Purification and characterization of a novel protease-resistant GH27 α-galactosidase from Hericium erinaceus.

A novel 57-kDa acidic α-galactosidase designated as HEG has been purified from the dry fruiting bodies of Hericium erinaceus. The isolation protocol involved ion-exchange chromatography and gel filtration on a Superdex75 column. The purification fold and specific activity were 1251 and 46 units/mg, respectively. A BLAST search of internal peptide sequences obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis suggested that the enzyme belonged to the GH27 family. The activity of the enzyme reached its maximum at a pH of 6.0 or at 60 °C. The enzyme was stable within an acidic pH range of 2.2-7.0 and in a narrow temperature range. The enzyme was strongly inhibited by Zn2+, Fe3+, Ag+ ions and SDS. The Lineweaver-Burk plot suggested that the mode of inhibition by galactose and melibiose were of a mixed type. N-bromosuccinimide drastically decreased the activity of the enzyme, whereas diethylpyrocarbonate and carbodiimide strengthened the activity slightly. Moreover, the isolated enzyme displayed remarkable resistance to acid proteases, neutral proteases and pepsin. The enzyme could also hydrolyse oligosaccharides and polysaccharides. In addition, acidic protease promoted the hydrolysis of RFOs by HEG. The Km values of the enzyme towards pNPGal, raffinose and stachyose were 0.36 mM, 40.07 mM and 54.71 mM, respectively. These favourable properties increase the potential of the enzyme in the food industry and animal feed applications.

Autophagy activated by duck enteritis virus infection positively affects its replication.

Duck enteritis virus (DEV) is an acute, septic, sexually transmitted disease that occurs in ducks, geese and other poultry. Autophagy is an evolutionarily ancient pathway that is important in many viral infections. Despite extensive study, the interplay between DEV and autophagy of host cells is not clearly understood. In this study, we found that DEV infection triggers autophagy in duck embryo fibroblast (DEF) cells, as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and decreased p62/SQSTM1 indicated complete autophagy flux. Heat-inactivated DEV infection did not induce autophagy, suggesting that the trigger of autophagy in DEF cells depended on DEV replication. When autophagy was pharmacologically inhibited by LY294002 or wortmannin, DEV replication decreased. The DEV offspring yield decreased when small interference RNA was used to interfere with autophagy related to the genes Beclin-1 and ATG5. In contrast, after treating DEF cells with rapamycin, an inducer of autophagy, DEV replication increased. These results indicated that DEV infection induced autophagy in DEF cells and autophagy facilitated DEV replication.

Immunomodulatory and antioxidative activity of Cordyceps militaris polysaccharides in mice.

To evaluate the immune activation and reactive oxygen species scavenging activity of Cordyceps militaris polysaccharides (CMP) in vivo, 24 male and 24 female Kunming mice were randomly divided into four groups. The mice in the four experimental groups were administered 0 (normal control), 50, 100, or 200mg/kg/d body weight CMP via gavage. After 30 days, the viscera index, leukocyte count, differential leukocyte count, immunoglobulin (IgG) levels, and biochemical parameters were measured. The effect of CMP on the expression of tumor necrosis (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-1ß in the spleens of experimental mice was investigated by real-time polymerase chain reaction. The results showed that the administration of CMP improved the immune function in mice, significantly increased the spleen and thymus indices, the spleen lymphocyte activity, the total quantity of white blood cells, and IgG function in mice serum. CMP exhibited significant antioxidative activity in mice, and decreased malondialdehyde levels in vivo. CMP upregulated the expression of TNF-α, IFN-γ, and IL-1ß mRNA in high-dose groups compared to that observed for the control mice. We can thus conclude that CMP effectively improved the immune function through protection against oxidative stress. CMP thus shows potential for development as drugs and health supplements.

[The Enhancement Research of Magnetic Stirring with Laser Irradiation Aqueous Solution on ICP Source Radiation].

At present, the way to introduce the sample into the inductively coupled plasma atomic emission spectrometry (ICP-AES) light source is still in the form of solution. In order to improve the treatment effect of the aqueous solution and change its physical properties, the surface tension and viscosity under different experimental conditions were measured with magnetic stirring combined with laser irradiation. . The treated samples were introduced into the inductively coupled plasma (ICP) to measure the spectral line intensity, signal-to-background ratio, excitation temperature and electron density emitted by the ICP source. The experimental results showed that: when the magnetic stirrer rotate speed was 1 197 r·min-1, the laser power density was 0.227 6 W·cm-2 and irradiation for 15 min, the surface tension and viscosity of the solution were decreased by 27.85% and 8.66% respectively than those of the untreated solution. As to the element spectral lines of As 188.980 nm, Cd 214.439 nm, Cr 267.716 nm, Cu 324.754 nm, Hg 253.652 nm and Pb 220.353nm: the intensity was enhanced 32.07%, 65.36%, 18.27%, 32.29%, 19.38% and 54.28%; the signal-to-background ratio increased by 25.13%, 60.97%, 18.18%, 27.69%, 21.11% and 48.93%, respectively. The enhancement of the plasma radiation was explained to a certain extent by measuring the excitation temperature and electron density of the plasma. The processing method of the aqueous solution can effectively improve the spectral intensity and signal-to-background ratio of the ICP. Compared with the laser irradiation aqueous solution separately, this method significantly shortened the processing time, improve the efficiency. This method is simple, with no secondary pollution in the treatment of the sample solution, convenient popularization and use.

[Enhancement effect of double-beam laser processed aqueous solution on ICP emission spectrum].

In order to change the physical properties of aqueous solution and improve the radiation intensity of the ICP emission spectrum, the effects of different laser power density and irradiation time on the surface tension and viscosity of aqueous solution were investigated by using near infrared laser at 976 nm and CO2 laser at 10. 6 µm to irradiate aqueous solution orthogonally, then the enhancement of ICP spectral intensity with processed solution was discussed. The results showed that the surface tension and viscosity of aqueous solution reduced by 42. 13% and 14. 03% compared with the untreated, and the atomization efficiency increased by 51.26% at the laser power density 0. 265 7 W . cm-2 of 976 nm and 0. 206 9 W . cm-2 of CO2 laser with 40 min irradiation time. With the optimized aqueous solution introduced into the ICP source, the spectral line intensity of sample elements As, Cd, Cr, Hg and Pb was enhanced by 46.29%, 94. 65%, 30. 76%, 33.07% and 94. 58% compared to the untreated aqueous solution, while the signal-to-background ratio increased by 43. 84%, 85. 35%, 28. 71%, 34. 37% and 90. 91%, respectively. Plasma temperature and electron density also increased by 5. 94% and 1. 18% respectively. It is obvious that the method of double-beam laser orthogonal irradiation on solution can reduce the surface tension and viscosity of aqueous solution significantly, and raise the radiationintensity of ICP source, and will provide a better condition for detecting the trace heavy metal elements in water samples.

[Enhancement effect of laser-processed aqueous solution on ICP source radiation].

To enhance the intensity of inductively coupled plasma-atomic emission spectrum and improve the detection level of trace heavy metal elements, the surface tension and viscosity of the aqueous solution processed by near-infrared laser at wave-length of 976 nm were studied in the present paper. The influences of the treated solution on the spectral line intensity and signal-to-background ratio of the ICP source were observed. The results showed that when the laser irradiation time was 60 min and the power density was 0.329 6 W x cm(-2), the surface tension and viscosity of the solution decreased by 36.73% and 9.73% respectively compared to the untreated solution. Under the optimum conditions, the aqueous solution treated by the laser irradiation was introduced into the ICP source. By measuring the intensity of emission spectrum of the sample elements, the spectral line intensity of Cd, Cr, Cu, Hg, and Pb was enhanced by about 73.52%, 22.97%, 33.86%, 24.44% and 65.59% compared to the untreated solution, while the signal-to-background ratio increased by 76.03%, 21.74%, 32.17%, 22.68% and 65.32%, respectively. Spectral line intensity and signal-to-background ratio of the ICP source were significantly improved so that the foundation was established for reducing the analysis detection limits. Further more, the surface tension and viscosity of the processed aqueous solution remain the same within 30 minutes standing time with the stable physical properties. This simple and easy method of laser-processed aqueous solution helps improve the detection capabilities of ICP spectrometry.

Purification and characterization of a ribonuclease from the wild edible mushroom Armillaria luteo-virens.

A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70 degrees C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U) > poly(C) > poly (G) > poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.

A laccase with antiproliferative and HIV-I reverse transcriptase inhibitory activities from the mycorrhizal fungus Agaricus placomyces.

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7'-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, K(m) values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC(50) of 1.8 µM, 1.7 µM, and 1.25 µM, respectively, signifying that it is an antipathogenic protein.

Nicotinic acids: liver-targeted SCD inhibitors with preclinical anti-diabetic efficacy.

An in vitro screening protocol was used to transform a systemically-distributed SCD inhibitor into a liver-targeted compound. Incorporation of a key nicotinic acid moiety enables molecular recognition by OATP transporters, as demonstrated by uptake studies in transfected cell lines, and likely serves as a critical component of the observed liver-targeted tissue distribution profile. Preclinical anti-diabetic oGTT efficacy is demonstrated with nicotinic acid-based, liver-targeting SCD inhibitor 10, and studies with a close-structural analog devoid of SCD1 activity, suggest this efficacy is a result of on-target activity.

SAR and optimization of thiazole analogs as potent stearoyl-CoA desaturase inhibitors.

Elevated stearoyl-CoA desaturase (SCD) activity has been linked to a number of metabolic disorders including obesity and type II diabetes. Compound 3j, a potent SCD inhibitor (human HepG2 IC(50)=1nM) was identified from the optimization of a lead thiazole compound MF-152 with over 100-fold improvement in potency. In a 4-week chronic oral dosing at 0.2mg/kg, 3j gave a robust 24% prevention of body weight gain in mice fed on a high fat diet accompanied with an improved metabolic profile on insulin and glucose levels.

The C3a anaphylatoxin receptor is a key mediator of insulin resistance and functions by modulating adipose tissue macrophage infiltration and activation.

OBJECTIVE: Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway. RESEARCH DESIGN AND METHODS: We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model. RESULTS: We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR(-/-) mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR(-/-) mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR(-/-) macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators. CONCLUSIONS: Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.

The effects of IL-5 on airway physiology and inflammation in rats.

BACKGROUND: There is evidence that the cytokine IL-5 is a prominent feature of airway inflammation in asthma. OBJECTIVE: The aim of this study was to determine whether exogenous IL-5 could cause changes in lung physiology, the early and late airway response after antigen challenge, and airway inflammation in rats that do not have a propensity to develop these changes after sensitization and challenge. METHOD AND RESULTS: Intratracheal administration of IL-5 to ovalbumin sensitized Brown Norway SSN rats increased the airway responsiveness to methacholine (AHR) 20 hours after administration of IL-5 at the same time as an increase in neutrophils occurred in the lung lavage. This effect was dose dependent and was not caused by endotoxin. Concurrent intratracheal administration of 50 ng of anti-IL-5 monoclonal antibody with 10 microg of recombinant human IL-5 decreased the AHR and neutrophil influx. Pretreatment with 3 microg of IL-5 had no effect on the early and late airway response or on AHR after ovalbumin challenge. However, IL-5 increased lung re-sistance 20 hours after antigen challenge. Although total lung cells and differential counts did not differ significantly 8 hours after antigen challenge, the blood lymphocyte CD4/CD8 ratio decreased in IL-5 pretreated rats (P <.05). In addition, in situ hybridization showed a significant increase in cells within the airway wall expressing IL-4 and IL-5 mRNA in IL-5 treated/challenged rats compared to controls (P <.05). CONCLUSION: The intratracheal administration of IL-5 causes only part of the physiologic changes that are associated with asthma. Other factors are necessary to obtain the complete asthma phenotype.

3,4-Diaryl-5-hydroxyfuranones: highly selective inhibitors of cyclooxygenase-2 with aqueous solubility.

The introduction of a hydroxyl group into the 5-position of the diaryl furanone system provides highly selective inhibitors of cyclooxygenase-2. These molecules can be converted into their sodium salts which are water soluble, facilitating intravenous formulation. These salts show excellent potency in rat models of pain, fever and inflammation.