Crystal structure of SARS-CoV-2 papain-like protease

The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC

Inhibitory effects of recombinant human Hespintor on proliferation, migration and invasion of hepatocellular carcinoma cells / 重庆医学

Objective To perfect the purification method of recombinant fusion protein of Hespintor (rHespintor) for increasing the protein extraction efficiency,and to investigate its effects on the proliferation,migration and invasion of hepatoblastoma cell line HepG2.Methods In the recombinant protein extraction,the inclusion body washing process was added and the protein purification buffer system was changed.BAPNA was used as the substrate.The inhibitory effect tof purified rHespintor on trypsin hydrolysis was detected.The blank group served as the control group.The MTT test,cell scratch wound healing test and tumor cell invasion test were performed to detect the effect of rHespintor on growth of hepatoblastoma HepG2 cells and its effect.Results The urea gradient washing on the inclusion body protein could effectively remove the vast majority of impure proteins from the targeted protein.After one-step purification,the target protein rHespintor exhibited a high inhibition effect of trypsin hydrolysis,and the inhibitory effect was exhibited a dose-dependent manner.After acting on hepatoblastoma HepG2 cells with rHespintor,the cell proliferation ability was inhibited,the migration ability was reduced and the number of invaded cells were significantly decreased.Conclusion rHespintor can significantly inhibit the proliferation,migration and invasion of hepatoblastoma cell line HepG2 cells in vitro.

Expression and significance of Maspin and IKKα in sinusoidal mucosa of rats with fungal rhinosinusitis / 中国比较医学杂志

Objective To investigate the expression and significance of Maspin and IKKα in nasosinusoidal mucosa of rats with fungal rhinosinusitis (FRS).Methods A total of 40 SD rats were used to establish the FRS model, and randomly divided into nasal obstruction group, FRS group, immunosuppressive group and invasive FRS group, 10 rats in each group.Another 10 normal rats were used as control group.Mice in the control group were fed with normal diet.In the nasal obstruction group, the mice had only hemostatic cotton stuffed in the nasal cavity and injection of 0.9% NaCl in the abdominal and nasal cavities.In the FRS group, the mice were injected Aspergillus fumigatus spore suspension into the nasal cavity and 0.9% NaCl i.p.The mice of the immunosuppressive group were given cyclophosphamide i.p.and 0.9% NaCl injection into the nasal cavity.The invasive FRS group was injected with cyclophosphamide i.p.and Aspergillus fumigatus spore suspension into the nasal cavity.The serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA).The expression of Maspin and IKKα in nasosinusoidal mucosa was detected by immunohistochemical staining.The expression of Maspin mRNA and IKKα mRNA in the nasosinusoidal mucosa was detected by fluorescence quantitative PCR.Results The serum levels of IL-6 and TNF-α in different groups were significantly different (P 0.05).Theresult of immunohistochemical staining showed that the protein expression of Maspin in the FRS group and invasive FRS group was significantly lower than that in the control group, nasal obstruction group and immunosuppressive group, while the expression of IKKα protein was significantly higher than that of control group, nasal obstruction group and immunosuppressive group (P< 0.05).The protein expression of Maspin in the invasive FRS group was significantly lower than that in the FRS group, by contrast, the expression of IKKα protein was significantly higher (P< 0.05).The PCRresult revealed that the expression levels of Maspin and IKKα mRNA were (0.217 ± 0.013) and (0.193 ± 0.012), significantly lower than that in the control, obstruction and immunosuppressive groups [(0.309 ± 0.021), (0.302 ± 0.017), and (0.293 ± 0.02)] (P< 0.05), while the expressions level of IKKα mRNA were significantly higher [(0.319 ± 0.043), (0.384 ± 0.048) vs (0.169 ± 0.015), (0.171 ± 0.018), and (0.175 ± 0.019)] (P< 0.05).Conclusions Down-regulation of Maspin expression after IKKα activation is the main cause of the onset of FRS, which may also be one of the mechanisms of invasive FRS.

A recombinant plasmid of composite cysteine proteinase inhibitor/ glyceraldehyde‑3‑phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host.

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.

Effects of Tamoxifen on proliferation and expression of serine proteinase inhibitor 9 in human gastric cancer cells / 重庆医学

Objective To observe the expression of estrogen receptors (ERαand ERβ) on gastric cancer cells and evaluate the effect of Tamoxifen(TAM) on the cell proliferation and expression of serine proteinase inhibitor 9(PI9) of gastric cancer cells . Methods PI9 positive expression(MNK45 ,SGC7901)and negative expression (BGC823)of gastric cancer cell lines were from pre‐liminary screened ,the expression of ERα and ERβ detected by immunofluorescence chemical method ,the cell proliferation and ex‐pression of PI9 were tested by CCK8 assay and reverse transcription‐PCR after intervention of TAM .Results ERαprotein expres‐sion was noted in MNK45 and SGC7901 ,ERβwas noted in BGC823 ,but the expression of ERαand ERβwere not appear to be obvi‐ous after the intervention of TAM .Tamoxifen could obviously inhibited cell proliferation of MNK45 and SGC7901 at concentration of 0 .1-100 .0 μmol/L ,the differences were statistically significant compared with negative control group (P<0 .05) ,but showed no dose‐dependent to the proliferation of BGC823 and MNK28 .After treating with TAM ,the expression of PI9 mRNA of SGC7901 (0 .402± 0 .020) and MNK45(0 .359 ± 0 .048) were obviously lower than that in the negatwe control group(P< 0 .05). Conclusion Tamoxifen could significantly inhibit the proliferation of PI9 positive expression than PI9 negative expression of gastric cancer cell lines ,and showed obviously dose‐dependent ,its role in inhibiting proliferation might closely related to immune tolerance improved by PI9 .

Overexpression of a cysteine proteinase inhibitor gene from Jatropha curcas confers enhanced tolerance to salinity stress

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.

Analysis of Tissue Specific Digestive and Antioxidant Enzymes from Cucurbita Pepo and Langenaria Siceraria (Molina) Standl.

Digestive enzymes and antioxidants present in food materials act as a surplus for our digestive system. In present study, five digestive enzymes namely amylase, protease, lipase, pectinase and α -glucosidase and seven antioxidant assays for Catalase, Peroxidase, H2O2, Super Oxide Dismutase, Malondialdehyde, Carotenoid & radical scavenging were carried out from two members of cucurbitaceae family namely Cucurbita pepo (Pumpkin) and Langenaria siceraria (bottle gourd). Amongst them amylase, lipase and pectinase activity were found in C. pepo and L. siceraria. The enzyme kinetics studies revealed their maximum velocities to be 0.2631 and 0.33557 mM min-1 for Amylase and Lipase respectively. Our further studies report the presence of proteinase inhibitors and α-gluosidase inducers, while absence of amylase inhibitors and α -glucosidase inhibitors in pumpkin and bottle gourd. Present findings suggest the use of these fruits as an attractive material for further study leading to possible development of digestive syrup.

Normativas de diagnóstico y tratamiento del déficit de alfa-1 antitripsina / Guidelines on Diagnosis and Treatment of Alpha-1 Antitrypsin Deficiency

El déficit de alfa-1 antitripsina (AAT) es una condición hereditaria rara y raramente diagnosticada en todo el mundo, incluida Argentina. El infradiagnóstico es fundamentalmente debido a que muchos médicos desconocen su existencia, diagnóstico y tratamiento. Por ello, la Asociación Argentina de Medicina Respiratoria encomendó a un grupo de expertos la elaboración de la presente normativa. La AAT es una glicoproteína secretada por el hígado, muy abundante en sangre, tejidos y fluidos corporales, cuya función principal consiste en inhibir la elastasa del neutrófilo y otras serin proteasas, confiriendo al suero humano más del 90% de su capacidad antiproteasa. El déficit de AAT deriva de mutaciones del gen de la SERPINA1, y se manifiesta clínicamente por enfisema pulmonar, cirrosis hepática y, con menor frecuencia, por paniculitis, vasculitis sistémicas y posiblemente otras enfermedades. El déficit grave de AAT afecta mayoritariamente a individuos de raza caucasiana y tiene su máxima prevalencia (1:2.000-1:5.000 individuos) en el norte, oeste y centro de Europa. En EEUU y Canadá, la prevalencia es de 1: 5.000-10.000, y es 5 veces menor en países latinoamericanos, incluida Argentina, donde se estima que puede haber unos 18.000 individuos con genotipos deficientes graves SZ y ZZ, la inmensa mayoría sin diagnosticar. Sospechar la enfermedad resulta clave para medir la concentración sérica de AAT y completar el diagnóstico con la determinación del fenotipo o genotipo ante concentraciones bajas. La detección de casos permite la puesta en práctica del consejo genético, el chequeo de familiares consanguíneos y, en casos seleccionados, la aplicación de terapia sustitutiva.

The alpha-1 antitrypsin (AAT) deficiency is a rare hereditary condition which is rarely diagnosed in the world, including Argentina. Underdiagnosis is mainly due to lack of knowledge of its diagnosis and treatment by many physicians. For this reason, the Argentine Association of Respiratory Medicine convened a group of experts to develop the present guidelines. AAT is a glycoprotein secreted by the liver; it reaches high levels in blood, body tissues and fluids. Its main function is to inhibit the neutrophil elastase and other serum proteases providing 90% of human serine antiprotease activity. The AAT deficiency is produced by mutations of the SERPINA1 gene. Its clinical manifestations are pulmonary emphysema, liver cirrhosis, and less often panniculitis, systemic vasculitis and possibly other conditions. The severe AAT deficiency affects mainly Caucasian individuals. The highest prevalence, ranging from 1 in 2000 to 1 in 5000 population is observed in northern, western and central Europe. In the USA and Canada, the prevalence varies from 1 in 5000 to 1 in 10000 population. It is 5 times less frequent in Latin American countries. It is estimated that in Argentina there may be 18000 cases with severe deficiency of SZ y ZZ genotypes, most of them undiagnosed. It is crucial to suspect the disease in order to measure the serum AAT concentration, and, if the concentrations are low, to confirm the diagnosis with the phenotype or genotype determinations. Case detection allows genetic advice, control of blood-related relatives and in selected cases, replacement therapy.

Analysis on clinical features and plasma SLPI levels in children with Mycoplasma pneumoniae pneumonia / 临床儿科杂志

Objective To analyze the clinical features and the plasma secretory leukocyte proteinase inhibitor (SLPI) levels in children with Mycoplasma pneumoniae pneumonia (MPP). Methods Clinical data and plasma SLPI levels of 136 children with MPP were retrospectively analyzed. Results From July 2011 to June 2013,136 children (male 80, female 56) with MPP were included in the study. The onset ages of all children ranged from 11 months to 14 years (mean age, 6.2±3.0 years), and 82.4%of the cases were at the age of 4 to 14 years. One hundred and twenty six cases (92.7%) with long-last high fever, 83.8%with cough, 74.3%with rale were found in the study. Small or large patchy shadows in chest X-ray radiography were found in all the cases. At the acute phase, 72.1%with low white blood cell count, 59.6%with normal neutrophil cell and 63.2%with higher high sensitive C-reactive protein (hs-CRP) were observed. The SLPI level at the acute phase in 85 cases was (9.3±8.8) ng/ml, which was signiifcant lower than that at the convalescent phase (11.8±8.0 ng/ml, Z=3.08, P=0.002). Conclusions The clinical features of MPP are usually presented with high fever, cough, higher hs-CRP, normal or lower white blood cell and neutrophil cell count, small or large patchy shadows in chest X-ray radiography. The plasma SLPI level at the acute phase was signiifcantly lower than that in convalescent phase in children with MPP.

In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae.

Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5% yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80°C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.

The positive detection rate of cystatin C in patients with sepsis and its prognostic significance / 中华急诊医学杂志

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.

Characterization of an Avian Protease Inhibitor.

Ovomucoid is a serine proteinase inhibitor in the egg whites of all avian species at a concentration of about 10 mg/ml. The involvement of proteinases in a multitude of control functions in an organism has created an interest in their physiological inhibitors. Regulation of proteolytic activity in tissues is a critical requirement in the maintenance of homeostasis. Egg white proteins possess ACE-inhibitory activity, & also high radical-scavenging activity. The combined antioxidant and ACE-inhibitory properties of egg white hydrolysates, or the corresponding peptides, would make a useful multifunctional preparation for the control of cardiovascular diseases. Proteases play key roles in several physiological processes, including intracellular protein degradation, bone remodeling, and antigen presentation, and their activities are increased in pathophysiological conditions such as cancer metastasis and inflammation. They are also required for invasion by microorganism. Four protease inhibitors have been identified in egg white: cystatin, ovomucoid, ovomacroglobulin (also known as ovostatin), and ovoinhibitor. Use of proteinase inhibitors in the treatment of certain diseases has renewed interest in their specificity and stability, both of which in turn depend on the tertiary structure of the inhibitor. Structural alteration to obtain molecules of desired properties requires knowledge of relationship between structure, function and stability. Aims: In view of its importance, in the present study duck ovomucoid was isolated and characterized for its physicochemical properties. Methodology: Duck ovomucoid was isolated and characterized for its physicochemical properties. Analytical gel filtration (Sephacryl S-100 HR column) was used for purification, determination of molecular weight (MW), carbohydrate content and Stokes radius.

Assessment of genetic variation in lucerne (Medicago sativa L.) using protease inhibitor activities and RAPD markers.

The aim of the present study is to identify and characterize lucerne lines resistance to weevil infestation. After three years of field screening for resistance to weevil infestation, 13 lines of lucerne were selected to assess the genotypic variations for lucerne weevil (Hypera postica Gyll.) at biochemical and molecular levels. Total phenols varied from 0.15 to 0.91 mg g-1 (DM) in these genotypes. The highest trypsin (11.11 unit mg-1 protein) and chymotrypsin (93.0 unit mg-1 protein) inhibitors activities were recorded in G-1-02 and B-4-03 lines respectively, whereas highest a-amylases inhibitor activity (14.2 unit mg-1 protein) in C-6-01. Zymogram patterns for trypsin inhibitor activity showed quantitative variations among the lines. In total 262 DNA fragments were generated when 45 deca-mer random primers were employed. Genetic variation in terms of genetic distance ranged from 0.65 to 0.85. Sequential Agglomerative Hierarchical and Nested (SAHN) clustering using the Un-weighted Pair Group Method with Arithmetic mean (UPGMA) algorithm yielded two clusters (cluster I and II) which converged at 72% similarity level. Cluster I contained most of the lines having low level of weevil infestation. High bootstrap values (>40) indicated the significance of nodes embodied in these two clusters. However, SDSPAGE analysis of the leaf proteins of these 13 lines showed no major variations except minor difference in the protein bands of molecular weights between 14 to 20 kD.

Analytical performance of three cysteine proteinase inhibitor c reagents applied in the automatic biochemistry analyzer / 中华检验医学杂志

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.

The present understanding of serine proteinase inhibitor-9 / 国际外科学杂志

Serine proteinase inhibitor9(P1-9),a charac-teristic member of serpins,has been identified as the only inhibitor of granzyme B(GrB).Accumulated evidence suggested that PI-9 inhibits GrB-induced apoptosis by blocking DNA fragmentation of target cell.Physiologically,PI-9 could protect cytotoxic lymphocytes from committing autolysis or fratricide,and play an important role in facilitating immunologic tolerance of immune-privileged sites.In addition,evidences in recent years suggest that PI-9 Was also involved in vailous pathologic processes,such as inflammation,trans plantation and immune tolerance of tumor.

Significance of Sera Cystatin C in Patients with Colorectal Cancer / 医学研究杂志

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.

Study on expression of Cystatin C in gastroenteric tumor / 中华检验医学杂志

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

Intralipid Infusions Effect on Nutrophil Elastase Level in Newborns

PURPOSE: Parenteral nutrition is given to infants who tempararily cannot take oral feeding adequately. A lipid emulsion is added to the parenteral to supply essential fatty acids. In neonatal sepsis, elastase from azuropilic granules of the neutrophils is released and rapidly bound to alpha1-Proteinase Inhibitor(alpha1-PI). The lipid emulsion has been noted to markedly inhibit chemotaxis of neutrophils, so we to measured the levels of Elastase-alpha1-Proteinase Inhibitor(E-alpha1-PI) complex to evaluate the effect of intralipid infusions on the neutrophil in newborns with sepsis. METHODS: This study evaluated 8 patients with neonatal sepsis and 12 normal newborns. We measured E-alpha1-PI complex levels in the serum of these patients by ELISA methods. RESULTS: Before infusion with lipid solution, patients with neonatal sepsis had significantly increased levels of E-alpha1-PI complex in comparison with those of vaginally delivered normal newborns. E-alpha1-PI complex levels were significantly decreased after lipid infusions of 0.5g/kg per day, but there was no further significant decrease with higher doses of the infusate. CONCLUSION: We observed the suppression neutrophil elastase levels by lipid infusions in newborn with sepsis. These results suggest that there were no appropriate chemotatic effects of neutrophil in newborn with sepsis. Therefore, we considered whether the lipid infusion was stopped if the newborn with sepsis was infused parenteral nutrition with intralipid.

Level of Plasma Elastase-α1- Proteinase Inhibitor in Patients with Behcet's Disease

BACKGROUND: The common histopathology of Behget's disease is vasculitis associated with activation of neutrophils. The level of plasma elastase a 1 proteinase inhibitor (E a 1 PI), which represents the activation of neutrophils, may be a marker of Behcet's disease. OBJECTIVE: We examined the level of plasma elastase al proteinase inhibitor to evaluate the degree of neutrophil activation in Behcet's disease. METHOD: We measured plasma elastase a 1 proteinase inhibitor in 34 cases of untreated Behcet's disease patients and 30 cases of normal individuals by an enzyme immunoassay. We also studied the differences between the levels in two clinical types of Behcet's disease, the complete and incomplete type. RESULTS: The plasma level of elastase a 1 proteinase inhibitor was significantly higher in untreated Behcet's disease patients than in healthy controls. However, there was no significant difference between the levels in the two clinical types. CONCLUSION: These data suggest that the elevated level of plasma elastase a 1 proteinase inhibitor may reflect a state of chronic activation of neutrophils in Behcet's disease immunologically and further studies will be needed to evaluate the clinical status of Behcet's disease patients by measuring levels of plasma elastase a 1 proteinase inhibitor.

The Role of Proteinase in Acanthamoeba keratitis and the Effect of Amniotic Membrane as a Proteinase Inhibitor

This study was performed to investigate the biochemical properties of Acanthamoeba proteinase, its role in the pathogenesis of Acanthamoeba keratitis and the therapeutic effect of the homogenate of amniotic membrane as a proteinase inhibitors. Acanthamoeba castellanii isolated from the keratitis patient was cultured in PYG medium, in which the excretory and secretory products were analysed. The secretory proteinases of A. castellanii wre identified using in vitro azocasein assay, activity-PAGE, and various protein substrate degradation assays, and one of them was purified and characterized. The pruified secretory proteinase was a kind of serine proteinase. Its molecular weight was 105 kDa and optimal pH was 8.5. It was able to degrade the various protein substrates such as fibronectin, IgA, IgG, fibrinogen. The various proteinase ingibitors and the amniotic membrane homogenates were tested in vitro against the purified seirne proteinase. The amniotic membrane homegenates markedly showed the inhibitory effect against the enzyme activity and this inhibitory effect was also revealed in animal study. In vivo study, this purified proteinase was infected into 14 pigmented rabbit corneas, pretreated with steroids. The corneal lesions induced by both of the purified proteinase and A. castellanii, showed similar clinical findings each other, in which the stromal infiltration and opacity with epithelial defect was revealed. These corneal lesions were significantly inhibited without any side effects of the amniotic membrane homogenates. Conclusively, Acanthamoeba proteinase was closely associated with the pathogenesis of Acanthamoeba keratitis. This study provides a successful animal model of Acanthamoeba keratitis using pigmented rabbit. And the fact that Acanthamoeba-induced corneal lesions were inhibited by the amniotic membrane homogenate, suggested that the amniotic membrane homogenate have the ability of the serine protinase inhibition further investigative studies are also necessary.