Nemaline Myopathy-Related Skeletal Muscle α-Actin (ACTA1) Mutation,Asp286Gly,Prevents Proper Strong Myosin Binding and Triggers Muscle Weakness

Many mutations in the skeletal muscle α-actin gene (ACTA1) lead to muscle weakness and nemaline myopathy. Despite increasing clinical and scientific interest, the molecular and cellular pathogenesis of weakness remains unclear. Therefore, in the present study, we aimed at unraveling these mechanisms using muscles from a transgenic mouse model of nemaline myopathy expressing the ACTA1 Asp286Gly mutation. We recorded and analyzed the mechanics of membrane-permeabilized single muscle fibers. We also performed molecular energy state computations in the presence or absence of Asp286Gly. Results demonstrated that during contraction, the Asp286Gly acts as a “poison-protein” and according to the computational analysis it modifies the actin-actin interface. This phenomenon is likely to prevent proper myosin cross-bridge binding, limiting the fraction of actomyosin interactions in the strong binding state. At the cell level, this decreases the force-generating capacity, and, overall, induces muscle weakness. To counterbalance such negative events, future potential therapeutic strategies may focus on the inappropriate actin-actin interface or myosin binding.     

Role of Calcium-independent Phospholipase A2�� in High Glucose-induced Activation of RhoA, Rho Kinase, and CPI-17 in Cultured Vascular Smooth Muscle Cells and Vascular Smooth Muscle Hypercontractility in Diabetic Animals

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A₂β (iPLA₂β) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA₂β mRNA, protein, and iPLA₂ activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA₂β protein up-regulation. Inhibiting iPLA₂β activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA₂β in iPLA₂β-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA₂β inhibition. Moreover, increases in iPLA₂ activity and iPLA₂β protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA₂β, but not iPLA₂γ, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA₂β up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.     

The Influence of 1800 MHz GSM-like Signals on Hepatic Oxidative DNA and Lipid Damage in Nonpregnant,Pregnant, and Newly born Rabbits

The aim of our study is to evaluate the possible biological effects of whole-body 1800 MHz GSM-like radiofrequency (RF) radiation exposure on liver oxidative DNA damage and lipid peroxidation levels in nonpregnant, pregnant New Zealand White rabbits, and in their newly borns. Eighteen nonpregnant and pregnant rabbits were used and randomly divided into four groups which were composed of nine rabbits: (i) Group I (nonpregnant control), (ii) Group II (nonpregnant-RF exposed), (iii) Group III (pregnant control), (iv) Group IV (pregnant-RF exposed). Newborns of the pregnant rabbits were also divided into two groups: (v) Group V (newborns of Group III) and (vi) Group VI (newborns of Group III). 1800 MHz GSM-like RF radiation whole-body exposure (15 min/day for a week) was applied to Group II and Group IV. No significant differences were found in liver 8 OHdG/10⁶ dG levels of exposure groups (Group II and Group IV) compared to controls (Group I and Group III). However, in Group II and Group IV malondialdehyde (MDA) and ferrous oxidation in xylenol orange (FOX) levels were increased compared to Group I (P < 0.05, Mann–Whitney). No significant differences were found in liver tissue of 8 OHdG/10⁶ dG and MDA levels between Group VI and Group V (P > 0.05, Mann–Whitney) while liver FOX levels were found significantly increased in Group VI with respect to Group V (P < 0.05, Mann–Whitney). Consequently, the whole-body 1800 MHz GSM-like RF radiation exposure may lead to oxidative destruction as being indicators of subsequent reactions that occur to form oxygen toxicity in tissues.     

Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase,GCN5

NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.     

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα_i1, Gα_i2 and Gα_i3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα_i subunit, and cp173Venus fused to the Gγ₂ subunit as acceptor. The Gα_i FRET biosensors constructs are expressed together with Gβ₁ from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα_i FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα_i FRET sensor in single cells upon stimulation of several GPCRs, including the LPA₂, M₃ and BK₂ receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα_i1, Gα_i2 and Gα_i3 activation will be valuable for live-cell measurements that probe Gα_i activation.     

134Cs and 137Cs levels in a grassland, 32 km northwest of the Fukushima 1 Nuclear Power Plant, measured for two seasons after the fallout

We measured the levels of radioactive caesium (RACs; ¹³⁴Cs and ¹³⁷Cs) in plants and soil in a grassland, 32 km northwest of the Fukushima 1 Nuclear Power Plant, from June 2011 to October 2012. In 2011, the highest RACs levels (¹³⁴Cs + ¹³⁷Cs) in plants and in the 0–5 cm soil layer were approximately 80 kBq per kg dry weight (DW). Forage grasses and clovers in this grassland showed similar RACs levels. On a DW basis, the levels of RACs in these plants tended to increase with increasing biomass over both years, but the absolute levels decreased in 2012. The RACs levels in the soil decreased sharply with soil depth; the RACs level in the 5–10 cm soil layer was only 3 % of that in the 0–5 cm layer. The transfer factor (ratio of radioactivity in plant parts on DW basis to that in the 0–10 cm soil layer) was 0.5 and 1.0 for the aboveground and belowground plant parts, respectively, in 2011, and these values decreased by approximately 50 % in 2012. We discuss the possible mechanisms underlying these trends, and strategies to decrease the level of RACs in plants to the permissible level for forage.     

Comparative Study of Bacillus thuringiensis Cry1Aa and Cry1Ac δ-Endotoxin Activation, Inactivation and In Situ Histopathological Effect in Ephestia kuehniella (Lepidoptera: Pyralidae)

A comparative study of different steps in the mode of action of the individual Bacillus thuringiensis kurstaki BNS3 Cry1Aa and Cry1Ac δ-endotoxins on E. kuehniella larvae was performed in order to investigate the origin of the difference in the response of this larvae to each of the latter. Proteolytic activation was shown to be one of the main steps impaired in E. kuehniella tolerance to Cry1Aa. The absence of two proteinase activities as well as an altered activity level observed in the case of Cry1Aa would be the consequence of proteinase-mediated tolerance of E. kuehniella to this toxin. In situ binding and histopathological effect analyses allowed concluding that the binding of the toxin to BBMV receptors is the key step in E. kuehniella tolerance to Cry1Aa toxin. The latter was slightly bound to apical membranes of epithelial cells that remained intact, whereas Cry1Ac was tightly bound to completely damaged cells basal membranes.