Effect of Metal Ions on Protease Activities in the Intestines and Hepatopancreas of Red-white Ornamental Carp (Cyprinus carpio L)

[Objective] The aim of this study was to study effects of metal ions on the protease activities in digestive tissues and gland of red-white ornamental carp（Cyprinus carpio L）.[Method] Effects of four kinds of metal ions （K＋,Na＋,Mg2＋ and Ca2＋） on protease activities in hepatopancreas,foregut,midgut,hindgut of red-white ornamental carp were studied by enzyme analysis method.[Result] Effects of four kinds of metal ions on protease activities of red-white ornamental carp were different in the range of experimental concentration from 25 mmol/L to 150 mmol/L.K＋ could promote protease activities in hepatopancreas and hindgut at different levels.Especially,K＋ had the promoting effect at low-concentration level,but the inhibitory effect at high-concentration level in midgut and the inhibitory effect in foregut.Na＋ had the promoting effect on protease activities in hepatopancreas,foregut and hindgut at different levels,but the inhibitory effect in midgut.Mg2＋ and Ca2＋ had the inhibitory effect on protease activities in intestinal and hepatopancreas at different levels.[Conclusion] This study provides basic data and theoretical foundation for researches on the digestive physiology of red-white ornamental carp or the development and optimization of compound feed.

Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation

Objective： To construct the point mutation plasmids expressing HCV NS3/4A with different secondary structures at the N-terminus, and to analyze their serine protease activities. Methods： The point mutation plasmid constructs were generated by using the QuickChange site-directed mutagenesis kit with the backbone of M-H05-5 （AI-1）, and were named as subgroup A1-2, A2-1, A2-2, BI-1, B1-2, B2-1, and B2-2 respectively. The transient expression of the constructs was investigated by immunofluorescence assay and Western blot analysis. The difference in in cis and in trans NS3 serine protease activity between each subgroup was determined by Western blot analysis. Luciferase reporter assay was used to observe the inhibitory effects of the constructs on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation. Results： The point mutation plasmid constructs were verified for the correct sequence by DNA sequencing. The immunofluorescence assay revealed 4 subcellular localization patterns of NS3, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blot analysis indicated that the incomplete cleavage of NS3/4A appeared in subgroups A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker when compared with the other subgroups. By using NS5A/SBAC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared the other subgroups. Differences in inhibitory effects of HCV NS3 on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion： The results suggest that subgroup A2-1 and B2-1 has weaker serine protease activities and weaker inhibitory activities on host cell functions than the other subgroups, which might be explained by the different secondary structure of the 120-aa sequence at N-terminus of NS3.

A dicistrovirus increases pupal mortality in Spodoptera frugiperda by suppressing protease activity and inhibiting larval diet consumption

Understanding interactions between viruses and their hosts is conducive to enabling better application of viruses as biocontrol agents.Certain viruses carried by parasitic wasps enhance the parasitic efficiency of wasp-larvae by protecting them against the immune system of their Lepidopteran host.However,the relationship between prey pests and viruses found in predatory natural enemies remains unclear.Herein,we report the interaction between Arma chinensis virus-1(AcV-1),originally isolated from a predatory natural enemy,Arma chinensis(Hemiptera:Pentatomidae),and one of its prey species,Spodoptera frugiperda(Lepidoptera:Noctuidae).The results showed that the AcV-1 virus appeared harmful to the novel host S.frugiperda by inhibiting larval diet consumption and increasing pupal mortality.Meanwhile,sequencing data indicated that the virus altered the gene expression profiles of S.frugiperda.KEGG analysis showed that the proteasome and phagosome pathways related to protein degradation and immune response were significantly enriched.Although the expression levels of digestive enzyme genes did not change significantly,the total protease activity of AcV-1 virus-positive individuals was significantly decreased,suggesting that the virus inhibited diet consumption of S.frugiperda via the down-regulation of digestive enzyme activities.These results indicate that a virus initially isolated in a predatory natural enemy can decrease the fitness of its prey species.The virus was found to impact the host proteasome and phagosome pathways related to protein degradation and immunity,providing a potential mechanism to enhance controlling efficiency.

Effects of temperature, pH and NaCl on protease activity in digestive tract of young turbot, Scophthalmus maximus

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish’s internal organs. The optimal activity in the fish’s stomach was at pH of 2.2, while that in the intes- tinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40℃ in stomach, intestine and hepato- pancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an op- posite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.

Comparison in effect of different metal ions, pH and reducing agent on the protease activity in human hyper mature and mature cataract

This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at -20 ℃. The total protein extract was isolated from 5 samples in each case （mature and hyper mature cataract） and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 ℃. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. β mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates.

Protease activated receptor 2 and epidermal growth factor receptor are involved in the regulation of human sperm motility

Aim： To investigate mechanisms of tryptase-induced reduction of sperm motility and explore whether epidermal growth factor receptor （EGF-R） and protease activated receptor 2 （PAR-2）- associated pathways are involved. Methods： Fresh semen was collected from healthy donors （n = 15）. Semen parameters and quality were assessed in accordance with the World Health Organization （WHO） criteria. Swim-up sperm were fixed and subjected to immunocytochemistry and immunoelectronmicroscopy with specific antibodies directed against PAR-2 and EGF-R. Protein extractions from swim-up spermatozoa were analyzed by Western blotting with antibodies for both receptors. Motility of spermatozoa was evaluated by computer-assisted semen analysis. Results： Immunocytochemistry found PAR-2 and EGF-R in approximately 30% of examined human ejaculated spermatozoa. Both receptors were localized in the plasma membrane. Like tryptase, the PAR-2 synthetic agonist SLIGKV reduced sperm motility, and this effect was inhibited by application of two specific EGF-R pathway blockers （AG1478 and PD168393）. Conclusion： The observed reduction of sperm motility by tryptase through the PAR-2 receptor involves EGF-R pathways.

Hippocampal expression of apoptotic protease activating factor-1 following diffuse axonal injury under mild hypothermia

The influence of mild hypothermia on neural cell apoptosis remains poorly understood. Therefore, the present study established rat models of diffuse axonal injury （DAI） at 33℃. Morris water maze results demonstrated significantly better learning and memory functions in DAI rats with hypothermia compared with DAI rats with normothermia. Expression of apoptotic protease activating factor-1 in the hippocampal CA1 region was significantly lower in the DAI hypothermia group compared with the DAI normothermia group. Expression of apoptotic protease activating factor-1 positively correlated with latency, but negatively correlated with platform location times and time of swimming in the quadrant area. Results suggested that post-traumatic mild hypothermia in a rat model of DAI could provide cerebral protection by attenuating expression of apoptotic protease activating factor-1.

Role of Protease Activated Receptor-2 Expression in Renal Interstitial Fibrosis Model in Mice

Summary： The role of protease activated receptor-2 （PAR-2） in the renal tubulointerstitial lesion induced by unilateral ureteral obstruction （UUO） was explored. Mice were sacrificed on the day 1, 3, 5, 7, 10, 14 and 21 after UUO. The expression of PAR-2 mRNA and protein and a-smooth muscle actin （α-SMA） protein in tubuloin,terstitium was detected by RT-PCR and immunohistochemistry at each time point, respedtively. The results showed that the PAR-2 expression in renal tubulointerstitium was increased progressively starting from 24 h to the day 14 post-ligation, and it was significantly associated with the relative volume of interstitium and the positive area of α-SMA. PAR-2 was mainly expressed in renal tubule epithelial cells, especially in proximal tubular cells. It also located in renal capillary ansa, interstitial infiltrate cells and fibroblasts. It was concluded that PAR-2 was active in interstitial and tubular cells in the early phase of fibrotic process and played an important role in mediating the tubulointerstitial lesion after UUO.

Purification and Biochemical Characterization of a Protease Inhibitor Ⅱ Family from Jalapeno Pepper(Capsicum annuum L.)

Capsicum annuum L. was initially domesticated in Mexico and northern Central America, and represented an ancient Neotropical plant food complex. The purpose of this paper is to report the isolation and purification of a novo-member of a protease inhibitor from jalape&ntildeo pepper (Capsicum annuum L.) (PIJP). The molecular weight of PIJP inhibitor is 5.95 kDa with 56 amino acids and 6 Cys residues with high inhibitory activity to trypsin with a Ki value of 95 nM. This inhibitor according to the alignment with homologous from NCBI and Pfam databases is a member of proteinase inhibitors II. It is worthwhile to mention a major compositional difference between the proteinase inhibitor II families which have 8 Cys residues. PIJP is the first purified proteinase inhibitor, member of this family with only 6 Cys residues.

Effects of Used Battery on Key Enzyme Activity during the Germination of Wheat Seeds

[Objective] This study aimed to explore the effects of used battery lixivium on wheat germination. [Method] The wheat seeds were treated with used battery lix- ivium at different concentrations to detect the change of activities of amylase, pro- tease, pyruvate dehydrogenase （PDH） and polyphenol oxidase （PPO） during the ger- mination period. [Result] The results showed that the used battery affected enzyme activity. With the increase of concentration of used battery lixivium, trends of the changes of amylase and protease activities were not different. The activities were en- hanced at low concentrations of lixivium, while were inhibited at high concentrations. The tends of changes of pyruvate dehydrogenase （PDH） and polyphenol oxidase （PPO） activities were not consistent with that of either amylase or protease, which showed continuous downward trends with the increasing concentration of used battery lixivium. [Conclusion] This study is of great practical significance for understanding the effects of used battery lixivium on the germination of wheat seeds.

Biochemical Changes Associated with Germinating Rice Grains and Germination Improvement

To determine biochemical changes during the germination of rice grains （Oryza sativa L. subsp, indica var. Mottaikaruppan） and to improve germination rate using gibberellic acid and surfactants [sodium dodecyl sulfate （SDS） （1.0 g/L） and Triton-X-100 （1.0 mL/L）], whole rice grains soaked in distilled water for 12 h at 30℃were germinated in the dark at 30℃ for five days. The highest germination rate （77.1%） was obtained on the 5th day. An increase in the content of reducing sugars from 7.3 to 58.1 mg/g DM （dry matter） was observed from the 1st day of germination. Free amino acids and soluble protein contents increased to 3.69 and 5.29 mg/g DM, respectively on the 5^th day of germination. Total protein content decreased from 100.5 to 91.0 g/kg DM during germination. Increases in amylolytic （1.1 to 190.0 U/g DM） and proteolytic （0 to 0.12 U/g DM） activities were observed during germination. Effects of different concentrations of gibberellic acid on the germination of rice grains were evaluated and 0.1 g/L was found to promote germination. When effects of gibberellic acid （0.1 g/L） and surfactants were evaluated individually and together, higher germination rate was observed in the control experiment （grains germinated in distilled water）, whereas giberellic acid and surfactants decreased the germination rate. Therefore, the flour obtained from the grains germinated for four days using distilled water to obtain high content of soluble materials and enzyme activities can be used in preparation of bakery items.

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.

Experimental Study on the PAR-1 Expression Around Hemotoma Following Intracerebral Hemorrhage in Rats

Summary: In order to explore the PAR-1 mRNA and protein expression around hemotoma following intracerebral hemorrhage and the relation between the PAR-1 expression and thrombin, collagenase Ⅶ was stereotaxically injected into right caudate nucleus in rats. The PAR-1 mRNA expression was detected by RT-PCR method and the PAR-1 protein expression by immunohistochemical method respectively. It was found that the PAR-1 mRNA and protein expression around hemotoma was increased at 6 h after intracerebral hemorrhage (P<0.05), peaked at 2 days (P<0.01), and then declined. The change pattern of the PAR-1 mRNA and protein expression was similar to that of intracerebral hemorrhage after thrombin intracerebral injection. The PAR-1 mRNA and protein expression in hirudin group showed no significant difference with control group. These results indicated that the PAR-1 mRNA and protein expression were markedly increased after intracerebral hemorrhage, which may be closely related to thrombin. Upregulation of the PAR-1 expression may involve in neurotoxic injury induced by thrombin.

cytoplasmic domain of tissue factor promotes liver fibrosis in mice

To evaluate the role of tissue factor (TF) and protease activated receptor (PAR)-2 in liver fibrosis. METHODSUsing CCl4 administration for eight weeks, we induced hepatic fibrosis in wild-type C57BL/6 mice and in mice with deletion of the cytoplasmic signalling domain of TF (TF§CT/§CT), deletion of PAR-2 (PAR-2-/-) and combined deletion of TF signalling domain and PAR-2 (TF§CT/§CT/PAR-2-/-). Hepatic fibrosis area was assessed by quantitative imaging of picrosirius red staining. Hepatic collagen content was assessed by hydroxyproline levels. Hepatic stellate cells (αSMA positive) and hepatic macrophages (CD68 positive) were identified by immunohistochemistry. Hepatic gene expression was determined by PCR and liver TGFβ1 content by ELISA. RESULTSCCl4 treated mice with deletion of the PAR-2 gene (PAR-2-/-) and the cytoplasmic domain of TF (TF§CT/§CT) developed significantly less hepatic fibrosis, characterised by reduced liver fibrosis area and hydroxyproline content, compared to control wildtype mice treated with CCl4. The observed reduction in histological fibrosis was accompanied by a significant decrease in the hepatic content of TGFβ, the prototypic fibrogenic cytokine, as well as fewer activated hepatic stellate cells and hepatic macrophages. Deletion of the TF cytoplasmic signalling domain reduced hepatic fibrosis to levels similar to those observed in mice lacking PAR-2 signalling but combined deletion provided no added protection against fibrosis indicating a lack of mutual modulating effects that have been observed in other contexts such as angiogenic responses. CONCLUSIONTissue factor cytoplasmic domain is involved in TF-PAR-2 signalling initiating hepatic fibrosis and is a potential therapeutic target, as its deletion would not impact coagulation.

Procaine Inhibiting Human Bladder Cancer Cell Proliferation by Inducing Apoptosis and Demethylating APAF1 CpG Island Hypermethylated

Studies have shown that aberrant DNA methylation of apoptotic protease activating factor-1（APAF1） is an important epigenetic mechanism of gene regulation in the progression of bladder cancer.In this article,we have proved that procaine,an inhibitor of DNA methyltransferases,could inhibit the proliferation of T24 and 5637 human bladder cancer cells by inducing their apoptosis.The mechanism studies reveal that procaine could induce demethylation of APAF1 gene in T24 or 5637 cells,subsequently activating caspase-3/9.It was also shown that the serum soluble fas ligand（sFasL） was activated,and the expression of matrix metallopeptidase 9（MMP-9） was down-regulated.Procaine seems to induce cell death by different pathways,and it might be used as a potential agent for bladder cancer treatment.

Up-regulation of interleukin-8 expressions induced by mast cell tryptase via protease activated receptor-2 in endothelial cell line

Background Protease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whetehr PAR-2 can mediate tryptase-induced inflammatory reaction. This study was conduct to investigate wheter PAR-2 could be the activated by mast cell tryptase and medicated the tryptase induced interleukin-8 expression in endothelial cells. Methods Protease activated receptor-2 expression was found in endothelial cell lines ECV304 cell by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. Interleukin-8 stimulated by purified human mast cell tryptase was determined by RT-PCR and enzyme linked immunosorbent assay （ELISA）. Data were analysed by the S-N-K one-way ANOVA test. Results The present study shows that mRNA and protein of protease activated receptor-2 could be expressed in ECV304 cells, and tryptase upregulated the expression levels of both interleukin-8 mRNA and protein. The increased expression of interleukin-8 was inhibited by an antiprotease activated receptor-2 monoclonal antibody, SAM11. An additional band was observed by Western blotting after the incubation of ECV304 cells with tryptase for 2 hours, which suggested that protease activated receptor-2 was activated. Conclusion Protease activated receptor-2 can mediate the mast cell tryptase stimulated expression of interleukin-8 in ECV304 cell.

Early detection of non-small cell lung cancer in liquid biopsies by ultrasensitive protease activity analysis

Aim:A significant fraction of mortalities from non-small cell lung cancer could be prevented,if the cancer would be diagnosed earlier.Nanobiosensors for the ultrasensitive detection of active proteases in serum were designed to detect a significant protease activity signature of non-small cell lung cancer(stage I and higher).Methods:We determined the activity of nine protease biomarkers in the sera of non-small cell lung cancer patients and compared them with the protease activities of a control group of healthy human subjects using optical nanobiosensors.They consist of a central Fe/Fe3O4 core/shell nanoparticle with an attached Fluorescence resonance energy transfer-pair[tetrakis-carboxyphenyl porphyrin(TCPP)and cyanine 5.5].TCPP is attached to the central nanoparticle via a protease-cleavable tether,whereas cyanine 5.5 is tethered permanently to the dopamine-layer surrounding the nanoparticle.Results:Based on the activity pattern of urokinase plasminogen activator,matrix metalloproteinases 1,2,3,7,9,and 13,and cathepsins B and L as well,non-small cell lung cancer could be detected at stage I by means of a liquid biopsy.Conclusion:This feasibility study,comprising 33 non-small cell lung cancer patients and 20 apparently healthy subjects,clearly demonstrated the feasibility of minimally invasive early diagnosis of non-small cell lung cancer,starting with stage I.

Domain-Swapping as a Mechanism to Lock the Active Conformation of SARS-CoV Main Protease

The main protease (Mpro) of SARS-CoV plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target

Polyoxometalate-based nanozyme： Design of a multifunctional enzyme for multi-faceted treatment of Alzheimer＇s disease

Proteolytic degradation of amyloid-β （Aβ） aggregates and clearance of Aβ- induced reactive oxygen species （ROS） have received significant attention for the treatment of Alzheimer＇s disease （AD）. However, it is difficult, and often unfeasible, to directly upregulate or transport intraceUular native enzymes. More importantly, penetration of the blood-brain barrier （BBB） has presented a major impediment. Herein, we report on the rational design of a polyoxometalate- based nanozyme with both protease-like activity for depleting A~ aggregates, and superoxide dismutase （SOD）-like activity for scavenging A[3-mediated ROS. Furthermore, this nanozyme acts as a metal chelator to remove Cu from Cu-induced Aβ oligomers. More intriguingly, the nanozyme can cross the BBB and exhibits low toxicity. This work provides new insights into the design and synthesis of inorganic nanozymes as multifunctional therapeutic agents in the treatment of AD.

Effect of thrombin on blood brain barrier permeability and its mechanism

Background Previous studies have indicated that thrombi n (TM) may play a major role in brain edema after intracerebral hemorrhages (ICHs). However, the mechanism of TM-induced brain edema is poorly understood. In this study, we explored the effect of TM on the permeability of the blood brain barrier (BBB) and investigated its possible mechanism, aiming at providing a potential target for brain edema therapy after ICHs.Methods TM or TM + cathepsin G (CATG) was stereotaxically injected into the right caudate nucleus of Sprague-Dawley rats in vivo. BBB permeability was measured by Evans-Blue extravasation. Brain water content was determined by the dry-wet weight method. Brain microvascular endothelial cells were then cultured in vitro. After TM or TM+CATG was added to the endothelial cell medium, changes in the morphology of cells were dynamically observed by phase-contrast light microscopy, and the expression of matrix metalloproteinase-2 (MMP-2) protein was measured by immunohistochemical method.Results BBB permeability increased at 6 hours after a TM injection into the ipsilateral caudate nucleus (P<0.05), peaked between 24 hours (P<0.01) and 48 hours (P<0.05) after the injection, and then declined. Brain water content changed in parallel with the changes in BBB permeability. However, at all time points, BBB permeability and brain water content after a TM+CATG injection were not significantly different from the respective parameters in the control group (P>0.05). TM induced endothelial cell contraction in vitro in a time-dependent manner and enhanced the expression of MMP-2 protein. After incubation with TM+CATG, cell morphology and MMP-2 expression did not change significantly as compared to the control group (P>0.05).Conclusions Increased BBB permeability may be one of the mechanisms behind TM-induced cerebral edema. TM induces endothelial cell contraction and promotes MMP-2 expression by activating protease activated receptor-1 (PAR-1), possibly leading to the opening of the BBB.