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At least 163 records · Page 9Linked to original sources
PubMed1980

In vivo localization of Ga-citrate in rat liver as determined by cell fractionation with isopycnic rate-zonal ultracentrifugation.

The in vivo behaviour of Ga-67 citrate was studied, using a rat liver model, and the mechanisms of cellular uptake were investigated by observing the time course of the radionuclide in subcellular liver cell fractions. Modified continuous ultracentrifugation was used to successively fractionate the homogenates according to isopycnic and rate-zonal principles. To collect the cell nuclei, a special technique, which consisted of supplementing the outer sucrose layer with cesium chloride to increase density, was applied to trap nuclei with isopycnic equilibrium. We isolated nuclei, mitchondria, lysosomes and peroxysomes, microsomes, and the cell supernatant in a sufficiently purified state. By this method, the in vivo localization of Ga-67 was studied. At 30 min after intravenous injection, radioactivity was found mainly in the cell supernatant, the radioactivity peak corresponded to 5 S. However, after 24 h, the radioactivity was localized in the heavier fraction containing lysosomes and heavy endoplasmic reticulum. No intranuclear localization was observed. In spite of the difficulty in completely separating the lysosomal fraction from heavy endoplasmic reticulum, our results suggest the participation of heavy endoplasmic reticulum in gallium localization.

Animals↗
PubMedDate not supplied

[Infectious activity of cell fractions isolated from the brain of mice with scrapie].

The agent of scrapie was found to induce clinically similar diseases in BALB/c and NIH mice but differing in the duration of the incubation period (155-180 days and 110-140 days, respectively) and pathomorphological manifestations. Differentiated examinations of a brain cell pool from mice with scrapie revealed more infectious activity not neutralized with nucleases and pronasa in membrane-free cell fractions than in fractions of slowly sedimenting membranes.

Animals↗
PubMed1978

Stimulation of human B lymphocytes by Listeria cell wall fraction.

Cell wall fraction of Listeria monocytogenes (LCWF), a B cell mitogen for mouse spleen cells, is also mitogenic for human adult and cord peripheral blood lymphocytes. Purified B-cell suspensions responded to LCWF in vitro proliferation, to a similar extent as the unfractionated suspensions. Furthermore, LCWF-induced B cell differentiation into IgM-containing cells and their percentage correlated significantly with the extent of lymphocyte proliferation.

Adult↗
PubMed1997

MIB-1 and S-phase cell fraction predict survival in non-Hodgkin's lymphomas.

The monoclonal antibody anti-Ki67 is used to detect proliferating cells, but its main limitation is the requirement of fresh-frozen material. On a series of patients with non-Hodgkin's lymphoma, we used a Ki67 equivalent monoclonal antibody, the recently proposed MIB-1, on formalin-fixed histopathological material using microwave antigen retrieval. MIB-1 expression was analysed in relation to other proliferation indices, such as autoradiographic H-thymidine labelling index (HTL1) and flow cytometric S-phase cell fraction (FCM-S) and to pathological status. Moreover, the prognostic relevance of the cell kinetic indices was defined in uni- and multivariate analyses including histology and tumour stage. The relationship between MIB-1 index and the other proliferation indices was statistically significant even though the correlation coefficient was around 0.6. The MIB-1 index was also related to the REAL (Revised European American Lymphoma) classification, but not to the Ann Arbor stage classification. Univariate analysis showed that the MIB-1 index was a significant predictor of 6-year survival in the overall series and in distinctly analysed low-grade and high-grade lymphoma subgroups. With regard to S-phase indices, HTLI was a powerful prognosticator in patients with high-grade histologies and FCM-S in patients with low-grade histologies. Multivariate analyses revealed that MIB-1 index, HTLI and FCM-S retained their prognostic significance independent of histology. In conclusion, the MIB-1 antibody provides prognostic information in non-Hodgkin's lymphomas and has the main advantage that it can be used in formalin-fixed, paraffin-embedded specimens.

Adolescent↗
PubMed1995

Using cell-fractionation and photochemical crosslinking methods to determine the cellular binding site(s) of the antitumor drug DMP 840.

In order to understand its mechanism of action we have begun an effort to better define the cellular target of action of the experimental antitumor agent DMP 840 (NSC D640430; (R,R)-2,2'-(1,2-ethanediylbis(imino-(1-methyl-2,1-ethanediyl)))-bi s(5- nitro-1H-benz(de)isoquinoline-1,3-(2H)-dione) dimethanesulfonate). Using a combination of gentle cell fractionation procedures and a previously unidentified photochemical crosslinking reaction, we have shown that after the drug is added to cultured Clone A cells, more than 80% of the drug that is found associated with cells partitions to the chromatin-containing structural framework of the cell and that the primary target after crosslinking with 360 nm light is DNA. While DMP 840 photoreacts quite efficiently with purified RNA in vitro, no photoattachment of the drug to RNA was observed in cells. In vitro photochemical studies also reveal that while GC-rich DNA is a preferred target for drug interaction, AT-rich DNA is more active in the photochemical crosslinking reaction. These results suggest that DMP 840 probably kills cells by interfering with DNA-metabolic processes, and that the drug and its derivatives are likely to be useful photoactive molecular probes for investigating higher order chromatin structures in cells.

Animals↗
PubMedDate not supplied

Acetylcholinesterase activity in several rat liver cell fractions after repeated poisoning with some organophosphates.

The reported investigations were carried out on female Wistar rats aged about 6 weeks, weighing 150 g. During 28 days the fats were given daily organophosphates (DFP, DDVP, Chlorfenvinphos, IPO-62, IPO-63, Phospholine) in a dose of 1/10 LD50 subcutaneously. The observations were conducted on groups of 8 rats. The body weight of the animals was noted on the 10th, 20th and 28th day of the experiment. On the 28th day the rats were killed and the activity of acetylcholinesterase (AChE) was determined by the biochemical method of Hestrin in liver homogenate and in the microsomal, mitochondrial and soluble fractions of liver cells. After 28 days of the experiment the body weight of the rats poisoned with these substances amounted to from 80.8% (DFP) to 90.7% (IPO-63) of that in control animals. The AChE activity was also reduced in relation to the control group ranging in the liver homogenate from 49.7% (DFP) to 75.6% (IPO-63), in the microsomal fraction from 33.0% (DFP) to 63.8% (IPO-63), in the mitochondrial fraction from 45.5% (DFP) to 72.9% (IPO-63), and in the soluble fraction from 52.8% (DFP) to 80.5% (DDVP).

Acetylcholinesterase↗
PubMed2006

Distribution of calcineurin activity in blood cell fractions and impact of tacrolimus inhibition.

The aim of this study was to investigate calcineurin (PP2B) activity in different blood cell fractions and its inhibition by tacrolimus. Basal PP2B activity was measured in each blood cellular fraction collected from healthy volunteers. The inhibition profile of PP2B activity was explored in isolated peripheral blood mononuclear cells (PBMC) and platelets exposed directly to tacrolimus and in PBMC and platelets isolated from whole blood previously exposed to tacrolimus. Contrasting with red blood cells (30%) and platelets (25%), PBMC represented only 8.7% of PP2B activity of unfractionated whole blood. After tacrolimus exposure of isolated PBMC and platelets, the concentration of tacrolimus required to inhibit 50% of PP2B activity (EC(50)) in PBMC was significantly lower than in platelets (0.26 ng/mL vs. 0.83 ng/mL, P < 0.001). EC(50) values were similar in PBMC and platelets isolated from whole blood previously exposed to tacrolimus (7.69 ng/mL vs. 7.42 ng/mL, respectively). These results suggest PBMC is a very suitable matrix for PP2B measurement in monitoring transplant recipients but clinical studies are necessary to solve clearly this issue.

Blood Platelets↗
PubMed1987

Analysis of protein patterns from different organs and cell fractions of trisomy 19 mice.

Proteins were extracted from liver, brain, and skin of 6-day-old mice with trisomy (Ts) 19 and fractionated into solubilized cell proteins and structure-bound cell proteins. The proteins were separated by two-dimensional electrophoresis, and protein patterns were compared in the combinations Ts/normal and normal/normal. Analysis of the protein patterns revealed protein spots (variants) with densities higher (h-type) or lower (l-type) in trisomies than in normal mice. Some of these variants were found in all Ts individuals investigated for a particular protein class. These variants, termed regular Ts-variants, constituted 0.8%-1.6% of the total number of spots. The proteins of the regular Ts variants were in most cases organ-nonspecific. However, in almost all cases a given quantitative variation was expressed in only one of the three organs investigated. To explain our results, we have presented models for the control of protein levels on the basis of gene regulation. New aspects in the conception of studies on trisomies in man could be gained.

Animals↗
PubMed1992

Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats.

The regulatory (R) subunits of adenosine 3',5',-cyclic monophosphate-dependent protein kinase were measured by photoaffinity labeling in heart tissue of rats flown on COSMOS 2044. Densitometric analyses showed a significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was unaffected by spaceflight or any of the simulation conditions. This was previously observed in heart muscle of rats flown on Spacelab 3 and COSMOS 1887 missions. A change in either the number or reactivity of the R subunits apparently results from some aspect of spaceflight. Proteins of the S2 fraction constitute a minor (less than 10%) component of the total, whereas the S1 fraction contained most of the cell proteins. No changes were seen when total incorporation of photoaffinity label was calculated on the basis of adrenal gland weights. A negative correlation resulted on comparison of controls with flight, synchronous control, and a tail-suspended simulation group of animals when incorporation of total counts due to azido labeling was based on body weights. Environmental factors that influence organismic responses may alter individual hormonal responses and may be reflected on the molecular level of organization. Conversely, changes in a relatively minor aspect of adenosine 3',5'-cyclic monophosphate-mediated reactions may be representative of a metabolic effect on an organismic level.

Affinity Labels↗
PubMed2000

Homing and engraftment potential of Sca-1(+)lin(-) cells fractionated on the basis of adhesion molecule expression and position in cell cycle.

Engraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1(+)lin(-) cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e(+) or CD43(+) Sca-1(+)lin(-) cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1(+)lin(-) cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential-colony forming cells, low proliferative potential-colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1(+)lin(-) AM(-) cells contained a higher percentage of cells in G(0)/G(1) than their AM(+) counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G(0)/G(1) following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1(+)lin(-) AM(+) or AM(-) cells were further fractionated by Hoechst 33342 into G(0)/G(1) or S/G(2)+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1(+)lin(-) engrafting cell as one that is mitotically quiescent, CD43(+), CD49e(+), CD11a(-), CD49d(dim), and CD62L(-). Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs. (Blood. 2000;96:1380-1387)

Animals↗
PubMed1992

Radiosensitivity, repair capacity, and stem cell fraction in human soft tissue tumors: an in vitro study using multicellular spheroids and the colony assay.

Radiation doses necessary to control 50% of spheroids (SCD50) were determined for five human soft tissue tumor lines after single dose and fractionated irradiation. Spheroids with 1000-1500 cells were used throughout. A similar number of cells per spheroid resulted in different sized spheroids for the respective cell lines. The parameters alpha, beta, and the number of regenerating cellular units per spheroid (SRU) were estimated from the spheroid control data using a direct fit according to the linear quadratic model assuming Poisson statistics. The number of spheroid regenerating cellular units was also determined from the growth delay at doses required for 10% spheroid control. In addition, alpha, beta, and the fraction of clonogenic cells of the five cell lines were obtained from a soft agar colony forming assay. The most precise parameter for radiation sensitivity was the SCD50, with a coefficient of variation smaller than 5%. SCD50 values ranged from 5.9 to 11.0 Gy for the five soft tissue tumor lines. Two of the five cell lines showed significantly higher alpha values and lower calculated survival fractions after 2 Gy (SF2) in the soft agar clonogenic assay than in the spheroid control assay. This points to a resistance-enhancing effect in the spheroid system. Whereas the fractions of SRU from the number of cells per spheroid, estimated from the spheroid control and growth delay assays, agreed well, no significant correlation existed between the fraction of SRU and the fraction of clonogenic cells in the soft agar colony forming assay. The alpha/beta ratios as a descriptive measure of the fractionation sensitivity of the tumor cell spheroids in the spheroid control assay corresponded well with those derived from the dose-cell survival data using a soft agar colony forming assay. Two of the five cell lines showed high fractionation sensitivities with alpha/beta values smaller than 5 Gy while those of the remaining three ranged from 7.8 to 10.8 Gy. Spheroids are structurally more similar to in vivo tumors than monolayer cultures. From the observed lack of correlation in the radiosensitivity parameters alpha and SF2 as well as in the fraction of SRU or clonogenic cells obtained from the spheroid control assay or the colony forming assay, one would expect even greater differences between results from colony forming assays and the radiosensitivity of in vivo tumors, at least for human soft tissue sarcomas.

Cell Aggregation↗
PubMed1994

Cell fractionation in non-ionic detergent distinguishes sub-populations of polyoma virus middle T antigen and reveals a novel form.

The transforming function of polyoma virus, middle T antigen (MT), interacts with several cellular enzymes, essential to its oncogenic activity. We have used cell fractionation to study the various MT/cellular protein complexes. We demonstrate that MT can be separated into three sub-species, dependent upon extraction in two buffers that we designate A and B: Antigen extracted from whole cells by both buffers (called MT1) is associated with most of the phosphorylated phosphatidyl-inositol kinase 85 kD subunit, pp85, and protein phosphatase 2A. Antigen (MT2), associated with the greater portion of pp60c-src, is extracted by buffer B, but not buffer A. A third population (MT3), resistant to extraction by either buffer, is not detectably associated with protein phosphatase 2A or pp85. It is, however, associated with a low level kinase activity. The interaction between pp60c-src and MT appears to influence the formation of both MT2 and MT3. MT2 fractionates with the cellular microtubule network, but does not appear to be directly associated with it. MT3, a previously undescribed population, comprises about one third of MT in wild type antigen-containing cells. It is missing in mutants incapable of interacting with pp60c-src, but exists in the absence of an interaction with pp85. We suggest that MT3 may be an intermediate in, or product of, one of the MT/pp60c-src signalling pathways, distinct from that involving pp85.

Animals↗
PubMed1992

The CD34+ cell fraction in bone marrow and blood is not universally predictive of CFU-GM.

We have measured the presence of granulocyte-macrophage colony forming cells (CFU-GM) and CD34+ cells in blood and bone marrow. We have compared these hematopoietic cell assays using regression analysis. We have found that under the limited and specific case of blood cells from an individual recovering from myelo-suppressive chemotherapy, the fraction of cells that is CD34 positive is predictive of the number of granulocyte-macrophage colonies (CFU-GM) which will grow. This result is in agreement with published data. We have found, however, that in bone marrow aspirates, or in the blood of individuals recovering from cyclophosphamide chemotherapy and receiving either granulocyte-macrophage colony stimulating factor (G-CSF) or folinic acid (FA) therapy, there is poor correlation between CD34+ cell fraction and CFU-GM. Accordingly, the use of CD34+ fraction cannot be relied upon to substitute for the CFU-GM assay in assessing the hematopoietic cell content of blood or bone marrow samples.

Antigens, CD↗