Polysome assembly and RNA synthesis during phytochrome-mediated photomorphogenesis in mustard cotyledons

The effect of phytochrome (high irradiance reaction, elicited by continuous far-red light) on cellular polysome levels was investigated using ribosome-isolation procedures which prevent the methodological artifacts inherent in previous studies on polysomes. By including the large pool of ribosomal subunits in the analysis and using the ratio (polysomes: monomers + subunits) as a quantitative estimate of the translational capacity of the ribosomes in mustard (Sinapis alba L.) cotyledons, we found the following results: 1) After a lag-phase of less than 30 min, phytochrome induces a massive increase in the relative amount of cytosolic (free) polysomes at the expense of ribosomal subunits. 2) Cytosolic and membrane-bound polysomes are increased by phytochrome in constant proportions (constant ratio of 65:35 in light and darkness). 3) Simultaneously with the light-mediated increase of the polysome level there is an increased incorporation of newly synthesized (labeled) non-ribosomal RNA, presumably mRNA, into the polysomes which can be kinetically discriminated from the slower incorporation of newly synthesized (labeled) rRNA. 4) Cordycepin strongly inhibits the synthesis of RNA and completely prevents the light-mediated increase of polysomes. 5) The electrophoretic patterns of the in-vitro translation products obtained with polysomal polyadenylated RNA from dark-grown and light-grown cotyledons showed no significant qualitative differences. We conclude from these results that photomorphogenesis of mustard cotyledons is related to a massive increase of newly synthesized mRNA leading to a correspondingly increased recruitment of ribosomal subunits into polysomes. The phytochrome-induced increase of translatable mRNA involves mainly quantitative changes in the production of mRNA species which are also present in the dark-grown cotyledons.     

The investigation of the effect of pollination on ribosomal RNA,transfer RNA and DNA contents in styles ofNicotiana alata.

Using the phenol extraction method and MAK column chromatography the contents of nucleic acids in styles ofNicotiana alata were estimated before and up to 48 h after compatible pollination. As a result of pollination, high-molecular-weight rRNA and DNA registered a significant increase in their content approximating 30% and 16% respectively. The change in sRNA (4-S tRNA and 5-S rRNA) level was very slight and non-significant. In pollen grains there is an unusually high amount of rRNA with respect to the content of other nucleic acids and the rRNA/tRNA ratio approximates 14 : 1. On the other hand, in non-pollinated styles the content of rRNA is only about 6 times higher than that of tRNA. The changes in nucleic acid level found in styles after pollination are at least in a major part the result of the addition of the nucleic acids present in the amount of pollen used for pollination. The high rRNA level relatively to tRNA being generally associated with rapidly growing cells, its significance in pollen may be related to the rapid growth of pollen tubes.     

Ribosomal RNA contents of maize genotypes with different ribosomal RNA gene numbers

Ribosomal RNA (rRNA) contents were determined in 16 maize genotypes whose individual rRNA gene numbers varied from 5000 to 23,000 per 2C nucleus. Analytical polyacrylamide gel electrophoresis of total RNA showed that no obvious relation existed between rRNA gene number and rRNA content. Only two of nine common inbred lines contained more rRNA than W-23, the inbred with the lowest rRNA gene number. Two of four lines with altered protein content (due to long-term experimental selection) had rRNA contents significantly reduced from those of W-23. A line with an apparent duplication of the nucleolus organizer region of chromosome 6 (called 2-NOR) was expected to possess an elevated quantity of rRNA because it possesses a larger nucleolus; however, we produced a 2-NOR isogenic version and found no difference in rRNA content. The rRNA genes in maize are distributed throughout the NOR-heterochromatin and the NOR-secondary constriction portions of the NOR. The absence of an obvious correlation between rRNA gene number and cellular rRNA content may reflect the presence of a large number of rRNA genes in an inactive state, at least during the stage of growth examined in these experiments.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Quercetin Potentiates the Antitumor Activity of Rituximab in Diffuse Large B-Cell Lymphoma by Inhibiting STAT3 Pathway

STAT3 pathway plays an important role in the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of Quercetin, a flavonoid compound, in combination with rituximab in DLBCL cell lines in vitro. We found that Quercetin synergistically enhanced rituximab-induced growth inhibition and apoptosis in DLBCL cell lines. Moreover, we found Quercetin exerted inhibitory activity against STAT3 pathway and downregulated the expression of survival genes. These results suggest that combining the Quercetin with rituximab may present an attractive and potentially effective way for the treatment of DLBCL.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

Circulating MicroRNA Biomarkers for Glioma and Predicting Response to Therapy

The need for glioma biomarkers with improved sensitivity and specificity has sparked research into short non-coding RNA known as microRNA (miRNA). Altered miRNA biogenesis and expression in glioma plays a vital role in important signaling pathways associated with a range of tumor characteristics including gliomagenesis, invasion, and malignancy. This review will discuss current research into the role of miRNA in glioma and altered miRNA expression in biofluids as candidate biomarkers with a particular focus on glioblastoma, the most malignant form of glioma. The isolation and characterization of miRNA using cellular and molecular biology techniques from the circulation of glioma patients could potentially be used for improved diagnosis, prognosis, and treatment decisions. We aim to highlight the links between research into miRNA function, their use as biomarkers, and how these biomarkers can be used to predict response to therapy. Furthermore, increased understanding of miRNA in glioma biology through biomarker research has led to the development of miRNA therapeutics which could restore normal miRNA expression and function and improve the prognosis of glioma patients. A panel of important miRNA biomarkers for glioma in various biofluids discovered to date has been summarized here. There is still a need, however, to standardize techniques for biomarker characterization to bring us closer to clinically relevant miRNA-based diagnostic and therapeutic signatures. A clinically validated biomarker panel has potential to improve time to diagnosis, predicting response to treatment and ultimately the prognosis of glioma patients.     

The action of exogenous gibberellic acid on polysome formation and translation of mRNA in germinating castor-bean seeds

The amount of protein synthesis in germinating castor-bean seeds has been estimated by the quantitative and qualitative exmainatin of polysomes from the seeds in the presence and absence of gibberellic acid (GA₃). Careful optimisation of polysome extraction procedures was required to minimise the ribonuclease activity in the extracts. Ribonuclease activity in seed extracts increased fourfold over the first 5 d of germination. Gibberellic acid stimulated polysome formation about twofold during the first 4 d of germination. It also stimulated the amount of mRNA associated with polysomes by about twofold during the first 3 d of germination. Between days 1 and 5 of germination, polysome formation was primarily limited by mRNA availability. During the period 0–24 h, polysome formation was independent of mRNA levles. The increase in enzyme activities stimulated by GA₃ was probably the result of an increase in the amount of cellular mRNA. No evidence was obtained for an action of GA₃ on translation other than on the increased production of RNA. Examination of the recruitment of isocitrate-lyase mRNA into polysomes showed that GA₃ did not specifically stimulate production of this enzyme.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.     

A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference

Allele-specific silencing by RNA interference (ASP-RNAi) is an atypical RNAi that is capable of discriminating target alleles from non-target alleles, and may be therapeutically useful for specific inhibition of disease-causing alleles without affecting their corresponding normal alleles. However, it is difficult to design and select small interfering RNA (siRNAs) that confer ASP-RNAi. A major problem is that there are few appropriate measures in determining optimal allele-specific siRNAs. Here we show two novel formulas for calculating a new measure of allele-discrimination, named “ASP-score”. The formulas and ASP-score allow for an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.