The deubiquitinating enzyme 13 retards non-alcoholic steatohepatitis via blocking inactive rhomboid protein 2-dependent pathway

Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis (NASH) have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated. Inactive rhomboid protein 2 (IRHOM2), a promising target for treatment of inflammation-related diseases, contributes to deregulated hepatocyte metabolism-associated nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanism underlying Irhom2 regulation is still not completely understood. In this work, we identify the ubiquitin-specific protease 13 (USP13) as a critical and novel endogenous blocker of IRHOM2, and we also indicate that USP13 is an IRHOM2-interacting protein that catalyzes deubiquitination of Irhom2 in hepatocytes. Hepatocyte-specific loss of the Usp13 disrupts liver metabolic homeostasis, followed by glycometabolic disorder, lipid deposition, increased inflammation, and markedly promotes NASH development. Conversely, transgenic mice with Usp13 overexpression, lentivirus (LV)- or adeno-associated virus (AAV)-driven Usp13 gene therapeutics mitigates NASH in 3 models of rodent. Mechanistically, in response to metabolic stresses, USP13 directly interacts with IRHOM2 and removes its K63-linked ubiquitination induced by ubiquitin-conjugating enzyme E2N (UBC13), a ubiquitin E2 conjugating enzyme, and thus prevents its activation of downstream cascade pathway. USP13 is a potential treatment target for NASH therapy by targeting the Irhom2 signaling pathway.

A study on differentially expressed gene screening of Chrysanthemum plants under sound stress.

Environmental stress can induce differential expression of genes of flower plants. It had been found that sound stimulation had an obvious effect on the growth and development of flower plants, but it is not reported on the differentially expressed genes and their expressing characteristics under sound stimulation. This is one of the few reports in terms of using the DDRT-PCR technique for screening the differentially expressed cDNA fragments responding to sound-wave stress on Chrysanthemum. Six differentially expressed cDNA fragments were obtained. Molecular weight of fragments was from 200 to 600 bp, respectively. Among differential fragments acquired, three of them (SA3, SG7-1, and CA2) were found to be positive fragments by northern dot hybridization, whose molecular weight are 270, 580 and 370 bp, respectively. SA3 was differentially expressed and SG7-1 was preferably expressed, while CA2 was restrained by the sound wave. These results indicated that expression of some genes was turned on, meanwhile the stress restrained some genes from expression under the mode of sound-stress stimulation.

Hydrolytic cleavage of DNA by quercetin manganese(II) complexes.

Quercetin manganese(II) complexes were investigated focusing on its DNA hydrolytic activity. The complexes successfully promote the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complexes with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in the presence of 100 microM of the complexes is found to be 1.32 x 10(-4) s(-1). The hydrolytic cleavage of DNA by the complexes was supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay and T4 ligase ligation.

Hydrolytic cleavage of DNA by quercetin zinc(II) complex.

Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.

Kinetics of degradation by immobilized cells with ultrasonic irradiation.

Linear alkylbenzene sulphonate (LAS) decompositions by immobilized cells with ultrasonic irradiation were investigated at the optimized condition in order to gain insight into the kinetics of the decomposition process. Firstly, by analyzing the decomposition process of LAS theoretically, showed the kinetic model of suspending cells and immobilized cells both followed the MONOD model (namely micro=micromaxs/(ks+s) during wastewater treatment, then discussed the kinetics model of LAS degradation by immobilized cells with ultrasonic irradiation at the presupposition conditions, and then the two unknown parameters ([See text]) in the gained model were researched at the condition of laboratory. Moreover, experiments have been done to validate the parameters ([See text]) in the kinetics equation, the result shows the valid kinetics equation of LAS degradation is at the LAS concentration of 30-80 mg/L:

Protoplast isolation of callus in Echinacea augustifolia.

Preparations from Echinacea are among the most widely used herbal medicines. Most uses of Echinacea are based on the reported immunological properties. In this paper, we used callus of Echinacea augustifolia for isolation and researched the factors influencing the process of protoplasts preparation, the result indicated it was easy to isolate protoplast from buff-green Callus in E. augustifolia which was looked like granule with symmetrical character. The result showed that the best enzyme solution concentration is composed of cellulase 2.0% (w/v), pectinase1.0% (w/v), hemicellulase 0.5% (w/v), 0.7 mol/L mannitol and 50.0 x 10(4) gFW(-1) protoplasts were obtained after 8 h later. The yield of protoplasts was significantly influenced by cellulase concentration and incubation time. Finally, we summarized an integrated approach including callus induction, callus culture, isolation, purification, identification and calculation of protoplasts.

Primary study on the application of Serum Pharmacology in Chinese traditional medicine.

In the paper, two main methods, which are Serum Pharmacology and Traditional Pharmacology, were adopted to study Chinese traditional medicine, such as Ginkgo biloba extract (GBE), ginsenosides (GS) and compound GG (GBE+GS), pharmacology in vitro. The results showed that there were evident difference between the results of Serum Pharmacology and that of Traditional Pharmacology. There was no significant difference between the drug effect of crude GS on nitric oxide (NO) production in ECV304 and that of crude GBE, and the drug effect of GG was superior to that of GS and GBE, respectively. But, compared with GBE serum, the GS serum up-regulation of NO production in ECV304 increased significantly, and the GG serum up-regulation of the NO production in ECV304 was inferior to that of GS serum and GBE serum significantly. The results suggested that Serum Pharmacological study should be adopted in the pharmacological investigation on the Chinese traditional medicine and the drug screening of the Chinese traditional medicine.

A method for isolating functional RNA from callus of Dendrobium candidum contented rich polysaccharides.

Isolation of high-quality RNA from Dendrobium candidum is particularly difficult. D. candidum contains considerable amounts of polysaccharides that coprecipitate with RNA, which render RNA unsuitable for either cDNA synthesis and/or PCR amplification. In this paper, a rapid and efficient method was described for functional RNA isolation from the callus of D. candidum. The procedure included: (i) an extraction with phenol and isopropyl alcohol, to remove proteins and polyphenols; (ii) purifications by lithium chloride, pre-cooled (-20 degrees C) ethanol successively to remove polysaccharides. The method resulted in high-quality RNA suitable for DDRT-PCR and cDNA library analysis finally.

Effect of compatibility on the pharmacokinetic characteristics of ginsenosides.

Pharmacokinetic research, which is one of the most important parts of preclinic research, plays an important role in guiding medicine compatibility and preparation improvement. In this paper, the influence of the compatibility of GGV on pharmacokinetics of ginsenosides (GS) was studied, which consisted of ginsenosides, ginkgo biloba extract (GBE) and a chemical monomer V. The result indicated that the addition of either GBE or V could influence the pharmacokinetic parameters of ginsenosides and the influence was different when different administering routes were adopted. Therefore, it could be concluded that there are interactions among GS, GBE and V, and the synergetic and inhibitory effects of the three ingredients contribute to the pharmacological effect of GGV.

Impacts of PEG-6000 pretreatment for barley (Hordeum vulgare L.) seeds on the effect of their mature embryo in vitro culture and primary investigation on its physiological mechanism.

In this paper, we studied polyetheneglycol (PEG) pretreatment effect on the mature embryo culture in vitro by using barley (Hordeum vulgare L.) seeds. Meanwhile, we analyzed and assayed its mineral element and endogenous hormone level. The experimental results were as follows: (1) PEG-6000 imbibition could obviously slow down the water timecourse absorbed by barley seeds; (2) 10% PEG-6000 treatment of barley seeds for 3 h had a positive effect on germination in vitro and callus induction of the barley seed mature embryos; (3) 10% PEG-6000 treatment inhibited soluble leakage from the seeds; (4) N leakage was mainly from the endosperms, Mn2+ leakage from embryos; (5) PEG-6000 treatment changed greatly the hormone level (ABA, IAA, GAs), which influenced the percentage of plantlets from the mature embryo callus. The results can provide some clues to scientific sowing of crop seeds, pretreatment for the purpose of uniform seedlings, and the explant response quality in plant tissue culture.

Linear alkyl benzene sulphonate (LAS) degradation by immobilized Pseudomonas aeruginosa under low intensity ultrasound.

We studied the LAS degradation of immobilized Pseudomonas aeruginosa with low-intensity ultrasonic and the influence of original LAS concentration, pH, rotary velocity and different conditions of low-intensity ultrasonic irradiation on the degradation of LAS. In our experiment, the degradation rate of LAS was the main index. We found that low-intensity ultrasonic irradiation could improve the metabolism of microorganism cells and promote the LAS biodegradation of immobilized cells. In the experiment, 50 mg/l LAS were used to simulate wastewater, and low-intensity ultrasonic was considered. We found the influence was obvious, and the optimal degradation rate was acquired when the conditions of ultrasonic were frequency 24 kHz, power 8 W, stimulation time 5 s, intermissive time 30 s, and total time 10 min. The LAS degradation rate of immobilized cells with ultrasonic were respectively 40% and 9.5% higher than that of the suspending cells and immobilized cells without irradiation.

Study of mRNA expression of Arabidopsis thaliana under stimulation using modified differential display RT-PCR with silver staining.

We studied differential expressions of Arabidopsis thaliana seedlings under sound stimulation using modified differential display RT-PCR with silver staining in this paper. Six differentially expressed cDNA fragments were obtained. Molecular weight of fragments was between 200 and 600 bp, respectively. On the base of the experimentation, we considered sound stress would have been a positive or negative influence on plant growth. The experimental results indicated some genes was activated, meanwhile some were restrained under after stimulation under sound stress.

Effect of chemical and physical factors to improve the germination rate of Echinacea angustifolia seeds.

Seeds of Echinacea angustifolia are known for their deep dormancy. In this paper, we studied the responses of E. angustifolia seeds to some chemical and physical factors, such as scarification, chilling (5 degrees C) period, light and applied BA (6-benzylaminopurine), GA3 (gibberellic acid) and sound stimulation. When the seed coat layers were removed, the germination rate grew up from 6 to 20% (incubated in light) and the mean time germination (MTG) was reduced from 18 to 6.6 days. On the basis of layers-removed, chilling and continuous light gave significantly higher germination rate (up to 70%). Compare the data of seeds chilled by 0, 6, 12, 18 and 24 days, the maximum germination rate (up to 70%) achieved at 18-days chilling treatment. Further increases in the chilling period could slightly improve germination. Exogenous application of 0.1, 0.2, 0.3 mg/L GA3 or BA in the previous pretreatment increased germination to 78, 90 and 84% or 76, 86 and 84%, respectively. Obviously, the best concentration of GA or BA is 0.3 mg/L. And the GA3 or BA treatment shortened the MTG to about 4 days. The influence of sound stimulation was also tested in the experiment. The result showed that one 100 dB and 1000 Hz sound wave (sine-wave) was beneficial to the germination of E. angustifolia seeds.

Soundwave stimulation triggers the content change of the endogenous hormone of the Chrysanthemum mature callus.

Hormones have been suggested to play a prominent role in the control of callus growth. In this paper, with the method of indirect enzyme-linked immunosorbent assays (ELISA), we investigated the induction effect of soundwave on the dynamic change of endogenous indole-3-acetic acid (IAA) and abscisic acid (ABA) in vitro during the differentiation process of Chrysanthemum synchronized mature Callus. These experiments showed that groups treated by optimal soundwave (1.4 kHz, 0.095 kdb) had significantly higher IAA levels and lower ABA than that of the control, which had been implicated activation of endogenous IAA and inhibition of ABA. Through the biochemical analysis, it revealed that the increased level of IAA as well as decreased levels of ABA correlated with soundwave stimulus. High rate of IAA/ABA was favorable to development of the callus and differentiation of mature callus. We conclude that soundwave contributes to endogenous hormone as well as the control of callus growth.

Effect of ultrasound stimulation on metabolic modulation of pyruvic acid.

Pyruvic acid lay on the center position of Eremothecium ashbyii metabolic fluxes, during fermentation, a high performance liquid chromatography (HPLC) method is presented for the simultaneous determination of pyruvic acid in the Eremothecium ashbyii cell culture of both with ultrasound stimulation and the control. Dry weight and riboflavin are also mentored. The results of this study show that the ultrasound stimulation can give rise to pyruvic acid change, at the same time; change of pyruvic acid concentration is correlative to that of dry weight of mycelium and content of riboflavin.

Extraction of total RNA from Chrysanthemum containing high levels of phenolic and carbohydrates.

Some standard methods are available in total RNA extraction of plant tissue, which are effective for the ordinary plants. But it is difficult to extract the total RNA from the plants with high levels of phenolic compounds, carbohydrates, or other compounds that bind and/or coprecipitate with RNA. In this paper, a method was described that used the soluble polyvinylpyrrolidone (PVP), ethanol precipitation, phenol extraction and LiCl precipitation. By this method, RNA capable of reverse-transcription and polymerase chain reaction (PCR) amplification was isolated from Chrysanthemum that contains high levels of phenolic compounds and carbohydrates. The OD260/OD280 absorbance ratio is 2, and the RNA is intact. Other specialized RNA extraction methods failed to deliver suitable product. Bands of PCR products are clearly appearing on the polypropylene acyl gel electrophoresis (PAGE) gel, which indicates that the purity, concentration and integrity of total RNA were suitable for the sequent researches.

Relationship between the minute structure and the lodging resistance of rice stems.

The aim of this experiment is to measure the morphological and mechanical properties, and to analyze the relationship between the morphology and lodging resistance of several different kinds of rice stem. Choose the typical species of tall, mid and short stem and hybrid rice, ten individual plants each species (all should be caulis), and when paddies are full heading, intercept the third internode above the ground. The minute structure characteristics include the size of outer diameter, Culm wall thickness, and the number of big and small vascular bundles. Mechanical properties include the tensile breaking point and the tensile elastic modulus. Based on the above characteristics it is concluded that the middle stem has the highest lodging resistance. Thus when we select the high-yield and lodging resistance species, the middle and rigid stem should be considered.

Investigation on the effects of diamide on NO production in vascular endothelial cells (VEC).

Nitric oxide (NO) controls several physiological functions of the cardiovascular system. The study on the effect of diamide (N(2)H(4).H(2)O) on NO production in vascular endothelial cells (VEC) may provide significant reference for VEC's modeling in studying cardiovascular diseases. The objective of this study was to elucidate how high concentration diamide (V(diamide)/V(culture miedium) = 5 ml/l) and low concentration diamide (V(diamide)/V(culture miedium) = 0.5 ml/l) affect NO production in a human endothelial cell line (ECV304). After cells were incubated with diamide (5 or 0.5 ml/l) for 4, 6, 8 or 10h, respectively, the amounts of NO metabolites released by the cells were quantitated and the degree of damage of VEC was observed using microscope. The results showed that NO production in VEC tended to decrease with the lapse of time in the 0.5 ml/l diamide group. In the 5 ml/l diamide group, on the contrary, NO production in VEC tended to increase with the lapse of time. At the same time, from the morphologic observation, the VEC were damaged severely after treated with 5 ml/l diamide. So it could be concluded that the severe damage induced by high concentration diamide would have triggered the express of inducible nitric oxide synthases (iNOS). Just for the expresssion of iNOS, NO production in VEC treated with high concentration diamide occurred abnormally in contrast to the 0.5 ml/l group.

Effect of low frequency ultrasonic stimulation on the secretion of siboflavin produced by Ecemothecium Ashbyii.

The structure of the cell membrane of Ecemothecium ashbyii is considered to be one of the main obstacles in the improvement of riboflavin productivity, which impedes the release of riboflavin from the cell into the fermentation broth. The results of the present study show that stimulation on growth and riboflavin biosynthesis phase, the content of riboflavin was most obviously enhanced, as compared with the control and the total riboflavin quantity released by ultrasonic treatment. The optimal stimulation time was from 104 to 112 h and ultrasonic should be loaded every 1.5 h.

Survivin and Bcl-2 modulate apoptosis in A549 cell induced by quercetin / 中国药理学通报

Aim To investigate the apoptosis of lung cancer cells A549 induced by quercetin and the regulation of survivin and Bcl-2 on A549 cells induced by quercetin.Methods MTT,fluorescence stain,flow cytometric analysis and immunocytochemistry stain were carried out.Results Quercetin had a significant inhibition on growth and proliferation of A549 cell in a concentration-and time-dependent manner.Evidence was provided that apoptosis occurred in A549 cells treated with quercetin using fluorescence microscopy.Quercetin arrested A549 cells at the G0/G1 phase by FCM analyses.Expression of survivin and Bcl-2 protein were decreased,and activity of caspase-3 were enhanced.Conclusion Quercetin could induce apoptosis of A549 cells.The arrested cell cycle and the down-regulation of survivin and Bcl-2 protein could activate caspase-3 resulting in cells apoptosis,which may contribute to the apoptosis mechanisms.The down-regulated survivin and Bcl-2 may play an important role in A549 cells apoptosis induced by quercetin.