Effect of MUC2 Antisense Oligodeoxynucleotide on Cell Proliferation,Adhesion,and Proteolytic Enzyme in Human Gastric Carcinoma in vitro

Objective：To investigate the effect of MUC2 antisense oligodeoxynucleotide （ASODN） on cell proliferation, adhesion and proteolytic enzyme inhuman gastric carcinoma cell line （SGC7901）. Methods： Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin, and then transfected to SGC7901 cells. The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemical method respectively, and the effect of MUC2 ASODN on cell proliferation, adhesion and proteolytic enzyme was determined by flow cytometry（FCM）, MTT method, Rose Bengal and immunohistochemical method. Results： Compared with the blank control group, ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection（P〈0.01）. Various concentrations of ASODN could significantly inhibit the growth of SGC7901, and the inhibition peaked at the 48th hour after transfection（P〈0.05）. The apoptosis rate of the experimental group was about 4.38%, and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase. In addition, cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro（P〈0.01）. By immunohistochemical method, the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed（P〈0.05）. Conclusion： MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation, reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.

STUDY ON THE IMMOBILIZATION OF PAPAIN WITH A MACROPOROUS BEAD CARRIER OF COPOLYMER CONTAINING MONOMER UNITS OF NAMINOETHYL ACRYLAMIDE AND VINYL ALCOHOI

A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups, hydrophilic amido links andhydroxyl groups, as well as macroporous structures based on its matrix (MR-AV matrix), furthermore, the activity recoveryof papain in the immobilization could reach 48%/～58%. In comparison with free papain, the resulting immobilized papainexhibits a remarkable thermostability and better reusability.

Slurry ice as an alternative cooling medium for fish harvesting and transportation:Study of the effect on seabass flesh quality and shelf life

The objective of the study was to investigate the efficiency of slurry ice during harvesting and transportation of European sea bass(Dicentrarchus labrax)to retain flesh quality and extend shelf life,compared with conventional flake ice.Fish was slaughtered and transported in different mixtures of slurry ice and conventional flake ice(C:slaughtered and transported in 100%flake ice-Control samples,SC:slaughtered in 100%slurry ice and transported in 100%flake ice,S50:slaughtered and transported in 50%slurry ice-50%flake ice,S100:slaughtered and transported in 100%slurry ice)and subsequently stored under controlled isothermal conditions at 0℃for shelf life modelling and flesh quality evaluation(proteolytic enzymes).The replacement of conventional flake ice with slurry ice as a slaughtering method led to improved quality stability during subsequent refrigerated storage and shelf life extension,in terms of microbial growth,flesh quality and sensory degradation of fish.Based on microbial growth,the shelf life of C samples was found to be 19 days,whereas the shelf life of S50/S100 and SC was 21 and 25 days,respectively,showing that the replacement of flake ice with slurry ice resulted in 2–6 days shelf life extension of whole sea bass stored at 0℃.The use of slurry ice at slaughter and flake ice in transportation was accompanied by low activities and late peaks of all four enzymes that is expected to lead to delayed proteolytic degradation and extended freshness.

Brain-derived neurotrophic factor inducing angiogenesis through modulation of matrix-degrading proteases

Background Recent studies have proved that brain-derived neurotrophic factor （BDNF） possesses angiogenic activity in vitro and in vivo. However, the proangiogenic mechanism of BDNF has not yet been provided with enough information. To explore the proangiogenic mechanism of BDNF, we investigated the effects of BDNF on extracellular proteolytic enzymes, including matrix metalloproteinases （MMPs） and serine proteases, particularly the urokinase-type plasminogen activator （uPA）-plasmin system in human umbilical vein endothelial cells （HUVECs） model.Methods Tube formation assay was performed in vitro to evaluate the effects of BDNF on angiogenesis. The HUVECs were treated with various concentrations of BDNF （25-400 ng/ml） for different （6-48 hours）, reverse transcriptase-polymerase chain reaction （RT-PCR） was used to assay MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA in HUVECs, and the conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography, respectively, uPA, plasminogen activator inhibitor （PAI）-1, tissue inhibitors of metalloproteinase （TIMP）-1, and TIMP-2 were quantified by western blotting analysis.Results BDNF elicited robust and elongated angiogeneis in two-dimensional cultures of HUVECs in comparison with control. The stimulation of serum-starved HUVECs with BDNF caused obvious increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation. However, BDNF had no effect on TIMP-1 and TIMP-2 production. BDNF increased uPA and PAI-1 production in a dose-dependent manner. Maximal activation of uPA and PAI-1 expression in HUVECs was induced by 100 ng/ml BDNF, while effects of 200 ng/ml and 400 ng/ml BDNF were slightly reduced in comparison with with those of 100 ng/ml. Protease activity for uPA was also increased by BDNF in a dose-dependent manner. BDNF also stimulated uPA and PAI-1 production beyond that in control cultures in a time-dependent manner from 12 hours to 48 hours after BDNF treatment.Conclusions BDNF stimulates MMP and uPA/PAI-1 proteolytic network in HUVECs, which may be important to the acquisition of proangiogenic potential.

Production, purification, characterization, immobilization, and application of Serrapeptase： a review

BACKGROUND： Serrapeptase is a proteolytic enzyme with many favorable biological properties like anti-inflammatory, analgesic, anti-bacterial, fibrinolytic properties and hence, is widely used in clinical practice for the treatment of many diseases. Although Serrapeptase is widely used, there are very few published papers and the information available about the enzyme is very meagre. Hence this review article compiles all the information about this important enzyme Serrapeptase. METHODS： A literature search against various databases and search engines like PubMed, SpringerLink, Scopus etc. was performed. RESULTS： We gathered and highlight all the published information regarding the molecular aspects, properties, sources, production, purification, detection, optimizing yield, immobilization, clinical studies, pharmacology, interaction studies, formulation, dosage and safety of the enzyme Serrapeptase. CONCLUSION： Serrapeptase is used in many clinical studies against various diseases for its anti-inflammatory, fibrinolytic and analgesic effects. There is insufficient data regarding the safety of the enzyme as a health supplement. Data about the anti- atherosclerotic activity, safety, tolerability, efficacy and mechanism of action of the Serrapeptase are still required.

Accurate, automatic annotation of peptidases with hotpep-protease

Peptidases are essential for intracellular protein processing,signaling and homeostasis,physiological processes and for digestion of food.Moreover,peptidases are important biotechnological enzymes used in processes such as industrial food processing,leather manufacturing and the washing industry.Identification of peptidases is a crucial step in their characterization but peptidase annotation is not a trivial task due to their large sequence diversity.In the present study short,conserved sequence profiles were generated for all peptidase families with more than four members in the comprehensive Merops peptidase database.The sequence profiles were combined with the Homology to Peptide Pattern(Hotpep)method for automatic annotation of peptidases.This method is a standalone software that annotates protease sequences to Merops family and subgroup and is suitable for large-scale sequence analysis.Compared to the Mammalian Degradome Database Hotpep-protease had an accuracy of 92%and a sensitivity of 96%for annotation of the human degradome.Annotation by commonly used methods(Blast and conserved domains)had an accuracy of 69%and a sensitivity of 78%.For fungal genomes,there were large differences between annotation with Hotpep-protease,Blast-and Hidden Markov Model-based annotation and the Merops annotation,which confirms the difficulty of large-scale peptidase annotation.Manual annotation indicated that Hotpep-protease had a positive prediction rate of 0.90 compared to a positive prediction rate of 0.67 for Blast search.Hence,Hotpep-protease is highly accurate method for fast and accurate annotation of peptidases.