Cyclophilin a protects Peg3 from hypermethylation and inactive histone modification

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. Disruption of normal imprinting leads to abnormal embryogenesis, certain inherited diseases, and is associated with various cancers. In the context of screening for the gene(s) responsible for the alteration of phenotype in cyclophilin A knockdown (CypA-KD) P19 cells, we observed a silent paternally expressed gene, Peg3. Treatment of CypA-KD P19 cells with the DNA demethylating agent 5-aza-dC reversed the silencing of Peg3 biallelically. Genomic bisulfite sequencing and methylation-specific PCR revealed DNA hypermethylation in CypA-KD P19 cells, as the normally unmethylated paternal allele acquired methylation that resulted in biallelic methylation of Peg3. Chromatin immunoprecipitation assays indicated a loss of acetylation and a gain of lysine 9 trimethylation in histone 3, as well as enhanced DNA methyltransferase 1 and MBD2 binding on the cytosine-guanine dinucleotide (CpG) islands of Peg3. Our results indicate that DNA hypermethylation on the paternal allele and allele-specific acquisition of histone methylation leads to silencing of Peg3 in CypA-KD P19 cells. This study is the first demonstration of the epigenetic function of CypA in protecting the paternal allele of Peg3 from DNA methylation and inactive histone modifications.     

Nucleic acids of Enterococcus faecalis strain EC-12 are potent Toll-like receptor 7 and 9 ligands inducing interleukin-12 production from murine splenocytes and murine macrophage cell line J774.1

In experiment 1 of this study, the interleukin-12 (IL-12)-inducing ability of six Enterococcus strains was evaluated in comparison with that of five Lactobacillus strains using murine splenocytes. At the same time, the involvement of Toll-like receptor (TLR) ligands in IL-12-inducing ability was assessed using splenocytes from TLR2-, TLR4- and MyD88-deficient mice. Most Enterococcus strains, especially Enterococcus faecalis strain EC-12, exerted higher IL-12-inducing ability compared with the Lactobacillus strains evaluated. Almost the same amount of IL-12 protein was produced by all lactic acid bacteria strains in splenocytes from TLR2- and TLR4-deficient mice, whereas splenocytes from MyD88-deficient mice showed no IL-12 production against all bacteria evaluated. In experiment 2, the role of TLR7, 8 and 9 ligands of E. faecalis strain EC-12 in the induction of IL-12 production was evaluated using murine macrophage cell line J774.1. A drastic decrease in IL-12-inducing ability was observed when heat-killed E. faecalis strain EC-12 was treated with nuclease, particularly RNase. In addition, less than one-tenth of IL-12 was produced by heat-killed E. faecalis strain EC-12 when both TLR7 and 9 were antagonized. These facts indicate that the nucleic acids of E. faecalis strain EC-12, particularly its RNA, are the potent TLR7 and 9 ligands that induce IL-12 production from antigen-presenting cells.     

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that in vivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.     

IRS-1 transgenic mice show increased epididymal fat mass and insulin resistance

Insulin receptor substrate-1 (IRS-1) is the major substrate of both the insulin receptor and the IGF-1 receptor. In this study, we created IRS-1 transgenic (IRS-1-Tg) mice which express human IRS-1 cDNA under control of the mouse IRS-1 gene promoter. In the IRS-1-Tg mice, IRS-1 mRNA expression was significantly increased in almost all tissues, but its protein expression was increased in very limited tissues (epididymal fat and skeletal muscle). IRS-1-Tg mice showed glucose intolerance and significantly enlarged epididymal fat mass, as well as elevated serum TNF-α concentrations. Importantly insulin signaling was significantly attenuated in the liver of IRS-1-Tg mice, which may contribute to the glucose intolerance. Our results suggest that excess IRS-1 expression may not provide a beneficial impact on glucose homeostasis in vivo.     

Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone--a potent NR2B-selective N-methyl D-aspartate (NMDA) antagonist for the treatment of pain

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index=4200 vs NR2B binding IC(50)). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.     

The Berg-Purcell Limit Revisited

Biological systems often have to measure extremely low concentrations of chemicals with high precision. When dealing with such small numbers of molecules, the inevitable randomness of physical transport processes and binding reactions will limit the precision with which measurements can be made. An important question is what the lower bound on the noise would be in such measurements. Using the theory of diffusion-influenced reactions, we derive an analytical expression for the precision of concentration estimates that are obtained by monitoring the state of a receptor to which a diffusing ligand can bind. The variance in the estimate consists of two terms, one resulting from the intrinsic binding kinetics and the other from the diffusive arrival of ligand at the receptor. The latter term is identical to the fundamental limit derived by Berg and Purcell (Biophys. J., 1977), but disagrees with a more recent expression by Bialek and Setayeshgar. Comparing the theoretical predictions against results from particle-based simulations confirms the accuracy of the resulting expression and reaffirms the fundamental limit established by Berg and Purcell.     

Identification of a caspase 3-independent role of pro-apoptotic factor Bak in TNF-alpha-induced apoptosis

By using our recently developed gene discovery system, we have identified Bak, a member of the Bcl-2 family, as a pro-apoptotic factor in the tumor necrosis factor (TNF)-alpha-induced apoptotic pathway in caspase 3-deficient cells. Unlike Bcl-2, Bak stimulates several apoptotic pathways, however the molecular mechanism(s) of its action remains unclear. For example, it is unclear whether Bak induces apoptosis in caspase 3-deficient cells. In this study, we examined the effects of overexpression of Bak in MCF-7 cells that lack caspase 3. We found that despite the absence of caspase 3 in MCF-7 cells, they were more sensitive to the cell death effects of Bak as compared to caspase 3-expressing HeLa S3 cells. The targeting of Bak function by ribozymes suggests that Bak is required for the TNF-alpha-induced apoptotic pathway in caspase 3-deficient cells. This study demonstrates the caspase 3-independent function of Bak in the TNF-alpha-induced apoptotic pathway.     

siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wild-type SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by over-expression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNA-based gene therapy of familial ALS with mutant SOD1.     

Preventive effect of egg yolk phosvitin on heat-insolubilization of egg white protein and its application to heat-induced egg white gel

The effects of phosvitin (PV) on insolubilization of egg white protein (EWP) and ovotransferrin (OT) were examined by measuring turbidity after heating at 80 degrees C in a pH range of 5 to 8. PV showed preventive ability against heat-insolubilization of EWP, especially heat-labile OT. The preventive ability of PV was reduced by adding NaCl to a PV-OT mixture on heating. Native PAGE and gel filtration analyses showed that PV prevented an insolubilization of heat-denatured OT through ionic interactions. The preventive effects of PV on insolubilization of EWP and OT resulted in the formation of a firm, transparent gel from EWP in coexistence with PV on heating. The addition of PV might make possible the preparation of liquid egg white without insoluble products even on heat-treatment at high temperatures.