共查询到20条相似文献,搜索用时 15 毫秒
1.
J M Whaun 《The Journal of protozoology》1984,31(3):381-384
In vitro culture of Plasmodium falciparum-infected human erythrocytes (RBC) has permitted systematic study of human host-parasite relations. In this study the effect of aspirin in the culture system was examined by using serum from blood of fasting, healthy male volunteers, before and after the ingestion of aspirin. The addition of aspirin-containing serum disturbed parasite growth and development: 0-1/2 dilutions of treated/control sera inhibited parasite development, with nuclear pyknosis, pyknotic extracellular parasites (trophozoites) in the media, decreased numbers and sizes of "rings" (early trophozoites), and an increased number of later trophozoites and schizonts. Paradoxically, while the incorporation of [3H]isoleucine into protein was not affected by the aspirin-containing sera, the incorporation [3H]hypoxanthine was significantly changed and did not correlate with morphological evidence of cytotoxicity. Thus, the so-called "incorporation" of a radioactive tracer is not a fully reliable index of parasite growth in the presence of certain compounds. The findings underscore the importance, in this culture system which employs human serum, of avoiding serum from donors who have recently ingested aspirin. 相似文献
2.
James B. Jensen Michael T. Boland Michael Hayes Mustafa A. Akood 《Experimental parasitology》1982,54(3):416-424
Tightly synchronized cultures of Plasmodium falciparum were exposed to various dilutions of human sera from healthy adults living in malarious areas before and after merozoite invasion was completed. All “immune” sera inhibited merozoite invasion to a greater or lesser degree, depending upon the dilution of serum added to the cultures. Additionally, most sera demonstrated moderate to severe retardation of parasite development, even when merozoite invasion was completed in nonimmune serum before developing parasites were exposed to immune serum dilutions. Both types of inhibition were discernable on Giemsa-stained thin films, and these observations were highly correlated with incorporation of [3H]hypoxanthine. Large numbers of serum samples could be examined using microliter quantities of immune serum in microtiter plate cultures processed with a cell harvester. 相似文献
3.
Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture
Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [3H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [3H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [3H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue. 相似文献
4.
E. Orozco R. Gharaibeh A. M. Riverón D. M. Delgadillo M. Mercado T. Sánchez E. Gómez Conde M. A. Vargas R. López-Revilla 《Molecular genetics and genomics : MGG》1997,254(3):250-257
We report here the presence of cytoplasmic DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica. Cytoplasmic DNA was detected in live trophozoites in a structure that we called EhkO, using the fluorescent dye acridine orange, and by in situ hybridization to trophozoites with a rDNA probe. The EhkO was found in the axenically grown clones A, L6 (strain HM1:IMSS) and MAVax (strain MAV) and in the polyxenically grown clone MAVpx (strain MAV). Bacteria present in MAVpx did not cross hybridize with the DNA probe neither in in situ hybridization or in Southern blot experiments. Autoradiography of metabolically [3H]thymidine-labeled trophozoites showed the presence of EhkO, and an EhkO-enriched fraction, purified from a nuclei-free extract and examined by light microscopy, exhibited [3H]thymidine incorporation into this structure. DNA was purified from the EhkO and enriched nuclear fractions and analyzed by transmission electron microscopy. The EhkO fraction contained DNA networks resembling those of trypanosome kDNA, whereas nuclear DNA was present mainly as linear molecules and some circles. Our findings imply that E. histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected. 相似文献
5.
《Molecular & general genetics : MGG》1997,254(3):250-257
We report here the presence of cytoplasmic DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica. Cytoplasmic DNA was detected in live trophozoites in a structure that we called EhkO, using the fluorescent dye acridine
orange, and by in situ hybridization to trophozoites with a rDNA probe. The EhkO was found in the axenically grown clones
A, L6 (strain HM1:IMSS) and MAVax (strain MAV) and in the polyxenically grown clone MAVpx (strain MAV). Bacteria present in
MAVpx did not cross hybridize with the DNA probe neither in in situ hybridization or in Southern blot experiments. Autoradiography
of metabolically [3H]thymidine-labeled trophozoites showed the presence of EhkO, and an EhkO-enriched fraction, purified from a nuclei-free extract
and examined by light microscopy, exhibited [3H]thymidine incorporation into this structure. DNA was purified from the EhkO and enriched nuclear fractions and analyzed
by transmission electron microscopy. The EhkO fraction contained DNA networks resembling those of trypanosome kDNA, whereas
nuclear DNA was present mainly as linear molecules and some circles. Our findings imply that E. histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected.
Received: 7 August 1996 / Accepted: 7 October 1996 相似文献
6.
Ariane Plet Yoheved Berwald-Netter 《Biochemical and biophysical research communications》1980,94(3):744-754
Culture conditions were defined allowing to demonstrate a stimulatory effect of both serum-contained and purified Somatomedin activity on incorporation of [3H]thymidine and replication of cultured normal human fibroblasts. The use of dialyzed human serum in MEM medium supplemented by 0.2 mM serine offered the necessary and sufficient culture conditions. A significant difference between normal and hypopituitary patients sera was found in their effect on the rate of [3H]thymidine incorporation (p < 0.0001) and on cell replication (p < 0.01). Purified Somatomedin-C, in MEM without serum, is a poor mitogen. Its activity was strongly enhanced by the addition of 0.1 % dialyzed serum and 0.2 mM serine without, however, exceeding the stimulatory level of 1 % whole normal serum. The requirement of concomitant presence, for optimal cell growth, of different low and high MW serum components is discussed. 相似文献
7.
The inhibition of human prostatic epithelial cell (MA-160) replication by cAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (cAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-cAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the cAMP phosphodiesterases, cAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl cAMP (db-cAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [3H]TdR and [3H]uridine than control cells. Treatment of cells with 0.5 mM cAMP for 24 h, on the other hand, resulted in both substantially increased [3H]TdR uptake and increased [3H]uridine incorporation into RNA. The effects of similar treatment with db-cAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine. 相似文献
8.
Plasmodium falciparum: assessment of in vitro growth by [3H]hypoxanthine incorporation 总被引:6,自引:0,他引:6
To evaluate rapidly Plasmodium falciparum growth in Vitro, [3H]hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, [3H]hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting [3H]hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro. 相似文献
9.
Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1 (HSV-1) 总被引:2,自引:0,他引:2
The envelope membrane glycoprotein gC of HSV-1 was purified from Triton X-100 extracts of virus-infected BHK-21 or HEp-2 cells by a single step immuno-affinity column using monoclonal anti-gC antibody. The analysis of the purified [3H]G1cN labeled glycoprotein gC (by gel filtration on Bio-Gel P4) before and after digestion with endo-β-N-acetylglucosaminidase (endo D) indicated that gC contains Asn-linked “complex type” oligosaccharides. No “high mannose” type oligosaccharides were detected. Fractionation of radio-labeled glycopeptides of gC on a column of concanavalin A-sepharose suggested that glycopeptides have “diantennary” and “triantennary” and/or “tetra antennary” structures. Tunicamycin inhibited the incorporation of [14C]GalN or [3H]GlcN into gC in HSV-1 infected BHK-21 or HEp-2 cells. Gel filtration analysis of [3H]GlcN labeled gC following β-elimination reaction failed to indicate O-glycosidically linked oligosaccharides. 相似文献
10.
R. S. PHILLIPS R. J. M. WILSON G. PASVOLS 《The Journal of eukaryotic microbiology》1978,25(3):394-398
SYNOPSIS. Gametocytes differentiated from ring-stage parasites in microcultures of human blood infected with Plasmodium falciparum. Immature gametocytes could be distinguished morphologically from late asexual trophozoites after ~ 40 h of culture. Differentiation into crescentic forms took several days and the incorporation of [3H]-isoleucine by developing gametocytes was demonstrated. About 1% of red cells contained gametocytes at the maximum densities attained. Differentiation of gametocytes occurred either directly from rings placed in culture or from the progeny of subsequent cycles of schizogony and invasion in vitro. The latter occurrence was confirmed by the development of gametocytes in marker fetal red cells added to cultures, although fetal red cells provide a less favorable environment than those with HbA for growth of the parasites. 相似文献
11.
Sang-Cheol Seong Takashi Matsumura Francis Youngin Lee Mary C. Whelan Xiao Qing Li Stephen B. Trippel 《Experimental cell research》1994,211(2)
Insulin-like growth factor I (IGF-I) is anabolic for chondrocytes and is thought to be important in regulating such normal cartilaginous tissues as the epiphyseal growth plate. In the present studies, we have investigated the role of IGF-I in the regulation of neoplastic cartilage. Chondrocytes cultured from a transplantable rat chondrosarcoma were analyzed for responsiveness to IGF-I with respect to DNA and glycosaminoglycan synthesis as determined by labeling with radioactive thymidine and sulfate, respectively. Stimulation of [3H]thymidine and [35S]sulfate incorporation by IGF-I was two to four times that in serum-free controls, with half-maximal stimulation at 1 × 10-9M. The efficacy of IGF-I was approximately one-half of that of serum in stimulating [3H]thymidine incorporation and was comparable to that of serum for [35S]sulfate incorporation. When Swarm rat chondrosarcoma chondrocytes were cultured in the presence of IGF-I and exposed to graded concentrations of anti-IGF-I antibody, [3H]thymidine incorporation and [35S]sulfate incorporation were attenuated in a dose-dependent fashion to 29 and 25% of antibody-free controls, respectively. Nonspecific antibody not raised against IGF-I was not inhibitory. These observations suggest that the majority of IGF-I action on these cells is susceptible to immunoinhibition. To estimate the contribution of IGF-I to the regulation of these cells by serum, Swarm rat chondrosarcoma chondrocytes were cultured with graded concentrations of either calf serum or fetal calf serum in the presence of anti-IGF-I antibody, nonspecific antibody, or no other additives. Specific antibody attenuated the effect of calf serum on both [3H]thymidine and [35S]sulfate incorporation with overall inhibition of 52% (P < 0.01) and 48% (P < 0.001), respectively. Nonspecific antibody superimposed small, variably stimulatory or inhibitory effects on those of calf serum. When chondrosarcoma chondrocytes were incubated with fetal calf serum, anti-IGF-I antibody exerted a minimal inhibitory effect, reducing both [3H]thymidine and [35S]sulfate incorporation by less than 25%. The immunoinhibition of both pre- and postnatal serum could be overcome in a dose-dependent fashion by increasing serum concentrations. These results suggest that the factors influencing Swarm rat chondrosarcoma chondrocytes may be developmentally regulated and that the contribution of IGF-I to the action of serum increases between fetal and postnatal life. These data support the hypothesis that chondrosarcoma is a somatomedin-responsive neoplasm and suggest that this tumor may be susceptible to interventions directed toward mechanisms that block insulin-like growth factor action. 相似文献
12.
Jean-Marc Lebel Wilfrid Giard Pascal Favrel Eve Boucaud-Camou 《Biology of the cell / under the auspices of the European Cell Biology Organization》1996,86(1):67-72
Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [3H]-leucine and [3H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [3H]-leucine or for [3H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells. 相似文献
13.
Kari Pietilä 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,677(2):318-325
This study sought to elucidate the optimal cell culture conditions for studies concerned with the incorporation of [3H]glucosamine into glycosaminoglycans by rabbit aortic smooth muscle cells. The incorporation of radioactivity into extracellular sulphated glycosaminoglycans was linear for at least 72 h and that into pericellular sulphated glycosaminoglycans for up to 24 h. The incorporation of radiolabel into hyaluronic acid was linear only up to 12 h. In the exponential growth phase the incorporation of [3H]glucosamine into sulphated glycosaminoglycans and hyaluronic acid proved to be less marked than in the stationary growth phase, but the highest values were nevertheless obtained immediately after trypsinisation. When studied in the stationary growth phase, cell density and incorporation of [3H]glucosamine were positively correlated in the case of hyaluronic acid, but in the case of sulphated glycosaminoglycans there was a negative correlation. The serum concentration of the incubation medium and the incorporation of radioactivity into hyaluronic acid were positively related. With sulphated glycosaminoglycans this was the case only after a 7-day preincubation in the different serum concentrations. when incorporation was studied without preincubation, the incorporation of radioactivity into sulphated glycosaminoglycans proved to be negatively associated with the serum concentration of the medium. The environmental pH of the cells was associated with the incorporation of radioactivity into hyaluronic acid and sulphated glycosaminoglycans in that between pH values 6.8 and 7.9 the incorporation of radioactivity increased when the pH of the medium was raised. 相似文献
14.
Summary In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation
in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme.
Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23–24 chick embryos and plated at various densities
(1, 5, 10, or 20 × 106 cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV.
Cell growth was assessed by the incorporation of [3H]leucine and [3H]thymidine. Chondrogenesis was determined by the incorporation of [35S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 × 106 cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis
equally well as FBS. In cultures plated at 5 × 106 cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation
of [35S]sulfate (2.6-fold), [3H]leucine (1.4-fold), and [3H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of
Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 × 106 cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed
with NuSerum or NuSerum IV. The observed effects of NuSerum and NuSerum IV may be attributed to a combination of factors,
including lower concentrations of serum and its associated proteins, as well as supplemented growth factors and hormones known
to promote cell proliferation and differentiation. Therefore, NuSerum and NuSerum IV are excellent, low-cost replacements
for FBS in maintaining cellular growth and promoting chondrogenesis in LB mesenchymal cell cultures in vitro. 相似文献
15.
16.
Cheryl A. Conover Ron G. Rosenfeld Raymond L. Hintz 《In vitro cellular & developmental biology. Plant》1989,25(6):521-527
Summary The biological actions of insulinlike growth factor I (IGF-I) measured under serum-free assay conditions were found to be
significantly influenced by prior subculture conditions for adult human fibroblasts. Glucocorticoids seemed to be the major
medium variable affecting IGF-I action. IGF-I added to serum-free cultures had little or no effect on [14C]aminoisobutyric acid uptake or [3H]thymidine incorporation in human fibroblasts previously maintained in media containing serum with low glucocorticoid levels
or in serum stripped of endogenous steroids. However, a 48-h preincubation with dexamethasone resulted in a marked synergistic
increase in IGF-I stimulation of [14C]aminoisobutyric acid uptake and [3Hthymidine incorporation in these cultures. In contrast, IGF-I in serum-free medium seemed to be a potent mitogenic and metabolic
stimulus for human fibroblasts which had been subcultured in media with a high glucocorticoid content, either endogenous,
or supplemented. After these culture conditions, a 48-h preexposure to dexamethasone had no further enhancing effect on IGF-I
action. Dexamethasone also potentiated IGF-I, insulin, and epidermal growth factor stimulation of fibroblast replication depending
on the earlier subcultivation conditions. Thus, glucocorticoids are important modulators of IGF-I bioactivity in cultured
human fibroblasts. Serum glucocorticoids can exert a profound influence on the biological phenomena measured in cell culture,
even when the serum has been removed before the actual experiment, and must be carefully taken into account for accurate evaluation
of the biological function of IGF-I and other growth factors.
This work was supported in part by grants DK28229, DK36054, CA42106, RCDA01275, and DK24085 from the National Institutes of
Health, Bethesda, MD. 相似文献
17.
Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [3H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [3H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression. 相似文献
18.
Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA 总被引:1,自引:0,他引:1
S A Schwartz D Horio W H Kirsten 《Biochemical and biophysical research communications》1974,61(3):927-933
Secondary cultures of rat embryo cells were exposed for 24 hrs. to 10-7M [3H] thymidine (TdR) or 10?7M [3H]5-bromodeoxyuridine (BrdU) in order to localize and compare the distribution of the isotopes in DNA. DNA was extracted, sheared, and centrifuged to equilibrium through neutral and alkaline CsCl density gradients. The DNA band from each gradient type was separated into a “heavy” and “light” fraction, and DNA-DNA reassociation hybridizations were performed on each sample. Renaturation profiles revealed that each fractionated DNA sample was representative of the complete rat cell genome, except for the “light” [3H]BrdU-DNA prepared by centrifugation through alkaline CsCl gradients. This fraction was predominantly depleted of labeled late repetitive and intermediate sequences. Uncentrifuged rat DNA was sequentially fractionated during reassociation into rapidly, intermediate, and slowly reassociating sequences by hydroxyapatite chromatography. Relative specific activities of each component revealed a non-uniform distribution of [3H]BrdU moieties as compared to [3H]TdR. These results suggest a nonrandom incorporation of 10?7M BrdU into rat cell DNA sequences. 相似文献
19.
B.E. Linebaugh J.A. Rillema 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(4):346-355
Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [3H]uridine incorporation into RNA and [3H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10−21 M). Insulin stimulated the rate of [3H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [3H]uridine, [3H]thymidine and [3H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis. 相似文献
20.
Summary The characteristics of hormone-dependent rat mammary tumors in response to serum and hormones were determined in collagen
gel matrix culture. Epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas were embedded
in collagen gel and the effect of estrogen, progesterone, prolactin, insulin, and serum was tested. The total cell number
and [3H]thymidine incorporation were used to determine the growth pattern of the cells in culture. It was found that in medium containing
20% porcine serum and supplemented with insulin, estrogen, progesterone and prolactin, both the cell number and [3H]thymidine labeling index increased with time, after an initial lag. Serum seemed to be essential to maintain growth of the
tumor cells, because hormones alone, in the absence of serum, were unable to sustain growth of the cells. When estrogen, progesterone,
prolactin, and insulin were tested individually in the presence of 20% porcine serum, only estrogen demonstrated a significant
stimulatory effect. 相似文献