离心法分离毕赤酵母活细胞和死细胞

本文以毕赤酵母为研究对象,探索出一种分离活酵母细胞的新方法。研究发现,通过改变淋巴细胞分离液和50%聚蔗糖溶液的比例,获得不同密度的酵母细胞分离液,进而通过离心分层的方法可使毕赤酵母活细胞主要存留于离心液的上层。当酵母细胞分离液的密度为1.1467 g/mL (27.5%淋巴细胞分离液+72.5%聚蔗糖溶液),分离液上下层中酵母活细胞的分配比例差别达到最大,分别为94.67%(分离液上层)和5.33%(分离液下层)。毕赤酵母细胞浓度为4.35×108~1.13×109/mL时,活细胞在分离液上下两层的分配比例约为95%和5%。低毕赤酵母细胞存活率有利于离心分离。本方法可用于有效分离培养液中的毕赤酵母活细胞。     

Endocytosis of chlamydiae by McCoy cells: measurement and effects of centrifugation

Abstract Chlamydia trachomatis strain 434 and C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) were compared for entry into McCoy cells and expression of productive infection (inclusion body formation). Entry was measured as the difference between extracellular cell-associated organisms, determined directly after fluorescence staining of live cells, and total cell-associated organisms (intracellular and extracellular); the latter were evaluated from radioactivity measurement and known particle: radioactivity ratios for stock radiolabelled suspensions. Under inoculation conditions of natural (spontaneous) infection, 69–82% of cell-associated organisms of both strains were internalised and entry was not enhanced by centrifugation of inocula with monolayers. For 434, inclusion bodies were seen in 10–20% of cells containing organisms and numbers were little influenced by mode of infection. For GPIC, productive infection initiated by centrifugation was comparable with that of 434 but some 15-fold reduced in spontaneous infection. The results suggest that unproductive infection by GPIC occurs, not because of defective entry, but from inhibition at an intracellular step which is circumvented when infection is initiated by centrifugation.     

Separation and subtyping of planarian neoblasts by density-gradient centrifugation and staining

A method has been devised to isolate neoblasts from planarians in high purity and high yield. Specimens of Dugesia polychroa and Dugesia tahitiensis were disintegrated mechanically. After several prepurification steps consisting in sequential filtering through increasingly fine meshes and differential centrifugation, the resulting cell suspension was separated by centrifugation in discontinuous density gradients formed from Percoll solutions. Isosmotic conditions were applied. Two gradients are recommended, one isopycnic four-step gradient (densities 1.03, 1.05, 1.07, 1.09) to obtain one single fraction with a high yield of neoblasts in high purity and one six-step gradient for preparing subfractions of neoblasts somewhat less pure, but in still greater amounts. This latter gradient (densities 1.04, 1.05, 1.06, 1.065, 1.07, 1.09) was run under conditions of rate zonal centrifugation and used for further subtyping of neoblasts by specific staining with azure A – eosin B. A first survey of tentative types based on morphological criteria is given. The viability of neoblasts isolated in the way described was tested in primary cell cultures. In current experiments, 6-week-old cultures had a viability of roughly 50%, with mitoses up to 1 week after the isolation of neoblasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

NAD+ levels control Ca2+ store replenishment and mitogen-induced increase of cytosolic Ca2+ by Cyclic ADP-ribose-dependent TRPM2 channel gating in human T lymphocytes

Intracellular NAD(+) levels ([NAD(+)](i)) are important in regulating human T lymphocyte survival, cytokine secretion, and the capacity to respond to antigenic stimuli. NAD(+)-derived Ca(2+)-mobilizing second messengers, produced by CD38, play a pivotal role in T cell activation. Here we demonstrate that [NAD(+)](i) modifications in T lymphocytes affect intracellular Ca(2+) homeostasis both in terms of mitogen-induced [Ca(2+)](i) increase and of endoplasmic reticulum Ca(2+) store replenishment. Lowering [NAD(+)](i) by FK866-mediated nicotinamide phosphoribosyltransferase inhibition decreased the mitogen-induced [Ca(2+)](i) rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores was greatly reduced in these cells in the presence of FK866. When NAD(+) levels were increased by supplementing peripheral blood lymphocytes with the NAD(+) precursors nicotinamide, nicotinic acid, or nicotinamide mononucleotide, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores as well as cell responsiveness to mitogens in terms of [Ca(2+)](i) elevation were up-regulated. The use of specific siRNA showed that the changes of Ca(2+) homeostasis induced by NAD(+) precursors are mediated by CD38 and the consequent ADPR-mediated TRPM2 gating. Finally, the presence of NAD(+) precursors up-regulated important T cell functions, such as proliferation and IL-2 release in response to mitogens.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

复方泛影葡胺速率区带密度梯度离心法纯化衣原体

目的利用速率区带密度梯度离心法建立纯化高纯度、高感染力衣原体的方法。方法采用HeLa229细胞扩增衣原体,制作含有大量衣原体的细胞裂解液,以国产复方泛影葡胺作为介质,利用速率区带密度梯度离心法纯化衣原体,负染纯化产物后使用透射电镜观察形态,并将纯化产物感染HeLa229细胞,测定纯化产物的感染力。结果在透射电镜下观察,证实纯化产物为直径300 nm左右的高纯度原体。纯化产物的感染力为8.62×10~8 IFU/mL,证实该纯化产物具有高感染力。结论通过复方泛影葡胺速率区带密度梯度离心法,可获得高纯度、高感染力的衣原体,为开展衣原体感染机制、免疫反应等研究提供了有效而可靠的保证。     

Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing

The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H₂DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.     

Seasonal variations in the immune system of the tench,Tinca tinca (Cyprinidae): proliferative response of lymphocytes induced by mitogens

The proliferation of tench lymphocytes induced by mitogens was studied during the four seasons of the year. Fish were maintained under natural conditions of photoperiod and temperature (mean ± SD: 12±2°C in winter, 22±3°C in spring, 30±3°C in summer and 21±3°C in autumn). Cultures were performed in vitro at 22°C in all seasons and the results were compared. Subsequently, in seasons with extreme water temperatures, cultures in vitro were performed at the same temperature as that of the water (12°C in winter and 30°C in summer) and the results were compared seasonally at the seasonal temperature, i.e. at 22°C in spring, 30°C in summer, 22°C in autumn and 12°C in winter. Phytohemagglutinin, concanavalin A, lipolisaccharide from E. coli and pokeweed mitogen were used as mitogens. Studies performed at 22°C as assay temperature in all seasons showed profound seasonal changes: while in spring, summer and autumn the mitogenic response of lymphocytes to phytohemagglutinin, concanavalin A, lipolisaccharide from E. coli and pokeweed mitogen was very low, during winter the results obtained were significantly higher. However, when the assays were performed at the corresponding seasonal temperature the differences were not as pronounced between the different seasons, and the mitogenic responses of lymphocytes were found to be the lowest during the winter and the highest during the summer with all mitogens used. This fact suggests that immunosuppression occurs in winter and an immunostimulation occurs in summer. However, the higher response found in winter when assaying at 22°C suggests that this property of lymphocytes needs an assay temperature higher than the in vivo temperature in order to observe accurate mitogenic responses.Abbreviations Con A concanavalin A - cpm counts per minute - LPS E. coli lipolisaccharide - MS222 tricainemethane sulphonate - PBS phosphate-buffered saline - PHA phytohemagglutinin - PWM pokeweed mitogen - SI stimulation index     

The composition and fluidity of normal and leukaemic or lymphomatous lymphocyte plasma membranes in mouse and man

The lymphocyte surface membranes from normal and leukaemic or lymphomatous cells from man and mouse were isolated, characterized, and analyzed both biochemically and by diphenyl hexatriene fluorescence polarization. The cholesterol/phospholipid molar ratio for all the pure lymphocyte plasma membranes was 0.45–0.50, and the fluorescence polarization results showed that values much higher than this were not credible. The lipid composition of all the plasma membranes was remarkably similar, except for the concentration of free fatty acids and glycerides.The latter two were particularly high in the mouse lymphoma membrane and these, rather than a low cholesterol concentration, were responsible for the increased fluidity of the cells.The most prominent protein in most of the plasma membrane preparations was actin. This is found only by some authors, and its presence probably depends on the method of lymphocyte disruption.     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.     

Evaluation of centrifugation parameters for density gradient experiments by means of a programmable pocket calculator

A calculation program is proposed suitable for programmable pocket calculators (e.g. HP series) to estimate s_20,w ∫ ω² dt values from density gradient centrifugation data. The program can be applied to linear or exponential density gradients prepared from sucrose or glycerol solutions spun in zonal rotors or swinging bucket rotors. A wide solute concentration range and temperature range is accounted for. Constants for empirical density calculation of glycerol and sucrose solutions concentrated in % (w/v) are estimated. Experiment verification of the program was carried out.     

Rapid purification of photosystem I chlorophyll-binding proteins by differential centrifugation and vertical rotor

Photosystem I (PSI), which consists of a core complex and light-harvesting complex I (LHCI), is an important multisubunit pigment-protein complex located in the photosynthetic membranes of cyanobacteria, algae and plants. In the present study, we described a rapid method for isolation and purification of PSI and its subfractions. For purification of PSI, crude PSI was first prepared by differential centrifugation, which was applicable on a large scale at low cost. Then PSI was purified by sucrose gradient ultracentrifugation in a vertical rotor to reduce the centrifugation time from more than 20 h when using a swinging bucket rotor to only 3 h. Similarly, for subfractionation of PSI into the core complex and light-harvesting complex I, sucrose gradient ultracentrifugation in a vertical rotor was also used and it took only 4 h to obtain the PSI core, LHCI-680, and LHCI-730 at the same time. The resulting preparations were characterized by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), absorption spectroscopy, and 77 K fluorescence spectroscopy. In addition, their pigment composition was analyzed by high-performance liquid chromatography and the results showed that each Lhca could bind 1.5-1.6 luteins, 1.0 Violaxanthins, and 0.8-1.1 beta-carotenes on average, demonstrating that fewer carotenoids were released than with the slower traditional centrifugation. These results showed that the rapid isolation procedure, based on differential centrifugation and sucrose gradient ultracentrifugation in a vertical rotor, was efficient, and it should significantly facilitate preparation and studies of plant PSI. Moreover, the vertical rotor, rather than the swinging bucket rotor, may be a good choice for isolation of some other proteins.     

Inhibition of transferrin receptor expression by interferon-alpha in human lymphoblastoid cells and mitogen-induced lymphocytes

125I-Transferrin binding to lymphoblastoid K562 and Daudi cells markedly increased after exposure of the cells to culture conditions that stimulated proliferation. Treatment of these cells with interferon-alpha (IFN-alpha) resulted in concurrent inhibition of cell growth and of the rise in transferrin binding. Scatchard analyses revealed that IFN reduced the number of transferrin receptors without altering the binding constant. When 125I-transferrin binding was measured using permeabilized cells, the IFN-induced reduction of binding was comparable to that observed with intact cells, indicating that IFN diminished the total number of cellular transferrin receptors. We also found that addition of IFN-alpha to phytohemagglutinin-stimulated human lymphocytes inhibited the mitogen-induced enhancement of [3H]thymidine incorporation as well as surface binding of 125I-transferrin. Our findings suggest that the decrease in transferrin receptor expression on IFN-alpha-treated cells may be one of the mechanisms responsible for the antiproliferative action of IFN.     

Nature of leukemia-associated antigenicity of dinitrophenylated human lymphocytes and lymphoblasts

Using dinitrophenylated human lymphocytes and phytohaemagglutinin-stimulated human lymphoblasts as antigens, antibodies were produced in rabbits. The immunological reactivities of the antisera so produced were tested against various types of leukemic cells after absorbing the sera with pooled normal leukocytes. Both the sera showed reactivity with all types of leukemic cells and no specific affinity for lymphoid leukemic cells was seen. This may suggest the presence of some common antigens on all types of leukemic cells or that dinitrophenylation brings about similar changes on all types of normal leukocytes.     

Enucleation of phagocytic cells with adenine, guanine, and their nucleosides in combination with centrifugation

Several purine compounds, such as adenine, guanine, adenosine, guanosine, and their related compounds, exhibited enucleation activity on adherent mouse peritoneal exudate cells (macrophages) during centrifugation at 25,000 and 35,000 g for 60 min at 34 degrees-36 degrees C in medium containing one of these compounds. Enucleation activity, however, did not occur in cells treated with adenine nucleotides, inosine, xanthine, or any of the tested pyrimidines. The purine compounds also had enucleation activity on mouse macrophage-like cell lines (P388D1 and RAW 264) and mouse polymorphonuclear leukocytes, but not on other typical cell lines such as a human epithelial cell line (HeLa S-3) or a mouse fibroblast cell line (L929). Cytochalasin B (CB) treatment, however, resulted in the enucleation of all cell types tested, even at a centrifugal force as low as 5,000 g. The process of macrophage enucleation was observed by both light microscopy and scanning electron microscopy. In enucleated macrophages that had been treated with purine compounds, but not with CB, a newly formed cytoplasmic crater-like structure (about 3-9 microns in diameter) was observed at the original site of the nucleus. Surface structures, such as microvilli and membrane ruffles, remained relatively intact in macrophages that had been enucleated by treatment with purine compounds. By contrast, these surface structures were markedly changed in CB-treated macrophages. Purine compounds may affect cytoskeletal elements in ways similar to the well characterized effects of CB, and thus result in the enucleation of phagocytes. However, the characteristic differences in the enucleation activity exhibited by purine compounds and CB may indicate that purines have a mechanism of action different from that of CB.     

Enzymatic and chemical analyses of pig platelet membrane subfractions isolated by zonal centrifugation.

1. A mixed membrane fraction prepared from pig platelets was subfractionated, using the "B 14" zonal rotor, into two distinct subpopulations of membrane vesicles, each associated with a different phosphodiesterase activity. 2. The lighter subfraction (MI) was enriched 7-8 fold with bis-(p-nitrophenyl) phosphate phosphodiesterase activity and the denser subfraction (MII) showed a similar degree of enrichment of 5'dTMP-p-nitrophenyl ester phosphodiesterase activity. 3. Assays for other enzyme activities revealed slight enrichement (approx. 2 fold) of acid phosphatase, 3'-dTMP-p-nitrophenyl ester phosphodiesterase and beta-glucuronidase activities in MI, and beta-galactosidase in MII. Cyclic AMP phosphodiesterase, lactate dehydrogenase and N-acetyl-beta-glucosaminidase showed negligible activity in both MI and MII, and succinate dehydrogenase activity could not be detected in either subfraction. 4. Chemical analyses of the membrane subfractions demonstrated that MI contained approx. twice as much cholesterol, phospholipid, sialic acid and hexosamine per unit weight of protein than MII. These results are consistent with our previously reported observations from surface-labelling experiments, which indicated that MI was derived principally from the platelet surface-exposed membranes and that MII was probably intracellular in origin. 5. Analysis of the membrane polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the presence of 12-15 components, in each subfraction, in the mol. wt. range 12000-200000, including a prominent band of approx. mol. wt. 46000, which has beeen identified to be actin. Qualitative as well as possible quantitative differences were apparent in that MII contained three components in addition to those present in MI. 6. Analysis of the periodate-Schiff staining components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis demonstrated the presence of 4 major glycoproteins in both subfractions with apparent mol. wt. ranging from approx. 95000 to 150000; in addition two minor components were also present. Further, a very fast-migrating band, which did not stain with Coomassie blue, was observed in both MI and MII and probably represents lipid material.     

Effects of combined centrifugation and ultraviolet irradiation of eggs on pattern formation in Chironomus embryos

Summary

Combined mild centrifugation and uv irradiation of Chironomus embryos modified the developmental types expected from centrifugation alone, somewhat differently from the combined strong centrifugation and uv irradiation of Smittia embryos. The modifications changed with the stages irradiated. The change caused by anterior irradiation may depend on whether or not a part of the cytoplasmic zone is irradiated simultaneously with the anterior yolky end; because most of the cytoplasm lies in the posterior half of egg at early irradiation, while the tip of the cytoplasm redistributes near the anterior end by the late irradiation. Early uv irradiation of the anterior end of centrifuged eggs, causing the formation of a double abdomen (DA) or an inverted embryo, is not photoreversible, while the uv damage to the anterior end of uncentrifuged eggs, inducing DA, is. These facts suggest that there is another photoirreversible uv target in addition to the photoreversible target for DA induction or the anterior determinant shown in Smittia. Other changes, such as the induction of a double cephalon by late irradiation of the centrifuged egg, are photoreversible, but in an unusual way in that the level of photorecovery is similar to the result of incubation in the dark after early irradiation, and not to that of the centrifuged controls. These modified results were then compared with those for Smittia embryos.     

Chemical composition and enzymic activity of some worm casts

Summary An investigation was carried out to analyze the chemical composition and enzymatic activity of five worm casts obtained from various animal dungs and municipal waste. The E₄/E₆ values for humic and fulvic acids were higher in all worm casts than in a typical Umbrian soil. Comparisons between worm cast, substrate and soil IR and NMR spectra demonstrated the humifying capacity of worms. Enzyme activity was high in all casts and only dehydrogenase seemed to be affected by lead concentration.Research work supported by CNR, Italy. Special grant I.P.R.A.-Sub-project 1. Paper N. 94.