A method for finding optimal RNA secondary structures using a new entropy model (vsfold)

We are developing a program to calculate optimal RNA secondary structures. The model uses di-nucleotide pairing energies as with most traditional approaches. However, for long-range entropy interactions, the approach uses an entropy-loss model based on the accumulated sum of the entropy of bonding between each base-pair weighted inversely by the correlation of the RNA sequence (the Kuhn length). Stiff RNA forms very different structures from flexible RNA. The results demonstrate that the long-range folding is largely governed by this entropy and the Kuhn length.     

dnc-1/dynactin 1 Knockdown Disrupts Transport of Autophagosomes and Induces Motor Neuron Degeneration

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. We previously showed that the expression of dynactin 1, an axon motor protein regulating retrograde transport, is markedly reduced in spinal motor neurons of sporadic ALS patients, although the mechanisms by which decreased dynactin 1 levels cause neurodegeneration have yet to be elucidated. The accumulation of autophagosomes in degenerated motor neurons is another key pathological feature of sporadic ALS. Since autophagosomes are cargo of dynein/dynactin complexes and play a crucial role in the turnover of several organelles and proteins, we hypothesized that the quantitative loss of dynactin 1 disrupts the transport of autophagosomes and induces the degeneration of motor neuron. In the present study, we generated a Caenorhabditis elegans model in which the expression of DNC-1, the homolog of dynactin 1, is specifically knocked down in motor neurons. This model exhibited severe motor defects together with axonal and neuronal degeneration. We also observed impaired movement and increased number of autophagosomes in the degenerated neurons. Furthermore, the combination of rapamycin, an activator of autophagy, and trichostatin which facilitates axonal transport dramatically ameliorated the motor phenotype and axonal degeneration of this model. Thus, our results suggest that decreased expression of dynactin 1 induces motor neuron degeneration and that the transport of autophagosomes is a novel and substantial therapeutic target for motor neuron degeneration.     

Selective Enhancing Effect of Early Mitotic Inhibitor 1 (Emi1) Depletion on the Sensitivity of Doxorubicin or X-ray Treatment in Human Cancer Cells

Chemotherapy and radiation in addition to surgery has proven useful in a number of different cancer types, but the effectiveness in normal tissue cannot be avoided in these therapies. To improve the effectiveness of these therapies selectively in cancer tissue is important for avoiding side effects. Early mitotic inhibitor 1 (Emi1) is known to have the function to inhibit anaphase-promoting complex/cyclosome ubiquitin ligase complex, which ubiquitylates the cell cycle-related proteins. It recently has been shown that Emi1 knockdown prevents transition from S to G₂ phase by down-regulating geminin via anaphase-promoting complex/cyclosome activation. At present, anticancer drugs for targeting DNA synthesis to interfere with rapidly dividing cells commonly are used. As Emi1 depletion interferes with completion of DNA synthesis in cancer cells, we thought that Emi1 knockdown might enhance the sensitivity for anticancer agents. Here, we confirmed that Emi1 siRNA induced polyploidy for preventing transition from S to G₂ phase in several cancer cell lines. Then, we treated Emi1 depleted cells with doxorubicin. Interestingly, increased apoptotic cells were observed after doxorubicin treatment in Emi1 siRNA-treated cancer cells. In addition, Emi1 depletion enhanced the sensitivity of x-ray irradiation in cancer cells. Importantly, synergistic effect of Emi1 knockdown in these combination therapies was not observed in normal cells. These results suggest that Emi1 siRNA can be a useful tool for enhancing of sensitivity of cancer cells to anticancer reagents and radiation.     

Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes

The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pH_out) of PCPECL liposomes, with an internal pH (pH_in) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK _a?~?6.9). Conversely, ΔpH generated by enhanced pH_in of PCPECL at a pH_out of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pH_in at a pH_out of 8.0. At bulk acidic pH, ΔΨ generated by Na⁺ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH_out, the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨ_M blocks inner mitochondrial membrane fusion during apoptosis.     

Creation of endoglucanase-secreting Streptomyces lividans for enzyme production using cellulose as the carbon source

We screened for high-activity endoglucanase (EG) as a first step toward the creation of cellulose-assimilating Streptomyces lividans transformants. EGs derived from Thermobifida fusca YX, Tfu0901, and S. lividans, cellulase B (CelB), were successfully expressed. Genes encoding Tfu0901 or CelB were introduced into S. lividans using the integrative vector pTYM18 and the high-copy-number vector pUC702, and EG activity was detected in the supernatant of each transformant. To achieve coexpression of EG and transglutaminase, the transglutaminase gene was introduced into EG-secreting S. lividans using pUC702. S. lividans coexpressing EG and transglutaminase effectively assimilated phosphoric acid swollen cellulose. The yield of Streptomyces cinnamoneus transglutaminase in the culture supernatant was 7.2 mg/L, which was 18 times higher than that of the control strain. To demonstrate the versatility of our system, we also created an EG-producing S. lividans transformant capable of coexpressing endoxylanase. The EG-secreting S. lividans transformants constructed here can be used to produce other useful compounds through cellulose fermentation.     

Polyvinyl alcohol and polyethylene glycol form polymer bodies in the periplasm of Sphingomonads that are able to assimilate them

Scanning electron microscopy (SEM) shows remarkable morphological surface changes in Sphingopyxis sp. 113P3 cells grown in polyvinyl alcohol (PVA) but not in Luria–Bertani medium (LB) (Hu et al. in Arch Microbiol 188: 235–241, 2007). However, transmission electron microscopy showed no surface changes in PVA-grown cells and revealed the presence of polymer bodies in the periplasm of PVA-grown cells, which were not observed in LB-grown cells. The presence of polymer bodies was supported by low-vacuum SEM observation of PVA- and LB-grown cells of strain 113P3, and the presence of similar polymer bodies was also found when Sphingopyxis macrogoltabida 103 and S. terrae were grown in polyethylene glycol (PEG). The extraction of PVA and PEG from the periplasmic fraction of cells using a modified Anraku and Heppel method and their analysis by MALDI–TOF mass spectrometry strongly suggested that the polymer bodies are composed of PVA and PEG, respectively, in Sphingopyxis sp. 113P3 (PVA degrader) and Sphingopyxis macrogoltabida 103 or S. terrae (PEG degraders). PEG-grown S. macrogoltabida 103 and S. terrae showed higher transport of ¹⁴C-PEG 4000 than LB-grown cells. Recombinant PegB (TonB-dependent receptor-like protein consisting of a barrel structure) interacted with PEG 200, 4000 and 20000, suggesting that the barrel protein in the outer membrane contributes to the transport of PEG into the periplasm.     

Survival analysis of mandibular complete dentures with acrylic‐based resilient liners

doi: 10.1111/j.1741‐2358.2012.00658.x Survival analysis of mandibular complete dentures with acrylic‐based resilient liners Objective: The purpose of this long‐term randomised controlled trial was to compare the longevity of dentures constructed using a conventional acrylic resin (CAR) to that of dentures constructed using an acrylic‐based resilient liner (ARL). Materials and methods: The follow‐up study was essentially carried out by annual telephone calls to each of the 67 participants. The Kaplan–Meier method and life‐table analysis were used for univariate analyses. The Cox proportional‐hazards test was used as a final model for statistically adjusting predictor variables such as sex, clinician type, mandibular denture type and age at denture delivery. Results: The denture type was likely to affect the survival time of the dentures, while the sex and clinician type were not. The group using acrylic‐based resilient denture liners had twice the risk of having shorter denture‐survival times than those using conventional acrylic resin dentures. Younger participants were likely to have a reduced risk of having shorter denture‐survival times than older participants. Conclusion: We conclude that mandibular complete dentures constructed using ARL are twice as likely as dentures constructed using CAR to have shorter denture survival times, mainly because of material deterioration.     

Grifola frondosa extract and ergosterol reduce allergic reactions in an allergy mouse model by suppressing the degranulation of mast cells

ABSTRACT

The increasing number of patients suffering from allergic diseases is a global health problem. Grifola frondosa is an edible mushroom consumed as a health food in Asia, and has recently been reported to have anti-allergic effects. We previously reported that G. frondosa extract (GFE) and its active components, ergosterol and its derivatives, inhibited the antigen-induced activation of RBL-2H3 cells. Here, we demonstrated that GFE and ergosterol also had an inhibitory effect on the degranulation of bone marrow–derived mast cells (BMMCs) and alleviated anaphylactic cutaneous responses in mice. Using an air pouch-type allergic inflammation mouse model, we confirmed that oral administration of GFE and ergosterol suppressed the degranulation of mast cells in vivo. Our findings suggest that G. frondosa, including ergosterol as its active component, reduces type I allergic reactions by suppressing mast cell degranulation in mice, and might be a novel functional food that prevents allergic diseases.