Acetylcholine increases Ca2+ influx by activation of CaMKII in mouse oocytes

IP3-induced Ca2+ release is the primary mechanism that is responsible for acetylcholine (ACh)-induced Ca2+ oscillation. However, other mechanisms remain to explain intracellular Ca2+ elevation. We here report that ACh induces Ca2+ influx via T-type Ca2+ channel by activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the ACh-induced Ca2+ influx facilitates the generation of Ca2+ oscillation in the mouse ovulated oocytes (oocytes(MII)). ACh increased Ca2+ current by 50+/-21%, and produced Ca2+ oscillation. However, the currents and Ca2+ peaks were reduced in Ca2+ -free extracellular medium. ACh failed to activate Ca2+ current and to produce Ca2+ oscillation in oocytes pretreated with KN-93, a CaMKII inhibitor. KN-92, an inactive analogue of KN93, and PKC modulators could not prevent the effect of ACh. These results show that ACh increases T-type Ca2+ current by activation of CaMKII, independent of the PKC pathway, in the mouse oocytes.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

Diversity of endophytic fungi associated with bryophyte in the maritime Antarctic (King George Island)

Bryophytes comprise one of the richest microfungal microhabitats in the Antarctic environment. The maritime Antarctic is very vulnerable to rapid environmental change caused by global warming. The aim of this study was to investigate the importance of bryophytes as a microhabitat for fungal species in the maritime Antarctic by surveying endophytic fungal diversity from several bryophytes including Andreaea sp., Barbilophozia hatcheri, Chorisodontium aciphyllum, Polytrichum alpinum, Polytrichum strictum, Sanionia uncinata, and Warnstorfia sarmentosa. We collected 13 bryophyte samples at four localities on Barton Peninsula, King George Island. In total, 31 endophytic fungi morphotypes were isolated from bryophyte tissues by a thorough surface sterilization method. Using internal transcribed spacer sequence analysis, 16 endophytic fungal strains belonging to Ascomycota (12), Basidiomycota (1), Oomycota (1), and Zygomycota (2) phyla were obtained. Our results suggest the presence of a diverse range of fungal species even in a very limited area, and those bryophytes play an important role in conserving fungal diversity in this harsh environment. Growth rate measurements at a wide range of temperatures confirmed that most of the fungal strains were both mesophilic and psychrotolerant. This is the first report of endophytic fungi in Antarctic moss tissue by fluorescence in situ hybridization.     

MicroRNA-27a Modulates HCV Infection in Differentiated Hepatocyte-Like Cells from Adipose Tissue-Derived Mesenchymal Stem Cells

Background and Aims

Despite the discovery of hepatitis C virus (HCV) entry factor, the mechanism by which it is regulated by miRNAs remains unclear. Adipose tissue-derived human mesenchymal stem cells (AT-hMSCs) have been widely used for differentiated hepatocyte-like cells (DHCs). Here, we established an in vitro HCV infection model using DHCs from AT-hMSCs and identified miRNAs that modulate HCV infectivity.

Methods

AT-hMSCs were differentiated into DHCs using the conditional media, and evaluated for hepatocyte characteristics using RT-PCR, immunocytochemistry, periodic acid-Schiff staining, and a urea synthesis assay. The expression of HCV candidate receptors was also verified using immunocytochemistry. The levels of candidate miRNAs targeting HCV receptors were then determined by relative quantitative RT-PCR (rqRT-PCR). Finally, DHCs were infected using HCVcc and serum from HCV-infected patients, and infectivity of the virus was measured by rqRT-PCR and transmission electron microscopy (TEM).

Results

The expected changes in morphology, function and hepatic gene expression were observed during hepatic differentiation. Moreover, the expression of candidate HCV entry factors and miR-27a were altered during hepatic differentiation. The infection and replication of HCV occurred efficiently in DHCs treated with HCVcc or infected with serum from HCV-infected patients. In addition, HCV infectivity was suppressed in miR-27a-transfected DHCs, due to the inhibition of LDLR expression by miR-27a.

Conclusions

Our results demonstrate that AT-hMSCs are a good source of DHCs, which are suitable for in vitro cultivation of HCV. Furthermore, these results suggest that miR-27a modulates HCV infectivity by regulating LDLR expression.     

Aortic Unfolding Determined Using Non-Contrast Cardiac Computed Tomography: Correlations with Age and Coronary Artery Calcium Score

Objective

Aortic unfolding occurs with aging and reflects proximal aortic dilation, aortic arch widening, and decreased curvature. This study 1) evaluated the relationship between aortic unfolding measured using non-contrast cardiac-gated computed tomography (CT) and age, 2) assessed factors influencing aortic unfolding, and 3) determined the association of this measurement with coronary artery calcium (CAC) score.

Methods

We reviewed the charts of 219 subjects (142 men, 77 women; mean age 54.2±9.3 years) who underwent coronary artery calcium scanning during routine health screening from December 2010 to May 2011. Multivariate regression analysis according to cardiovascular risk factors was performed. We also analyzed the relationship between aortic unfolding measurements and CAC score using stepwise multiple linear regression.

Results

Mean aortic unfolding was 103.7±13.9 mm (men, 106.5±13.5 mm; women, 98.4±12.9 mm). Age, body surface area, and hypertension were exclusively associated with aortic unfolding. The association between aortic unfolding and CAC score was significant after adjustment for age and gender (β = 1.89, p = 0.017) and for Framingham risk score (β = 2.83, p<0.001).

Conclusions

Aortic unfolding defined by measuring aortic width was a reproducible and practical method with non-contrast cardiac CT and associated with age, body surface area, and hypertension. CAC score, a well-established surrogate marker of cardiovascular disease, is positively associated with aortic unfolding. Further study to evaluate aortic unfolding as a potential predictor of cardiovascular risk is warranted.