Complex Topology of Ubiquitin Chains Mediates Lysosomal Degradation of MrgC Proteins.

BACKGROUND: Activation of Mas-related G protein-coupled receptor C (MrgC) receptors relieves pain, but also leads to ubiquitination of MrgC receptors. Ubiquitination mediates MrgC receptor endocytosis and degradation. However, MrgC degradation pathways and ubiquitin-linked chain types are not known. METHODS: N2a cells were treated with cycloheximide (CHX, protein synthesis inhibitor), Mg132 (proteasome inhibitor), 3-Methyladenine (3MA, autophagy lysosome inhibitor) and Chloroquine (CQ, autophagy lysosome inhibitor) to observe the half-life and degradation pathway of MrgC. The location of internalized MrgC receptors and lysosomes (Lyso-Tracker) was observed by immunofluorescence staining. N2a cells were transfected with Myc-MrgC and a series of HA-tagged ubiquitin mutants to study the ubiquitin-linked chain type of MrgC. RESULTS: The amount of MrgC protein decreased with time after CHX treatment of N2a cells. Autophagy lysosome inhibitors can inhibit the degradation of MrgC. The amount of MrgC protein decreased with time after CHX treatment of N2a cells. 3-MA and CQ inhibited the degradation of MrgC protein, whereas Mg-132 did not inhibit it. Partially internalized MrgC receptors were co-labeled with lysosomes. MrgC proteins have multiple topologies of ubiquitin-modified chains. CONCLUSION: As a member of the G protein-coupled receptor family, MrgC receptors can be degraded over time. The complex topology of the ubiquitin-linked chain mediates the lysosomal degradation of MrgC proteins.

Effect of CaCl2 on 2 heat-induced whey protein concentrate fibrillation pathways: Spontaneous and nuclear induction.

Amyloid fibrils have many excellent functional properties that facilitate their applications in the food industry. There are 2 pathways for whey protein concentrate (WPC) to form amyloid fibril aggregates: spontaneous pathway and nuclear induction pathway. Low ionic strength is a necessary condition for the spontaneous pathway to proceed successfully. In this paper, the effect of salt ions on 2 WPC fibrillation pathways was investigated by adding CaCl2. The results demonstrated WPC fibrils were unable to form normally through spontaneous pathway as adding CaCl2; but still could form through nuclear induction pathway with 20 to 30 mM CaCl2, the nuclei accelerated the fibrillation process led to the resistance to the disordered aggregation brought by CaCl2. Moreover, divalent cations (Ca2+, Mg2+) had much stronger effects than monovalent cations (Na+) on fibril formation, and the results of X-ray photoelectron spectrum together with Fourier-transform infrared spectroscopy suggested that Ca2+ had a greater effect on the fibril formation than Cl-.

Chemical Constituents of the Deep-Sea-Derived Penicillium solitum.

A systematic chemical investigation of the deep-sea-derived fungus Penicillium solitum MCCC 3A00215 resulted in the isolation of one novel polyketide (1), two new alkaloids (2 and 3), and 22 known (4-25) compounds. The structures of the new compounds were established mainly on the basis of exhaustive analysis of 1D and 2D NMR data. Viridicatol (13) displayed moderate anti-tumor activities against PANC-1, Hela, and A549 cells with IC50 values of around 20 µM. Moreover, 13 displayed potent in vitro anti-food allergic activity with an IC50 value of 13 µM, compared to that of 92 µM for the positive control, loratadine, while indole-3-acetic acid methyl ester (9) and penicopeptide A (10) showed moderate effects (IC50 = 50 and 58 µM, respectively).

Mini-Review on the Redox Additives in Aqueous Electrolyte for High Performance Supercapacitors.

Supercapacitors, also known as electrochemical capacitors, are attracting much research attention owing to their high power density, long-term cycling stability, as well as exceptional safety compared with rechargeable batteries, although the globally accepted quantitative benchmarks on the power density, cycling stability, and safety are yet to be established. However, it should be noted that the supercapacitors generally exhibit low energy density, which cannot satisfy the demands where both high energy density and power density are needed. To date, various methods have been employed to improve the electrochemical performances of supercapacitors. Among them, introducing redox additives (or redox mediators) into conventional aqueous electrolyte is regarded as one of the most promising strategies. The redox additives in aqueous electrolyte are widely demonstrated to be able to increase the charge storage capability via redox transformation and thus enhance the electrochemical performances. Herein, we present a brief review on the classification, state-of-the-art progress, challenges, and perspectives of the redox additives in aqueous electrolyte for high performance supercapacitors.

Chitin deacetylases Cod4 and Cod7 are involved in polar growth of Aspergillus fumigatus.

Chitin is one of the key components of fungal cell wall, and chitin deacetylases (CDAs) have been found in fungi; however, their functions remain unknown. Aspergillus fumigatus is known to cause fatal invasive aspergillosis (IA) among immunocompromised patients with a high mortality rate. Although the A. fumigatus cell wall has long been taken as a unique target for drug development, its dynamic remodeling is complicated and not well understood. Seven putative CDAs are annotated in the A. fumigatus genome. In this study, we analyzed the function of the putative CDAs, Cod4 and Cod7, in A. fumigatus. Biochemical analysis of recombinant proteins showed that Cod4 preferentially deacetylated (GlcNAc)4 and was less active on chitooligosaccharides with DP > 5, whereas Cod7 was unable to catalyze deacetylation. Simulation of three-dimensional structure revealed that both Cod4 and Cod7 shared a similar folding pattern with HyPgdA from Helicobacter pylori and, similar to HyPgdA, a substitution of Thr8 by Ala8 in Cod7 abolished its CDA activity. Deletion of the cod4, cod7, or both in A. fumigatus led to polarity abnormality and increased conidiation. Furthermore, the expression level of the genes related to polarity was upregulated in the mutants. Our results demonstrated that Cod4 and Cod7 were involved in polarity, though Cod4 was inactive.

Comparative experiments of fibril formation from whey protein concentrate with homogeneous and secondary nuclei.

Two types of special structures, homogeneous and secondary nuclei, form during fibril formation. The structural and functional properties of amyloid fibrils in whey protein concentrate (WPC) with different ratios of added homogeneous nuclei to secondary nuclei were investigated. Thioflavin T fluorescence analysis and kinetic equations indicated that two types of nuclei could accelerate WPC fibrillation compared with WPC self-assembling into amyloid fibrils, thereby reducing the lag time and increasing the number of fibrils. However, there were considerable differences in the nucleation-inducing capability of WPC fibrillation between homogeneous and secondary nuclei. The number of fibrils formed by adding homogeneous nuclei was higher than that obtained with secondary nuclei, the increase in the Th T fluorescence intensity induced by homogeneous nuclei was 1.83-fold much than secondary nuclei. Meanwhile, secondary nuclei yielded a 2.71-fold faster aggregation rate of WPC than homogeneous nuclei, particularly during the first hour of thermal treatment (protein mass ratio of nuclei to WPC 1:1). The gelation time of WPC after secondary nuclei addition was shorter, from 10 h (WPC (2.0/6.5)) to 4 h (WPC + HN) to 2 h (WPC + SN); however, the gel microstructure of WPC after the addition of homogeneous nuclei was denser, yielding a preferred water holding capacity.

The structure and amphipathy characteristics of modified γ-zeins by SDS or alkali in conjunction with heating treatment.

γ-Zein was modified by SDS or alkali combined with heating treatments in water and in 70% ethanol to change its amphipathic properties and explore the relationship between amphipathic characteristic and structure. γ-Zein water-dispersibility was dramatically increased via alkali or SDS combined with heating treatments, but their ethanol-dispersibilities were significantly different during ethanol evaporation. High both water-dispersibility and ethanol-dispersibility were found from alkali modified γ-zein while high water-dispersibility but low ethanol-dispersibility were obtained from SDS modified γ-zein, indicating that alkali modified γ-zein had better amphipathic characteristic compared with SDS modified γ-zein. Alkali modified γ-zein with higher amphipathic characteristic possessed higher structural inversion ability since it was easy to recover its native state as solvent changing from water to ethanol, contrary to SDS modified γ-zeins whose amphipathic characteristic was not improved. Moreover, the higher structural inversion ability of alkali modified γ-zein depended on the recovery capability of α-helix structure as solvent altering.

[Characterization of Phosphorus Forms in Organic Composts and Their Effects on Leaf Phosphorus Content of Castanea mollissima in Chinese Chestnut Producing Area].

Characterization of the forms of phosphorus (P) in four organic composts including compost of hulls and leaves of Castanea mollissima, edible fungi residues, corn stalks and chicken manure (BYZ), compost of edible fungi residues and chicken manure(ZF), composts of hulls and leaves of C. mollissima, edible fungi residues and chicken manure (BZ1 and BZ2) in Chinese chestnut producing area was conducted by sequential P fractionation developed by Dou et al. Field trial was conducted to study the effects of compost application on leaf phosphorus content of Castanea mollissima. The results showed that total phosphorus content(TP) of organic composts ranked as follows:BZ1(10.61 g·kg-1) > ZF(9.03 g·kg-1) > BYZ(8.56 g·kg-1) > BZ2(7.68 g·kg-1), and the inorganic phosphorus(Pi) accounted for 62.88%-73.62% of the total phosphorus in the organic composts. The content and proportion of HCl-P in the total fractionated phosphorus(Prt) was the highest among P forms in ZF, while that of H2O-P was the highest in BYZ, BZ1 and BZ2. The content of NaOH-P was very low in all the composts. Active H2O-Por NaHCO3-P had the highest inorganic phosphorus proportion(89.17%-96.00%) of all the P forms. The major Piforms in BZ2 were H2O- and HCl-extractable P fractions, while H2O-Pi, HCl-Pi and NaHCO3-Pi were major Pi forms in BYZ, BZ1 and ZF. Residual-P was the predominant organic phosphorus form, and BZ2 showed the highest Residual-P proportion of the total fractionated phosphorus(Prt) among all the composts. Leaf phosphorus contents per unit area of Castanea mollissima by field trial on the added phosphorus-equivalent basis were expressed as:BZ2>BZ1>BYZ>ZF>CK. Increment of leaf phosphorus content per unit area in Julyand per month on average showed positive relationship with the content and proportion of H2O-Pi in the total fractionated phosphorus of composts, which presumably played a key role in leaf phosphorus absorption of C. mollissima measured in the year of deep concentrated fertilization. The results suggested that composts of hulls and leaves of C. mollissima, edible fungi residues and chicken manure (BZ2) was a good choice in given area for aerobic composting raw material system in which organic wastes from Chinese chestnut industry accounted for 80% (hulls and leaves of Castanea mollissima and edible fungi residues accounted for 60% and 20% respectively), which had a high proportion of H2O-Pi and organic Residual-P pool and led to the highest leaf phosphorus content of C. mollissima.

Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro.

In vitro generation of functional gametes is a promising approach for treating infertility, although faithful replication of meiosis has proven to be a substantial obstacle to deriving haploid gamete cells in culture. Here we report complete in vitro meiosis from embryonic stem cell (ESC)-derived primordial germ cells (PGCLCs). Co-culture of PGCLCs with neonatal testicular somatic cells and sequential exposure to morphogens and sex hormones reproduced key hallmarks of meiosis, including erasure of genetic imprinting, chromosomal synapsis and recombination, and correct nuclear DNA and chromosomal content in the resulting haploid cells. Intracytoplasmic injection of the resulting spermatid-like cells into oocytes produced viable and fertile offspring, showing that this robust stepwise approach can functionally recapitulate male gametogenesis in vitro. These findings provide a platform for investigating meiotic mechanisms and the potential generation of human haploid spermatids in vitro.

Generation of fertile offspring from Kit(w)/Kit(wv) mice through differentiation of gene corrected nuclear transfer embryonic stem cells.

Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kit(w)/Kit(wv) mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kit(w)/Kit(wv) somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kit(w)/Kit(wv) infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.

[Effects of 7.5% hypertonic saline pre-injection on postoperative cognitive dysfunction in senile rats].

OBJECTIVE: To explore the effects of hypertonic saline (7.5% NaCl) pre-injection on postoperative cognitive function in senile rats. METHODS: A total of 60 healthy male Sprague-Dawley rats, aged 18 months, weighting 450-500 g, were randomly divided into 3 groups of control (C); model (M) and hypertonic saline (HS) (n = 20 each). At 30 min pre-operation, the rats of groups C and HS received an injection of 7.5% hypertonic sodium 4 ml/kg via tail vein. And the same volume of saline was injected in group M. Rats of groups M and HS were anesthetized by inhaling 3% sevoflourane and underwent splenectomy while group C inhaled merely pure oxygen. Escape latency and frequency of crossing original platform were assessed by Morris water maze on 1 day pre-operation and 1, 3, 7 days post-operation. The rats were randomly taken for detecting the intracellular [Ca²âº]i and apoptotic rate of hippocampal neuron with flow cytometry. And ultrastructures of hippocampal neurons were observed with transmission electron microscope at 1 day pre-operation and 1, 7 days post-operation. RESULTS: Compared with pre-dosing value, escape latency was significantly prolonged, the frequency of crossing original platform decreased and apoptotic rate and [Ca²âº]i increased at each time point post-operation in groups M and HS. And no significant changes were found in the above-mentioned parameters in group C. Compared with group M, escape latency was significantly shortened, the frequency of crossing original platform increased and apoptotic rate and [Ca²âº]i decreased at each time point post-operation in group HS. Pathological changes were found in groups M and HS and the damage was more severe in group M than that in group HS. And no significant pathological change was found in group C. CONCLUSION: Infusing 7.5% hypertonic saline can improve postoperative cognitive function of senile rats. And it may be due to a decreased apoptotic rate of hippocampal neurons.

Detection of carbohydrates using a pre-column derivatization reagent 1-(4-isopropyl) phenyl-3-methyl-5-pyrazolone by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry.

A reagent, 1-(4-isopropyl) phenyl-3-methyl-5-pyrazolone (PPMP) has been synthesized and used for high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) determination of pre-column labeled carbohydrates. Monosaccharides have been quantitatively converted into mono-PPMP-labeled derivatives with 28% aqueous ammonia as a catalyst at 80 degrees C during 70 min. Mono-PPMP derivatives have been demonstrated to exhibit better chemical stability than bis-PMP ones. PPMP-labeled mixture of twelve monosaccharides (galactosamine, glucosamine, galacturonic acid, glucuronic acid, galactose, glucose/N-acetylgalactosamine, N-acetylglucosamine, xylose, arabinose, mannose, fucose, and rhamnose) has been well separated by a reverse-phase HPLC and detected by on-line ESI-MS method under optimized conditions. The data on characteristic fragment ions of the 13 PPMP-labeled monosaccharides with MS(2) data have been collected. The suggested method exhibits good linearity (correlation coefficients > 0.9975) between the peak areas and the concentration of monosaccharides in a broad concentration range and good reproducibility (RSD < 3.19%). The developed method has been successfully applied to analyze the monosaccharide composition of natural Spirulina polysaccharide SPPB-1.