Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MTT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG (2 μM) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

We investigated free radical reactions in lung of living mice using an in vivo electron spin resonance (ESR) spectrometer and nitroxyl radical as a probe. When an aqueous solution of nitroxyl probe was trans-tracheally administered into lung of living mice, a sharp triplet signal was observed at the chest of the mice. The signal showed a gradual decrease with time, obeying first-order kinetics. Signal decay rates of carbamoyl-PROXYL and carboxy-2,2,6,6-tetramethyl-piperidine-N-oxyl were faster than those of CAT-1 and carboxy-PROXYL. The mechanism of signal decay may be attributed to (i) reaction with reactive oxygen species such as ·OH, (ii) transfer into blood circulation, or (iii) reduction by compounds continuously supplied. However, little is known about the clearance mechanism of the nitroxyl probe in lung. To evaluate the disappearance of the ESR signal of the nitroxyl probe in lung, in vivo ESR spectra in chest of mice was recorded after trans-tracheal administration of an aqueous high concentrate solution of nitroxyl probe. A broad signal from the chest was observed immediately after administration due to Heisenberg spin exchange interaction. A sharp triplet signal was superimposed on the broad signal and the appearance of a triplet signal was followed by disappearance of the broad one. Peak-to-peak line width of the sharp signal was almost the same as that after intravenous administration. A distinct signal was detected in blood collected 10 min after trans-tracheal administration of nitroxyl probe. The observations indicate the transfer from lung to blood circulation and its contribution to clearance of probe in lung. Appearance of a sharp signal in blood after trans-tracheal administration was dependent on the kind of nitroxyl probe, showing a different transfer rate from lung to blood.

Emodin is a bioactive compound isolated from the root and rhizomes of Rheum plamatum L. (polygenaceae), which is known as a traditional Chinese and Japanese medicine. In the present study, the effect of emodin on YD-15 mucoepidermoid carcinoma cells and its molecular mechanism were investigated. This study shows that emodin significantly inhibits the growth of YD-15 cells. Activation of caspase-3 and PARP is triggered by emodin and it increases sub-G1 population and the number of YD-15 cells with nuclear condensation and fragmentation. In addition, we found that emodin significantly decreases myeloid cell leukemia 1(MCL-1). These results suggest that MCl-1 is an important molecule for emodin-induced apoptosis. Taken together, emodin inhibits cell viability and induces apoptosis via down-regulation of MCL-1 and it can be a new potent anticancer drug candidate for the treatment of mucoepidermoid carcinoma

Only an optimum number of viable spermatozoa in a frozen-thawed insemination dose can ensure conception at artificial insemination (AI). We report here the percentages of normal, abnormal and viable spermatozoa present in the frozen-thawed semen of 20 Black Bengal bucks used for commercial AI. Bucks in this experiment were of 19.3~46.1 months old and 25~42 kg body weight. Four semen straws (0.25 ml) from each buck were collected for evaluation of their kinetic parameters. Scrotal circumference was measured by using a scrotal tape, sperm motility was estimated on eye estimation and sperm concentration was determined by using a haemocytometer. Sperm morphology was studied in paraformaldehyde fixed spermatozoa under differential interference contrast (DIC) microscope. To determine the proportion of live (plasma membrane intact) spermatozoa, semen was stained with SYBR-14 and propidium iodide and examined under fluorescent microscope. Scrotal circumference, post-thaw sperm motility, sperm concentration per insemination dose and proportion of normal spermatozoa were , , million and , respectively. The percentages of spermatozoa with head shape and acrosome abnormalities were lower ( and , respectively), whereas higher percentages of abnormalities () were observed in mid piece and tail portion. The proportion of live spermatozoa was . It is concluded that although a good number of morphologically normal spermatozoa are present in the insemination dose, the proportion of live spermatozoa is low, which warrants further improvements of buck semen freezing procedures to ensure good quality at AI.

Mushrooms including Mycoleptodonoides aitchisonii are used as foods and employed as folk remedies for diabetes and inflammatroy diseases. This study analyzed anti-diabetic effects of Mycoleptodonoides aitchisonii, which grows in some areas such as Gangwon-do and Jeju-do in Korea, as insulin-derived phosphorylation. When 100 ng/ml of IL-6, inflammatory cytokine was given to SK-hep1, HepG2, Akt phosphorylation by insulin was found to be remarkably reduced. In addition, metformin, Antidiabetics serving as positive control in liver was used. Mycoleptodonoides aitchisonii used for analyzing anti-diabetic effects in this test didn’t give a great impact on the decrease in Akt phosphorylation by IL-6 at high concentration. However, fruit body in Mycoleptodonoides aitchisonii inhibited the decrease in Akt phosphorylation by IL-6 according to the concentration. At the highest concentration 100㎍/ml, it had an effect of increasing Akt phosphorylation to 77%, which was decreased by 50% compared to the insulin treated group by IL-6. Therefore, Mycoleptodonoides aitchisonii fruit body used in this test activated Akt phosphorylation inhibited by IL-6 and showed a possibility of significant anti-diabetic effects compared to positive control(metformin).

Lentinula edodes (shiitake mushroom) is a very popular edible, cultivated mushroom in Japan. There are post-storage problems with shiitake mushrooms, such as gill browning and cell wall lysis of the fruiting body, which can result in loss of fresh food quality and consequent loss of value. Lentinan is a cell wall component of beta-1, 3-linked-D-glucan with beta-1, 6 branches, which was isolated as an anti-tumor active-substance from L. edodes. Lentinan content decreases following harvest as a result of increased glucanase activity. We isolated one exo-glucanase encoding genes, exg21) and two endo-glucanase encoding gene tlg12) and glu1 from L. edodes. Transcription level of the exg2, tlg1 and glu1 gene increased after harvesting. Enzymes encoded by the genes have lentinan degrading activity, therefore, these genes are involved in lentinan degradation after harvesting. We also identified several cell wall degradation- related enzyme-encoding genes3), such as mixed-linked glucanase (mlg1), chitinases (chi1, chi29), chitin deacetylase (chd1), and chitosanase (cho1). It is revealed that transcriptional levels of these genes increased after harvesting, by real-time PCR. Glucanase and chitinase activity increased following harvest as results of increased transcription of these cell wall degradation-related enzyme-encoding genes. Increase of these cell wall degradation- related enzyme activities would cause cell wall lysis and lentinan degradation during post-harvest preservation. We identified laccase and tyrosinase encoding genes (lcc4 and tyr, respectively) by PCR-subtraction. The lcc4 was a novel laccase-encoding gene in L. edodes. Transcription levels of lcc4 and tyr increased after harvesting, and these genes would be involved in browning of the fruiting body. 1) Sakamoto et al. (2005) Current Genetics, 48: 195-203 2) Sakamoto et al. (2006) Plant Physiology 141: 793-801 3) Sakamoto et al. (2009) Current Genetics 55: 409-423

Phellinus gilvus(PG) is a medicinal mushroom belonging to the Hymenochaetaceae basidiomycetes, and has advantages over many Phellinus species due to its short growth period (3 mo), making it cheaper to produce. In the current investigation, we determined the major components of the ethyl acetate extract of PG responsible for its biological activities and further compared the magnitude of the antioxidant/anti-inflammatory activities of components with the various fractional extracts of PG. As the results, the average total DPPH radical scavenging activities of both Fd and Fc of PG was 10 mg/mL, > 95%. Among the fractional extracts of PG, Fd had the greatest inhibitory activity with an IC50 value of 36.70㎍/mL, whereas Fb showed the lowest activity. PCA had even greater activity of NO inhibition than Fd with an IC50 value of 19.46㎍/mL. The mRNA expression of iNOS or COX-2 was nearly undetectable in the absence of LPS. However, LPS- stimulation markedly increased the expression of both iNOS and COX-2 genes. Fd inhibited the effect of LPS in a concentration-dependent manner. Six major compounds were identified from the ethyl acetate extract of PG, and protocatechualdehyde (PCA) was supposed to be the major phenolic compound of PG responsible for its DPPH free radical scavenging activity and its inhibitory effects on LPS-induced NO production in RAW264.7 cells. Further in vitro and in vivo experiments are currently underway to confirm this observation and to investigate the detailed molecular mechanisms involved in the process as well as the biological activities of other fractions of Fd.

Laccase (Lcc; EC 1.10.3.2) belongs to a group of polyphenol oxidases, which catalyzed the oxidation of single-electron from phenolic substrates or aromatic amines. Many organisms possess several lcc encoding genes, and their biological functions diverge into many branches. There are many studies on biochemical function of Lccs, however, there are few studies about biological functions of one Lcc in detail. We researched on biological function of Lcc1, which is most abundantly secreted enzyme from vegetative mycelia into liquid culture in L. edodes. In our previous study, lcc1 gene was down regulated by RNAi method in L. edodes, and then ivrL1#32 was selected as a completely lcc1 dowaregulated transformant. We revealed that fruiting body development in ivrL1#32 was significantly suppressed compared to wild type strain. In this study, we observed the hyphal morphology of ivrL1#32. IvrL1#32 did not form thick aerial mycelium mat when grown on MYPG agar plate. From the observation using scanning electron microscope (SEM), hyphae of ivrL1#32 had many abnormal short branches and their mycelial density was lower than that of wild type strain. From transmission electron microscope observation (TEM), ivrL1#32 lacked obviously distinguishable outer and inner layer in their cell wall. In addition, the fibrous layer of ivrL1#32, which connected hyphae, obviously decreased. These morphological phenotypes would be caused by the absence of Lcc1 in L. edodes. Our results provide a clue to resolve of the biological function of Lcc1 in L. edodes.

Singapore will soon submit a national report to and subsequently appear before the UN Human Rights Council for a universal periodic review of its human rights laws and practices. This review will elicit a rare and unprecedented expression of whether and how Singapore feels it has adhered to international human rights law, and ways in which it may further refine or calibrate its domestic practices. This article seeks to identify Singapore’s human rights achievements; highlight challenges it should be prepared to address; and recommend measures it should adopt to promote human rights.

Magnetars are neutron stars possessing a magnetic field of about 1014-1015 G at the surface. Thermodynamic properties of neutron star matter, approximated by pure neutron matter, are considered at finite temperature in strong magnetic fields up to 1018 G which could be relevant for the inner regions of magnetars. In the model with the Skyrme effective interaction, it is shown that a thermodynamically stable branch of solutions for the spin polarization parameter corresponds to the case when the majority of neutron spins are oriented opposite to the direction of the magnetic field (i.e. negative spin polarization). Moreover, starting from some threshold density, the self-consistent equations have also two other branches of solutions, corresponding to positive spin polarization. The influence of finite temperatures on spin polarization remains moderate in the Skyrme model up to temperatures relevant for protoneutron stars. In particular, the scenario with the metastable state characterized by positive spin polarization, considered at zero temperature in Phys. Rev. C 80, 065801 (2009), is preserved at finite temperatures as well. It is shown that, above certain density, the entropy for various branches of spin polarization in neutron matter with the Skyrme interaction in a strong magnetic field shows the unusual behavior, being larger than that of the nonpolarized state. By providing the corresponding low-temperature analysis, we prove that this unexpected behavior should be related to the dependence of the entropy of a spin polarized state on the effective masses of neutrons with spin up and spin down, and to a certain constraint on them which is violated in the respective density range.