Density Gradient Centrifugation Technique Research Articles

Effect of alkyltins on rabbit articular and growth-plate chondrocytes in monolayer culture.

The effect of four different alkyltins (trimethyltin, triethyltin, dibutyltin, and dioctyltin) on the metabolism of rabbit articular and growth-plate chondrocytes was investigated using a monolayer cell-culture system. In most instances the compounds tested exhibited a general cytotoxic effect on these cells, inhibiting the synthesis of both DNA and sulfated proteoglycans. The effect of these compounds on proteoglycan synthesis was both quantitative and qualitative, as demonstrated by CsCl isopycnic density gradient centrifugation and gel exclusion chromatographic techniques. However, certain tin compounds tested, at specific concentrations, exerted a stimulatory effect on chondrocyte proliferation. Regarding DNA synthesis, growth-plate chondrocytes were more sensitive to the effect of the triethyltin, dibutyltin, and dioctyltin than were articular chondrocytes. The data are discussed in relation to the possible effects of the alkyltins on skeletal growth and development as well as the mechanism of action of the alkyltins at the molecular level.

Density-gradient enrichment of newly-formed mouse erythrocytes: Application to the micronucleus test

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080–1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60–80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.

Estrogen and progesterone receptors in meningiomas.

Two-thirds of all meningiomas and four-fifths of intraspinal and sphenoidal meningiomas occur in women. Meningiomas frequently enlarge or become symptomatic during pregnancy or during the luteal phase of the menstrual cycle. There is an increased incidence of meningiomas in women with breast carcinoma. In a series of 23 patients with meningiomas, the authors assayed biopsy specimens of the tumor for the presence of estrogen (ER) and progesterone (PR) receptors, using glycerol density gradient centrifugation and dextran-coated charcoal techniques. Significant levels of ER were found in only 17% of the patients, while significant PR levels were detected in 39%. Only one of the 16 tumors from female patients had significant ER levels, whereas three of the seven tumors from men had significant ER levels. Eight of the 16 tumors in women had significant PR levels, whereas only one of the seven tumors in men had a significant PR level. Thus, three out of four tumors with definite ER were from men, whereas eight of nine tumors with definite PR were from women. Of the eight women whose tumors contained PR, three were premenopausal and five postmenopausal. The single tumor with high levels of PR in the male patient was histologically atypical. The results of this series were compared with six published series of sex steroid assays in meningiomas. These seven series were divided into two groups: one group included two reports from the same laboratories in France, and the other the remaining five reports. Much higher percentages of both ER- and PR-positive tumors were reported from the French group. The authors suggest that this discrepancy may be due to the use of preoperative glucocorticoid therapy in the series from the United States. Since meningiomas are known to enlarge during periods when levels of circulating progestins are high, the presence of significant quantities of PR in a high percentage of tumors may have therapeutic implications for recurrent, malignant, or incompletely excised tumors, or for medically fragile patients. Conversely, since meningiomas are not known to enlarge during the proliferative phase of the menstrual cycle or with exogenous estrogen therapy, the small number of tumors positive for ER may indicate that ER lacks clinical significance. High levels of PR found in a small group of histologically aggressive tumors in several series may indicate that hormonal therapy may be especially useful in this difficult subset of patients.

Intracellular localization and properties of particulate hexokinase in the Novikoff ascites tumor. Evidence for an outer mitochondrial membrane location.

A detailed investigation concerned with localizing hexokinase in the Novikoff ascites tumor is presented. At least 50% of the total hexokinase activity was shown by differential and density gradient centrifugation techniques to be associated with tumor mitochondria. None of this activity was latent. Fractionation of isolated tumor mitochondria with digitonin revealed an outer membrane location for this enzyme. Treatment of tumor mitochondria with glucose 6-phosphate released about 80 to 85% of the hexokinase activity without disrupting the intermembrane compartment. This suggests that at least this proportion of the activity is bound to the outer surface of the outer membrane. Successive treatments did not remove the remaining hexokinase activity. At 30 degrees C, an incubation time of about 10 min with glucose 6-phosphate was required to achieve maximal release. No solubilization occurred at 0-4 degrees C. The isozymes derived from Novikoff mitochondria were identified by anion exchange chromatography as types I and III. Glucokinase activity was not detectable. Evidence is also presented which indicates that the hexokinase obtained from Novikoff mitochondria binds to the outer membrane of rat liver mitochondria. In contrast, the low endogenous hexokinase activity present in isolated liver mitochondria was found not to fractionate with outer membrane markers, but rather with contaminating microsomal membrane markers. Results described here provide the first direct evidence for the submitochondrial location of hexokinase in a tumor. They reveal an outer membrane location and an involvement of two hexokinase isozymes. Because these findings are characteristic of the hepatoma and not observed in control liver preparations, it is suggested that they may be very relevant to the general property of rapidly growing tumors to catabolize large amounts of glucose.

An improved purification method and a physical characterization of phage T7 DNA polymerase.

The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin. Elution of the enzyme is made by a pulse of buffer at pH 12.0. This decreases the binding capacity of the column. Binding experiments with [3H]thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers. T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength. Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form. The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques. The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.

Isolation and characterisation of insulin secretory granules from a rat islet cell tumour.

Density gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour. These procedures were readily completed within 7 h and from each animal yielded approximately 1 mg of granule protein. The isolated granules were essentially free of other subcellular organelles as evaluated by their contents of marker proteins, electron microscopy and by electrophoretic analyses. Their susceptibilities to lysis at low osmotic strength, at pH values above 7 or in media containing sodium ions were similar to those of granules partially purified from islets. Insulin comprised 50-60% of the total granule protein when determined by immunoassay or by densitometry of electrophoretic profiles. The proinsulin content was marginally higher than that of islets, as was the ratio of insulins I to II. Electrophoretic analyses revealed that the secretory granules contained 150 or more proteins besides insulin-related peptides. The majority of these had acidic isoelectric points and were located both within the granule interior and its enveloping membrane.

Carbon-13 NMR spectra of macerals separated from individual coals

The density gradient centrifugation technique has recently been applied to the separation of macerals from whole coals. Sufficient quantities have been separated to permit examination of pure exinite, vitrinite and inertinite fractions by combined cross polarization and magic angle sample spinning (CP/MASS) carbon-13 NMR techniques. The similarities and differences observed in the maceral groups of two coals are discussed. Diversity in precursors or in differential chemical maturation or a combination of both of these factors can be used to account for subtle spectral differences in the two coals and macerals studied even though the gross spectral features are very similar for a given maceral type. The data show that CP/MASS is a technique that can address such interesting geochemical questions.

Mechanism of toxicity of platinum(II) compounds in repair-deficient strains of Escherichia coli

The survival of wild-type and repair-deficient Escherichia coli treated with cis-Pt(NH 3) 2Cl 2, trans-Pt(NH 3) 2Cl 2 and [Pt(dien)Cl]Cl (dien = H 2NCH 2CH 2NHCH 2CH 2NH 2) was inversely correlated with the ability of these compounds to inhibit DNA synthesis in different bacterial strains. The relative amounts of these 3 compounds covalently bound to DNA immediately after treatment with the same dose were, respectively, 1:⩾2:1, their relative abilities to inhibit DNA synthesis were 6:1:0 and their relative toxicities toward the wild-type and uvrA strains were 3–5:1:0. More repair synthesis, as measured by density-gradient centrifugation techniques, was observed in wild-type bacteria after treatment with the cis than with the trans isomer whereas no repair synthesis was detected after exposure to [Pt(dien)Cl]Cl. These results are consistent with the hypothesis that cis-Pt(NH 3)Cl 2 binds to DNA and inhibits DNA synthesis thereby killing the cell. The lower toxicity of this compound toward wild-type bacteria compared with repair-deficient strains is in part a consequence of DNA repair. trans-Pt(NH 3) 2Cl 2 and [Pt(dien)Cl]Cl are less toxic than the cis isomer; this lesser toxicity is not a consequence of low levels of DNA binding or enhanced repair of the lesions but appears to reflect a weaker inhibition of DNA synthesis by these Pt-DNA adducts.

Isolation and characterization of autolysosomes which appeared in rat liver after leupeptin treatment.

Autolysosomes were isolated from rat livers treated with leupeptin by a combination of differential and Percoll density gradient centrifugation techniques. The purified autolysosome fraction was verified by morphological analysis to be highly purified and to contain contaminants which were scarcely detectable. The enrichment of the lysosomal enzyme activities in the purified autolysosomes over the homogenate was 12-, 14-, 22-, and 24-fold for beta-glucuronidase, acid phosphatase, beta-N-acetylglucosaminidase, and cathepsin D, respectively. Measurement of the activity of the marker enzymes for various subcellular organelles also proved that the purified autolysosome fraction was essentially free from contamination by other organelles. When the autolysosomes isolated from rat livers treated with leupeptin for 1 h were disrupted by osmolysis, acid hydrolases were easily solubilized. Acid phosphatase, however, became membrane bound in the autolysosomes prepared at longer periods of time after the leupeptin treatment. The autolysosomes exhibited enhanced permeability of the membranes after a short duration of time after the leupeptin treatment (30 and 60 min) and became stabilized later. These changes in the properties of the autolysosomes with time after the leupeptin treatment may be interpreted as meaning that progressive rearrangement of the lysosomal constituents occurred within the autolysosomes with time after the genesis.

Separation of coal macerals

The conditions necessary for efficient, small-scale separation of coal macerals are investigated. The method developed takes advantage of known density differences of the various macerais, but unlike most separations, an isopycnic density gradient centrifugation (DGC) technique is used to isolate pure macerals. The technique consists of grinding the coal in a fluid energy mill to ≈ 3 μm average particle size, separating macerals in an aqueous CsCI density gradient, and analysing the products for maceral composition as a function of density. Excellent separations can be achieved in a single run if the coal is first demineralized and then well dispersed with a wetting agent. Sink-float separations also benefit substantially from the addition of a surfactant. The density gradient centrifugation technique is limited more by inability to liberate macerals from each other on comminution than by any inherent errors in the gradient technique itself. Resolution of pure macerals using the DGC technique is much better than would be obtained from sink-float techniques. Data for three coals of differing constitution are presented.

CAMP-dependent protein kinase from [formula omitted]: Physical evidence of a ternary complex holoenzyme-cAMP

Gel electrophoresis and sucrose density gradient centrifugation techniques permitted the visualization for the first time of the ternary complex formed by the binding of cAMP to Mucor rouxii cAMP-dependent protein kinase holoenzyme. The addition of 0.5 M NaCl or histone plus ATP-Mg ++, together with cAMP, dissociates the holoenzyme into free regulatory (R) and catalytic (C) subunits. At 4°C, cAMP bound to the holoenzyme is readily exchangeable with unlabeled cAMP (half life 2.5 min), while the nucleotide bound to the R subunit has a very slow exchange rate (half life 210 min). The amount of cAMP bound to R subunit is approximately twice the amount bound to holoenzyme at saturation.

The major components of the mouse and human genomes. 1. Preparation, basic properties and compositional heterogeneity.

Main-band DNA from mammals and birds can be resolved by density gradient centrifugation techniques into three or four families of fragments of different dG + dC contents. These major DNA components are similar in their buoyant densities and relative amounts in all species tested and are observed in DNA preparations ranging in Mr from 2 X 10(6) to over 200 X 10(6). In the present work, the four major components of mouse and human DNAs were prepared and characterized in several basic properties: relative amounts, dG + dC contents, buoyant densities and compositional heterogeneity. The results obtained lead to the following conclusions: (a) the major DNA components of mouse and man form at least 85% and possibly the totality of the main bands of these DNAs; (b) they have very low compositional heterogeneities over a wide molecular weight range; (c) they derive from very large chromosomal DNA segments of fairly homogeneous base composition, for which the name 'isochores' is proposed. A comparison of the compositional heterogeneity of main-band DNAs from warm-blooded and cold-blooded vertebrates confirms our previous conclusion that these DNAs are characterized by a different sequence organization.

1654 ISOLATION OF ALVEOLAR TYPE II CELLS WITH A LASER FLOW CYTOMETER

Isolation of relatively pure populations of alveolar type II epithelial cells has previously only been possible by density gradient centrifugation techniques which exploit the low buoyant density conferred on such cells by lung surfactant containing lamellar bodies. We have used a multiparameter laser flow cytometer (cell sorter) to obtain similar populations of lamellar body containing cells from single cell suspensions made from rabbit lungs subjected to enzymatic dissociation by trypsin and elastase. The lamellar body cells are sorted on the basis of two parameters: low angle light scatter, and green fluorescence from the lipid seeking probe phosphine-3R. Cells do not separate well on the sorter when either of these two parameters is used alone. For example,alveolar macrophages scatter light in an overlapping range with lamellar body cells, and various kinds of cell debris stain with phosphine-3R in the same intensity region as viable cells with lamellar bodies. However, when both light scatter and fluorescence are considered simultaneously, the multiparameter cell sorter gives a highly purified population of lamellar body cells in a regime of high light scatter and high fluorescence. Presently we sort at a rate slightly less than 107 lamellar body cells per hour, certainly less than obtainable with gradient separation methods. However, the multiparameter cell sorter has the advantage of near absolute purity of type II cell populations obtained, and has the potential to isolate developing lung cells with few lamellar bodies which would not readily segregate by buoyant density.

Separation of cytotoxic leukocyte populations of human peripheral blood and colostrum on PVP-silica (Percoll®) density gradients

PVP-silica (Percoll) did not inhibit antibody-dependent cellular cytotoxicity in an 18 h 51Cr-release assay. By the use of a simple discontinuous density gradient centrifugation technique, leukocytes from peripheral blood and colostrum were separated into morphologic and cytotoxic functional populations. The major cytotoxic effector cells in colostrum are lipid-free macrophages and probably a small lymphocyte subpopulation.

Centrifugal separation of cells in sputum specimens from patients with adenocarcinoma.

A Ficoll density gradient centrifugation technique was used to separate fixed cells from the sputa of patients with bronchogenic adenocarcinoma. The selective enrichment of leukocytes, squamous or cancerous cells was achieved while retaining the diagnostically significant cell morphologic features. This technique is shown to typically eliminate most of the leukocytes (96.9%) macrophages (86.7%), squamous cells (70.0%) and necrotic debris from specimen fractions containing the majority of cancerous cells. The maximum purity of cancer cells (14.6%) occurs in high density (p = 1.35 g/ml) gradient fractions and results in an average 12.2-fold enrichment for cancer cells in contrast to their relative frequency of occurrence in unprocessed specimens. Differential cell count analyses of serial density centrifugation fractions show that this technique produces comparable enrichment rates for material from bronchogenic adenocarcinoma and squamous cell carcinoma.

A Solid-phase Immunosorbent Technique for the Rapid Detection of Rubella IgM by Haemagglutination Inhibition

The rubella haemagglutination inhibition (HAI) test has been modified for the detection of rubella-specific IgM. The rubella HAI test is performed in microtitre plate wells in which IgM from patients' sera has been selectively retained by anti-human IgM bound to the polystyrene surface. The test requires only anti-human IgM-coated microtitre plates, in addition to the standard rubella HAI reagents. The results obtained in this test, a solid-phase immunosorbent technique (SPIT), are in good agreement with results obtained by the sucrose density gradient centrifugation technique (DGCT). Advantages include the essentially unrestricted number of sera which can be tested daily, the availability of results within 24 h and the lack of interference by rheumatoid factor and by rubella-specific IgG.

Calciferol 25-hydroxylase activity in smooth and rough endoplasmic reticulum of rat liver

Calciferol 25-hydroxylase activity of vitamin D-deficient rats was measured in both liver microsomes and submicrosomal fractions. The smooth and rough-surfaced microsomes were prepared by a density gradient centrifugation technique in the presence of cesium chloride. Purity of the isolated microsomal membranes was ascertained by electron microscopy, RNA determination, measurement of enzyme markers, and by labeling of the cytoplasmic RNA with [5- 3H]orotic acid. Calciferol 25-hydroxylase activity was present in both smooth and rough-surfaced microsomes. The specific activity of the enzyme was greater in the rough fraction. There was a linear relation between enzymic activity and the concentration of enzyme for both total and submicrosomal fractions. These data show the presence of calciferol 25-hydroxylase activity in both smooth and rough-surfaced microsomes isolated from livers of vitamin D-deficient rats.

Intracellular location of isocitrate lyase in leaf tissue

Density gradient centrifugation techniques were to establish that the isocitrate lyase present in pea leaves is compartmented in the mitochondria and not the microbodies.

Isolation and characterization of four plasmids from Bacillus subtilis

Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6). The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.

Specific binding protein for 17β-estradiol in prostate with adenocarcinoma

A specific binding protein for 17β-estradiol has been detected in prostates of patients with prostatic adenocarcinoma, who had no endocrine manipulation prior to the removal of their prostates. Both the sucrose density gradient centrifugation and dextran-coated charcoal techniques were employed. The 17β-estradiol binding protein has an approximate sedimentation coefficient of 3.6S and is distinct from cytosolic dihydrotestosterone binding protein.