A series of experiments based on electrophoretical and biochemical assays were conducted to partially characterize proteinases present in the hepatopancreas and intestine of black carp （Mylopharyngodon piceus）, a...A series of experiments based on electrophoretical and biochemical assays were conducted to partially characterize proteinases present in the hepatopancreas and intestine of black carp （Mylopharyngodon piceus）, and investigate enzymatic activity and protein digestibility in vitro. Casein digestion assays revealed the presence of acidic proteinases with optimum activity in the range of pH 2.0-2.5 and alkaline proteinases with significantly higher activities both in the range of pH 8.1-8.6 and near pH 9.5. The inhibition and substrate specificity assays showed that trypsin and chymotrypsin are the main active components of the alkaline proteinases. The SDS-substrate-PAGE showed that the crude extract of black carp intestine had eight types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa while the crude extract of black carp hepatopancreas had six types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa. These enzymes were characterized as trypsin （27.5 kDa, 30.1 kDa）, chymotrypsin （40.5 kDa, 42.5 kDa）, serine proteinases （32.1 kDa, 33.2 kDa） and non-serine proteinase （61.5 kDa, 78.5 kDa）.In vitro protein digestibility assays showed that black cardcan be able to utilize a wider range of proteins.展开更多
A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression c...A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.展开更多
In this study, feed-grade proteinase was added into conventional diets of three-line crossbred （Duroc x Landrace x Large White） piglets, to investigate the effects of feed-grade proteinase on anti-diarrhea capacity,...In this study, feed-grade proteinase was added into conventional diets of three-line crossbred （Duroc x Landrace x Large White） piglets, to investigate the effects of feed-grade proteinase on anti-diarrhea capacity, daily weight gain, feed intake and feed conversion ratio of piglets. The results showed that adding feed-grade proteinase in diets enhanced anti-diarrhea capacity of piglets and improved signifi- cantly production performance and breeding efficiency of piglets. This study provided the reference for rational utilization of feed-grade proteinase in actual production.展开更多
By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE...By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.展开更多
The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 ℃ for 30 min, recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their mol...The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 ℃ for 30 min, recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.展开更多
A cathepsin B-like proteinase was purified from eggs of Antheraea pernyi by 5 steps of purification. The molecular mass of the proteinase are estimated to be 47 kDa by using SDS-polyacrylamide gel electro-phoresis. Th...A cathepsin B-like proteinase was purified from eggs of Antheraea pernyi by 5 steps of purification. The molecular mass of the proteinase are estimated to be 47 kDa by using SDS-polyacrylamide gel electro-phoresis. The activity of the purified proteinase were strongly inhibited by E-64 and Leupeptin. The optimum pH is 3.5 as determined by using the bovine hemo-globin as substrate. The activity and quantity of the proteinase during embryo development were studied. The results suggested that the proteolytic activities during embryo development at least partially came from this proteinase.展开更多
Women are more likely than men to develop cancer of the breast.Most breast cancer drugs are highly toxic,and treatment can cause side effects.It is imperative to find safe alternative medicines in the pursuit of a cur...Women are more likely than men to develop cancer of the breast.Most breast cancer drugs are highly toxic,and treatment can cause side effects.It is imperative to find safe alternative medicines in the pursuit of a cure for breast cancer.An extract of pineapple contains cysteine proteases known as bromelains.In general,pineapples are regarded as safe foods.From the fruit,stem,and of pineapples,bromelain is produced by multiple endopeptidases.As well as reducing the growth of tumors locally,bromelain severely impaired the cytotoxicity of monocytes in the immune system in the fight against cancer.Specifically,we investigated pineapple’s possible mechanisms of action and its bioactive compounds in breast cancer.展开更多
Numerous reports have identified a dysbiosis in the intestinal microbiota in patients suffering from inflammatory bowel diseases(IBD),yet the mechanism(s)in which this complex microbial community initiates or perpetua...Numerous reports have identified a dysbiosis in the intestinal microbiota in patients suffering from inflammatory bowel diseases(IBD),yet the mechanism(s)in which this complex microbial community initiates or perpetuates inflammation remains unclear.The purpose of this review is to present evidence for one such mechanism that implicates enteric microbial derived proteases in the pathogenesis of IBD.We highlight and discuss studies demonstrating that proteases and protease receptors are abundant in the digestive system.Additionally,we investigate studies demonstrating an association between increased luminal protease activity and activation of protease receptors,ultimately resulting in increased intestinal permeability and exacerbation of colitis in animal models as well as in human IBD.Proteases are essential for the normal functioning of bacteria and in some cases can serve as virulence factors for pathogenic bacteria.Although not classified as traditional virulence factors,proteases originating from commensal enteric bacteria also have a potential association with intestinal inflammation via increased enteric permeability.Reports of increased protease activity in stools from IBD patients support a possible mechanism for a dysbiotic enteric microbiota in IBD.A better understanding of these pathways and characterization of the enteric bacteria involved,their proteases,and protease receptors may pave the way for new therapeutic approaches for these diseases.展开更多
Tumor invasion and metastasis is a highly complicated, multi-step phenomenon. In the complex event of tumor progression, tumor cells interact with basement membrane and extracellular matrix components. Proteolytic enz...Tumor invasion and metastasis is a highly complicated, multi-step phenomenon. In the complex event of tumor progression, tumor cells interact with basement membrane and extracellular matrix components. Proteolytic enzymes(proteinases) are involved in the degradation of extracellular matrix, but also in cancer invasion and metastasis. The four categories of proteinases(cysteine-, serine-, aspartic-, and metalloproteinases) are named and classified according to the essential catalytic component in their active site. We and others have shown that proteolytic enzymes play a major role not only in colorectal cancer(CRC) invasion and metastasis, but also in malignant transformation of precancerous lesions into cancer. Tissue and serumplasma antigen concentrations of proteinases might be of great value in identifying patients with poor prognosis in CRC. Our results, in concordance with othersindicate the potential tumor marker impact of proteinases for the early diagnosis of CRC. In addition, proteinases may also serve as potential target molecules for therapeutic agents.展开更多
Gastric cancer(GC)is the fourth most common cancer in the world and the second cause of cancer-related death.Gastric carcinogenesis is a multifactorial process,in which environmental and genetic factors interact to ac...Gastric cancer(GC)is the fourth most common cancer in the world and the second cause of cancer-related death.Gastric carcinogenesis is a multifactorial process,in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells.One such a pathway is regulated by proteinase activated-receptors(PARs),seven transmembrane-spanning domain G protein-coupled receptors,which comprise four receptors(i.e.,PAR-1,PAR-2,PAR-3,and PAR-4)activated by various proteases.Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways,which sustain gastric carcinogenesis.There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients.Consistently,data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients.In contrast,PAR-4levels are down-regulated in GC and correlate inversely with the aggressiveness of GC,thus suggesting a negative role of this receptor in the control of GC.In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior.展开更多
Six soil enzymes (invertase, acid phosphatase, proteinase, catalase,peroxidase and polyphenoloxidase) were chosen for investigation under different spruce forests withrestoration ages of 4, 10, 16 years and an old-gro...Six soil enzymes (invertase, acid phosphatase, proteinase, catalase,peroxidase and polyphenoloxidase) were chosen for investigation under different spruce forests withrestoration ages of 4, 10, 16 years and an old-growth spruce forest over 400 yearsold in the easternQinghai-Tibet Plateau, China. Results showed that the activities of invertase, phosphatase,proteinase, catalase and peroxidase decreased in newly restored forests except forpholyphenoloxidase. With the development of forests after restoration, the activities of invertase,acid phosphadase, proteinase increased gradually. Our study also indicated that the soil enzymeactivities were associated with surface soils and decreased with depths. This result suggested thatin the earlier restoration stage the application of organic fertilizer may be more effective bysurface addition to soils than deep addition.展开更多
Proteinase A （PrA）, encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. ...Proteinase A （PrA）, encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial PEP4 gene deletion mutant （36 h） and PrA-negative mutant （48 h） was significantly extended, compared with the wild type strain （24 h） （P〈0.05）, but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking cultivation. The effects of glucose starvation on cell growth of partial PEP4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption ofS. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 ℃ the reduced cell viability rate （RCVR） was 10% for the wild type S. cerevisiae and 90% for both mutant strains （P〈0.01）, suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 ℃ to 70℃, the wild type S. cerevisiae had significantly lower relative glucose consumption rate （RGCR） （61.0% and 80.0%） than the partial mutant （78.0% and 98.5%） and the complete mutant （80.0% and 98.0%） （P〈0.05）, suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.展开更多
Total RNA was isolated from mycelium of T. harzianum by Total RNA extraction kit, and two clear bands of rRNA （28S and 18S） were observed in agarose electrophoresis. By joining the 3＇end sequence with the known SA7...Total RNA was isolated from mycelium of T. harzianum by Total RNA extraction kit, and two clear bands of rRNA （28S and 18S） were observed in agarose electrophoresis. By joining the 3＇end sequence with the known SA76 EST from cDNA library of T. harzianum, a full-length cDNA sequence of 2019bp was obtained, whose open reading frame contained 1593bp, a stop codon TAA, a 5＇untranslated region （5＇UTR） of 266bp, a 3＇untranslated region （3＇UTR） of 201bp, and poly （A） 29 encoded a protein of 530 amino acids, had a signal peptide. T. harzianum shared 53% identity of secreted aspartic proteinase gene with G. zeae, 37% with N. crassa and 36% with C. globosum. The full-length cDNA sequence of secreted aspartic proteinase gene from T. harzianum was cloned for the first time by using BD SMART RACE technique, which provides a foundation to obtain and validate functional genes of T. harzianum.展开更多
Hyaluronic acid （HA） had been prepared from pigskin residues with neutral proteinase. The preparing results and conditions were studied. After extracting and purification, HA was detected through ultraviolet spectra...Hyaluronic acid （HA） had been prepared from pigskin residues with neutral proteinase. The preparing results and conditions were studied. After extracting and purification, HA was detected through ultraviolet spectra and infraction spectrum, and its content and purity were tested by carbazole and Elason-Morgan, respectively. This results indicated that significant quantities of HA could be prepared in fresh pigskin with biologic enzyme, and the pure HA was cosmetic grade and food grade.展开更多
AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (D...AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.展开更多
Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3（PR3）, a serine protease of the chymotrypsin family,...Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3（PR3）, a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay.A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries（P,0.01）; a positive correlation（r50.87; P,0.01; Pearson’s correlation analysis） was also observed between salivary p H and PR3 concentration. In an antibacterial test,a PR3 concentration of 250 ng？m L21 or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation（P,0.05）. These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3.展开更多
To investigate the effect of the Ginkgo Biloba Extract （GBE） on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticaso...To investigate the effect of the Ginkgo Biloba Extract （GBE） on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticasone propionate for 2 weeks or 4 weeks, or treated with fluticasone propionate plus GBE for 2 weeks or 4 weeks. Fifteen healthy volunteers served as healthy controls. Sputum inhalation with inhaling hypertonic saline （4%-5%） was performed. Lung ventilatory function and forced expiratory volume in one second （FEVI） were measured. The numbers of different cells in induced sputum were calculated. The expression of PKCα in the cells was immunocytochemically detected and the percentages of positive cells in different cells were counted. Interleukin-5 （IL-5） in sputum supernatants was detected with enzyme-linked immunosorbent assay. The percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the concentration of IL-5 in asthmatic patients were higher than those in the controls （P〈0.05）, and the eosinophils, lymphocytes, positive expression of PKCα and the level of IL-5 were significantly decreased in asthmatic patients after they were treated with fluticasone propionate or fluticasone propionate plus GBE. However, they were still significantly higher than those of the controls. Compared to the group treated with glucocorticosteroid for 2 weeks, no significant decrease was found in the percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the IL-5 in the supernatant of induced sputum. Compared with the group treated with glucocorticosteroid for 2 or 4 weeks, significant decrease in the same parameters was observed in the group treated with fluticasone propionate and GBE for 4 weeks. The IL-5 level in the supernatant of induced sputum was positively correlated with the percentage of PKCα-positive inflammatory cells and the percentage of eosinophils in the induced sputum in asthma patient groups respectively （n=150, r=0.83, P〈0.01; n=150, r=0.76, P〈0.01）. The FEVI was negatively correlated with the percentage of PKCα-positive inflammatory cells and the IL-5 levels in supernatant of induced sputum in asthma patients respectively （n=150, r=-0.77, P〈0.01; n=150, r=- 0.64, P〈0.01）. It is concluded that GBE could significantly decrease the infiltration of inflammatory cells such as eosinophils and lymphocytes in the asthmatic airway and relieve the airway inflammation. GBE may decrease the activation of the PKCα in the inflammatory cells and thereby decrease the IL-5 level in induced sputum. GBE may be used as a complement to the glucocorticosteroid therapy for asthma.展开更多
The effect of tea plant Camellia sinensis induced by exogenous methyl jasmonate （MJA） on lipoxygenase （LOX）, polyphenol oxidase （PPO） and proteinase inhibitor （PI） activity in the leaves of tea plants, as well...The effect of tea plant Camellia sinensis induced by exogenous methyl jasmonate （MJA） on lipoxygenase （LOX）, polyphenol oxidase （PPO） and proteinase inhibitor （PI） activity in the leaves of tea plants, as well as the growth and midgut proteinase activity of the geometrid Ectropis obliqua larvae were studied. MJA significantly induced LOX, PPO and PI activity in leaves of tea plants. When geometrid larvae have fed on leaves of tea plants treated with MJA, the activities of the high alkaline trypsin-like enzyme and chymotrypsin-like enzyme in their midgut were significantly inhibited, but the activities of the low alkaline trypsin-like enzyme in their midgut were unaffected, leading to imbalance between different types of proteinase activity in the midgut of the larvae and in turn, the growth were inhibited. These chains of response may be an important mechanism of the direct resistance induced by MJA-treatment of tea plant on geometrid larvae.展开更多
A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further co...A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further confirmed by ELISA, PCR and PCR Southern assays. Results of bioassays show that transgenic plants display notably inhibitory effects to larvae development and survival of Heliothis armigera Hubner.展开更多
文摘A series of experiments based on electrophoretical and biochemical assays were conducted to partially characterize proteinases present in the hepatopancreas and intestine of black carp （Mylopharyngodon piceus）, and investigate enzymatic activity and protein digestibility in vitro. Casein digestion assays revealed the presence of acidic proteinases with optimum activity in the range of pH 2.0-2.5 and alkaline proteinases with significantly higher activities both in the range of pH 8.1-8.6 and near pH 9.5. The inhibition and substrate specificity assays showed that trypsin and chymotrypsin are the main active components of the alkaline proteinases. The SDS-substrate-PAGE showed that the crude extract of black carp intestine had eight types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa while the crude extract of black carp hepatopancreas had six types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa. These enzymes were characterized as trypsin （27.5 kDa, 30.1 kDa）, chymotrypsin （40.5 kDa, 42.5 kDa）, serine proteinases （32.1 kDa, 33.2 kDa） and non-serine proteinase （61.5 kDa, 78.5 kDa）.In vitro protein digestibility assays showed that black cardcan be able to utilize a wider range of proteins.
文摘A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.
文摘In this study, feed-grade proteinase was added into conventional diets of three-line crossbred （Duroc x Landrace x Large White） piglets, to investigate the effects of feed-grade proteinase on anti-diarrhea capacity, daily weight gain, feed intake and feed conversion ratio of piglets. The results showed that adding feed-grade proteinase in diets enhanced anti-diarrhea capacity of piglets and improved signifi- cantly production performance and breeding efficiency of piglets. This study provided the reference for rational utilization of feed-grade proteinase in actual production.
文摘By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.
文摘The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 ℃ for 30 min, recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.
文摘A cathepsin B-like proteinase was purified from eggs of Antheraea pernyi by 5 steps of purification. The molecular mass of the proteinase are estimated to be 47 kDa by using SDS-polyacrylamide gel electro-phoresis. The activity of the purified proteinase were strongly inhibited by E-64 and Leupeptin. The optimum pH is 3.5 as determined by using the bovine hemo-globin as substrate. The activity and quantity of the proteinase during embryo development were studied. The results suggested that the proteolytic activities during embryo development at least partially came from this proteinase.
文摘Women are more likely than men to develop cancer of the breast.Most breast cancer drugs are highly toxic,and treatment can cause side effects.It is imperative to find safe alternative medicines in the pursuit of a cure for breast cancer.An extract of pineapple contains cysteine proteases known as bromelains.In general,pineapples are regarded as safe foods.From the fruit,stem,and of pineapples,bromelain is produced by multiple endopeptidases.As well as reducing the growth of tumors locally,bromelain severely impaired the cytotoxicity of monocytes in the immune system in the fight against cancer.Specifically,we investigated pineapple’s possible mechanisms of action and its bioactive compounds in breast cancer.
基金Supported by The national institutes of health(DK092330)to Carroll IM
文摘Numerous reports have identified a dysbiosis in the intestinal microbiota in patients suffering from inflammatory bowel diseases(IBD),yet the mechanism(s)in which this complex microbial community initiates or perpetuates inflammation remains unclear.The purpose of this review is to present evidence for one such mechanism that implicates enteric microbial derived proteases in the pathogenesis of IBD.We highlight and discuss studies demonstrating that proteases and protease receptors are abundant in the digestive system.Additionally,we investigate studies demonstrating an association between increased luminal protease activity and activation of protease receptors,ultimately resulting in increased intestinal permeability and exacerbation of colitis in animal models as well as in human IBD.Proteases are essential for the normal functioning of bacteria and in some cases can serve as virulence factors for pathogenic bacteria.Although not classified as traditional virulence factors,proteases originating from commensal enteric bacteria also have a potential association with intestinal inflammation via increased enteric permeability.Reports of increased protease activity in stools from IBD patients support a possible mechanism for a dysbiotic enteric microbiota in IBD.A better understanding of these pathways and characterization of the enteric bacteria involved,their proteases,and protease receptors may pave the way for new therapeutic approaches for these diseases.
文摘Tumor invasion and metastasis is a highly complicated, multi-step phenomenon. In the complex event of tumor progression, tumor cells interact with basement membrane and extracellular matrix components. Proteolytic enzymes(proteinases) are involved in the degradation of extracellular matrix, but also in cancer invasion and metastasis. The four categories of proteinases(cysteine-, serine-, aspartic-, and metalloproteinases) are named and classified according to the essential catalytic component in their active site. We and others have shown that proteolytic enzymes play a major role not only in colorectal cancer(CRC) invasion and metastasis, but also in malignant transformation of precancerous lesions into cancer. Tissue and serumplasma antigen concentrations of proteinases might be of great value in identifying patients with poor prognosis in CRC. Our results, in concordance with othersindicate the potential tumor marker impact of proteinases for the early diagnosis of CRC. In addition, proteinases may also serve as potential target molecules for therapeutic agents.
文摘Gastric cancer(GC)is the fourth most common cancer in the world and the second cause of cancer-related death.Gastric carcinogenesis is a multifactorial process,in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells.One such a pathway is regulated by proteinase activated-receptors(PARs),seven transmembrane-spanning domain G protein-coupled receptors,which comprise four receptors(i.e.,PAR-1,PAR-2,PAR-3,and PAR-4)activated by various proteases.Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways,which sustain gastric carcinogenesis.There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients.Consistently,data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients.In contrast,PAR-4levels are down-regulated in GC and correlate inversely with the aggressiveness of GC,thus suggesting a negative role of this receptor in the control of GC.In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior.
文摘Six soil enzymes (invertase, acid phosphatase, proteinase, catalase,peroxidase and polyphenoloxidase) were chosen for investigation under different spruce forests withrestoration ages of 4, 10, 16 years and an old-growth spruce forest over 400 yearsold in the easternQinghai-Tibet Plateau, China. Results showed that the activities of invertase, phosphatase,proteinase, catalase and peroxidase decreased in newly restored forests except forpholyphenoloxidase. With the development of forests after restoration, the activities of invertase,acid phosphadase, proteinase increased gradually. Our study also indicated that the soil enzymeactivities were associated with surface soils and decreased with depths. This result suggested thatin the earlier restoration stage the application of organic fertilizer may be more effective bysurface addition to soils than deep addition.
基金Project (No. 2007AA10Z315) supported by the Hi-Tech Research and Development Program (863) of China
文摘Proteinase A （PrA）, encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial PEP4 gene deletion mutant （36 h） and PrA-negative mutant （48 h） was significantly extended, compared with the wild type strain （24 h） （P〈0.05）, but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking cultivation. The effects of glucose starvation on cell growth of partial PEP4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption ofS. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 ℃ the reduced cell viability rate （RCVR） was 10% for the wild type S. cerevisiae and 90% for both mutant strains （P〈0.01）, suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 ℃ to 70℃, the wild type S. cerevisiae had significantly lower relative glucose consumption rate （RGCR） （61.0% and 80.0%） than the partial mutant （78.0% and 98.5%） and the complete mutant （80.0% and 98.0%） （P〈0.05）, suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.
基金This paper was supported by Development Program (863) of China (No. 2003AA241140)
文摘Total RNA was isolated from mycelium of T. harzianum by Total RNA extraction kit, and two clear bands of rRNA （28S and 18S） were observed in agarose electrophoresis. By joining the 3＇end sequence with the known SA76 EST from cDNA library of T. harzianum, a full-length cDNA sequence of 2019bp was obtained, whose open reading frame contained 1593bp, a stop codon TAA, a 5＇untranslated region （5＇UTR） of 266bp, a 3＇untranslated region （3＇UTR） of 201bp, and poly （A） 29 encoded a protein of 530 amino acids, had a signal peptide. T. harzianum shared 53% identity of secreted aspartic proteinase gene with G. zeae, 37% with N. crassa and 36% with C. globosum. The full-length cDNA sequence of secreted aspartic proteinase gene from T. harzianum was cloned for the first time by using BD SMART RACE technique, which provides a foundation to obtain and validate functional genes of T. harzianum.
文摘Hyaluronic acid （HA） had been prepared from pigskin residues with neutral proteinase. The preparing results and conditions were studied. After extracting and purification, HA was detected through ultraviolet spectra and infraction spectrum, and its content and purity were tested by carbazole and Elason-Morgan, respectively. This results indicated that significant quantities of HA could be prepared in fresh pigskin with biologic enzyme, and the pure HA was cosmetic grade and food grade.
基金National Natural Science Foundation of China,No.39770300,30070873the Overseas Chinese Affairs Office of the State Council Foundation,No.98-33
文摘AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.
基金the support of the National Natural Science Foundation of China (No. 81372892)Sichuan Province Science and Technology Innovation Team Program (JCPT 2011-9)
文摘Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3（PR3）, a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay.A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries（P,0.01）; a positive correlation（r50.87; P,0.01; Pearson’s correlation analysis） was also observed between salivary p H and PR3 concentration. In an antibacterial test,a PR3 concentration of 250 ng？m L21 or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation（P,0.05）. These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3.
基金This project was supported by a grant from Foundation for outstanding University Teacher by the Ministry of Education (2000 year)
文摘To investigate the effect of the Ginkgo Biloba Extract （GBE） on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticasone propionate for 2 weeks or 4 weeks, or treated with fluticasone propionate plus GBE for 2 weeks or 4 weeks. Fifteen healthy volunteers served as healthy controls. Sputum inhalation with inhaling hypertonic saline （4%-5%） was performed. Lung ventilatory function and forced expiratory volume in one second （FEVI） were measured. The numbers of different cells in induced sputum were calculated. The expression of PKCα in the cells was immunocytochemically detected and the percentages of positive cells in different cells were counted. Interleukin-5 （IL-5） in sputum supernatants was detected with enzyme-linked immunosorbent assay. The percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the concentration of IL-5 in asthmatic patients were higher than those in the controls （P〈0.05）, and the eosinophils, lymphocytes, positive expression of PKCα and the level of IL-5 were significantly decreased in asthmatic patients after they were treated with fluticasone propionate or fluticasone propionate plus GBE. However, they were still significantly higher than those of the controls. Compared to the group treated with glucocorticosteroid for 2 weeks, no significant decrease was found in the percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the IL-5 in the supernatant of induced sputum. Compared with the group treated with glucocorticosteroid for 2 or 4 weeks, significant decrease in the same parameters was observed in the group treated with fluticasone propionate and GBE for 4 weeks. The IL-5 level in the supernatant of induced sputum was positively correlated with the percentage of PKCα-positive inflammatory cells and the percentage of eosinophils in the induced sputum in asthma patient groups respectively （n=150, r=0.83, P〈0.01; n=150, r=0.76, P〈0.01）. The FEVI was negatively correlated with the percentage of PKCα-positive inflammatory cells and the IL-5 levels in supernatant of induced sputum in asthma patients respectively （n=150, r=-0.77, P〈0.01; n=150, r=- 0.64, P〈0.01）. It is concluded that GBE could significantly decrease the infiltration of inflammatory cells such as eosinophils and lymphocytes in the asthmatic airway and relieve the airway inflammation. GBE may decrease the activation of the PKCα in the inflammatory cells and thereby decrease the IL-5 level in induced sputum. GBE may be used as a complement to the glucocorticosteroid therapy for asthma.
基金the National NaturalScience Foundation of China(30170631)Key Laboratory Foundation of Tea Chemical Engineering,Ministry of Agriculture,P.R.China(2003-2).
文摘The effect of tea plant Camellia sinensis induced by exogenous methyl jasmonate （MJA） on lipoxygenase （LOX）, polyphenol oxidase （PPO） and proteinase inhibitor （PI） activity in the leaves of tea plants, as well as the growth and midgut proteinase activity of the geometrid Ectropis obliqua larvae were studied. MJA significantly induced LOX, PPO and PI activity in leaves of tea plants. When geometrid larvae have fed on leaves of tea plants treated with MJA, the activities of the high alkaline trypsin-like enzyme and chymotrypsin-like enzyme in their midgut were significantly inhibited, but the activities of the low alkaline trypsin-like enzyme in their midgut were unaffected, leading to imbalance between different types of proteinase activity in the midgut of the larvae and in turn, the growth were inhibited. These chains of response may be an important mechanism of the direct resistance induced by MJA-treatment of tea plant on geometrid larvae.
文摘A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further confirmed by ELISA, PCR and PCR Southern assays. Results of bioassays show that transgenic plants display notably inhibitory effects to larvae development and survival of Heliothis armigera Hubner.
