Purification of ovarian theca-interstitial cells by density gradient centrifugation

The study of the biochemical mechanisms regulating the differentiation of the ovarian androgen-producing cells has been difficult because of the lack of a method for isolating the theca-interstitial cells (TIC) from the granulosa and other cell types. We report here a simple, rapid and reproducible method for obtaining large numbers of highly enriched TIC using density gradient centrifugation. When dispersed cells from ovaries of hypophysectomized immature rats were centrifuged in a Percoll gradient (20-70%), the cells were separated into five incompletely resolved bands. Of these, band V contained the androgen-producing cells. The band V cells produced 3.2- and 3.9-fold more cAMP and androgen, respectively, than the whole ovarian cells and contained 3.8- and 3.5-fold more 125I-hCG binding and 3 beta-HSDH labeled cells, respectively. These data indicated that the TIC were 65% pure. In order to improve the degree of purification, a discontinuous density procedure was used. When the ovarian cells were centrifuged in d = 1.055 g/ml Percoll, the purified TIC produced 4.7- and 5.9-fold more cAMP and androgen than the whole ovarian cells and they contained 5.3-fold more 125I-hCG binding. The TIC were cleanly separated from the granulosa cells and histochemical staining for 3 beta-HSDH activity revealed that the TIC were 93.0 +/- 1.3% pure. This method provides, for the first time, the opportunity to study TIC differentiation under defined conditions without granulosa cell contamination.     

Isolation of human monocytes by ficoll density gradient centrifugation

Summary The separation of human blood monocytes by a two step density gradient centrifugation using Ficoll is reported. From 10 ml venous blood 0.5×10⁶ monocytes could be harvested with a purity of 72 ± 11%. Cells characteristics typical of monocytes (positive -naphthylesterase staining, phagocytosis of polyacrylic acid beads, antigen presenting, adherence on plastic surfaces) confirmed the identity and the intact function of these cells.Supported by the Stiftung Volkswagenwerk (Az I 35 229)     

Assessment of efficacy of cell separation techniques used in the enrichment of foetal erythroblasts from maternal blood: triple density gradient vs. single density gradient

The aim of this study was to determine the efficacy of cell separation with single density and triple density-gradient techniques in the yield of foetal erythroblasts isolated from maternal blood. Maternal blood was obtained from 20 singleton pregnancies at 11-14 weeks of gestation immediately before foetal karyotyping by chorionic villus sampling. In each woman, the blood sample was divided into two portions; one portion was used for single density-gradient separation and the other, for triple density-gradient separation. Magnetic cell sorting (MACS) was subsequently performed with anti-CD71/antiglycophorin-A. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the gamma, epsilon and zeta globin chains. The percentage of foetal cells positive for each stain was calculated. Fluorescence in situ hybridization (FISH) for X- and Y-chromosomes was also performed. Comparison was made in the proportion of enriched foetal cells between the two separation methods for each CD71 and glycophorin-A (GPA) antibody. The percentage of erythroblasts enriched from maternal blood that stained positive for gamma, epsilon and zeta globin chains and with Kleihauer-Giemsa was significantly higher in the triple density-gradient separation fractions compared with the single density-gradient fractions with both anti-CD71 and GPA MACS. FISH analysis for the Y-chromosome confirmed the increase in foetal cell proportion in the triple density-gradient samples. Isolation of foetal erythroblasts from maternal blood using triple density-gradient separation and MACS is more effective with regard to foetal cell yield and purity than single density-gradient separation and MACS.     

Nonpolar density gradient ultracentrifugation in the direct determination of myocardial subcellular calcium

A new method is described for making direct measurements of compartmentalized subcellular stores of calcium in the isolated perfused dog heart. Cellular calcium was immobilized by freezing the myocardium in diastole with an extremely cold fluorocarbon fluid (-125 degrees C). All subsequent procedures were conducted under conditions which prevented ionic diffusion, either at temperatures well below the freezing point of water or in the absence of water. Sarcolemmal and mitochondrial enriched fractions were segregated from dessicated, homogenized, myocardial tissue by ultracentrifugation utilizing density-gradients composed of blends of silicone and halocarbon on fluids within which physiological salts are insoluble. The total calcium content of these isolated fractions were then determined by atomic absorption spectrophotometry. Ouabain and epinephrine were subsequently used to alter the contractility of the perfused hearts and such contractile alterations were then related to changes noted in the calcium activity of the isolated subcellular fractions. In this study the calcium levels of the enriched mitochondrial fractions were elevated by both ouabain and epinephrine, while the calcium levels of the enriched sarcolemmal fractions were elevated only by ouabain. The advantage of this segregative procedure is that it prevents artifactual intercompartmental calcium rearrangement and preserves calcium levels to those initially fixed in situ at the time of freezing.     

The optimization of large-scale density gradient isolation of human islets

The use of the COBE 2991 cell processor (COBE Laboratories, Colorado) for large-scale islet purification using discontinuous density gradients has been widely adopted. It minimizes many of the problems such as wall effects, normally encountered during centrifugation, and avoids the vortexing at interfaces that occurs during acceleration and deceleration by allowing the gradient to be formed and the islet-containing interface to be collected while continuing to spin. We have produced cross-sectional profiles of the 2991 bag during spinning which allow the area of interfaces in such step gradients to be calculated. This allows the volumes of the gradient media layers loaded on the machine to be adjusted in order to mazimize the area of the gradient interfaces. However, even using the maximal areas possible (144.5 cm²), clogging of tissue at such interfaces limits the volume of digest which can be separated on one gradient to 15 ml. We have shown that a linear continuous density gradient can be produced within the 2991 bag, that allows as much as 40 ml of digest to be successfully purified. Such a system combines the intrinsic advantages of the 2991 with those of continuous density gradients and provides the optimal method for density-dependent islet purification.     

Insights into the nature of the transition zone from physically constrained inversion of long-period seismic data

Imposing a thermal and compositional significance to the outcome of the inversion of seismic data facilitates their interpretation. Using long-period seismic waveforms and an inversion approach that includes constraints from mineral physics, we find that lateral variations of temperature can explain a large part of the data in the upper mantle. The additional compositional signature of cratons emerges in the global model as well. Above 300 km, we obtain seismic geotherms that span the range of expected temperatures in various tectonic regions. Absolute velocities and gradients with depth are well constrained by the seismic data throughout the upper mantle, except near discontinuities. The seismic data are consistent with a slower transition zone and an overall faster shallow upper mantle, which is not compatible with a homogenous dry pyrolite composition. A gradual enrichment with depth in a garnet-rich component helps to reduce the observed discrepancies. A hydrated transition zone would help to lower the velocities in the transition zone, but it does not explain the seismic structure above it.     

Sucrose density gradient analysis of erythrocyte membranes in hemolytic anemias

To investigate the membrane abnormalities that may play a pathophysiologic role in several hemolytic anemias we determined the density distribution on sucrose density gradients of human red blood cell (RBC) membranes from patients with these disorders, from normal controls, and from incubated normal RBC. We analyzed the fractions for membrane-adsorbed hemoglobin (Hb), globin, and nonglobin cytoplasmic proteins. The relationship between the cytoplasmic proteins adsorbed on the membranes and the specific gravity (SG) of the membranes was linear. An increase in SG of the entire membrane population was seen in Hb C disease due to adsorbed Hb. Subpopulations of membranes with increased SG due to adsorption of nonglobin protein were evident in the membranes from two splenectomized patients with hemolytic glucose-6- phosphate dehydrogenase (G6PD) variants. Dense membrane subpopulations found in RBC membranes from three splenectomized patients with Hb Koln were associated with adsorbed globin, while similar subpopulations in RBC membranes from three splenectomized patients with hereditary spherocytosis demonstrated increased SG due to adsorbed Hb. Splenectomized normals had no such abnormality in membrane density. Sucrose density gradients demonstrate that membrane bound cytoplasmic protein is characteristic of the RBC membranes in several hemolytic disorders. Additionally, gradients are useful for the isolation and further analysis of those subpopulations of RBC membranes with abnormal SG and exaggerated membrane protein abnormalities.     

Purification of thyroid lysosomes by colloidal silica density gradient centrifugation

A procedure was devised for fractionating crude thyroid lysosomal particles (P750-15,000) by self-forming density gradient centrifugation with colloidal silica. Two discrete particle-containing peaks were observed, based on 131I-labeling and acid phosphatase activity: a heavy peak (density, 1.11-1.12) and a light peak (density, 1.05). Ultrastructural analysis revealed that the heavy peak consisted almost entirely of lysosomes, whereas the light peak represented a heterogeneous mixture of small vesicles and fragments of other intracellular organelles. In thyroids removed from rats 30 min after 131I injection, almost all of the 131I was present in the low density peak. This 131I appeared on sucrose density gradient centrifugation as a 19S peak, and it was almost completely insoluble in trichloroacetic acid. This was interpreted as indicating that the low density peak contained pinocytotic vesicles. In thyroids removed 4 days after 131I injection, the radioactivity appeared largely in the high density peak. Both the trichloroacetic acid solubility and the pattern on sucrose density gradient centrifugation indicated that the [131I] thyroglobulin had undergone extensive proteolysis. Thyroglobulin proteolytic activity was found primarily in the high density particles and to only a small extent in the low density particles. Studies performed at intervals after 131I injection combined with double labeling (131I and 125I) experiments provided evidence that radioactivity was transferred from the low density to the high density particles. Heterogeneity existed within the dense peak, related to the degree of thyroglobulin degradation, as it was observed that thyroid lysosomes become denser with increasing proteolysis of thyroglobulin. The acid phosphatase in the low density particles could be distinguished from that in the high density (lysosomal) particles by its elution pattern on Sephadex G-200 column chromatography, its response to freezing and thawing, and its reactivity with p-nitrophenylphosphate. It was concluded, therefore, that the acid phosphatase in the low density fraction was derived from prolysosomal structures such as vesiculated Golgi-endoplasmic reticulum-lysosomes. The prolysosomal acid phosphatase associated with the low density fraction appeared to be a large membrane-bound molecule which could be transformed into lysosomal acid phosphatase by incubation at pH 5.0.     

A new density gradient system for the separation of human red blood cells

A new density system for the separation of human red blood cells by density-gradient centrifugation is described. The gradient medium is made with colloidal silica particles coated with polyvinylpyrrolidone suspended in aqueous solution of meglamine diatrizoate. By this method, more than 10 red-cell fractions can be separated. These show different ages (by creatine content and ⁵⁹Fe in vivo labelling) and different characteristics (ie, potassium content). A 40-fold enrichment in reticulocytes can be obtained in the top layers, with a great improvement of specific activity in labelling procedures of newly synthesized globin chains. The method is simple, rapid, inexpensive, reliable, nontoxic for erythrocytes, and is suitable for globin synthesis and other studies of erythrocyte metabolism.     

Rapid isolation of low density lipoprotein (LDL) subfractions from plasma by density gradient ultracentrifugation

High resolution density gradient ultracentrifugation (DGUC) and non-denaturing gradient gel electrophoresis (GGE) indicate that low density lipoprotein (LDL) in both normal and hyperlipidaemic subjects is composed of overlapping particle populations. A new centrifugation procedure has been developed which permits the separation of LDL subspecies directly from plasma within 24 h. The profiles obtained were analogous to those seen on gradient gel electrophoresis. LDL was divided into 3 fractions. The plasma concentration of LDL-I seen in young females was twice that in men (85.6 +/- 28.8 vs. 42.3 +/- 25.7 mg/dl, P less than 0.005). LDL-II was not significantly different in any group while LDL-III was specifically elevated in coronary artery disease (CAD) patients (207.1 +/- 92.6 mg/dl in CAD vs. 87.4 +/- 79.6 mg/dl in normal men, P less than 0.05). The presence of small, dense LDL detected either by density gradient centrifugation or gel electrophoresis was associated with raised triglyceride (TG) and low high density lipoprotein (HDL) cholesterol and may be a risk marker for coronary artery disease.     

Isolation of highly purified Leydig cells by density gradient centrifugation.

Purification of specific endocrine cells from mixed populations after dispersion of target tissues is important for detailed analysis of mechanisms of hormone action. A simple method for rapid isolation of endocrine cells with retention of biological integrity, has been developed by centrifugation in density gradients formed with Metrizamide. By this procedure, highly purified Leydig cells retaining morphological and biochemical characteristics were obtained. Such preparations bound 20, 300+/-3, 100 molecules of hCG per cell with affinity of 1.1+/-0.25 X 10(10) M-1. During incubation with hCG, cyclic AMP and testosterone responses of purified Leydig cells were considerably increased, and hCG concentrations as low as 0.2 pM caused activation of cAMP-dependent protein kinase.     

Functional integrity of anterior pituitary cells separated by a density gradient

Using a continuous Percoll density gradient, endocrine cells of the anterior pituitary were separated. The cells were obtained from adult female Sprague-Dawley rats which had been ovariectomized for 7 days. The gradient revealed two equally sized populations of cells with densities of about 1.02 and 1.09 g/ml. Ninety-two per cent of the cellular GH content, 64% of LH, and 60% of TSH were found in the high density peak. Sixty-one per cent of the cellular Prl appeared in the low density peak. Immunocytochemical staining of the LH containing cells showed that 74% of the gonadotrophs were in the high density peak. After separation, the cells retained their responsiveness to LRH, TRH and GRF. Culture conditions influenced stimulated hormone release. Before stimulation, the cells were cultured either in tissue culture flasks (attached cells) or in Petri dishes (cells in suspension) for 3 days. After TRH-stimulation, suspended thyrotrophs released more TSH than attached thyrotrophs. Comparing the cells of both peaks, attached thyrotrophs of the high density peak showed higher stimulated TSH-release than those of the low density peak. The response of the gonadotrophs and somatotrophs to stimulation did not differ when culture conditions were changed. The present results demonstrate that the secretory activity of endocrine cells is influenced by culture conditions and should be evaluated fore each cell type.     

Effects of explicit atmospheric convection at high CO2

The effect of clouds on climate remains the largest uncertainty in climate change predictions, due to the inability of global climate models (GCMs) to resolve essential small-scale cloud and convection processes. We compare preindustrial and quadrupled CO₂ simulations between a conventional GCM in which convection is parameterized and a “superparameterized” model in which convection is explicitly simulated with a cloud-permitting model in each grid cell. We find that the global responses of the two models to increased CO₂ are broadly similar: both simulate ice-free Arctic summers, wintertime Arctic convection, and enhanced Madden–Julian oscillation (MJO) activity. Superparameterization produces significant differences at both CO₂ levels, including greater Arctic cloud cover, further reduced sea ice area at high CO₂, and a stronger increase with CO₂ of the MJO.Clouds play an important role in the climate system by reflecting incoming shortwave solar radiation (cooling), intercepting outgoing longwave radiation from the surface (warming), and influencing temperature and circulation. Their net radiative impact at the surface is about −20 W/m² cooling in the global mean, and regional impacts can approach ∼40 W/m². Understanding how clouds will respond to rising CO₂ concentrations is thus a critical issue in climate science. Progress has been complicated by the hundred-kilometer horizontal grid spacing of most global circulation models (GCMs), which remain unable to directly resolve the much smaller-scale turbulent motions involved in atmospheric moist convection, the corresponding cloud-formation processes, and their radiative effects (1, 2).Current treatment of convection in global climate models relies on parameterizations and therefore suffers significant uncertainties, particularly relating to the representation of convection and clouds in a changing climate. Model results are sensitive to formulation and parameter choices in parameterized convection schemes. As a result, the magnitude of cloud feedbacks remains uncertain and inconsistently predicted by different models (2). An alternative approach, “superparameterization,” attempts to reduce the uncertainties of parameterization by running a higher resolution cloud-permitting model in a small domain within each grid cell of the atmospheric GCM, simulating the convection and cloud motions more explicitly (3, 4). Superparameterized GCMs have been shown to have a more realistic representation of convective variability, including the diurnal cycle (5) and intraseasonal variability such as the Madden–Julian oscillation (MJO) (6) and the Australian and Indian monsoons. They are beginning to be used to project future climate changes (7), although such work has been limited due to computational costs of about 100 times that of a standard GCM.Here we present the results of running a global coupled ocean–atmosphere model [the Community Earth System Model (CESM; ref. 8)], and its superparameterized variant (SP-CESM; refs. 3, 4, 9) at a preindustrial CO₂ concentration, as well as at 4 times higher concentration. We run CESM to near steady state for both preindustrial CO₂ concentration and 4 times this value (×1CO₂ and ×4CO₂), and then run shorter simulations of SP-CESM starting from these steady states (Materials and Methods). We choose to examine a rather significant (although not necessarily unrealistic) ×4CO₂ increase scenario because the equilibrium climate sensitivity of CESM to CO₂ doubling is on the low side of the warming range of 2.1–4.7 K seen in a recent model intercomparison (10), and to maximize the signal-to-noise ratio in the model response to superparameterization.CESM and SP-CESM are nearly identical except for their convection and cloud representation and related physics (Materials and Methods), but they show significant differences in their simulations at ×1 and ×4CO₂. Concerns have been raised that convection and cloud parameterizations may lead to either artificial amplification or weakening of the response to CO₂ increase. We find the global climate responses of CESM and SP-CESM to be broadly similar, a reassuring result in terms of present projections that are based on parameterized models. However, we find significant regional differences for Arctic sea ice and the tropical Madden–Julian oscillation on which we focus in this paper. Specifically, we find that SP-CESM shows (i) significantly less sea ice at ×1CO₂ and a larger area reduction at ×4CO₂, and (ii) a stronger MJO at ×1CO₂ and a larger increase at ×4CO₂. We analyze these differences and discuss the implications for uncertainties in climate change projections.The Arctic, and Arctic sea ice melting in particular, is strongly affected by the presence of low clouds that reduce solar heating in summer and by high clouds that induce warming in winter. Arctic sea ice has undergone rapid recent changes (11, 12), and is believed to have played a major role in past abrupt climate changes (13). Sea ice has a major impact on climate due to its high albedo and ability to insulate the atmosphere from the warmer ocean. Arctic sea ice change impacts local ecosystems (14), modulates extreme weather events in the sub-Arctic and midlatitudes (15), and has implications for shipping routes (16).Our focus on the MJO is motivated in part by numerous studies showing that present-day MJO simulations with SP-CESM are significantly improved relative to results from conventional GCMs, which have historically struggled to simulate it realistically. The MJO is characterized by an envelope of convective anomalies with a 30–70-day timescale that forms episodically over the Indian Ocean, propagates slowly eastward at around 5 m/s, and dissipates over the central Pacific (17, 18). The MJO affects the monsoons and Atlantic tropical cyclogenesis, modulates westerly wind bursts that can help trigger El Niño events, dramatically impacts tropical rainfall, and contributes to extreme precipitation events globally (18, 19). There is observational (20–23) and model (24–27) evidence of enhanced MJO activity with warming, although not all models agree on the sign of MJO change (28), and the change may be sensitive to the spatial pattern of warming (29).     

A new density gradient for the separation of large quantities of rosette-positive and rosette-negative cells

Allogeneic transplantation of peripheral blood stem cells will not be feasible until new techniques are developed for large-scale depletion of T-lymphocytes. Small quantities of cells can be depleted of T-lymphocytes by sheep erythrocyte rosetting and Ficoll-diatrizoate discontinuous gradient fractionation. However, when processing 10(9)-10(10) cells, the fractionation step is inefficient because of the limited capacity of Ficoll-diatrizoate. We therefore developed a new discontinuous gradient for the separation of large numbers of sheep red blood cell (RBC) rosette-positive and negative cells. The gradient was designed so that sheep cells and rosettes would not interfere with the banding of rosette-negative cells. In that way, nonspecific entrapment was reduced, and high cell capacity and yield were achieved. The system fractionated rosetted cell suspensions into four populations: free sheep RBC, rosetted T cells, nonrosetted T cells, and low-density non-T cells. The 15-ml gradient routinely separated 3-24 X 10(8) cells. Progenitor yield ranged from 83% to 99%, with 95% depletion of T-lymphocytes. The method is rapid, reproducible, and inexpensive. This preparative technique could prove useful clinically when large-scale separation of E-rosette-positive and negative cells is required.     

国产泛影葡胺密度梯度沉降法纯化Q热立克次体

Q热立克次体Henzerling株鸡胚培养的感染卵黄囊在蔗糖-磷酸缓冲生理盐水中制成悬液,以差速离心法提取,继用复方泛影葡胺密度梯度离心法排除鸡胚卵黄囊成分,提取和纯化活的立克次体。在线性密度梯度液中段,立克次体形成乳白色界限明晰的区带。 经光学显微镜及电子显微镜检查,立克次体的形态完整。在豚鼠、小白鼠及鸡胚感染实验中表明仍保留着活力及感染性。免疫荧光试验,单克隆抗体保护试验及补体结合试验证明对Ⅰ相及Ⅱ相的抗原性无变化。此提纯的制剂中未查出污染的鸡胚卵黄囊成分,其平均回收量为0.868mg/g卵黄囊或0.3472mg/g卵黄囊。