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81.
本文报告一种为适于杂交瘤细胞生长“无血清”培养基-适量的成牛血清低分子成分超滤液、10mg/L转铁蛋白(T)、10mg/L胰岛素(I)、20μmol/L乙醇胺(E)、40nmol/L硒酸钠(S)等诸补充成分替代胎牛血清加到基础液中-也完全适用于培养肿瘤细胞,且观察到与之相似的规律性结果,癌细胞在本“无血清”培养若的生长水平达到在含胎牛血清培养基中生长的水平。对于癌细胞的生长,LMW-CS与T、I、 相似文献
82.
Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro 总被引:1,自引:1,他引:0
JEROME F. LA PEYRE DORIS Y. SCHAFHAUSER ESAM H. RIZKALLA MOHAMED FAISAL 《The Journal of eukaryotic microbiology》1995,42(5):544-551
ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases. 相似文献
83.
The heterobasidiomycetous yeastRhodotorula glutinis was able to grow in medium containing a high concentration of LiCl. This character ofR. glutinis was presumed to be attributable to its ability to incorporate [14C]-adenine and [14C]-leucine into nucleic acids and proteins, respectively, in the presence of LiCl. Intracellular levels of Li+ and Cl– ions, production and accumulation of glycerol as an osmoregulator, and respiration in the LiCl-stressed condition were almost the same in the tolerant yeastR. glutinis and the sensitive yeastRhodosporidium sphaerocarpum. 相似文献
84.
Koichiro Yoshihara Motokatsu Tsuyuki Asako Itaya Yasuharu Tanaka Tomoya Kamiya 《Molecular and cellular biochemistry》1994,135(2):143-151
HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition. 相似文献
85.
A new simple method for the preparation of chemically crosslinked chitosan beads is presented. It consists of the dropwise addition of 2-3% (w/v) low molecular weight chitosan solution containing 2% (w/v) glyoxal in 1% (w/v) tetrasodiumdiphosphate, pH 8.0. Immobilized viable baker's yeast (Saccharomyces cerevisiae) could be obtained via gel entrapment within the new beads when means preventing their direct contact with soluble chitosan were provided, "disguising" the cells until gelation and crosslinking were completed. Such means included cell suspension in castor oil or mixing with carboxymethyl-cellulose powder. Application of these means was shown to be necessary, as cells exposed to soluble chitosan immediately lost their viability and glycolytic activity. Yeast disguised in castor oil was also protected from bead reinforcement by glutaraldehyde treatment, significantly strengthening bead stability while operating under acidic conditions. This capability was demonstrated by continuous ethanol production by chitosan entrapped yeast. (c) 1994 John Wiley & Sons, Inc. 相似文献
86.
We describe a screen to isolate cDNAs encoding Drosophila mitosis inhibitors capable of suppressing the mitotic catastrophe phenotype resulting in Schizosaccharomyces pombe from the combination of the weel-50 mutation with either a deletion allele of mil1, or with overexpression of cdc25
+. One plasmid was isolated which could suppress the temperature sensitive lethality of both these strains. The cDNA in this plasmid encodes a protein highly homologous to the DEAD-box family of ATP-dependent RNA helicases, rather than to protein kinases as might be expected. It is possible that the RNA helicase described here may regulate entry into mitosis by down regulating the expression of other genes whose activity may be rate-limiting for entry into mitosis. 相似文献
87.
几种濒危植物及其近缘类群总DNA的提取与鉴定 总被引:122,自引:0,他引:122
用低pH 介质,高盐沉淀蛋白质方法成功地从银杉(Cathaya argyrophylla Chun etKuang)、矮牡丹(Paeonia suffruticosa var. spontanea)、南川升麻(Cim icifuga nanchuanensisHsiao)、裂叶沙参(Adenophora lobophylla)的同属种泡沙参(A. potaninii)等植物中提取和部分纯化了细胞总DNA,并对其产率、质量和纯度作了鉴定。此方法的关键是用了一个低pH提取介质,它能有效防止组织破碎及沉淀大量材料时的电离化作用及酚化合物的进一步氧化。所得DNA 不需经氯化铯梯度离心或柱层析,直接可用于限制性片断长度多态性(RFLP)及随机扩增的DNA多态性(RAPD)等分子水平的遗传标记。为检测濒危植物的遗传多样性提供了一套迅速、简便和可靠的技术方案 相似文献
88.
Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells 总被引:2,自引:0,他引:2
The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C12FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG
ortho-phenyl 2--D-galactopyranoside
- OUR
oxygen uptake rate (-mol O2/liter/hour)
- qglucose
specific glucose uptake rate (mg glucose/106cell/hour)
- qglutamine
specific glutamine uptake rate (mg glutamine/106cell/hour)
- qO2
specific oxygen uptake rate (-mol O2/106cell/hour)
- MOI
virus multiplicity of infection (viral plaque forming units/cell) 相似文献
89.
Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×106 cells mL–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium. 相似文献
90.
Daniel Keppler Patrice Waridel Magnus Abrahamson Daniel Bachmann José Berdoz Bernard Sordat 《生物化学与生物物理学报:疾病的分子基础》1994,1226(2):117-125
The lysosomal cystein proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-β, TNF-α, and IL-1β. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-β, TNF-α, and IL-1β, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastate at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor comples. In addition, our data from neutrophil elastate activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo. 相似文献