Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the βγ Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C β1 Isoform

Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca²⁺ currents (I_Ca,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5′-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist {"type":"entrez-nucleotide","attrs":{"text":"CH132799","term_id":"45009640","term_text":"CH132799"}}CH132799 blocked the U-II-induced I_Ca,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid abolished the U-II-induced responses, whereas PKCα inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKCβ₁ expression, whereas inhibition of PKCβ₁ pharmacologically or by shRNA targeting abolished the U-II-induced I_Ca,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKCβ inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates I_Ca,L through the βγ subunits of G_i/o-protein and downstream activation of the class I PI3K-dependent PKCβ₁ isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes.     

Brucella suis vaccine strain S2-infected immortalized caprine endometrial epithelial cell lines induce non-apoptotic ER-stress

Brucella, which is regarded as an intracellular pathogen responsible for a zoonotic disease called brucellosis, survives and proliferates within several types of phagocytic and non-phagocytic cells. Brucella infects not only their preferred hosts but also other domestic and wild animal species, inducing abortion and infertility. Therefore, the interaction between uterine cells and Brucella is important for understanding the pathogenesis of this disease. In this study, we describe the Brucella suis vaccine strain S2 (B.suis.S2) infection and replication in the immortalized caprine endometrial epithelial cell line hTERT-EECs and the induced cellular and molecular response modulation in vitro. We found that B.suis S2 was able to infect and replicate to high titers and inhibit the proliferation of EECs and induce non-apoptotic pathways, as determined by B.suis.S2 detection using MTT and acridine orange/ethidium bromide (AO/EB) staining and flow cytometry. We explored the evidence of non-apoptotic pathways using real-time quantitative RT-PCR and by western blot analysis. Finally, we discovered the over-expression of GRP78, ATF4, ATF6, PERK, eIF2α, CHOP, and cytochrome c (Cyt-c) but not IRE1, xbp-1, and caspase-3 in B.suis.S2 (HK)-attacked and B.suis.S2-infected cells, suggesting that the molecular mechanism of ER stress sensor activation by B.suis.S2 is basically concomitant with that by B.suis.S2 (HK) and that ER stress, especially the PERK pathway, plays an important role in the process of B.suis.S2 infecting EEC, which may, in part, explain the role of the uterus in the pathogenesis of B.suis.S2.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0564-x) contains supplementary material, which is available to authorized users.     

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (IT) versus intravenous (IV) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the IT (5×10⁵) or IV (2×10⁶) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both IT and IV transplantations. However, IT administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to IV administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the IT group, but not in the IV group. Although the IT group received only one fourth of the number of MSCs that the IV group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the IT group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the IT group, but not in the IV group. Thus, local IT MSC transplantation was more effective than systemic IV MSC administration in protecting against neonatal hyperoxic lung injury.     

Antimicrobial peptide scolopendrasin VII,derived from the centipede Scolopendra subspinipes mutilans,stimulates macrophage chemotaxis via formyl peptide receptor 1

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484]     

Modeling the Transmission of Middle East Respirator Syndrome Corona Virus in the Republic of Korea

The 2015 epidemic of Middle East respiratory syndrome (MERS) in the Republic of Korea has been the largest outbreak outside Middle East. This epidemic had caused 185 laboratory-confirmed cases and 36 deaths in the Republic of Korea until September 2, 2015, which attracted public’s attention. Based on the detailed data of patients released by World Health Organization (WHO) and actual propagation of the epidemic, we construct two dynamical models to simulate the propagation processes from May 20 to June 8 and from June 9 to July 10, 2015, respectively and find that the basic reproduction number R₀ reaches up to 4.422. The numerical analysis shows that the reasons of the outbreak spread quickly are lack of self-protection sense and targeted control measures. Through partial correction analysis, the parameters β₁ and γ have strong correlations with R₀, i.e., the infectivity and proportion of the asymptomatic infected cases have much influence on the spread of disease. By sensitivity analysis, strengthening self-protection ability of susceptible and quickly isolating or monitoring close contacts are effective measures to control the disease.     

Computational modeling of the arterial wall based on layer-specific histological data

Arterial walls typically have a heterogeneous structure with three different layers (intima, media, and adventitia). Each layer can be modeled as a fiber-reinforced material with two families of relatively stiff collagenous fibers symmetrically arranged within an isotropic soft ground matrix. In this paper, we present two different modeling approaches, the embedded fiber (EF) approach and the angular integration (AI) approach, to simulate the anisotropic behavior of individual arterial wall layers involving layer-specific data. The EF approach directly incorporates the microscopic arrangement of fibers that are synthetically generated from a random walk algorithm and captures material anisotropy at the element level of the finite element formulation. The AI approach smears fibers in the ground matrix and treats the material as homogeneous, with material anisotropy introduced at the constitutive level by enhancing the isotropic strain energy with two anisotropic terms. Both approaches include the influence of fiber dispersion introduced by fiber angular distribution (departure of individual fibers from the mean orientation) and take into consideration the dispersion caused by fiber waviness, which has not been previously considered. By comparing the numerical results with the published experimental data of different layers of a human aorta, we show that by using histological data both approaches can successfully capture the anisotropic behavior of individual arterial wall layers. Furthermore, through a comprehensive parametric study, we establish the connections between the AI phenomenological material parameters and the EF parameters having straightforward physical or geometrical interpretations. This study provides valuable insight for the calibration of phenomenological parameters used in the homogenized modeling based on the fiber microscopic arrangement. Moreover, it facilitates a better understanding of individual arterial wall layers, which will eventually advance the study of the structure–function relationship of arterial walls as a whole.     

盐爪爪耐盐相关基因的cDNA-AFLP分析

以不同浓度NaCl(0、100、200、300、400和500 mmol/L)处理生长了4周的盐爪爪(Kalidium foliatum),48h后取其幼嫩叶片用于RNA提取,并进行了cDNA-AFLP表达差异分析.结果表明,用256对引物扩增出连续性差异片段155条,按照NaCl浓度升高的顺序,划分为8种差异带类型,即强→弱"、弱→强"、有→无"、无→有"、有→无→有"、无→有→无"、强→弱→强"和弱→强→弱".测序后获得176个新表达序列标签(ESTs),已将长度大于100 bp的162个ESTs登录到GenBank,登录号为HO205290~HO205450.经BLAST序列比对分析发现,盐爪爪的耐盐性可能与能量、新陈代谢、防御、转运、转录和翻译等相关蛋白有关.     

Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes

It has been shown that aquaporin-3, a water channel, is expressed in mouse embryos. This type of aquaporin transports not only water but also neutral solutes, including cell-permeating cryoprotectants. Therefore, the expression of this channel may have significant influence on the survival of cryopreserved embryos. However, permeability coefficients of aquaporin-3 to cryoprotectants have not been determined except for glycerol. In addition, permeability coefficients under concentration gradients are important for developing and improving cryopreservation protocols. In this study, we examined the permeability of aquaporin-3 to various cryoprotectants using Xenopus oocytes. The permeability of aquaporin-3 to cryoprotectants was measured by the volume change of aquaporin-3 cRNA-injected oocytes in modified Barth's solution containing either 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% DMSO (1.51-1.83 Osm/kg) at 25 degrees C. Permeability coefficients of aquaporin-3 for ethylene glycol and propylene glycol were 33.50 and 31.45 x 10(-3) cm/min, respectively, which were as high as the value for glycerol (36.13 x 10(-3) cm/min). These values were much higher than those for water-injected control oocytes (0.04-0.11 x 10(-3) cm/min). On the other hand, the coefficients for acetamide and DMSO were not well determined because the volume data were poorly fitted by the two parameter model, possibly because of membrane damage. To avoid this, the permeability for these cryoprotectants was measured under a low concentration gradient by suspending oocytes in aqueous solutions containing low concentrations of acetamide or DMSO dissolved in water (0.20 Osm/kg). The coefficient for acetamide (24.60 x 10(-3) cm/min) was as high as the coefficients for glycerol, ethylene glycol, and propylene glycol, and was significantly higher than the value for control (6.50 x 10(-3) cm/min). The value for DMSO (6.33 x 10(-3) cm/min) was relatively low, although higher than the value for control (0.79 x 10(-3) cm/min). This is the first reported observation of DMSO transport by aquaporin-3.     

Multiple steps determine the overall rate of the reduction of 5alpha-dihydrotestosterone catalyzed by human type 3 3alpha-hydroxysteroid dehydrogenase: implications for the elimination of androgens

Human type 3 3alpha-hydroxysteroid dehydrogenase, or aldo-keto reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5alpha-dihydrotestosterone (DHT, potent androgen) to form 3alpha-androstanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The k(cat) for the NADPH-dependent reduction of DHT catalyzed by AKR1C2 is 0.033 s(-1). We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low k(cat) value. Stopped-flow experiments to measure the formation of the AKR1C2.NADP(H) binary complex indicated that two slow isomerization events occur to yield a tight complex. A small primary deuterium isotope effect on k(cat) (1.5) and a slightly larger effect on k(cat)/K(m) (2.1) were observed in the steady state. In the transient state, the maximum rate constant for the single turnover of DHT (k(trans)) was determined to be 0.11 s(-1) for the NADPH-dependent reaction, which was approximately 4-fold greater than the corresponding k(cat) x k(trans) was significantly reduced when NADPD was substituted for NADPH, resulting in an apparent (D)k(trans) of 3.5. Thus, the effects of isotopic substitution on the hydride transfer step were masked by slow events that follow or precede the chemical transformation. Transient multiple-turnover reactions generated curvilinear reaction traces, consistent with the product formation and release occurring at comparable rates. Global fitting analysis of the transient kinetic data enabled the estimate of the rate constants for the three-step cofactor binding/release model and for the minimal ordered bi-bi turnover mechanism. Results were consistent with a kinetic mechanism in which a series of slow events, including the chemical step (0.12 s(-1)), the release of the steroid product (0.081 s(-1)), and the release of the cofactor product (0.21 s(-1)), combine to yield the overall observed low turnover number.