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Objectives: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine‑elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription‑polymerase chain reaction in muscle tissues. The stimulation index (SI) of T‑lymphocyte proliferation and the levels of interferon‑gamma (INF‑γ) and interleukin‑4 (IL‑4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme‑linked immunosorbent assays. Results: The pcDNA3.1(+)‑BmCPI/ BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF‑γ and IL‑4 of pcDNA3.1(+)‑BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF‑γ of pcDNA3.1(+)‑BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)‑BmCPI/CpG group (P < 0.05). Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.
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Background Cysteine proteinase inhibitor (cystatin, CPI) is one of the most important molecules involved in plant development and defense, especially in the regulation of stress responses. However, it is still unclear whether the Jatropha curcas CPI (JcCPI) gene functions in salinity response and tolerance. In this study, the sequence of the JcCPI gene, its expression pattern, and the effects of overexpression in Escherichia coli and Nicotiana benthamiana were examined. The purpose of this study was to evaluate the regulatory role of JcCPI in salinity stress tolerance. Results The CPI gene, designated JcCPI, was cloned from J. curcas; its sequence shared conserved domains with other plant cystatins. Based on a transcription pattern analysis, JcCPI was expressed in all tissues examined, but its expression was highest in the petiole. Additionally, the expression of JcCPI was induced by salinity stress. A potential role of JcCPI was detected in transgenic E. coli, which exhibited strong CPI activity and high salinity tolerance. JcCPI was also transferred to tobacco plants. In comparison to wild-type plants, transgenic plants expressing JcCPI exhibited increased salinity resistance, better growth performance, lower malondialdehyde (MDA) contents, higher anti-oxidase activity, and higher cell viability under salinity stress. Conclusions Based on the results of this study, overexpression of JcCPI in E. coli and N. benthamiana conferred salinity stress tolerance by blocking cysteine proteinase activity. The JcCPI gene cloned in this study will be very useful for the development of stress-tolerant crops.
Subject(s)
Cysteine Proteinase Inhibitors/metabolism , Jatropha , Salt Tolerance , Sequence Analysis , Computational Biology , Cysteine Proteases , Real-Time Polymerase Chain Reaction , Salt StressABSTRACT
ObjectiveThe difference of Cys-C ( serum cysteine proteinase inhibitor C) among sepsis group,systemic inflammatory response syndrome (SIRS) group,and non -SIRS group were explored in this study.The significance of mortality and the relationship between Cys-C and acute physiology and chronic health evaluation (APACHE)Ⅱ score were under discussed. Methods After excluding the individual whose survival less than 24 hours,two hundred and fifty patients sought medical care in the emergency department of Beijing Chaoyang Hospital of the Capital Medical University were selected as samples from October 2008 to October 2009.They were classified into three groups:SIRS group ( n =121 ),non-SIRS group (n =74) and sepsis group ( n =55 ).The serum Cys-C level and APACHE Ⅱ score were determined for each patient.The positive detection rate of Cys-C ( ＞ 830 ng/ml) was calculated and then a 28-day mortality was recorded according to this study result.The positive detection rate and 28-day mortality were also compared with chi-square test.The prognostic values of Cys-C,APACHE Ⅱ score for the 28-daymortality were evaluated by logistic regression analysis.Results There was significant change observed between sepsis group and non-SIRS group (41.38％ vs. 13.57％,P =0.007 ) for the positive detection rate of Cys-C,as well as that between SIRS group and non-SIRS group ( 32.79％ vs. 13.57％,P =0.005).However,a contrary result was obtained when compared sepsis group with SIRS group (41.38％ vs.32.79％,P =0.346) ).Significant difference was noticed in the 28-day mortality of the patients from sepsis group and SIRS group in comparison to those of non-SIRS group (41.6％ vs. 17.2％,P ＜ 0.01 ;36.91％ vs. 17.2％,P ＜ 0.05).Cys-C level in patient with sepsis indicated a positive correlation to APACHE Ⅱ score ( P ＜0.0001 ).ConclusionsThe positive rate of Cys-C in SIRS group and septic group were significantly higher than that of non-SIRS patients,and this is an index for poor prognosis in sepsis patients.
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Objective To validate the analytical performance of three Cys C reagents with particle-enhanced turbidimetric immunoassay(PETIA) method used on the automatic biochemistry analyzer for preliminary clinical application.Methods The performance of three Cys C reagents (labeled as A, B, C) with PETIA method from Shanghai Jing Yuan Co., Beijing Leadman Co. and Beijing Jiuqiang Co. on OlympusAU2700 automatic biochemistry analyzer were assessed.According to the standard of CLSI EP6-A, EP15-A and EP7-P, the precision, linearity range, disturbance (bilirubin, hemoglobin, chyle) were assessed, and compared with those of Cys C reagent based on particle-enhanced nephelometric immunoassay(PENIA) from Dade Behring Co.. The reference ranges for Cys C in serum of 120 healthy individual were evaluated.Results The within-run CVs of the three reagents (A, B and C) were 3.08%-3.2%, 2.3%-4.15% and 1.38%-1.53% respectively.The total CV in A, B and C were 3.29%-3.44%, 2.65%-5.18% and 1.67%-1.69% respectively, lower than the stated.Limits of quantitative determination (LOQ) of the three reagents were 0.41, 0.23 and 0.07 mg/L, basically meeting the testing requirement.The linearity range was 0.22-7.26 mg/L(r=0.996), 0.20-7.72 mg/L(r=0.999)and 0.20-7.62 mg/L(r=0.997)in the three reagents, which demonstrated a sound linear correlation. For interference tests, no remarkable interference (<±10%) of reagent C was detected when bilirubin≤684 μmol/L, hemoglobin≤9.7 g/L and Chyle turbidity≤6 200 FTU; and no significant interference of reagent B was found when bilirubin≤684 μmol/L, hemoglobin≤6.79 g/L and Chyle turbidity≤6 200 FTU; when bilirubin≤684 μmol/L, hemoglobin≤4.85 g/L and Chyle turbidity≤1 240 FTU reagent A was not interfered significantly. The comparison afte and before the high-speed centrifugation reveals that the average percentage of bias for reagents A, B, C measured Cys C in chylous serum samples of patients was -8.31%, 1.52%, 1.32%, respectively.In method comparison tests, the regression equations of the three reagents compared with Dade Behring PENIA Cys C reagent were as follows:Y=0.787X+0.492 (R2=0.976), Y=1.098X+0.137 (R2=0.982) and Y=1.037X+0.249 (R2=0.996), respectively. Agreement rates of the high Cys C in reagent A, B, C and Dade Behring Cys C reagent were 80% (Kappa=0.615,P=0.000), 100% (Kappa=1.000,P=0.000), 91.2% (Kappa=0.824,P=0.000); While for reference range of preliminary clinical assessment, diagnosis coincidence rate of reagent A increased to 98.8% (Kappa=0.974,P=0.000). Conclusions When used in automatic biochemical analyzer, the three Cys C reagent with PETIA showed high precision,sensitivity, and sound correlation with Dade Behring PENIA reagents.The three reagents are all able to meet clinical test requirements, nevertheless, anti-interference capability were diffierent and the reference range should be further validated.
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Objective To study the clinical significance by detecting the levels of sera cystatin C(Cys C)in patients with colorectal cancer.Methods We compared the levels of sera cys C detected with the immune rate nephelometry(IRN)in 71 colorectal cancer patients.Then we compared it in 40 healthy persons and 48 colorectal disease patients without cancer to investigate the relationship between the level of cys C and the clinicopathological characteristics.Results The levels of cys C in colorectal cancer patients were much higher than those in the other control groups,and were significantly related to Dukes stages and the tumor differentiated degree.Conclusion The results provide convincing evidence that cys C may contribute to occurrence and development of colorectal cancer.
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Objective To investigate the contribution of Cystatin C to the preoperative diagnosis and clinical therapy of gastroenteric tumor.Methods Using surgical materials from patients with stomach, colon and rectum cancer, immunohistochemisty, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis were performed with antibodies against Cystatin C.Cystatin C of the cancerous and paired noncancerous lesions were determined by enzyme-linked immunosorbent assay (ELISA).Results (1)Immunohistochemical staining of Cystatin C was evident expression in cancer cells and associated stromal tissues, this was not the case in paired noncancerous tissues,there were also statistical relationship between the expression of Cystatin C in cancerous and that in noncancerous tissues (?~2=6.825,P