Ficoll and dextran enhance adhesion of Sendai virus to liposomes containing receptor (ganglioside GD1a)

Previous work has shown that high-speed centrifugation (300,000 g) of Sendai virus and liposomes in 40% (w/v) sucrose layered under a discontinuous sucrose gradient removes Sendai virus bound to liposomes containing the ganglioside GD1a, a Sendai virus receptor. Centrifugation also removes virus bound to liposomes containing other negatively charged lipids. This work shows that centrifugation of virus through a discontinuous ficoll gradient does not remove virus bound to liposomes containing GD1a but does remove virus from liposomes containing various other negatively charged lipids including the ganglioside GM1, which is not a Sendai virus receptor. The amount of virus that adheres to liposomes increases with increasing content of GD1a in the liposomes. The adhesion of virus to receptor-containing liposomes during centrifugation through a ficoll gradient results from the presence of ficoll and increases with increasing ficoll concentration. Virus also adheres to receptor-containing liposomes during centrifugation in the presence of dextran. These data indicate that caution should be used in interpreting associations demonstrated by centrifugation through dextran and ficoll gradients. They also indicate that binding of virus by ganglioside receptors can be modulated by carbohydrate polymers, which are thought not to have any specific interaction with either viruses or gangliosides.     

Population of L-forms of Bacillus subtilis studied in Ficoll density gradients]

The distribution of cells in the population of L-forms of Bas. subtilis was analysed by isopicnic centrifugation in density gradient of ficoll. Two main fractions of L-forms different in their density were found. The study of the fractions by various methods indicated that a considerable part of the L-forms population is presented by unviable cells of diverse size with fragments of genome or without DNA.     

Comparison between 2 density gradients for separation of CFU

We separated 40 bone marrow samples by centrifugation on two density gradients, ficoll-metrizoate sodium (lymphoprep) and percoll. There is no difference between the two methods when expressing the colony forming cells (CFU-C) per 2.10(5) bone marrow cells plated. But the absolute number of recovered CFU-C is significantly greater with percoll than with ficoll metrizoate sodium.     

Number of nucleoli in Ehrlich tumor cells during interphase.

Fractions of mouse Ehrlich ascites tumor cell populations with a high percentage of cell either in early or in late interphase were separated by centrifugation on ficoll gradients. Nucleoli were studied by light or electron microscopy in these cell subpopulations. It was shown that, in these cells, the number of nucleoli per nucleus does not vary significantly during interphase. This result is discussed and an anlysis of the relationships between the number and the volume of the nucleoli in these cells is present.     

Preparation and properties of thyroid cell membranes

Summary Calf and human thyroids have been disrupted by nitrogen microcavitation, and the thyroid membranes prepared by repeated centrifugation in low ionic strength buffers. Two classes of membranes were prepared by centrifugation on a discontinuous gradient of ficoll. A lighter fraction was comprised of somewhat larger vesicles; they were higher in Na⁺–K⁺-activated ATPase, phosphodiesterase, and 5-nucleotidase than was the heavier fraction. The heavier fraction had a higher nicotinamide adenine nucleotide dehydrogenase-diaphorase activity. Thus the lighter fraction appears to have been enriched in fragments derived from the plasma membrane.     

Influence of molecular configuration on the passage of macromolecules across the glomerular capillary wall

The influence of molecular configuration on the filtration of macromolecules across glomerular capillary walls was examined by comparing fractional clearances of two uncharged polysaccharides of distinctly different molecular configuration in the Munich-Wistar rat. The macromolecules employed were dextran, a slightly branched polymer of glucopyranose, and ficoll, a highly cross-linked copolymer of sucrose and epichlorohydrin. Differences in effective shape between these two polymers were determined from measurements of several physical properties of aqueous solutions containing either dextran or ficoll. It was found that dextran is best represented as a prolate ellipsoid with axial ratios of 4, 9, and 16 for molecules with Stokes-Einstein radii of 22, 32, and 40 A, respectively. On the other hand, ficoll is more closely approximated as spherical since the axial ratio was found to be between 1 and 2 for all molecular sizes. Fractional clearances of dextran and ficoll ranging in effective radius from 18 to 44 A were determined in each of seven Munich-Wistar rats. Fractional clearances of dextran were found to be greater than those of ficoll, the difference being significant for molecular radii ranging from 24 to 44 A. In addition, as shown previously for dextran, ficoll was found to be neither secreted nor reabsorbed by the renal tubules. These results, therefore, suggest that in addition to molecular size and charge, molecular configuration is also a determinant of the filtration of macromolecules across the glomerular capillary wall.     

RNA metabolism in rabbit brain: study with neuron-glia and subcellular fractions

—Uridine incorporation into RNA of rabbit brain was studied by using an in vitro system for incubation of brain slices for up to 180 min. Neuron-enriched and glia-enriched fractions were prepared by ficoll density gradient centrifugation, and various subcellular fractions were prepared by sucrose density gradient centrifugation. Although the difference was not as great as in the case of l -leucine incorporation into protein, the neuron-enriched fraction consistently showed a higher specific radioactivity than the glia-enriched fraction. The specific radioactivity of the nuclei increased promptly and remained high at 180 min; the increase in the microsomes was gradual. Comparison of these data suggests that both neuron-enriched and glia-enriched fractions retain high radioactivities in their nuclei at 180 min when a considerable portion of the ribosomal RNA in these fractions is not labeled. The sharp diffusion gradient of nucleotides is discussed in relation to the acid-soluble radioactivity.     

Cytochemical and biochemical identification of lysosomes in cryptococcus neoformans

Normaski optics, fluorescence and electron microscopy were employed to demonstrate the occurrence of lysosomes in capsulated, enzymatically decapsulated, and dewalled cells of a human isolate ofCryptococcus neoformans. Fluorescent studies, using acridine orange as a lysosomal indicator, revealed the presence of variously sized, spherical, reddish-orange fluorescing bodies. Electron microscopy studies demonstrated the presence of acid phosphatase (AP), a lysosome marker enzyme, in single-membrane bound organelles. Lysosomes were removed from dewalled cells and separated by differential centrifugation on ficoll gradients. That fraction indicating the highest assay for AP was centrifuged at high speed, and the resulting pellet was fixed for electron microscopy and stained by the Gomori procedure for AP. Sections of the pellets revealed AP stained vesicles of the same size range as those within intact cells.     

Activity of cell-detached 5'-nucleotidase in thymocytes of various densities

The rat thymocytes submitted to heating at 45 degrees C for 1 hr liberate plasma membrane fragments containing 5'-nucleotidase activity in the supernatant. The thymocytes were separated by ficoll density gradient centrifugation. High activity of 5'-nucleotidase per 10(6) cells was found in the supernatant of low density (1.069) subset of thymocytes. Thymocyte supernatant of rats treated with hydrocortisone demonstrated higher 5'-nucleotidase activity per 10(6) cells than in intact animals. This is due to an increase of the low density population of thymocytes in treated rats since the 5'-nucleotidase activity per 10(6) cells of the supernatant obtained from this density fraction is the same both in treated with hydrocortisone and intact rats. Hydrocortisone seems to induce a selection of the thymocytes with high 5'-nucleotidase activity.     

The effect of somatotropin on adenosine deaminase activity in tissues of rats with hypo- and hyperthyroidism

It has been shown that under hypothyrosis (thyroidectomy) the adenosine deaminase activity in the total suspension of thymocytes does not change as compared with that of intact rats. When suspension of thymocytes was separated by centrifugation in the ficoll-urografin density gradient into 6 fractions the enzyme activity has been found to increase by 83 and 31%, respectively, in light fractions with the ficoll density of 1.065 and 1.071 and to decrease by 21% in the fraction with density of 1.095 as compared with the intact rats. The enzyme activity in spleen homogenate of hypothyroid rats is higher by 72% than the norm. This activity in thymocytes and spleen of hyperthyroid rats is lower by 37% and 30% respectively. Somatotropin administration to rats normalizes these changes.     

Bovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term

The objective of the present study was to determine if oocytes vitrified by the open pulled straw (OPS) method could subsequently be used to produce somatic cell cloned cattle. Post-thaw survival rates were 77.0, 79.1, 97.2 and 97.5% for oocytes vitrified with EDFS30 (15% ethylene glycol, 15% dimethyl sulfoxide, ficoll and sucrose), EDFS40 (20% ethylene glycol, 20% dimethyl sulfoxide, ficoll and sucrose), EDFSF30 (15% ethylene glycol, 15% dimethyl sulfoxide, ficoll, sucrose and FBS) and EDFSF40 (20% ethylene glycol, 20% dimethyl sulfoxide, ficoll, sucrose and FBS), respectively. The parthenogenetic blastocyst rates of the vitrified-thawed oocytes activated with 5 microM of the calcium ionophore A23187 for 5 min and 2 microM of 6-dimethylaminopurin (6-DMAP) for 4h ranged from 10.3 to 23.0%, with the highest group not significantly differing from that of the controls (33.2%). In total, 722 vitrified-thawed oocytes were used as recipients for nuclear transfer, of which 343 fused (47.6%). Fifty-six (16.3%) of the reconstructed embryos reached the blastocyst stage after 7d of in vitro culture. Twenty-four blastocysts derived from vitrified-thawed oocytes were transferred to six Luxi yellow cattle recipients. Two recipients (33%) were diagnosed pregnant; one aborted 97 d after transfer, whereas the other delivered a cloned calf after 263 d. As a control, 28 synchronous Luxi yellow cattle recipients each received a single blastocyst produced using a fresh oocyte as a nuclear recipient; 10 recipients were diagnosed pregnant, of which 6 (21.4% of the original 28) delivered cloned calves. In conclusion, bovine oocytes vitrified by the OPS method and subsequently thawed supported development (to term) of somatic cell cloned embryos.     

Functional properties of ficoll and their influence on anther culture responses of wheat

The effects of ficoll in liquid culture media have been contradictory in previous reports. The objective of this study was to determine the functional properties of ficoll in potato 4 (P4) liquid induction medium and their influence on anther culture responses of wheat. Ficoll addition significantly (p0.01) reduced callus production from the anthers of spring wheat cv. Pavon 76. The reduction was directly related to the concentration of ficoll added within the range of 50 to 200 g l^-1 medium. Although the addition of ficoll significantly (p0.01) increased the percentage of regenerable calli and the ratio of green vs. albino plants, the final yield of green plants per 100 anthers was significantly lower. Consistent results also were obtained with four other spring wheat genotypes (Chris, Butte 86, WA 6916, and Edwall). Ficoll concentration affected the density, viscosity, and osmolality of the liquid media. The higher medium density caused by ficoll addition increased the percentage of floating calli, as well as the percentage of regenerable calli and the ratio of green vs. albino plants. However, the increased medium viscosity by ficoll addition significantly (p0.01) reduced callus production. Ficoll addition also increased medium osmolality, which affected callus production by interacting with the sugar concentration of the induction media. Using response functions, the estimated maltose concentration for maximum callus production was 105 g l^-1 for the standard P4 media, compared with 68 g l^-1 for the ficoll-containing P4 media. These results clearly demonstrate that ficoll addition to the liquid P4 induction medium containing high sucrose concentration (90 g l^-1) is deleterious to the maximum production of green plants from wheat anther culture.     

Isolation of dictyosomes fromEuglena gracilis

Summary A method for the isolation of dictyosomes fromEuglena gracilis Klebs strain Z (Pringsheim) is described. An extensive Golgi system, with the individual dictyosomes commonly containing ten to twenty cisternae is present. Log phase cells are broken in a French pressure cell at 105 to 120 kg/cm₂ in a breaking mix containing sucrose, sorbitol and ficoll. Addition of 0.3% of glutaraldehyde or formaldehyde to the breaking mix increases the number of stacked cisternae present in the final preparation. In addition to membrane stacks, the fractions contain numerous smooth vesicles. Swollen cisternae, which are also present, may account for these vesicles. Three dictyosome-enriched fractions are obtained by centrifugation in a discontinuous sucrose gradient. Fractions differ morphologically in the degree of stacking of cisternae. Further identification of the membrane fractions was accomplished by measuring IDPase activities in each of the fractions. Inosine diphosphatase activity is enriched 8–10-fold relative to the initial homogenate. The highest IDPase activity was present in the fraction containing the greatest number of stacked cisternae.     

Intermediates in adenovirus assembly.

Three intermediates in adenovirus assembly have been defined; nuclear intermediates, young virions, and mature virions. The nuclear intermediates are fragile and heterogenous in size (550S-670S) and withstand separation on ficoll gradients but fall apart upon CsCl gradient centrifugation unless prefixed with glutaraldehyde. They contain both capsid and core structures, and the core structures are preferentially released during purification in CsCl. The precursor polypeptides pVI and pVII are present in the intermediates without any corresponding mature polypeptide. The young virions (Ishibashi and Maizel, 1974) are stable and preferentially confined to the nuclei after cell fractionation. They contain both uncleaved precursor polypeptides and their cleavage products. The mature virions accumulate in the cytoplasm during cell fractionation and contain the final mature polypeptides. Pulse-chase labeling kinetics, focusing on the precursor polypeptides, suggest that these three classes participate in assembly of adenovirus. Tryptic peptide maps establish that polypeptide pVI is the precursor of polypeptide VI, but only a small fraction of polypeptide 26K can in vivo account for polypeptide VIII.     

Separation of human urinary proximal tubular cells from familial hypercholesterolemic homozygotes by Ficoll gradient centrifugation. Morphological and biochemical characteristics

The isolation of renal proximal tubular (PT) cells from the fresh urinary sediment ( UrS ) of familial hypercholesterolemic (FH) homozygotes using discontinuous Ficoll gradient centrifugation was studied. For comparative purposes, cultured cells derived from normal human UrS or kidney were also studied. Unfractionated PT cells were nucleated and 90-99% of the cells excluded trypan blue. Both the unfractionated and fractionated cultured normal PT cells contained numerous empty cytoplasmic vesicles. In contrast, similar preparations from the UrS of FH homozygotes contained membrane-enclosed cytoplasmic vesicles that stained with the Papanicolaou (Pap) reagent and were strongly positive with a fluorescein-labeled antibody against lactosylceramide. The PT cells of FH homozygotes contained 2.0 to 2.5 fold higher activity of gamma-glutamyltransferase and alkaline phosphatase, respectively, than the unfractionated UrS cells. We conclude that human PT cells can be separated from other UrS cells by ficoll gradient centrifugation, and that most, if not all of the LacCer present in the UrS of FH homozygotes is associated with the PT cells. Purified PT cells should provide a useful model to test the biochemical mechanisms of LacCer accumulation in FH.     

In vitro survival of murine morulae after quick freezing in the presence of chemically defined macromolecules and different cryoprotectants

We studied the ability of frozen-thawed mouse morulae to develop in vitro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and ficoll. We also determined how these agents interacted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the above factors was measured on the basis of post-thaw morphological appearance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovulated mice. Those classified as good or excellent were distributed among the 12 different freezing solutions, obtained by combining the 3 cryoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). Embryos frozen in PVA, PVP and ficoll tended to be a little difficult to recover from the straws. Development to the expanded blastocyst stage was significantly lower (P<0.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol provided a higher survival rate when combined with glycerol (90.3) or ethylene glycol (95.0), but when it was combined with propylene glycol, only 56.5% of embryos survived after thawing. A positive interaction was observed between glycerol and PVA and between ethylene glycol and PVA or ficoll. The results indicate that fetal serum could be successfully substituted for any of the 3 chemically defined macromolecules. However, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freezing method.     

Macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins

Structural and molecular properties extracted from circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the high concentration of synthetic crowding agents (dextran 40, dextran 70 and ficoll 70) stabilizes and refolds the base-denatured ferricytochrome c (Ferricyt c) and lysozyme (Lyz) at pH 12.9 (±0.1) to molten globule (MG) states (C_B-states). These results further revealed that the C_B-states resemble the generic properties of MG-states. Thermodynamic analysis of thermal denaturation curves of base-denatured Ferricyt c and Lyz at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence increases the thermal stability of base-denatured proteins and also prevents the cold denaturation of Ferricyt c. The results further showed that the nature, size and shape of crowder influence the crowding-mediated increase in secondary structure stabilization and thermal stability of base-denatured Ferricyt c and Lyz. Analysis of kinetic and thermodynamic parameters measured for CO association reaction of alkaline ferrocytochrome c (Ferrocyt c) at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence reduces the level of structural fluctuation of M80-containing Ω-loop that control CO association to alkaline Ferrocyt c.     

Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N

We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA. For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U. The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters. Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme. The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks). Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU. This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS)     

Method for selecting cells during various phases of the mitotic cycle

Exponentially growing L5178Y cells in suspension culture were separated according to their position in the cell cycle on the basis of their volume with a velocity sedimentation method in which a linear and continuous ficoll gradient was used. Highly purified populations of G1 and S cells were obtained, containing about 90% G1 phase cells and 80% S phase cells. The method is rapid and a larger number of cells can be easily processed with no loss of viability.     

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)