3-(2-氯-4-三氟甲基苯氧基)苯甲酰腙的合成与生物活性

以3-(2-氯-4-三氟甲基苯氧基)苯甲酸(Ⅰ)为起始原料,经酯化、肼解后得3-(2-氯-4-三氟甲基苯氧基)苯甲酰肼(Ⅲ),再与不同的芳香醛缩合,合成了4个未见文献报道的3-(2-氯-4-三氟甲基苯氧基)苯甲酰腙(Ⅳa~Ⅳd)。目标化合物经1 HNMR、IR、MS和元素分析进行结构表征和确认。测试了目标化合物对油菜和稗草的生长抑制活性。结果表明:化合物Ⅳa和Ⅳb在100μg·g^(-1)浓度下,对油菜根和稗草根的生长具有较好的抑制活性,且经化合物Ⅳa处理后的植株表现出明显的白化现象,植株叶绿素和类胡萝卜素生物合成受到明显抑制。

Purification and Immunocompetence Analysis of GP5 Protein in Porcine Reproductive and Respiratory Syndrome

[Objective] The purification and immunocompetence of GPS protein in porcine reproductive and respiratory syndrome (PRRS) were analyzed in this study, which provided basis for establishing the corresponding serological method. [Method] The recombinant expression plasmid pGEX-6P-5 was transformed into BL21 and expressed after being induced with IPTG. The solubility analysis of expression products was carried out, and then the recombinant protein was purified for SDS-PAGE identification and Western-blot analysis. Finally, the recombinant antigen was used in the immune experiment of guinea pigs. [Result] The target protein content accounted for 30% of the total cells protein content according to the chromatography scanning, and the purity of target protein after purification reached 80%. The purified protein was analyzed by Western-blot and immune experiment of guinea pigs, and the results showed that the expressed protein had good reactionogenicity and immunogenicity. [Conclusion] This study provides materials for further studies on the function between PRRSV ORF5 gene and its editing protein, which also lays a foundation for porcine reproductive and respiratory syndrome virus genetic engineering products.

Antioxidase Activities of Tetraploid Lines and Diploid Control Line of Chrysanthemum cinerariifolium

Objective The SOD, POD and APX enzyme activities of tetraploid lines and diploid control line of Chrysanthemum cinerariifolium in test-tube and field were determined in order to provide a reference for the selective breeding of high resistance good tetraploid varieties of C. cinerariifolium. Method The SOD, POD and APX activities of each sample were determined. Every material should have 3 replicates which been determined and calculated mean value to analyze. Result The various kinds of antioxidase activities in protective enzyme system of tetraploid lines were increased generally in comparison with that of diploid control line. And there is certain relativity between the samples in field and in test-tube of these lines. Conclusion The antioxidase activities of each line in test-tube could be considered as reference index to screen high resistance good tetraploid varieties of C. cinerariifolium.

Purification and Characterization of Flammulin,a Basic Protein with Anti-tumor Activities from Flammulina velutipes

Aim To purify and characterize flammulin, a basic protein with anti-tumoractivities. Methods Ammonium sulfate, ethanol fractionation and column chromatography were used forseparation and purification. Electrophoretic analysis, amino acid analysis, and MS of flammulin werecarried out. Results Flammulin was purified to electrophoretic homogeneity and crystallized. With amolecular mass of 19891.13 Da, pI 8.9, λ_(max) = 276 - 278 nm, λ_(min) = 250 nm, flammulin wascharacterized by its lack of methionine. Fingerprint mapping of flammulin was determined by MALDI-MSfollowing in-gel protease digestion; no close matches were identified. Conclusion Flammulin waspurified to electrophoretic homogeneity, and its characteristics are discussed for the first time.

Extraction, Purification and Identification of Bone Morphogenetic Protein in Conditioned Medium of Osteosarcoma Cell (MG-63)

Objective: To find out a method of extraction and purification of bone morphogenetic protein (BMP) from osteosarcoma cell conditioned medium, and evaluate the biological activity of BMP.Methods: Conditioned medium of osteosarcoma cell lines (MG-63) was collected, concentrated and dialyzed. The concentrated protein was purified through gel chromatography on Sephcryl-S-100. The purified protein was tested by BMP monoclonal antibody (McAb), its molecular weight (MW) was determined by SDS-PAGE and its biological activity was demonstrated by heterotopic ossification.Results: The purified protein was proved to be BMP by BMP McAb, had a satisfactory heterotopic ossification, and its MW was about 21 kD.Conclusion: BMP existed in the conditioned medium of osteosarcoma cell and had a satisfactory biological activity after purification. Because osteosarcoma cell can be cultured and grew for a long timein vitro, this method will be helpful to a vast extraction of BMP and clinical application. Key words osteosarcoma cell - conditioned medium - bone morphogenetic protein - protein purification This project was a key scientific and technological program of Hubei Provicial Scientific and Technological Committee (No. 002p1503).

Antibacterial Activity of Bacillus subtilis and Properties of Protein Crude Extract

In order to get biological drugs with no resistance or toxic side effects and to reduce the use of antibiotics, a strain of Baci//us subtilis was isolated from animal intestine, and the isolate was identified by molecular biological method; in vitro an- tibacterial test of the isolate was performed using agar diffusion method; the optimal fermentation condition of the isoJate was screened by conventional culture method; the antibacterial crude protein of the isolate was extracted by saturated ammonium sulfate method; the physicochemical properties of antibacterial crude protein was de- tected by comparison method; The results showed that the isolate was B. subti/is, which had antibacterial effects on Staphy/ococcus aureus, streptococcus and swine erysipelas. The fermentation effect of the isolate was the best under the condition of temperature 30 ~C, pH 7, liquid volume 75 ml/250 ml, inoculation volume 20% and culture time 48 h. The antibacterial effect of the isolate was the best when extract- ed by 80% saturated ammonium sulfate. The antibacterial crude protein had strong resistance to heat and acid. Organic solvent and UV irradiation had some influences on antibacterial crude protein. Proteases had hydrolytic effects on antibacterial crude protein. The isolated B. subti/is can be used to prevent and control the diseases caused by S. aureus, streptococcus and swine erysipelas, and can regulate intesti- nal microecology by adding into expanded feeds.

Involvement of MAPK/ERK kinase-ERK pathway in exogenous bFGF-induced Egr-1 binding activity enhancement in anoxia-reoxygenation injured astrocytes

Objective Intravenous administration of basic fibroblast growth factor （bFGF） is effective to reduce the volume of cerebral infract due to ischemia. This study was designed to investigate the molecular mechanism, especially the signal transduction pathways, involved in this protective role of bFGF. Methods Anoxia-reoxygenation treated atrocytes were used to study the role of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase （MAPK/ERK kinase, MEK）-ERK signaling pathway after exogenous bFGF administration by Western blot. Electrophoretic mobile shift assay was used to detect the binding activity of early growth response factor-1 （Egr-1）, an important transcription factor for endogenous bFGF. Results bFGF could protect some signal transduction proteins from the oxygen-derived free radicals induced degradation. ERK1/2 was activated and involved in Egr-1 binding activity enhancement induced by exogenous bFGF. Conclusion MEK-ERK MAPK cascade may be an important signal transduction pathway contributed to bFGF induced enhancement of Egr-1 binding activity in anoxia-reoxygenation injured astrocytes.

Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

Abstract The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR （FQ-PCR） methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.

Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,respectively.The protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.

Antioxidation Activities of Low-Molecular-Weight Gelatin Hydrolysate Isolated from the Sea Cucumber Stichopus japonicus

Gelatin extracted from the body wall of the sea cucumber （Stichopus japonicus） was hydrolyzed with flavourzyme. Low-molecular-weight gelatin hydrolysate （LMW-GH） of 700-- 1700 Da was produced using an ultrafiltration membrane bioreaetor system. Chemiluminescence analysis revealed that LMW-GH scavenges high free radicals in a concentration-dependent manner; IC50 value for superoxide and hydroxyl radicals was 442 and 285 μgmL-1, respectively. LMW-GH exhibited excellent inhibitory characteristics against melanin synthesis and tyrosinase activity in B16 cells. Furthermore, LMW-GH notably increased in- traeellular glutathione （GSH）, which in turn suppressed melanogenesis. LMW-GH performs antioxidation activity, holding the potential of being used as a valuable ingredient in function foods, cosmetics and pharmaceuticals or nutriceuticals.

Stability and Refolding of Prophenol Oxidase Protein with 2-Propanol in Drosophila melanogaster

Phenol oxidase in Drosophila melanogaster occurs as folded phase precursors designated as prophenol oxidase A1 and A3, and prophenol oxidase is activated with alcohol, especially 2-propanol, within a few minutes as unfolded-phase in vitro. To clarify a common effect of alcohols on proteins and peptides, the extract containing prophenol oxidase protein was prepared. Phenol oxidase activity activated with 2-propanol has been maintained stable at least 24 hours remains as it is. Protein of prophenol oxidase was not denatured opposite hypnoses known as the instability of protein with alcohol. Activated prophenol oxidase with 2-propanol remain enzyme activity with no aggregation, stable, renaturation, and the refolding phenomena occurred around the active phase within the catalytic active center of prophenol oxidase protein in Drosophila melanogaster. This study is important to induce the wide range applications of the effect in many fields for rational drag design.

Effect of stocking density on performances of juvenile turbot (Scophthalmus maximus) in recirculating aquaculture systems

Limited information has been available about the influence of loading density on the performances of Scophthalmus maximus, especially in recirculating aquaculture systems （RAS）. In this study, turbot （13.84±2.74 g; average weigh±SD） were reared at four different initial densities （low 0.66, medium 1.26, sub-high 2.56, high 4.00 kg/m^2） for 10 weeks in RAS at 23±1℃ Final densities were 4.67, 7.25, 14.16, and 17.47 kg/m^2, respectively, which translate to 82, 108, 214, and 282 percent coverage of the tank bottom. Density had both negative and independent impacts on growth. The final mean weight, specific growth rate （SGR）, and voluntary feed intake significantly decreased and the coefficient of variation （CV） of final body weight increased with increase in stocking density. The medium and sub-high density groups did not differ significantly in SGR, mean weight, CV, food conversion rate （FCR）, feed intake, blood parameters, and digestive enzymes. The protease activities of the digestive tract at pH 7, 8.5, 9, and 10 were significantly higher for the highest density group, but tended to be lower （not significantly） at pH 4 and 8.5 for the lowest density group. The intensity of protease activity was inversely related to feed intake at the different densities. Catalase activity was higher （but not significantly） at the highest density, perhaps because high density started to induce an oxidative effect in turbot. In conclusion, turbot can be cultured in RAS at a density of less than 17.47 kg/m^2. With good water quality and no feed limitation, initial density between 1.26 and 2.56 kg/m^2 （final： 7.25 and 14.16 kg/m^2） would not negatively affect the turbot cultured in RAS. For culture at higher density, multi-level feeding devices are suggested to ease feeding competition.

Antitumor and Antifungal Activities of Organic Extracts of Seacucumber Holothuria atra from the Southeast Coast of India

In phylum Echinodermata, the family Holothuridae is distinguished by its capacity of bioactive compounds. Sea cu- cumber Holothuria atra is commonly known as the lollyfish. The antifimgal activity was detected using agar well diffusion method against the various fungal strains such as Trichoderma viride, Aspergillus niger, Aspergillus flavis, Candida albicans and Penicillium chrysogenum. Relatively high antifungal activity was seen against Candida albicans at 100 μL-1 concentration of extracts. Zone of inhibition was measured at 18 mm of diameter. The anti-tumor activities were detected against the Vero and Hep2 cell lines using MTT assay. The cells were treated with H. atra extract at concentrations 0.078-10mg mL-1. The extract showed high proliferative activity against the Hep2 cells. The body wall extracts of sea cucumber （H. atra） showed effective antifungal and antitumor activities All these findings suggest that the extracts could be used for the development of drugs.

Inactivation of digestive proteases: Another aspect of gut bacteria that should be taken into more consideration

TO THE EDITOR Protein has been one of the main components of our diet, and a large amount of digestive proteases is released into the gut for their digestion. However, these proteases can digest not only the proteins we eat, but also the structural proteins built in our body. To protect against this damage, our body has taken a variety of measures. For instance, these digestive proteases are stored and secreted in the form of zymogen and only activated in gut lumen. These luminal digestive proteases are further prevented from direct contact with epithelial cells by the mucus layer that is incessantly secreted by the goblet cells in gut mucosa. In addition, large quantities of protease inhibitors are produced in the body to inactivate the digestive proteases that have entered the body. Despite these measures, the protection seems still weak and can be easily compromised. For instance,

PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro

Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.

AOF1 is a histone H3K4 demethylase possessing demethylase activity-independent repression function

LSD1 （KDM1 under the new nomenclature） was the first identified lysine-specific histone demethylase belonging to the flavin-dependent amine oxidase family. Here, we report that AOF1 （KDM1B under the new nomenclature）, a mammalian protein related to LSD1, also possesses histone demethylase activity with specificity for H3K4mel and H3K4me2. Like LSD1, the highly conserved SWIRM domain is required for its enzymatic activity. However, AOF1 differs from LSD1 in several aspects. First, AOF1 does not appear to form stable protein complexes containing histone deacetylases. Second, AOF1 is found to localize to chromosomes during the mitotic phase of the cell cycle, whereas LSD1 does not. Third, AOF1 represses transcription when tethered to DNA and this repression activity is independent of its demethylase activity. Structural and functional analyses identified its unique N-terminal Zf-CW domain as essential for the demethylase activity-independent repression function. Collectively, our study identifies AOF1 as the second histone demethylase in the family of flavin-dependent amine oxidases and reveals a demethylase-independent repression function of AOF1.

IN VITRO ANALYSIS OFτPHOSPHORYLATION SITES AND ITS BIOLOGICAL ACTIVITY

To explore the association between the abnormal phosphorylation sites found in Alzheimer disease (AD) 蚲 and the inhibition of its biological activit y. Methods. Ultracentrifugation, chromatography, manual Edman degradation and autos equence techniques were used to prepare and phosphorylate human recombinant 蚲, isolate and purify 32P 蚲 peptides and determine phosphorylation sites. Results. Phosphorylation of 蚲 by casein kinase 1 (CK 1), cyclic AMP dependent protein kinase (PKA) and glycogen synthetase kinase 3 (GSK 3) separately inhi bited its biological activity and the inhibition of this activity by GSK 3 was significantly increased if 蚲 was prephosphorylated by CK 1 or PKA. The most po tent inhibition was seen by a combined phosphorylation of 蚲 with PKA and GSK 3 . The treatment of 蚲 by PKA and GSK 3 combination induced phosphorylation of 蚲 at Ser 195, Ser 198, Ser 199, Ser 202, Thr 205, Thr 231, Ser 235, Ser 262, Ser 356, Ser 404, whereas Thr 181, Ser 184, Ser 262, Ser 356 and Se r 400 were phosphorylated by GSK 3 alone under the same condition. Conclusion. Phosphorylation of 蚲 by PKA plus GSK 3 at Thr 205 might play a ke y role in 蚲 pathology in AD.

Expression and proteolytic activity of calpain in lens epithelial cells of oxidative cataract

Objective: To study the role of calpain in the mechanism of oxidative cataract through detecting the level of intracellular free Ca2+, the expression and proteolytic activity of calpain in the lens epithelial cells (LECs) of H2O2-induced cataract. Methods: Rat lenses were cultured in vitro and cataract was induced by H2O2. The level of intracellular free Ca2+ was measured by fluorescence determination with fura-2/AM. The expression of m-calpain protein in LECs was detected with immunohistochemical method. The proteolytic activity in LECs was measured using a fluorogenic synthetic substrate. Results: There were significant differences of the level of intracellular free Ca2+ (P=0.001, 0.000, 0.000), the expression of m-calpain (P=0.001, 0.000, 0.000) and the proteolytic activity of calpain (P=0.001, 0.000, 0.000) between H2O2-induced and control group at 6, 12 and 24 h, respectively. Conclusions: H2O2 can increase intracellular free Ca2+, then enhance the expression and proteolytic activity of calpain which may play a role in the mechanism of oxidative cataract of rat.

Purification and Photodynamic Bioactivity of Phycoerythrin and Phycocyanin from Porphyra yezoensis Ueda

Phycoerythrin and phycocyanin were purified from Porphyra yezoensis Ueda with their bioactivity determined in this study. Continuous precipitation with ammonium sulfate at different concentrations(10%, 20%, 40% and 50%) increased the purity(A564:A280) of phycoerythrin to 1.49, 3.92 fold of the raw extract(0.38) and the purity(A615:A280) of phycocyanin to 0.70, 3.33 fold of the raw extract(0.21). Two more times of chromatography with hydroxylapatites finally made the purity of phycoerythrin and phycocyanin reach 5.50, 14.47 fold of the raw extract, and 5.10, 24.29 fold of the raw extract, respectviely. The yield of high purity phycoerythrin and phycocyanin were 0.21% and 0.09% of dried P. yezoensis blade, respectively. The photodynamic cytotoxic experiment showed that both phycoerythrin and phycocyanin inhibited the growth of liver tumor cells significantly. It was found that 250 mg L-1 purified phycoerythrin and phycocyanin inhibited the growth of hepatocellular carcinoma cells 24 h after laser-irradiation by 80% and 59%, respectively, and 100 mg L-1 purified phycoerythrin and phycocyanin induced the apoptosis of 31.54% and 32.54% of the cells, respectively, 8 h after photodynamic therapy. Oue findings demonstrated that P. yezoensis can serve as photosensitizer(phycoerythrin and phycocyanin) producer.

Expression profiles of two small heat shock proteins and antioxidant enzyme activity in Mytilus galloprovincialis exposed to cadmium at environmentally relevant concentrations

Small heat shock proteins encompass a widespread but diverse class of proteins, which play key roles in protecting organisms from various stressors. In the present study, the full-length cDNAs of two small heat shock proteins （MgsHSP22 and MgsHSP24.1） were cloned from Mytilus galloprovincialis, which encoded peptides of 181 and 247 amino acids, respectively. Both MgsHSP22 and MgsHSP24.1 were detected in all tissues examined by real-time PCR, with the highest expression being observed in muscle and gonad tissues. The real-time PCR results revealed that Cd significantly inhibited MgsHSP22 expression at 24 h and MgsHSP24.1 at 24 and 48 h under 5 ug/L Cd2＋ exposure. MgsHSP24.1 expression was also significantly inhibited after 50 ug/L Cd2＋ exposure for 48 h. With regard to antioxidant enzymes, increased GPx and CAT activity were detected under Cd2＋ stress （5 and 50 ug/L）, while no significant difference in SOD activity was observed throughout the experiment. Overall, both MgsHsps and antioxidant enzymes revealed their potential as Cd stress biomarkers in M. galloprovincialis.