Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice.

Impairment of skeletal muscle function has been associated with changes in ovarian hormones, especially estradiol. To elucidate mechanisms of estradiol on skeletal muscle strength, the hormone's effects on phosphorylation of the myosin regulatory light chain (pRLC) and muscle contractility were investigated, hypothesizing an estradiol-specific beneficial impact. In a skeletal muscle cell line, C2C12, pRLC was increased by 17ß-estradiol (E2) in a concentration-dependent manner. In skeletal muscles of C57BL/6 mice that were E2 deficient via ovariectomy (OVX), pRLC was lower than that from ovary-intact, sham-operated mice (Sham). The reduced pRLC in OVX muscle was reversed by in vivo E2 treatment. Posttetanic potentiation (PTP) of muscle from OVX mice was low compared with that from Sham mice, and this decrement was reversed by acute E2 treatment, demonstrating physiological consequence. Western blot of those muscles revealed that low PTP corresponded with low pRLC and higher PTP with greater pRLC. We aimed to elucidate signaling pathways affecting E2-mediated pRLC using a kinase inhibitor library and C2C12 cells as well as a specific myosin light chain kinase inhibitor in muscles. PI3K/Akt, MAPK, and CamKII were identified as candidate kinases sensitive to E2 in terms of phosphorylating RLC. Applying siRNA strategy in C2C12 cells, pRLC triggered by E2 was found to be mediated by estrogen receptor-ß and the G protein-coupled estrogen receptor. Together, these results provide evidence that E2 modulates myosin pRLC in skeletal muscle and is one mechanism by which this hormone can affect muscle contractility in females.

Interaction mechanisms of peanut protein isolate and high methoxyl pectin with ultrasound treatment: The effect of ultrasound parameters, biopolymer ratio, and pH.

Ultrasound could effectively change molecular structure of proteins, polysaccharides, and their interactions, and was used to treat the peanut protein isolate-high methoxy pectin (PPI-HMP) complexes in this study. Effects of different ultrasound parameters, PPI-HMP mixing ratio (40:1-5:2), and pH (2.0-8.0) on the PPI-HMP interactions were investigated. Turbidity, solution appearance, and Zeta-potential analysis revealed an electrostatic interaction between PPI and HMP from pH 2.0 to pH 6.0. Ultrasound changed the tertiary structure conformation of PPI according to the surface hydrophobicity analysis. Increased ultrasound power density and pH broke the hydrogen bonds between the complexes according to Fourier transform infrared spectroscopy analysis. Apparent viscosity and confocal laser scanning microscopy analysis showed that appropriate ultrasound treatment (5.43 W/cm3, 25 min, 25 °C) reduced the viscosity of the complexes, and enhanced the electrostatic and hydrophobic interactions between PPI and HMP. These findings will contribute to the application of PPI-HMP complexes in the food industry.

The droplet breakup model and characteristics of pH-shifted peanut protein isolate-high methoxyl pectin stabilised emulsions under ultrasound.

The effect of pH on the occurrence states of peanut protein isolate (PPI) and high methoxyl pectin (HMP), and droplet breakup model of the emulsions under ultrasound were studied. Particle size distribution and scanning electron microscopy results showed that PPI-HMP existed a soluble complex at pH 5.0, had no interaction at pH 7.0, and was co-soluble at pH 9.0. Droplet breakup model results revealed that the characteristics of emulsion stabilised by PPI-HMP treated at pH 5.0 was different from that at pH 7.0 and 9.0. The average diameter of the droplet well satisfied the model. According to rheological properties, interface tension, and microstructure, the formation mechanism and characteristics of emulsion stabilised by PPI-HMP treated at pH 5.0 was different from that at pH 7.0 and pH 9.0. The research provided a reference for constructing emulsions using pH-shifted PPI-HMP under ultrasound.

14-3-3 Binding to ataxin-1(ATXN1) regulates its dephosphorylation at Ser-776 and transport to the nucleus.

Spinocerebellar ataxia type 1 (SCA1) is a lethal neurodegenerative disorder caused by expansion of a polyglutamine tract in ATXN1. A prominent site of pathology in SCA1 is cerebellar Purkinje neurons where mutant ATXN1 must enter the nucleus to cause disease. In SCA1, phosphorylation of ATXN1 at Ser-776 modulates disease. Interestingly, Ser-776 is located within a region of ATXN1 that harbors several functional motifs including binding sites for 14-3-3, and splicing factors RBM17 and U2AF65. The interaction of ATXN1 with these proteins is thought to be regulated by the phosphorylation status of Ser-776. In addition, Ser-776 is adjacent to the NLS in ATXN1. Although pS776-ATXN1 is enriched in nuclear extracts of cerebellar cells, the vast majority of 14-3-3 is in the cytoplasmic fraction. We found that dephosphorylation of cytoplasmic pS776-ATXN1 is blocked by virtue of it being in a complex with 14-3-3. In addition, data suggest that binding of 14-3-3 to cytoplasmic ATXN1 impeded its transport to the nucleus, suggesting that 14-3-3 must disassociate from ATXN1 for transport of ATXN1 to the nucleus. Consistent with this hypothesis is the observation that once in the nucleus pS776 is able to be dephosphorylated. Evidence is presented that PP2A is the pS776-ATXN1 phosphatase in the mammalian cerebellum. In the nucleus, we propose that dephosphorylation of pS776-ATXN1 by PP2A regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65.

Sandwich Ct real-time PCR identifies single-copy T-DNA integration accumulating in backbone-free transgenic T1 Arabidopsis.

A sandwich Ct real-time PCR (SC-PCR) was used to detect single-copy T-DNA plants by visualizing Ct patterns of T-DNA and two reference amplicons. Detecting the T-DNA copy number directly by visualizing the Ct pattern eliminates the errors introduced by multistep calculations of relative Ct values. Using SC-PCR, we found that single-copy T-DNA integrations were more frequent in transgenic T1 Arabidopsis without a vector backbone. On the basis of this phenomenon, we combined the negative screen of the vector backbone and SC-PCR to efficiently identify single-copy T-DNA plants. We found that T-DNA copy number detection was underestimated in transgenic plants containing inverted T-DNA repeats due to hairpin structures formed during PCR, indicating that PCR-based methods for detecting T-DNA copy number should be reevaluated. We solved this problem by releasing T-DNA from the complex structures using restriction enzymes before performing SC-PCR. We also demonstrated that latent Agrobacterium contamination in the T1 transgenic Arabidopsis generated by the floral dip method was exceedingly low and may not affect the detection of T-DNA copy number. Overall, our method provides a whole-set procedure for detecting single-copy T-DNA plants more efficiently than other screening methods including Southern blotting.

Comparative study on the gel properties and nanostructures of gelatins from chicken, porcine, and tilapia skin.

To clarify the feasibility of replacing commercial gelatin with chicken skin gelatin, we investigated the gel properties and nanostructures of chicken skin gelatin (CG), commercial porcine skin gelatin (PG), and tilapia skin gelatin (FG). Compared with PG and FG, CG exhibited the better gel strength, hardness, chewiness, melting point, gelling temperature, and thermostability. The different physicochemical properties of CG might be caused by its higher imino acid content (25.43 residues/100 total residues), which make it more liable to form intramolecular H-bonds (lower amplitude of amide A wave number). In addition, atomic force microscopy (AFM) result was shown that CG contained larger spherical aggregates (483 nm) than PG and FG (334 and 224 nm, respectively), and the lack of chain and ring-like structure promoted the formation of a dense rigid gel. These results revealed that the intramolecular H-bond and the aggregation behavior are the fundamental explanations for the different gel properties of gelatins from three sources. PRACTICAL APPLICATION: This research provides guidance for the application of chicken skin gelatin as a replacer for commercial gelatin. And the results provide a theoretical basis for the modification of chicken skin gelatin.

Effect of chitosan coatings on the evolution of sodium carbonate-soluble pectin during sweet cherry softening under non-isothermal conditions.

The inhibiting effect of chitosan coating (2%) on the softening and sodium carbonate-soluble pectin (SSP) evolution of sweet cherries during non-isothermal storage was investigated. Chitosan coating significantly extend the softening (6.4% greater than the control group), maintained the SSP content (6.6% greater than the control group), and reduced the degradation of SSP by inhibiting the expression of the paPME1-5 genes, which regulating pectin methylesterase activity of sweet cherries under temperature variation. In addition, the results of methylation and monosaccharide composition indicated that the chitosan coating reduced demethylation of SSP and the loss of RG-I main and side chain neutral sugars. Atomic force microscopy images revealed that the coated sweet cherries contained more linked, branched, and long SSP chains and maintained the width of the pectin backbone (>140 nm). These results indicated that a chitosan coating is feasible to preserve postharvest fruit under non-isothermal conditions.

Calcium permeation property and firmness change of cherry tomatoes under ultrasound combined with calcium lactate treatment.

This study aimed to investigate the effect of ultrasound combined with calcium lactate (2%, w/v) treatment (U + Ca) on calcium permeation and firmness of cherry tomatoes. Calcium distribution and fruit pectin nanostructure were also analysed by transmission electron microscope (TEM) and atomic force microscopy (AFM), respectively. The firmness (31.45 N) was maintained when ultrasound energy density was 20 W/L for 15 min at 15 °C. The Ca content increased in U + Ca treated fruit. Meanwhile, the Peleg's model could be used to express the change of solid gain in cherry tomatoes under ultrasound treatment at 15, 20, and 25 °C. According to the AFM results, the width (≥40 nm) and length (≥2 µm) of chelate-soluble pectin (CSP) and sodium carbonate-soluble pectin (SSP) chains with large frequency was observed in U + Ca treated fruit. Under desirable conditions (15 °C, 15 min, 20 W/L), ultrasound combined with calcium lactate could maintain the quality of cherry tomatoes.

Phosphorylation of ATXN1 at Ser776 in the cerebellum.

Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative disorders caused by a mutant protein with an expanded polyglutamine tract. Phosphorylation of ataxin-1 (ATXN1) at serine 776 is implicated in SCA1 pathogenesis. Previous studies, utilizing transfected cell lines and a Drosophila photoreceptor model of SCA1, suggest that phosphorylating ATXN1 at S776 renders it less susceptible to degradation. This work also indicated that oncogene from AKR mouse thymoma (Akt) promotes the phosphorylation of ATXN1 at S776 and severity of neurodegeneration. Here, we examined the phosphorylation of ATXN1 at S776 in cerebellar Purkinje cells, a prominent site of pathology in SCA1. We found that while phosphorylation of S776 is associated with a stabilization of ATXN1 in Purkinje cells, inhibition of Akt either in vivo or in a cerebellar extract-based phosphorylation assay did not decrease the phosphorylation of ATXN1-S776. In contrast, immunodepletion and inhibition of cyclic AMP-dependent protein kinase decreased phosphorylation of ATXN1-S776. These results argue against Akt as the in vivo kinase that phosphorylates S776 of ATXN1 and suggest that cyclic AMP-dependent protein kinase is the active ATXN1-S776 kinase in the cerebellum.

Characterization of the zebrafish atxn1/axh gene family.

In mammals, ataxin-1 (ATXN1) is a member of a family of proteins in which each member contains an AXH domain. Expansion of the polyglutamine tract in ATXN1 causes the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1) with prominent cerebellar pathology. Toward a further characterization of the genetic diversification of the ATXN1/AXH gene family, we identified and characterized members of this gene family in zebrafish, a lower vertebrate with a cerebellum. The zebrafish genome encodes two ATXN1 homologs, atxn1a and atxn1b, and one ATXN1L homolog, atxn1l. Key biochemical features of the human ATXN1 protein not seen in the invertebrate homologs (a nuclear localization sequence and a site of phosphorylation at serine 776) are conserved in the zebrafish homologs, and all three zebrafish Atxn1/Axh proteins behave similarly to their human counterparts in tissue-culture cells. Importantly, each of the three homologs is expressed in the zebrafish cerebellum, which in humans, is a prominent site of SCA1 pathogenesis. In addition, atxn1a and atxn1b are expressed in the developing zebrafish cerebellum. These data show that in zebrafish, a lower vertebrate, the complexity of the atxn1/axh gene family is more similar to higher vertebrates than invertebrates with a simple central nervous system and suggests a relationship between the diversification of the ATXN1/AXH gene family and the development of a complex central nervous system, including a cerebellum.

Effects of calcium and pectin methylesterase on quality attributes and pectin morphology of jujube fruit under vacuum impregnation during storage.

Calcium chloride (1% w/w, CaCl2) and pectin methylesterase (PME) (15 U/mL) were vacuum impregnated (VI) into jujubes to preserve their quality. The nanostructure of jujube pectin was investigated using atomic force microscopy (AFM) to determine the degradation mechanism of pectin. CaCl2 with PME under VI treatment (VI + Ca + PME) maintained jujubes' quality. Weight loss in VI + Ca + PME group at day 56 was only 60.36% of that in control group (CK). Firmness, soluble solids content, and ascorbic acid content of jujubes in VI + Ca + PME group were higher than those in CK. Firmness was highly positively correlated with sodium carbonate-soluble pectin (SSP) content. According to AFM results, frequencies of molecules with a width ≥60 nm of water-soluble pectin (WSP), chelate-soluble pectin (CSP), and SSP were the highest in VI + Ca + PME group at the end of storage. WSP, CSP, and SSP degradation was delayed by VI + Ca + PME treatment. The quality of jujubes was effectively maintained by VI + Ca + PME treatment.

Combined effects of ultrasound and calcium on the chelate-soluble pectin and quality of strawberries during storage.

The combined effects of ultrasound and calcium on the water migration, quality, and chelate-soluble pectin (CSP) properties of strawberries were investigated using nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). The relationship among water migration, firmness, and CSP properties was also determined. Treatment with ultrasound and calcium (U + Ca) prevented the decrease in firmness of strawberries during storage (17 days). Measurements of physicochemical parameters (titratable acidity (TA), soluble solid content (SSC), CSP and Ca content) showed that U + Ca treatment maintained better fruit quality. AFM showed a larger percentage of wider and longer CSP molecules in the U + Ca group (width ≥90 nm; length ≥800 nm). These results, together with the HPLC results, confirmed that U + Ca treatment effectively inhibits CSP degradation. This study revealed that the application of ultrasound and calcium could preserve the quality of stored strawberries.

Effect of exogenous ATP on the postharvest properties and pectin degradation of mung bean sprouts (Vigna radiata).

The effects of exogenous ATP on the postharvest quality, browning and softening of mung bean (Vigna radiata) sprouts were evaluated. ATP treatment significantly alleviated the quality loss and browning events during the storage of 3 days. It also reduced the oxidant damage by inducing high activities of peroxidase (9.3-13.9%) and superoxide dismutase (8.8-10.3%) which scavenged the reactive oxygen species (ROS) effectively. Transcriptional results indicated that ATP treatment decreased VrPL1, VrPME and VrPG1 gene expression levels more than 2 folds at some time points. Furthermore, the atomic force microscope (AFM) images revealed that the pectin degradation was notably slowed by ATP treatment and the width and height of pectin backbone were better maintained (47.1% and 45.6% higher than control without ATP treatment). The cooperative effects of ROS scavenging and decreased expressions of pectin-related genes might contribute to the deferred pectin deterioration and firmness loss by ATP treatment.

Energy Regulated Nutritive and Antioxidant Properties during the Germination and Sprouting of Broccoli Sprouts ( Brassica oleracea var. italica).

The role of energy status in germination and sprouting of broccoli seeds was investigated by exogenous ATP and DNP treatments. With the synthesis of adenylates from 38.82 to 142.69 mg·100 g-1 DW, the nutritive components (soluble sugar, proteins, pigments, and phenolics) and AAs were increased during germination and early sprouting (day 5). Elements of the BoSnRK2 pathway were down-regulated by more than 2 fold under the energy charge feedback inhibition. At the end of sprouting (day 7), energy depletion resulted in slowdown or reduced nutritional accumulation and antioxidant capacities. Exogenous ATP depressed the BoSnRK2 pathway by maintaining the energy status at high levels and further promoted the nutrition and antioxidant levels. It also prevented the energy depletion at day 7. On the contrary, DNP reduced the ATP contents (16.10-26.86%) and activated the BoSnRK2 pathway. It also notably suppressed the energy-consuming activities including germination, sprouts growth, and secondary metabolic synthesis.

Metabolite profiling of Listeria innocua for unravelling the inactivation mechanism of electrolysed water by nuclear magnetic resonance spectroscopy.

Bactericidal effects of low concentration electrolysed water (LcEW) on microorganisms are previously well reported; however, the inactivation mechanism of EW is not understood. The lethal and sublethal injuries of L. monocytogenes and L. innocua by EW treatments were determined and the metabolic profile changes for L. innocua were characterised using nuclear magnetic resonance (NMR). Microbial metabolomics approach combined with multivariate data analyses was used to interpret the cellular chemical fingerprints of L. innocua. The relative amount of intracellular reactive oxygen species (ROS) was assayed using 2',7-dichlorodihydrofluorescein diacetate (H2DCFDA). The results showed that the proportion of the sublethally injured microbial cells L. monocytogenes and L. innocua increased from 40% to 70% and from 35% to 65%, respectively, when the free available chlorine (FAC) of LcEW increased from 2 to 8 mg/L. Overall, 36 low-molecular-weight metabolic compounds in L. innocua extracts were characterised by NMR spectroscopy. EW perturbation resulted in a drastic and multitude disruption across a wide range of biochemical process including peptidoglycan synthesis, nucleotides biosynthesis and amino acid metabolism. Elevated levels of α-ketoglutarate and succinate implicated the enhanced glutamate decarboxylase (GAD) system and γ-aminobutyric acid (GABA) shunt for the protection against oxidative stress. These findings provided the comprehensive insights into the metabolic response of Listeria to EW oxidative stress and can serve as a basis for better utilisation for sanitisation.

Effects of calcium treatment and low temperature storage on cell wall polysaccharide nanostructures and quality of postharvest apricot (Prunus armeniaca).

Cell wall polysaccharides play an important role in postharvest fruit texture softening. Effects of calcium treatment combined with cold storage on the physical properties, polysaccharide content and nanostructure of apricots were investigated. Apricots were immersed in distilled water, 1% or 3% w/v calcium chloride, then stored at 5°C or 10°C. Storage at 5°C significantly improved apricot quality and shelf life. Significant changes in the concentration and nanostructure of cell wall pectins and hemicelluloses revealed their disassembly and degradation during apricot storage. These modifications could be retarded by 1% w/v calcium chloride treatment. Meanwhile, the basic width units of apricot cell wall polysaccharide chains were 11.7, 31.2 and 39.1nm for water-soluble pectin, 11.7, 17.6 and 19.5nm for chelate-soluble pectin, and 15.6 and 23.4nm for hemicellulose. The results suggest that texture of apricots can be effectively maintained by 1% calcium chloride treatment and storage at 5°C.

Structure and Antioxidant Activities of Proanthocyanidins from Elephant Apple (Dillenia indica Linn.).

UNLABELLED: Proanthocyanidins were isolated and purified from fruits of elephant apple (Dillenia indica Linn.) and their structural and bioactive properties were examined. Bate-Smith alcoholysis, FTIR, and (13) C NMR spectra revealed that elephant apple proanthocyanidins (EAPs) contained a dominant amount of B-type procyanidins (PC) with a minor amount of B-type prodelphinidins (PD) but no A-type interflavan linkage. (13) C NMR spectrum indicated that the cis isomer was dominant in EAPs. The electron spray ionization and matrix-assisted laser desorption ionization time of flight mass spectra of EAPs showed the clear ion peaks corresponding to B-type PC dimer to B-type PD with degree of polymerization of 11. EAPs had strong antioxidant activity, which was evidenced by the high oxygen radical scavenging capacity at 1.06 × 10(4) µmol TE/g and ferric reducing antioxidant power of 2320 µmol Fe(II)/g. The results suggest that EAPs could be extracted to be used as promising functional food materials. PRACTICAL APPLICATION: In this study, the elephant apple proanthocyanidins (EAPs) with a yield of 0.23% were identified for the first time as dominant B-type poly(catechin/epicatechin) but no A-type interflavan linkage. EAPs had higher ORAC and FRAP values compared to commercial grape seed proanthocyanidins, suggesting that EAPs may be used as promising functional food materials.

Preparation of organic tofu using organic compatible magnesium chloride incorporated with polysaccharide coagulants.

Organic tofu using organic compatible coagulants of magnesium chloride and three polysaccharides including carrageenan, guar gum and gum Arabic were generated. For MgCl2 coagulated tofu, carrageenan significantly increased the hardness from 969.5 g to 1210.5 g whereas guar gum (0.6g) decreased the hardness to 505.5 g. Interestingly, gypsum and guar gum (0.6g) increased the yield of tofu significantly. These organic compatible coagulants didn't affect most of 7S and 11S protein subunits. Importantly, the overall-acceptability of organic tofu prepared with MgCl2 combined with guar gum or gypsum was almost the same as conventional tofu made with gymsum while having more beany-flavour. Among these organic coagulants, tofu made from 0.6g guar gum and MgCl2 mixture was the most similar to that coagulated by conventional gypsum. Thus this mixture is promising as coagulant for making organic tofu.

Nanostructural difference of water-soluble pectin and chelate-soluble pectin among ripening stages and cultivars of Chinese cherry.

Nanostructure of water-soluble pectin (WSP) and chelate-soluble pectin (CSP) of two Chinese cherry (Prunus pseudocerasus L.) cultivars (soft cultivar 'Caode' and crisp cultivar 'Bende') with two different ripening stages were characterised using atomic force microscopy. Both cultivars shared some common values of chain widths for WSP or CSP, and both pectins shared several values of chain widths including 37, 55 and 61 nm. The results indicate that different cultivars shared similar components of pectin, and cultivar textural difference might be related to the interaction between pectin and other cherry components or the dissociation of pectin. During ripening, the wide WSP and CSP gradually dissociate in width. The results demonstrated that the changes of WSP and CSP of Chinese cherry in widths were a dissociation process.

Insulin resistance, serum visfatin, and adiponectin levels are associated with metabolic disorders in chronic hepatitis C virus-infected patients.

OBJECTIVE: The aim of this study was to determine the serum levels of visfatin, adiponectin, and insulin resistance (IR) in patients with hepatitis C virus (HCV) infection and their relations to the biochemical markers of hepatitis C. MATERIALS AND METHODS: This study was carried out on 40 HCV-infected patients and 40 sex/age/BMI-matched healthy adults. Lipid profile, liver function tests, IR, serum adiponectin, and visfatin of all patients were examined. Correlations between IR, adiponectin, visfatin, and other variables were analyzed. RESULTS: The levels of visfatin and adiponectin were significantly lower in HCV patients compared with healthy controls. However, IR of HCV patients were higher than those of healthy controls. IR was significantly correlated to triglycerides, visfatin was closely related to low-density lipoprotein cholesterol, whereas adiponectin was associated with high-density lipoprotein cholesterol. These results suggest that IR, serum visfatin, and adiponectin levels are associated with metabolic disorders in chronic HCV-infected patients. CONCLUSION: IR, adiponectin, and visfatin were related to several metabolic markers of HCV, suggesting the characteristics of HCV-related metabolic abnormalities.