Evidence obtained in resent years suggests that proteasomes are one of the major components of a new regulatory cell system. Successive cell-cycle phases, differentiation, apoptosis, signal transduction, and immune response are controlled by 26S proteasomes that are involved in the specific proteolysis of regulatory proteins [1]. Programmed proteolysis is not the only function of proteasomes in a cell. For example, 20S proteasomes exhibit ribonuclease activity [2]. The specific endoribonuclease activity of 26S proteasomes has also been found [3], and its regulation has been studied during the differentiation of K562 cell [4], induction of K562 apoptosis [5], and signal transduction from the receptor of epidermal growth factor (EGF) in A431 cells [6]. The obtained results suggest that proteasomes control the half-life or stability of specific messenger RNAs during the above processes.According to current views, the composition and specific proteolytic activity of the cell proteasome population is heterogeneous; it is represented by a mixture of several subtypes of particles [7,8].It was previously shown that cells excreted proteasomes and proteasome-like particles into the culture medium or extracellular space, and the number of exported proteosomes changed upon malignant transformation [9][10][11]. However, the properties of excreted proteasomes, their enzymatic activity, and specificity remain unknown.The evidence obtained in this study testifies to the specificity of 26S proteasomes excreted by cells into the culture medium. The excreted proteasomes were found to retain their characteristic enzymatic activities, although they differed from intracellular proteasomes in the specificity of proteolytic activity and in characteristics of RNase activity. In addition, the activity of extracellular particles activity depended on the cell functional state. It should be emphasized that changes in cytoplasmic and excreted proteasome activities were different, which suggests that the excretion of the specific proteasome population from cells is part of a regulatory mechanism.Proteasomes were isolated from A431 human epidermoid carcinoma cells and K562 human proerythroleukemic human cells (Russian Collection of Cell Cultures, Institute of Cytology, Russian Academy of Science).The cells of line A431 were grown in DMEM containing 10% fetal calf serum. Subconfluent cells were transferred onto a medium containing 0.5% serum and incubated for 24 h (the control cells); then, EGF (100 ng/ml) was added, and the cells were incubated for another 15 min. The cells of line K562 were grown on RPMI 1640 containing 10% fetal calf serum. Programmed cell death was induced by the addition of either diethylmaleate to a final concentration of 1 mM or doxorubicine to a final concentration of 4 µ M. After 24 h of incubation, the rate of apoptosis was assessed from changes in nuclear morphology as determined upon staining with Hoehst 33258 and from internucleosome DNA fragmentation.Cytoplasmic 26S proteasomes were isolated from the postmitochondr...
For the first time small nuclear ribonucleoprotein particles (K K-RNP) tightly bound to chromatin as well as cytoplasmic K K-RNP are shown to possess strong and regulated endonuclease activity specific for mRNAs and hnRNAs. The enzymatic nature of this activity is confirmed, and the optimal conditions detected. This RNase activity is controlled by the action of a differentiating stimulus, dimethylsulfoxide, in human K562 cells. Small K K-RNP involvement in the coordinated control of stability of pre-messenger RNA and messenger RNA molecules is suggested.z 1999 Federation of European Biochemical Societies.
Aim.To use the ability of potato leafroll virus (PLRV) to infect and multiply in mammalian continuous cell lines to purify PLRV isolates from the vegetative plant material, and to study the pathogenicity of those isolates for plants (after culturing in mammalian continuous cell line), to investigate morphological, physical-chemical, biological and antigen properties of PLRV isolates from mammalian cells and to study an alternative diagnostic method -the neutralization test in the mammalian continuous cell lines. Methods. The methods of cultivating animal viruses in the mammalian continuous cell line, microscopical biochemical, and serological methods, the method of arti¿ cial nutrition of aphids are detailed under Material and Methods. Results. It was demonstrated that successful cultivation of PLRV in mammalian continuous cell line allowed obtaining pure virus isolates from potato plants and aphids and preserving them for a long time (over a period of 7 years). The cultivation of PLRV in the mammalian continuous cell line did not impact its pathogenic properties and allowed transmitting the virus to plants. Continuous cells lines of pig embryonic kidney (PEKV), of kidney Syrian hamster (BHK-21), of testicles of piglets (PTP), of kidneys of the bull (MDBC), and of carcinoma rabbit kidney (RK-13) were found to be sensitive to PLRV, Con tinuous cell lines of human (HeLa, Hep-2 and of African green monkey kidney (Vero) were not infected by the virus. The infectious activity of PLRV in the sensitive continuous cell lines was 20-8.5 lg TCD 50 /ml depending on the cell line. The isolates of PLRV were resistant to lipiddissolving solvents, multiplied in a pH range from 4.0 till 10.0 and were thermoresistant at 50 ºɋ in the absence of bivalent ions of magnesium, ɌIP was in the range of 60-65 ºɋ under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 °ɋ. The use of neutralization test in the mammalian continuous cell line allowed isolation in pure culture and identi¿ cation of PLRV reliably in a time span of c. 14 days. Conclusions. It was proven that PLRV can be cultivated in the mammalian continuous cell lines of PEKV, ȼɇɄ-21, PTV, MDȼɄ and RK-13. It was established that the cultivation of PLRV in these continuous cell lines did not impact its biological, pathogenic, antigenic and physical-chemical properties. The identi¿ cation of pure cultures of PLRV obtained in mammalian cells can be reliably performed by the use of neutralization reaction.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.