胰蛋白酶消化法和组织块法原代培养人包皮成纤维细胞的比较

小鼠胚胎成纤维细胞(MEFs)是目前国际上公认的人胚胎干细胞(hESCs)体外培养的饲养层细胞,但MEFs存在寿命短、动物源性污染等问题,需要探索适于hESCs体外培养且寿命长的人源性饲养层.采用胰蛋白酶消化法和组织块法分别原代培养人包皮成纤维细胞(hFFs),探索hFFs原代培养的最佳方法,为hFFs作为饲养层在hESCs研究中的应用提供可靠的科学依据;通过倒置显微镜下观察其生长状态和免疫细胞化学染色鉴定,结果显示两种原代培养方法获得的hFFs的形态及生物学特性均符合成纤维细胞;通过测定细胞生长曲线及MTT法检测,结果显示两种原代培养方法获得的不同代数的hFFs均具有较高的增殖活性,传代10余代仍能保持较好的细胞形态,可以制备成饲养层用于hESCs的研究.     

Poliovirus 2A(Pro) increases viral mRNA and polysome stability coordinately in time with cleavage of eIF4G

Poliovirus (PV) 2A protease (2A(Pro)) cleaves eukaryotic initiation factors 4GI and 4GII (eIF4GI and eIF4GII) within virus-infected cells, effectively halting cap-dependent mRNA translation. PV mRNA, which does not possess a 5' cap, is translated via cap-independent mechanisms within viral protease-modified messenger ribonucleoprotein (mRNP) complexes. In this study, we determined that 2A(Pro) activity was required for viral polysome formation and stability. 2A(Pro) cleaved eIF4GI and eIF4GII as PV polysomes assembled. A 2A(Cys109Ser) (2A(Pro) with a Cys109Ser mutation) protease active site mutation that prevented cleavage of eIF4G coordinately inhibited the de novo formation of viral polysomes, the stability of viral polysomes, and the stability of PV mRNA within polysomes. 2A(Cys109Ser)-associated defects in PV mRNA and polysome stability correlated with defects in PV mRNA translation. 3C(Pro) activity was not required for viral polysome formation or stability. 2A(Pro)-mediated cleavage of eIF4G along with poly(rC) binding protein binding to the 5' terminus of uncapped PV mRNA appear to be concerted mechanisms that allow PV mRNA to form mRNP complexes that evade cellular mRNA degradation machinery.     

Comparison of the Effect of Intravenous Administration of d-Lysergic Acid Diethylamide on Free and Membrane-Bound Polysomes in the Rabbit Brain

Abstract: The intravenous administration of LSD to young adult rabbits resulted in the disaggregation of both free and membrane-bound classes of brain polysomes. Based on the analysis of LSD dosage and the time course of the LSD-induced brain polysome shift, it was found that free polysomes were more sensitive to the drug than the membrane-bound polysome fraction. LSD-induced hyperthermia may be involved in the disaggregation of free and membrane-bound polysomes, since a correlation was found between the extent of LSD-induced hyperthermia and the degree of brain polysome shift. Prevention of LSD-induced hyperthermia by maintaining the animal at 4°C blocked the disaggregation of both polysome classes. Induction of hyperthermia by elevation of ambient temperature also resulted in a shift in free and membrane-bound polysomes. In all cases the disaggregation of polysomes to monosomes was not caused by RNase activation. During polysome disaggregation, polyadenylated mRNA associated with both free and membrane-bound polysomes was not degraded but was relocalized from polysomes to monosomes.     

Interaction of plant polysomes with the actin cytoskeleton

Protein composition and functional activity of various polysome subpopulations isolated from Vicia faba L. leaves and Triticum aestivum L. and Hordeum vulgare L. seedlings were studied. Membrane- and cytoskeleton-bound polysomes were more active in the wheat germ cell-free translational system than free polysomes. Several non-ribosomal proteins were detected in the polysome preparations by gel electrophoresis and Western blot analysis: (1) a canonical actin of mol wt 42 kDa; (2) a 40 kDa protein, demonstrating affinity for ribosomes, sharing some determinants with actin, and present predominantly in the subpopulations of bound polysomes; and (3) an acidic ribosome-associated p40 evenly distributed between free and bound polysomes. The possibility of involvement of these proteins in interactions between polysomes and the actin cytoskeleton is discussed.     

Translation of mRNA associated with monosomes and residual polysomes following disaggregation of brain polysomes by LSD and hyperthermia

Intravenous administration of LSD to young adult rabbits induces a transient disaggregation of brain polysomes and a relocalization of mRNA from polysomes to monosomes. To analyze the spectrum of mRNA molecules which were associated with either the residual polysomes or the translationally inactive monosome complex, these two fractions were isolated on sucrose gradients and translated in a reticulocyte cell-free system. Analysis of [³⁵S]methionine labeled translation products by one and two dimensional gel electrophoresis revealed that a full spectrum of mRNA molecules was relocalized from polysomes to monosomes following drug induced polysome disaggregation. The only exception was the mRNA coding for the LSD-induced 74K protein which was associated with the residual polysome fraction and not with the monosome complex. This brain protein is similar in molecular weight to one of the major heat shock proteins which are induced in tissue culture cells following elevation of ambient temperature and disaggregation of existing polysomes. The mRNA coding for the 74K brain protein was not observed in polysomes isolated following blockage of LSD-induced hyperthermia but it was noted when hyperthermia was induced by elevation of ambient temperature. The mRNA species coding for the 74K protein was polyadenylated.     

Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation

LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.     

Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes

The kinetics of labeled histone mRNA entry into polysomes was studied in nuclease-treated reticulocyte lysates. Added mRNA rapidly bound 1 or 2 ribosomes. However, the formation of full size polysomes required at least 16 min. The amount of mRNA bound to ribosomes reached a maximum (73%) within 2 min after mRNA addition and then declined slowly for the remainder of the experiment. Two initiation inhibitors, aurintricarboxylic acid and 7-methylguanosine 5'-triphosphate, were found to affect polysome size and the fraction of mRNA in polysomes in an opposite manner. These results suggest that initiation and reinitiation events may be intrinsically different. The relatively long time period required for the formation of large polysomes can be explained by large polysomes having higher initiation and/or reinitiation rates or slower elongation rates. These possibilities are not mutually exclusive. The results suggest that there exist several levels of control which can regulate polysome size and the fraction of mRNA in polysomes.     

Dissociation of polysome aggregates by protease k

Apparent large size-classes of zein-synthesizing polysomes from developing kernels of Zea mays L. were converted to smaller polysomes after treatment with Protease K. The reduction in polysome size was not a result of ribonuclease activity, inasmuch as the enzyme did not affect the free polysomes or the size of the mRNA from the membrane-bound polysomes. High concentrations of MgCl(2) in polysome buffer inhibited ribonuclease activity and appeared to cause protein interaction between nascent zein polypeptides. Although Protease K inhibited the polysome's capacity for protein synthesis, it was a useful reagent for determining if polysomes were aggregated by protein.     

Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay.

Polysome and messenger ribonucleoprotein (mRNP) preparations from various mammalian cells contain tightly bound nuclease activity that causes degradation of the mRNA in the preparations. This activity was found to cosediment with all polysome size classes as well as with free mRNPs and to remain associated with the mRNPs released from polysomes by treatment with EDTA. No association with ribosomal subunits was evident. The rates of mRNA degradation were not affected by serial dilution, an indication that enzyme and substrate are tightly associated. beta-Globin mRNA in purified reticulocyte polysomes was cleaved at AU sequences in the 3'-terminal region. Cleavages at the same sites occurred when deproteinized reticulocyte RNA was incubated with mouse sarcoma 180 (S-180) polysomes. The S-180 preparations caused additional cleavages, primarily at UG sequences. A P40 mRNA in S-180 polysomes was cleaved primarily in the 3' noncoding region, but the cleavages in a P21 mRNA were seen in the 5' noncoding region only. Actin mRNA was cleaved in an internal region, yielding large relatively stable 3'- and 5'-terminal fragments. These data suggest the occurrence of highly specific interactions between one or more mRNA-bound nucleases and individual mRNA species.     

Translational control mechanism of ornithine decarboxylase by asparagine and putrescine in primary cultured hepatocytes

Asparagine stimulated the translation of ornithine decarboxylase (ODC) mRNA more than 10-fold in cultured hepatocytes which had been pretreated with glucagon in simple salt/glucose medium. Putrescine suppressed the increase in the rate of ODC synthesis caused by asparagine without significant change in the amount of ODC mRNA, suggesting that putrescine inhibited the effect of asparagine at least in part at the level of translation. Polysomal distribution of ODC mRNA was analyzed to examine the site of translational regulation by these effectors. In uninduced hepatocytes, most of the ODC mRNA was sedimented slightly after the 40 S ribosomal subunit. This ODC mRNA was sequestered from translational machinery since it was not shifted to the polysome fraction when peptide elongation was specifically inhibited by a low concentration of cycloheximide. In asparagine-treated cells, 40% of total ODC mRNA was in the polysomal fraction and formed heavier polysomes, indicating that asparagine stimulated both recruitment of ODC mRNA from the untranslatable pool and the initiation steps of translation. Putrescine did not change the distribution pattern of ODC mRNA on polysomes significantly. Thus, 30% of ODC mRNA remained on polysomes even when ODC synthesis was completely inhibited by putrescine. Paradoxically more than 70% of ODC mRNA was shifted into polysomes by putrescine in the presence of low concentrations of cycloheximide. These results, together with changes in the polysome profile, suggested that putrescine nonspecifically stimulated the recruitment of ODC mRNA from the untranslatable pool, whereas it specifically inhibited its translation at both the initiation and the elongation steps.     

S6 phosphorylation accompanies recruitment of ribosomes and mRNA into polysomes in response to dichlororibofuranosyl benzimidazole

Dichlororibofuranosyl benzimidazole (DRB), a potent inhibitor of nuclear RNA synthesis and messenger RNA (mRNA) accumulation, produces a paradoxical mobilization of rRNA and mRNA from the subpolysomal pool into polysomes in HeLa cells during the first 40 min of treatment. S6 is phosphorylated concurrently with polysome accumulation, and ribosomal subunits containing phosphorylated S6 are preferentially localized in polysomes, indicating that they form initiation complexes more readily than their non-phosphorylated counterparts.     

The characterization of free,cytoskeletal and membrane-bound polysomes in Krebs II ascites and 3T3 cells

Summary Polysomes from Krebs II ascites and 3T3 cells were separated into three populations by using a sequential extraction method. Free polysomes were released by using a combination of low salt (25 mM KCl) and NP-40 detergent in the lysis buffer. The cytoskeletal bound polysomes were subsequently released by raising the salt concentration to 130 mM and finally, polysomes bound to the membranes of the endoplasmic reticulum were extracted by the combined treatment with Triton X-100 and deoxycholate. The results presented here illustrate that the three polysome-containing fractions differ in many parameters such as polysome profiles, cytoskeletal components and phospholipid content. When polyA-containing mRNA was isolated from the three polysome fractions and translated in an in vitro system, some differences were observed in the patterns of proteins being synthesized.     

La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.     

Direct association of messenger RNA-containing ribonucleoprotein particles with membranes of the endoplasmic reticulum in ethionine-treated rat liver.

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The fate of the mRNA molecules after polysome breakdown was investigated by measuring the amount of poly(A)-containing mRNA in membranous and non-membranous fractions obtained from the cytoplasm of ethionine-treated rat liver. The amount of poly(A)-containing mRNA in the membrane fraction of ethionine-treated liver was found to be the same as that of normal liver. When poly(A)-containing mRNAs from various fractions were translated in a wheat germ system and the products were isolated by immunoprecipitation, the albumin-specific mRNA was found exclusively in the membrane fraction of both normal and ethionine-treated livers. The membrane-bound mRNA in ethionine-treated liver, selectively labeled with [14C]orotate, was banded in CsCl gradient centrifugation at 1.42 g/ml which corresponds to the previously reported mRNA-containing ribonucleoprotein particles. From these results, we concluded that even after the polysome disaggregation by ethionine, most of the mRNA of membrane-bound polysomes remains attached to the endoplasmic reticulum membranes independently of ribosomes and the nascent polypeptide chains.