In order to clarify material basis of effective parts of liangxue tongyu prescription, blood-heat and blood-stasis rat model induced by dry yeast was established. The changes of rectal temperature, blood viscosity and plasma viscosity were used to evaluate the cooling-blood and activating-blood effects of liangxue tongyu prescription and its parts. Compared with the model group, the extract from liangxue tongyu prescription, its volatile oil and n-butanol part could significantly reduce rectal temperature (P<0.01), and also reduce blood viscosity and plasma viscosity to various degrees (P<0.01 or P<0.05). So volatile oil and n-butanol part were primarily identified as effective parts of liangxue tongyu prescription. By using GC-MS with normalization method of area to analyze volatile oil of liangxue tongyu prescription, 70 compounds were identified, accounting for about 92.54%, mainly as β-asarone, paeonol, α-asarone and shyobunone. 42 compounds such as peony glycosides, tannins, and iridoid glycosides were identified by HPLC-MS techniques and standard comparison. The study determined the effective parts of liangxue tongyu prescription and clarified the chemical composition providing the foundation for further studies on material basis of liangxue tongyu prescription.
Acetophenones ; chemistry ; Animals ; Anisoles ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Gas Chromatography-Mass Spectrometry ; Oils, Volatile ; chemistry ; Rats ; Tannins ; chemistry

An enzyme-displaying yeast as a whole-cell biocatalyst seemed an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, we tried to use a recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) to catalyze the synthesis of short chain flavor esters in n-heptane. We studied some major influential factors of esterification reactions, such as carbon chain length of the substrates, alcohol structure, enzyme concentration, substrates concentration, molar ratio of the substrates. The acid conversions were determined by titration and gas chromatography analysis. About ten kinds of esters were synthesized successfully, and the acid conversions of eight esters reached as high as 90% after reaction for 6 h. The result also indicated that ethanol and hexanoic acid were the most suitable substrates for this whole-cell catalyst. Under the optimal reaction conditions (the amount of lipase 20 g/L (306.0 U/g-dry cell), hexanoic acid concentration 0.8 mol/L, the molar ratio of hexanoic acid to ethanol 1:1.1), hexanoic acid conversion reached 97.3% after reaction for 1.5 h. To our knowledge, the CALB-displaying P. pastoris whole-cell biocatalyst showed good tolerance for high substrates concentration and exhibited high reaction rate on esterification of short chain flavor esters among the present enzyme/cell reported. Thus, CALB-displaying P pastoris whole-cell biocatalyst was promising in commercial application for flavor esters synthesis in non-aqueous phase.
Biocatalysis ; Candida ; enzymology ; Enzymes, Immobilized ; Esters ; metabolism ; Fungal Proteins ; Lipase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Adolescent ; Adult ; Aged ; Cerebellar Neoplasms ; physiopathology ; Cerebellopontine Angle ; Child ; Humans ; Male ; Middle Aged ; Vestibular Evoked Myogenic Potentials ; Young Adult

Today, the understanding of the sequence and structure of biologically relevant targets is growing rapidly and researchers from many disciplines, physics and computational science in particular, are making significant contributions to modern biology and drug discovery. However, it remains challenging to rationally design small molecular ligands with desired biological characteristics based on the structural information of the drug targets, which demands more accurate calculation of ligand binding free-energy. With the rapid advances in computer power and extensive efforts in algorithm development, physics-based computational chemistry approaches have played more important roles in structure-based drug design. Here we reviewed the newly developed computational chemistry methods in structure-based drug design as well as the elegant applications, including binding-site druggability assessment, large scale virtual screening of chemical database, and lead compound optimization. Importantly, here we address the current bottlenecks and propose practical solutions.
Computational Biology ; Drug Design ; Drug Discovery ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Quantitative Structure-Activity Relationship

Emerging data indicated that HCV subverts the antiviral activity of interferon (IF); however,whether HCV core protein contributes to the process remains controversial. In the present study, we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway. Our results showed that, following treatment with IFN-α, the transcription of PKR, MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein. In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased. Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1, whereas the level of STAT1 expression was unchanged.Accordingly, SOCS3, the negative regulator of the Jak/STAT pathway, was induced by HCV core protein. These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.

Objective To investigate the effects of hydrogen sulfide (H2S) on the tubular interstitial fibrosis and on the levels of kidney injury molecule-1 (KIM-1) and ectodermal dysplasia-1 (ED-1) in the process of renal interstitial fibrosis in rats with unilateral ureteral occlusion(UUO).Methods Ninety-six Sprague-Dauley (SD) male rats were divided into 4 groups randomly:sham-operated group (n =24),model group (n =24),low-dose therapy group (n =24) and high-dose therapy group(n =24).The rats in the model group and treatment groups were ligated at the left ureter and UUO was induced,meanwhile,the rats in the control group were free from the left ureter ligation.Rats received sodium hydrosulfide(NaHS) in traperitoneatly(1.4 μmol/kg,twice a day),and NaHS(7.0 μmol/kg,twice a day),respectively.Rats in sham-operated group and the model group were injected intraperitoneally with identical voluminal 9 g/L saline.Eight rats in each group were sacrificed randomly at the 7 days,14 days and 21 days,respectively.The concentration of plasma H2S was detected.Renal tubular interstitial damage and interstitial fibrosis were evaluated with routine HE staining and Masson staining under microscope,and both of them were used to evaluate the obstruction of renal histopathological changes.The expressions of ED-1 and KIM-1 were measured with immunohistochemical staining.Results 1.The pathological findings showed that the renal tubular interstitial changes were not obvious in the shamoperated group.The tubular epithelial cells in the model group and treatment groups showed swelling and vacuoles degeneration,with renal interstitial broadening,interstitial cells and extracellular matrix increasing.The renal tubular interstitial pathological injury of model group and treatment group were more serious than those in the sham-operated group at each time point(P ＜ 0.05).The renal tubular interstitial pathological injury of the treatment groups were obviously lower than that in the model group(P ＜0.05).However,there was no statistically significant difference between highdose therapy group and low-dose therapy group(P ＞ 0.05).2.Immunohistochemical analysis showed that expressions of ED-1 and KIM-1 in renal tubular interstitices in the model group and the treatment groups were higher than those in the sham-operated group at each time point(P ＜ 0.05).The expressions of ED-1 and KIM-1 in renal tubular interstitices in the treatment groups were obviously lower than those in the model group(P ＜ 0.05).There was no statistically significant difference between the high-dose therapy group and the low-dose therapy group(P ＞0.05).3.Plasma H2S levels in model group and treatment group were lower than those in the sham-operated group at each time point (P ＜ 0.05).The plasma H2S level of the treatment group was obviously higher than that in the model group(P ＜ 0.05).However,there was no statistically significant difference between high-dose therapy group and low-dose therapy group (P ＞0.05).Conclusions H2S implements renal protection effect partly by reducing the expression of ED-1 and KIM-1 in tubule interstitices to ease tubular interstitial fibrosis.

Yeast surface display involves that the exogenous protein, which was fused with the yeast outer shell cell wall protein, was genetically anchored on the yeast cell surface. It has been widely used in expression and screening of functional protein. Here, we focused on the construction of lipase-displaying systems and its application in enzymatic biosynthesis, such as fatty acid methyl esters, short-chain flavour esters and sugar esters applications, and so on.
Candida ; enzymology ; genetics ; Lipase ; biosynthesis ; genetics ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Solvents ; Yeasts ; enzymology ; genetics

OBJECTIVETo observe the level in plasma hydrogen sulfide (H2S) and the expression of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) (two key synthetases for endogenous H2S generation in the kidney) in obstructed kidney tissue among rats with tubulointerstitial fibrosis (TIF) induced by unilateral ureteral obstruction (UUO), and to explore the role of H2S in TIF.

METHODSNinety-six male Sprague-Dawley rats were randomly divided into sham-operated, model, low-dose NaHS and high-dose NaHS groups (n=24 each). TIF was induced by UUO in the model, low-dose NaHS and high-dose NaHS groups. The low-dose and high-dose NaHS groups were intraperitoneally injected with NaHS (1.4 and 7.0 μmol/kg respectively) twice daily immediately after operation, and the sham-operated and model groups were intraperitoneally injected with an identical volume of normal saline. In each group, 8 rats were randomly selected and sacrificed at 7, 14 or 21 days after operation. Plasma H2S concentration was measured by deproteinization. The obstructed kidney tissue was subjected to hematoxylin and eosin staining and Masson staining, and the renal tubulointerstitial injury was evaluated under a microscope. mRNA and protein expression of CBS and CSE in the obstructed kidney tissue was measured by RT-PCR and immunohistochemistry respectively.

RESULTSThe degree of UUO-induced renal tubulointerstitial injury was negatively correlated with plasma H2S concentration in (r=-0.891, P<0.01). With H2S supplementation, renal tubulointerstitial injury was reduced (P<0.01), the expression of mRNA and protein of CBS and CSE in the kidney tissue and plasma H2S level were upregulated (P<0.01), and the degree of TIF was reduced (P<0.01). There were no significant differences in plasma H2S level and mRNA and protein expression of CBS and CSE between the low-dose and high-dose NaHS groups (P>0.05).

CONCLUSIONSH2S is involved in the development of UUO-induced TIF, and the CBS/H2S and CSE/H2S systems play key roles in this process. H2S supplementation can delay the progression of TIF.

Animals ; Cystathionine beta-Synthase ; analysis ; genetics ; Cystathionine gamma-Lyase ; analysis ; genetics ; Dietary Supplements ; Fibrosis ; Hydrogen Sulfide ; administration & dosage ; blood ; Kidney Tubules ; pathology ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Ureteral Obstruction ; blood ; pathology

Objective To investigate the effect of fluid shear stress (FSS) on the expression of B lymphoma MoMLV insertion region 1 (Bmi-1) in bone mesenchymal stem cells (BMSCs) and possible signal transduction mechanism.Methods BMSCs were isolated from SD rats and FSS at different magnitude (0.5,1.5,3.0 Pa)and under different time phase (1,2,6,24 h) were loaded by parallel-plate flow chamber system.The expression of Bmi-1 was measured by real-time RT-PCR at mRNA level and the levels of phosphorylated Akt (p-Akt)and extracellular signalregulated kinase 1/2 (p-ERK1/2) were detected by Western blotting.The signaling inhibitors,wortmannin (PI3K specific inhabitor) and PD98059 (ERK1/2 specific inhabitor),were used to investigate possible mechanical signal transduction pathway.Results Bmi-1mRNA expression increased when BMSCs were exposed to 1.5 Pa FSS for 1 h and reached the peak at 24 h.All FSS with different magnitude could increase Bmi-1 expression,especial at high FSS (3.0 Pa).Meanwhile,FSS resulted in a significant activation of p-Akt and p-ERK1/2 in BMSCs.After treated with wortmannin,the expression of Bmi-1 was inhibited prominently,however,PD98059,the expression of Bmi-1 did not change.Conclusions FSS can activate the expression of Bmi-1,the amount of Bmi-1 expression was closely related to the stimulating time and the magnitude of FSS,and Akt signal molecule plays an important role during the process.These findings provide significant references for studying the mechanical biological mechanisms of stem cell differentiation.

Objective:To explore the key factors affecting plasma clot retraction and optimize the experimental method of plasma clot retraction,in order to study the regulation of platelet function and evaluate the modulatory effects of drugs on plasma clot retraction.Methods:The effects of different concentrations of thrombin,Ca2+and platelets on plasma clot retraction were studied,and the detection system of plasma clot retraction was optimized.The availability of the detection system was then validated by analyzing the regulatory effects of multiple signaling pathway inhibitors on plasma clot retraction.Results:Through the optimization study of multiple factors,platelet rich plasma(PRP)containing 0.5 mmol/L Ca2+and 40 × 109/L platelets was treated with 0.2 U/ml thrombin to perform plasma clot retraction analysis.After treatment with thrombin for 15 min,plasma clot retracted significantly.After treatment with thrombin for 30 min,the percentage of plasma clot retraction was more than 50％.The regulatory effects of multiple signaling pathway inhibitors on plasma clot retraction were studied in this detection system.PKC inhibitor Go 6983 exhibited a significant inhibitory effect on plasma clot retraction,while PI3K inhibitor Ly294002 and p38 MAPK inhibitor SB203580 slightly suppressed plasma clot retraction.Conclusion:PRP containing 0.5 mmol/L Ca2+and 40 × 109/L platelets can be induced with 0.2 U/ml thrombin to conduct plasma clot retraction analysis,which can be used to study the regulation of platelet function and evaluate the modulatory effects of drugs on plasma clot retraction.