PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.     

Metabolite profiling reveals tissue- and temperature-specific metabolomic responses in thermoregulatory male florets of Dracunculus vulgaris (Araceae)

The male part of the spadix of Dracunculus vulgaris exhibits a degree of temperature regulation by inversely controlled heat production over a 20–35 °C range of tissue temperature. To clarify the effects of temperature on cellular metabolism, comparative analysis was performed using 51 metabolites from two distinct tissues (florets and pith) of thermogenic male spadices that had been temperature clamped at either 20 (to produce high respiration) or 35 °C (to produce low respiration). Principal component analysis and hierarchical clustering analysis showed that changes in metabolites in the florets, but not in the pith, were associated with temperature change. The energy charge in the florets treated at 20 °C was significantly higher than that of the florets treated at 35 °C. This indicated the presence of an increased energy-producing pathway that ultimately led to an increased level of thermogenesis at 20 °C. Intriguingly, succinate, a direct substrate for complex II in the mitochondrial respiratory chain, was the metabolite most significantly affected in our analysis, with its concentration in the florets 3.5 times higher at 20 than at 35 °C. However, the mitochondria fed with succinate showed that state 2 and 3 respirations and the capacity of the alternative and cytochrome pathways were all significantly higher at 35 than at 20 °C. Taken together, the results show that the male florets are the primary sites for temperature-induced changes in metabolomic pathways, although succinate-stimulated mitochondrial respiration, per sé, is not the control mechanism for thermoregulation in D. vulgaris.     

Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2′-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients.     

Correlation between dielectric property by dielectrophoretic levitation and growth activity of cells exposed to electric field

The purpose of this study is to develop a system analyzing cell activity by the dielectrophoresis method. Our previous studies revealed a correlation between the growth activity and dielectric property (Re[K(ω)]) of mouse hybridoma 3-2H3 cells using dielectrophoretic levitation. Furthermore, it was clarified that the differentiation activity of many stem cells could be evaluated by the Re[K(ω)] without differentiation induction. In this paper, 3-2H3 cells exposed to an alternating current (AC) electric field or a direct current (DC) electric field were cultivated, and the influence of damage by the electric field on the growth activity of the cells was examined. To evaluate the activity of the cells by measuring the Re[K(ω)], the correlation between the growth activity and the Re[K(ω)] of the cells exposed to the electric field was examined. The relations between the cell viability, growth activity, and Re[K(ω)] in the cells exposed to the AC electric field were obtained. The growth activity of the cells exposed to the AC electric field could be evaluated by the Re[K(ω)]. Furthermore, it was found that the adverse effects of the electric field on the cell viability and the growth activity were smaller in the AC electric field than the DC electric field.     

Different Mechanisms of Inhibition of DNA Synthesis by (E)-5-(2-Bromovinyl)-2′-deoxyuridine in Cells Transfected with Gene for Thymidine Kinase of Herpes Simplex Virus Type 1 and in Cells Infected with the Virus

Abstract

The effect of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) on deoxyribonucleoside 5′-triphosphate pools was studied in cells transfected with gene for thymidine kinase of herpes simplex virus type 1 and cells infected with the virus. When infected cells were treated with BVDU, the triphosphate form of the nucleoside analog was detected. When transfected cells were treated with BVDU, the triphosphate form was not detected and the pattern of changes in the pools was the same as after 5-fluoro-2′-deoxyuridine treatment. BVDU seems to inhibit DNA synthesis differently in the two cell lines and nucleotide metabolism in the transfected cells was not the same as in the infected cells.     

Oral Behaviors of Beef Steers in Pen and Pasture Environments

This study observed the behavioral characteristics of 122 steers in eight pens and 1,136 steers at six pastures. Nonhuman animals kept in pens performed less nutritive oral behaviors and more nonnutritive oral behaviors than animals kept at pasture. Although these could not be described as stereotypies, they did represent a replacement of nutritive oral behaviors by nonnutritive oral behaviors, rather than simply an increase in resting time. This could be indicative of a level of oral frustration. At pasture, there was a greater proportion of oral behaviors in animals with low pasture availability as compared to high availability, but this was an increase in nutritive oral behaviors rather than nonnutritive oral behaviors. Factors other than oral frustration—for example, rumen fill—probably drove this increase.     

Diethylstilbestrol Attenuates Antioxidant Activities in Testis from Male Mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( &#110 -GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of &#110 -GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Engineering Fructosyl Peptide Oxidase to Improve Activity Toward the Fructosyl Hexapeptide Standard for HbA1c Measurement

The recently discovered fructosyl peptide oxidase from Phaeosphaeria nodorum (PnFPOX) was demonstrated to react with the glycated hexapeptide measurement standard of hemoglobin A1c, fVHLTPE. The highly reactive Coniochaeta FPOX (FPOX-C) showed no detectable activity with the hexapeptide. Two loop regions were identified as having important effects on the enzymatic properties of FPOX. The first loop has a strong influence on the ability to bind larger glycated peptides, while the second loop has a significant effect on catalytic activity. Loop-substitution mutants showed that the highest activity against fVHLTPE resulted from the combination of the first loop from PnFPOX and the second loop from FPOX-C. The most promising engineered FPOX created, which showed 17-fold greater dehydrogenase activity against fVHLTPE than wild-type PnFPOX, was the FPOX-C mutant with a PnFPOX-derived loop 1 region and an Asn56Ala substitution.     

How plants grow under gravity conditions besides 1 g: perspectives from hypergravity and space experiments that employ bryophytes as a model organism

Plant Molecular Biology - Plants have evolved and grown under the selection pressure of gravitational force at 1 g on Earth. In response to this selection pressure, plants have acquired...