light and heavy fractions from phototrophic cells were 1.1004 to 1.1006 and 1.1478, respectively, and the densities of the light and heavy fractions from chemotrophic cells were 1.0957 to 1.0958 and 1.1315, respectively. Both fractions were active in photochemical and respiratory functions and in electron transport-coupled phosphorylation. The light membrane fraction isolated from chemotrophic cells contained the reaction center and the light-harvesting pigment-protein complex B 870, but not the variable light-harvesting complex B 800-850. A small amount of the complex B 800-850 was present in the light fraction isolated from phototrophically grown cells, but it was not energetically coupled to the photosynthetic apparatus. From inhibitor studies, difference spectroscopy, and measurement of enzyme activities it was tentatively concluded that the light membrane fraction contains only the reduced nicotinamide adenine dinucleotide-oxidizing electron transport chain having a KCN-insensitive, low-potential cytochrome c oxidase, whereas the heavy fraction contains additionally the succinate dehydrogenase and a high-potential cytochrome b terminal oxidase sensitive to KCN. The light membrane fraction was more labile than the heavy fraction in terms of phosphorylating activity.

Cell Fractionation↗
PubMed1986

Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells.

Adenoviridae Infections↗
PubMed1979

Interactions of gonadotropins with corpus luteum surface membranes. V. Differential effects of digitonin on the buoyant densities of light and heavy rat ovarian membrane fractions.

Previous studies have indicated that rat luteal cells at certain stages of development can be fractionated so as to obtain two plasma membrane fractions with different densities and different profiles of marker enzymes. The light membrane fractions (density 1.13) contain the majority of hCG-binding sites and little or no cyclase enzyme, while the heavy membranes (density 1.17) contain the majority of cyclase enzyme and lesser quantities of hormone-binding sites. These membrane fractions were further compared with respect to their susceptibility to perturbation by digitonin. The buoyant density of luteal cell light membrane fractions, as marked by [125I]iodo-hCG binding, Mg2+-dependent ATPase, and 5'-nucleotidase, were highly perturbable by digotonin (delta density, greater than 0.05), while adenylate cyclase activity and phosphodiesterase activity associated with this fraction were only slightly perturbed (delta density, less than 0.02). The buoyant density of luteal cell heavy membrane fractions, as marked by adenylate cyclase, ATPase, and nucleotidase, was not significantly perturbed by digotonin. The hCG binding associated with the heavy membrane fraction was not perturbed by digitonin. From these studies, we conclude that the adenylate cyclase activity associated with light membrane fractions is due to contamination by heavy membranes, while the hCG-binding activity in heavy membrane fractions is intrinsic to that membrane. Except for the lysosomal marker (glucuronidase), which was solubilized by digitonin, the detergent had no significant effect on the density of mitochondrial, Golgi, GERL (Golgi, endoplasmic reticulum, and lysomal), or endoplasmic reticulum membranes. Plasma membranes from isolated granulosa cells and ovaries obtained 24 h after priming with PMS gonadotropin-hCG behaved as heavy membranes (density, 1.17) which contained hCG-binding sites, adenylate cyclase, nucleotidase, and Mg2+-dependent ATPase. These were not significantly perturbed by digitonin. The appearance of light membranes and the segregation of adenylate cyclase from the majority of hCG-binding sites is a development feature of the luteal cell.

Adenylyl Cyclases↗