A tobacco calmodulin-related protein suppresses sense transgene-induced RNA silencing but not inverted repeat-induced RNA silencing

A tobacco calmodulin-related protein, rgs-CaM, interacts with viral suppressors of RNA silencing and modulates host RNA silencing. Plants overexpressing the rgs-CaM gene were crossed with plants exhibiting sense transgene-induced RNA silencing (S-PTGS) or inverted repeat-induced RNA silencing (IR-PTGS). S44 plants harboring a sense transgene encoding a tobacco microsomal ω-3 fatty acide desaturase (NtFAD3) exhibited the S-PTGS phenotype. The frequency of the S-PTGS phenotype incidence was nearly 100 % in the hemizygous S44 plants, but was reduced to 30 % in crossbred plants with an rgs-CaM-overexpressing transgenic line. The remaining 70 % of crossbred plants successfully overexpressed the NtFAD3 transgene, and the amount of NtFAD3 small interfering RNAs (siRNAs) was largely decreased. In contrast, overexpression of rgs-CaM did not suppress siRNA production in the IR-PTGS that targeted the NtFAD3 gene. These results indicated that rgs-CaM suppresses RNA silencing at a step upstream of siRNA production and does not interfere with the later steps of RNA silencing, including siRNA-mediated RNA degradation.     

Oleic acid promotes adaptability against oxidative stress in 3T3-L1 cells through lipohormesis

Although fatty acids are important components of biological membranes, energy sources, and signal transducers or precursors of lipid mediators, excess intake of fatty acids and their accumulation cause obesity and metabolic syndrome. Thus, fatty acid quantity is known to be an important factor for obesity-related diseases, but the effects of different types of fatty acids (i.e., fatty acid quality) on human health are not completely understood. We here focused on the relationship between fatty acid quality and oxidative stress by investigating whether resistibility to tert-butyl hydrperoxide (t-BuOOH)-induced oxidative stress in 3T3-L1 cells varied according to the fatty acid type. Among eight fatty acids (both saturated and unsaturated) tested, oleic acid (OA) exerted the most pronounced cytoprotective effects, with efficacy over a wide range of concentrations. OA treatment markedly enhanced the intracellular levels of lipid peroxidation markers, including N ^ε-(hexanoyl)lysine, 4-hydroxy-2-nonenal, and acrolein. The levels of these markers in OA-treated cells were decreased after t-BuOOH exposure, whereas the levels in untreated control cells were notably increased after t-BuOOH exposure. Our results suggested that unsaturated fatty acids, particularly OA, could promote an adaptive response and enhance cell tolerance through increased cellular antioxidative capacity via OA-induced mild lipid peroxidation (lipohormesis), and thus protect cells against subsequent oxidative stress-related injury.     

The Role of Arabidopsis ABCG9 and ABCG31 ATP Binding Cassette Transporters in Pollen Fitness and the Deposition of Steryl Glycosides on the Pollen Coat

The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9_pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen.     

Analysis of <Emphasis Type="Italic">COQ2</Emphasis>gene in multiple system atrophy

Background

Loss of function COQ2 mutations results in primary CoQ10 deficiency. Recently, recessive mutations of the COQ2 gene have been identified in two unrelated Japanese families with multiple system atrophy (MSA). It has also been proposed that specific heterozygous variants in the COQ2 gene may confer susceptibility to sporadic MSA. To assess the frequency of COQ2 variants in patients with MSA, we sequenced the entire coding region and investigated all exonic copy number variants of the COQ2 gene in 97 pathologically-confirmed and 58 clinically-diagnosed MSA patients from the United States.

Results

We did not find any homozygous or compound heterozygous pathogenic COQ2 mutations including deletion or multiplication within our series of MSA patients. In two patients, we identified two heterozygous COQ2 variants (p.S54W and c.403?+?10G?>?T) of unknown significance, which were not observed in 360 control subjects. We also identified one heterozygous carrier of a known loss of function p.S146N substitution in a severe MSA-C pathologically-confirmed patient.

Conclusions

The COQ2 p.S146N substitution has been previously reported as a pathogenic mutation in primary CoQ10 deficiency (including infantile multisystem disorder) in a recessive manner. This variant is the third primary CoQ10 deficiency mutation observed in an MSA case (p.R387X and p.R197H). Therefore it is possible that in the heterozygous state it may increase susceptibility to MSA. Further studies, including reassessing family history in patients of primary CoQ10 deficiency for the possible occurrence of MSA, are now warranted to resolve the role of COQ2 variation in MSA.

     

Effects of tryptophan-rich breakfast and light exposure during the daytime on melatonin secretion at night

Background

The purpose of the present study is to investigate effects of tryptophan intake and light exposure on melatonin secretion and sleep by modifying tryptophan ingestion at breakfast and light exposure during the daytime, and measuring sleep quality (by using actigraphy and the OSA sleep inventory) and melatonin secretion at night.

Methods

Thirty three male University students (mean ± SD age: 22 ± 3.1 years) completed the experiments lasting 5 days and 4 nights. The subjects were randomly divided into four groups: Poor*Dim (n = 10), meaning a tryptophan-poor breakfast (55 mg/meal) in the morning and dim light environment (<50 lx) during the daytime; Rich*Dim (n = 7), tryptophan-rich breakfast (476 mg/meal) and dim light environment; Poor*Bright (n = 9), tryptophan-poor breakfast and bright light environment (>5,000 lx); and Rich*Bright (n = 7), tryptophan-rich breakfast and bright light.

Results

Saliva melatonin concentrations on the fourth day were significantly lower than on the first day in the Poor*Dim group, whereas they were higher on the fourth day in the Rich*Bright group. Creatinine-adjusted melatonin in urine showed the same direction as saliva melatonin concentrations. These results indicate that the combination of a tryptophan-rich breakfast and bright light exposure during the daytime could promote melatonin secretion at night; further, the observations that the Rich*Bright group had higher melatonin concentrations than the Rich*Dim group, despite no significant differences being observed between the Poor*Dim and Rich*Dim groups nor the Poor*Bright and Rich*Bright groups, suggest that bright light exposure in the daytime is an important contributor to raised melatonin levels in the evening.

Conclusions

This study is the first to report the quantitative effects of changed tryptophan intake at breakfast combined with daytime light exposure on melatonin secretion and sleep quality. Evening saliva melatonin secretion changed significantly and indicated that a tryptophan-rich breakfast and bright light exposure during the daytime promoted melatonin secretion at this time.     

Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection

This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.     

A role of the sphingosine-1-phosphate (S1P)–S1P receptor 2 pathway in epithelial defense against cancer (EDAC)

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)–S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.     

Genetic and environmental sources of egg size, fecundity and body size in the migrant skipper, Parnara guttata guttata (Lepidoptera: Hesperiidae)

Genetic and environmental sources of egg size, fecundity and body size (forewing length) were examined in the butterfly, Parnara guttata guttata. Phenotypic and genetic correlation and heritability were estimated for these traits under different day-length and temperature conditions. Egg size and fecundity had relatively high heritabilities, and body sizes in males and females had moderate and high heritability, respectively. Negative phenotypic and genetic correlations between egg size and fecundity were estimated in treatments corresponding to the natural conditions during larval development of the first and second generations. Phenotypic and genetic correlations between body size and egg size differed considerably between insects reared under long and short day-lengths. Next, genotype–environment interactions were estimated by comparing reaction norms to day-length or temperature of these traits among families. ANOVA analysis revealed significant genotype–environment interactions in egg size and forewing length in both sexes for day-length and temperature. These results suggested that a large additive genetic variance for egg size might have been maintained by a genetic trade-off and/or by genotype–environment interactions in P. g. guttata.     

Effects of spirulina, a blue-green alga, on bone metabolism in ovariectomized rats and hindlimb-unloaded mice

The safety and effectiveness were examined of the spirulina alga on bone metabolism in ovariectomized estrogen-deficient rats and hindlimb-unloaded mice. The dosage range was from an amount equal to that recommended in so-called health foods for humans (0.08 g/kg BW/day) to a 100-fold higher dose. The bone mineral density (BMD) of the whole femur and tibia of ovariectomized rats in the any spirulina-treated groups was not significantly different from that of the ovariectomized group, although BMD of the distal femur and proximal tibia was significantly lower in the spirulina-treated groups than in the ovariectomized group after a 6 week-experimental period. BMD of the femur and tibia was not affected by treatment with any dose of spirulina in hindlimb-unloaded mice. These results suggest that the intake of spirulina decreased BMD in the trabecular bone of rodents under estrogen-deficient conditions.