Protein kinase A determines timing of early differentiation through epigenetic regulation with G9a

Timing of cell differentiation is strictly controlled and is crucial for normal development and stem cell differentiation. However, underlying mechanisms regulating differentiation timing are fully unknown. Here, we show a molecular mechanism determining differentiation timing from mouse embryonic stem cells (ESCs). Activation of protein kinase A (PKA) modulates differentiation timing to accelerate the appearance of mesoderm and other germ layer cells, reciprocally correlated with the earlier disappearance of pluripotent markers after ESC differentiation.?PKA activation increases protein expression of G9a, an H3K9 methyltransferase, along with earlier H3K9 dimethylation and DNA methylation in Oct3/4 and Nanog gene promoters. Deletion of G9a completely abolishes PKA-elicited acceleration of differentiation and epigenetic modification. Furthermore, G9a knockout mice show prolonged expressions of?Oct3/4 and Nanog at embryonic day 7.5 and delayed development. In this study, we demonstrate molecular machinery that regulates timing of multilineage differentiation by linking signaling with epigenetics.     

MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation

MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affects pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.     

Oxygen evolution in the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421

The oxygen-evolving reactions of the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 were compared with those of Synechocystis sp. PCC 6803. Four aspects were considered: sequence conservation in three extrinsic proteins for oxygen evolution, steady-state oxygen-evolving activity, charge recombination reactions, i.e., thermoluminescence and oscillation patterns of delayed luminescence on a second time scale and delayed fluorescence on the nanosecond time scale at − 196 °C. Even though there were significant differences between the amino acid sequences of extrinsic proteins in G. violaceus and Synechocystis sp. PCC 6803, the oxygen-evolving activities were similar. The delayed luminescence oscillation patterns and glow curves of thermoluminescence were essentially identical between the two species, and the nanosecond delayed fluorescence spectral profiles and lifetimes were also very similar. These results indicate clearly that even though the oxygen-evolving reactions are carried out in the periplasm by components with altered amino acid sequences, the essential reaction processes for water oxidation are highly conserved. In contrast, we observed significant changes on the reduction side of photosystem II. Based on these data, we discuss the oxygen-evolving activity of G. violaceus.     

Replication Study in a Japanese Population to Evaluate the Association between 10 SNP Loci,Identified in European Genome-Wide Association Studies,and Type 2 Diabetes

AimWe performed a replication study in a Japanese population to evaluate the association between type 2 diabetes and 7 susceptibility loci originally identified by European genome-wide association study (GWAS) in 2012: ZMIZ1, KLHDC5, TLE1, ANKRD55, CILP2, MC4R, and BCAR1. We also examined the association of 3 additional loci: CCND2 and GIPR, identified in sex-differentiated analyses, and LAMA1, which was shown to be associated with non-obese European type 2 diabetes.MethodsWe genotyped 6,972 Japanese participants (4,280 type 2 diabetes patients and 2,692 controls) for each of the 10 single nucleotide polymorphisms (SNPs): rs12571751 in ZMIZ1, rs10842994 near KLHDC5, rs2796441 near TLE1, rs459193 near ANKRD55, rs10401969 in CILP2, rs12970134 near MC4R, rs7202877 near BCAR1, rs11063069 near CCND2, rs8108269 near GIPR, and rs8090011 in LAMA1 using a multiplex polymerase chain reaction invader assay. The association of each SNP locus with the disease was evaluated using a logistic regression analysis.ResultsAll SNPs examined in this study had the same direction of effect (odds ratio > 1.0, p = 9.77 × 10^-4, binomial test), as in the original reports. Among them, rs12571751 in ZMIZ1 was significantly associated with type 2 diabetes [p = 0.0041, odds ratio = 1.123, 95% confidence interval 1.037–1.215, adjusted for sex, age and body mass index (BMI)], but we did not observe significant association of the remaining 9 SNP loci with type 2 diabetes in the present Japanese population (p ≥ 0.005). A genetic risk score, constructed from the sum of risk alleles for the 7 SNP loci identified by un-stratified analyses in the European GWAS meta-analysis were associated with type 2 diabetes in the present Japanese population (p = 2.3 × 10^-4, adjusted for sex, age and BMI).ConclusionsZMIZ1 locus has a significant effect on conferring susceptibility to type 2 diabetes also in the Japanese population.     

Effects of hydrogen-rich water and ascorbic acid treatment on spontaneously hypertensive rats

Hydrogen-rich water (HW) has been suggested to possess antioxidant properties of value in treatments of lifestyle diseases and for prevention of latent pathologies. To date, the potential benefits of HW against the deleterious effects of excessive salt intake and hypertension have not been investigated. Here, we first examined the effects of HW or HW supplemented with 0.1% ascorbic acid (HWA) on spontaneously hypertensive rats (SHR) that had been fed a normal diet. In comparison to control rats given distilled water (DW), we found that HW did not significantly influence systolic blood pressure (SBP) or diastolic blood pressure (DBP) in SHR; however, the increase in SBP and DBP were inhibited in the HWA group. Next, four groups of SHR were given DW, 0.1% ascorbic acid-added DW (DWA), HW, or HWA in combination with a 4% NaCl-added diet. SHR fed the 4% NaCl-added diet showed increased hypertension; HWA treatment resulted in a significant reduction in blood pressure. The HWA group tended to have lower plasma angiotensin II levels than the DW group. In addition, urinary volumes and urinary sodium levels were significantly lower in the HWA group than the DW group. Urinary isoprostane, an oxidative stress marker, was also significantly lower in the HWA group, suggesting that the inhibitory effect of HWA on blood pressure elevation was caused by a reduction in oxidative stress. These findings suggest a synergistic interaction between HW and ascorbic acid, and also suggest that HWA ingestion has potential for prevention of hypertension.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Improvement of Intracellular Traffic System by Overexpression of KDEL Receptor 1 in Antibody‐Producing CHO Cells

The localization of soluble endoplasmic reticulum (ER) chaperones in the cell organelle is mediated by the C‐terminal KDEL (lysine, aspartic acid, glutamic acid and leucine) motif. This motif is recognized by the KDEL receptor, a seven‐transmembrane protein that cycles between the ER and cis‐Golgi to capture missorted KDEL chaperones from post‐ER compartments in a pH‐dependent manner. The KDEL receptor's target chaperones have a substantial role in protein folding and assembly. In this study, the gene expression level of KDEL receptor 1 shows a moderate upregulation during either ER stress or growth of Chinese hamster ovary (CHO) cells in batch culture, while the ER chaperones show higher upregulation. This might indicate the possibility of saturation of the ER retention machinery or at least hindered retention during late stage batch culture in recombinant CHO cells. KDELR1 is overexpressed in a monoclonal antibody‐producing CHO cell line to improve the intracellular chaperone retention rate in the ER. An increase in the specific productivity of IgG1 by 13.2% during the exponential phase, and 23.8% in the deceleration phase of batch culture is observed. This is the first study to focus on the ER retention system as a cell engineering target for enhancing recombinant protein production.     

Different effects of mating group size as male and as female on sex allocation in a simultaneous hermaphrodite

Sex allocation theory predicts that the optimal sexual resource allocation of simultaneous hermaphrodites is affected by mating group size (MGS). Although the original concept assumes that the MGS does not differ between male and female functions, the MGS in the male function (MGSm; i.e., the number of sperm recipients the focal individual can deliver its sperm to plus one) and that in the female function (MGSf; the number of sperm donors plus one) do not always coincide and may differently affect the optimal sex allocation. Moreover, reproductive costs can be split into “variable” (e.g., sperm and eggs) and “fixed” (e.g., genitalia) costs, but these have been seldom distinguished in empirical studies. We examined the effects of MGSm and MGSf on the fixed and variable reproductive investments in the sessilian barnacle Balanus rostratus. The results showed that MGSm had a positive effect on sex allocation, whereas MGSf had a nearly significant negative effect. Moreover, the “fixed” cost varied with body size and both aspects of MGS. We argue that the two aspects of MGS should be distinguished for organisms with unilateral mating.