Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/- mice

Obesity is a life-threatening factor and is often associated with dysregulation of gene expression. Here, we show that the CNOT3 subunit of the CCR4-NOT deadenylase complex is critical to metabolic regulation. Cnot3(+/-) mice are lean with hepatic and adipose tissues containing reduced levels of lipids, and show increased metabolic rates and enhanced glucose tolerance. Cnot3(+/-) mice remain lean and sensitive to insulin even on a high-fat diet. Furthermore, introduction of Cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs is not altered in Cnot3(+/-) vis-à-vis wild-type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism-related PDK4 and IGFBP1, are increased in Cnot3(+/-) hepatocytes, having poly(A) tails that are longer than those seen in control cells. We provide evidence that CNOT3 is involved in recruitment of the CCR4-NOT deadenylase to the 3' end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decrease upon fasting, we propose that CNOT3 responds to feeding conditions to regulate deadenylation-specific mRNAs and energy metabolism.     

Effects of Age and Gender on Sleep Habits and Sleep Trouble for Aged People

The healthy 455 subjects above 60 years of age were questioned on their sleep habit inventory and the morningness-eveningness questionnaire. We analyzed the effects of age and sex on sleep habits and sleep-related trouble. Bedtimes on weekdays and weekends became earlier with aging, and women went to bed significantly later than men did. The length of sleep on weekdays slightly increased with aging, and it was longer for men than for women. The number of urinations and awakenings during nocturnal sleep and the amount of daytime napping increased with aging. The score on morningness-eveningness shifted toward the morning type with aging. In comparison with men, women had significantly longer sleep latency; and a higher percentage of subjects who reported that they sleep for only a short time, have sleep trouble, have received medical treatment for their sleep trouble, and take sleep medication. From these results, we deduced that the phase of sleep shifted forward in subjects above 60 years of age, and they showed frequent interruptions during nocturnal sleep and long daytime napping. We discussed the factor of gender difference in sleep in relation to social and cultural factors, particularly the household activities of women.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

FAT10 protein binds to polyglutamine proteins and modulates their solubility

Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.     

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.     

Fe-type nitrile hydratase

The characteristic features of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys-SO2H) and -sulfenic acids (Cys-SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli. The results of the studies on several recombinant NHases have shown that the Cys-SO2H oxidation of alphaC112 is indispensable for the catalytic activity of Fe-type NHase.