A genetic variant in the immune-related gene ERAP1 affects colorectal cancer prognosis / 中华医学杂志(英文版)

BACKGROUND@#Findings on the association of genetic factors and colorectal cancer (CRC) survival are limited and inconsistent, and revealing the mechanism underlying their prognostic roles is of great importance. This study aimed to explore the relationship between functional genetic variations and the prognosis of CRC and further reveal the possible mechanism.@*METHODS@#We first systematically performed expression quantitative trait locus (eQTL) analysis using The Cancer Genome Atlas (TCGA) dataset. Then, the Kaplan-Meier analysis was used to filter out the survival-related eQTL target genes of CRC patients in two public datasets (TCGA and GSE39582 dataset from the Gene Expression Omnibus database). The seven most potentially functional eQTL single nucleotide polymorphisms (SNPs) associated with six survival-related eQTL target genes were genotyped in 907 Chinese CRC patients with clinical prognosis data. The regulatory mechanism of the survival-related SNP was further confirmed by functional experiments.@*RESULTS@#The rs71630754 regulating the expression of endoplasmic reticulum aminopeptidase 1 ( ERAP1 ) was significantly associated with the prognosis of CRC (additive model, hazard ratio [HR]: 1.43, 95% confidence interval [CI]: 1.08-1.88, P = 0.012). The results of dual-luciferase reporter assay and electrophoretic mobility shift assay showed that the A allele of the rs71630754 could increase the binding of transcription factor 3 (TCF3) and subsequently reduce the expression of ERAP1 . The results of bioinformatic analysis showed that lower expression of ERAP1 could affect the tumor immune microenvironment and was significantly associated with severe survival outcomes.@*CONCLUSION@#The rs71630754 could influence the prognosis of CRC patients by regulating the expression of the immune-related gene ERAP1 .@*TRIAL REGISTRATION@#No. NCT00454519 ( https://clinicaltrials.gov/ ).

Characterization of digestive enzymes from captive Brazilian flounder Paralichthys orbignyanus / Caracterização de enzimas digestivas do linguado brasileiro de cativeiro Paralichthys orbignyanus

Abstract Knowledge of specific enzyme activity, along with animal habits and digestive capacity is essential in formulating an appropriate diet for any species. In this study, we evaluated and characterized the activity of digestive enzymes present in the liver, intestine, and stomach of Paralichthys orbignyanus. The effects of pH and temperature on enzyme activity were also evaluated via the use of specific substrates. The use of specific substrates and inhibitors showed strong evidence of the presence of trypsin (BApNA= 0.51 ± 0.2 mU mg-1), chimotrypsin (SApNA= 2.62 ± 1.8 mU mg-1), and aminopeptidases (Leu-p-Nan =0.9709 ± 0.83 mU mg-1) in the intestine. Optimum pH for the activity of trypsin, chemotrypsin, leucino aminopeptidase, amilase, and pepsin were 9.5, 9.0, 8.0, 7.5, and 3.5, respectively, while optimum temperatures were 50, 50, 50, 40, and 45 °C, respectively. These results provide additional information regarding the biology of Brazilian flounder and can be used as a basis for further studies regarding fish feeding physiology.

Resumo O conhecimento da atividade enzimática é essencial para formular uma correta dieta específica para espécie, além de estarem correlacionadas com o hábito da alimentação e capacidade digestive. Neste estudo determinamos e caracterizamos a atividade enzimática presente no intestino, estômago e fígado do linguado Paralichthys orbignyanus. Os efeitos da temperatura e pH sobre a atividade enzimática também foram avaliados utilizando substratos específicos. O uso de substratos e inibidores específicos mostrou uma forte evidência da presença da tripsina (BApNA = 0,51 ± 0,2 mU mg-1), quimotripsina (SAPNA = 2,62 ± 1,8 mU mg-1), e as aminopeptidases (Leu-p-Nan = 0,97 ± 0,83 mU mg-1) no intestino. O pH ótimo observado para a atividade de tripsina, quimotripsina, leucino aminopeptidase, amilase e pepsina foi 9,5, 9,0, 8,0, 7,5 e 3,5, respectivamente. A temperatura ótima observada foi 50, 50, 50, 40 e 45 °C, respectivamente. Estes resultados fornecem informações adicionais sobre a biologia do linguado brasileiro e pode ser usado como base para novos estudos sobre fisiologia alimentar.

Desenvolvimento de um modelo preditivo para identificação de perda de estabilidade e ocorrência de proteólise em leite UAT / Development of a predicative model for identification of loss of stability and proteolysis occurrence in UHT milk

Este trabalho se propôs a desenvolver um modelo preditivo para identificação de perda de estabilidade e de sedimentação em leite UAT por determinação da atividade enzimática de aminopeptidase no leite por espectrofotometria. Foram analisadas amostras de leite cru, pasteurizado e UAT após envase durante seis meses, na região Sul do Brasil. Acidez, crioscopia, gordura, extrato seco total, extrato seco desengordurado e densidade foram analisados nos leites cru e pasteurizado. Amostras de leite cru foram ainda submetidas à análise de contagem de psicrotróficos e à atividade de aminopeptidase, e amostras de leite UAT estocadas foram analisadas quanto ao grau de proteólise mediante análises sensoriais e atividade de aminopeptidase. Alterações sensoriais foram observadas em tempos de estocagem menores para amostras originadas de leite cru com contagem de psicrotróficos acima de 107 UFC mL-1. Não houve correlação entre a atividade de aminopeptidase e proteólise e também não foi observada correlação significativa entre os parâmetros físico-químicos e a ocorrência de proteólise no leite estocado. O modelo estudado não foi apto para predizer perda de estabilidade e ocorrência de proteólise no leite UAT.(AU)

The aim of this work was to develop a predictive model for identifying loss of stability and sedimentation in UHT milk by determining the enzymatic activity of aminopeptidasis in milk by spectrophotometry. Samples of raw milk, pasteurized and UHT after filling for 6 months in Southern Brazil were analyzed. Acidity, freezing point, fat, total solids, nonfat solids and density were analyzed in raw and pasteurized milk. Raw milk samples were also subjected to psychrotrophic count analysis and aminopeptidasis activity and UAT samples of stored milk were analyzed for degree of proteolysis through sensory analysis and aminopeptidasis activity. Sensory changes were observed in smaller storage time for samples of raw milk originated with psychrotrophic count above 107 CFU ml-1. There was no correlation between aminopeptidasis activity and proteolysis and there was also no significant correlation between physicochemical parameters and the occurrence of proteolysis in stored milk. The model was unable to predict loss of stability and occurrence of proteolysis in UHT milk.(AU)

Comparative effects of angiotensin II and angiotensin-(4-8) on blood pressure and ANP secretion in rats

Angiotensin II (Ang II) is metabolized from N-terminal by aminopeptidases and from C-terminal by Ang converting enzyme (ACE) to generate several truncated angiotensin peptides (Angs). The truncated Angs have different biological effects but it remains unknown whether Ang-(4-8) is an active peptide. The present study was to investigate the effects of Ang-(4-8) on hemodynamics and atrial natriuretic peptide (ANP) secretion using isolated beating rat atria. Atrial stretch caused increases in atrial contractility by 60% and in ANP secretion by 70%. Ang-(4-8) (0.01, 0.1, and 1 µM) suppressed high stretch-induced ANP secretion in a dose-dependent manner. Ang-(4-8) (0.1 µM)-induced suppression of ANP secretion was attenuated by the pretreatment with an antagonist of Ang type 1 receptor (AT₁R) but not by an antagonist of AT₂R or AT₄R. Ang-(4-8)-induced suppression of ANP secretion was attenuated by the pretreatment with inhibitor of phospholipase (PLC), inositol triphosphate (IP₃) receptor, or nonspecific protein kinase C (PKC). The potency of Ang-(4-8) to inhibit ANP secretion was similar to Ang II. However, Ang-(4-8) 10 µM caused an increased mean arterial pressure which was similar to that by 1 nM Ang II. Therefore, we suggest that Ang-(4-8) suppresses high stretch-induced ANP secretion through the AT₁R and PLC/IP₃/PKC pathway. Ang-(4-8) is a biologically active peptide which functions as an inhibition mechanism of ANP secretion and an increment of blood pressure.

Biased expression, under the control of single promoter, of human interferon alpha-2b and Escherichia coli methionine amino peptidase genes in E. coli, irrespective of their distance from the promoter

Human interferon alpha-2b and Escherichia coli methionine amino peptidase genes were cloned independently as well as bicistronically in expression plasmid pET-21a [+]. Production of human interferon alpha-2b was comparable to that of E. coli methionine amino peptidase when these genes were expressed independently in E. coli BL21-CodonPlus [DE3]-RIL. However, human interferon alpha-2b was produced in a much less amount whereas there was no difference in the production of methionine amino peptidase when the encoding genes were expressed bicistronically. It is important to note that human interferon alpha-2b was the first gene in order, after the promoter and E. coli methionine amino peptidase was the next with a linker sequence of 27 nucleotides between them

Construction of RGD10-NGR9 dual-targeting superparamagnetic iron oxide and its magnetic resonance imaging features in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct angiogenesis-specific RGD10-NGR9 dual-targeting superparamagnetic iron oxide nanoparticles, and to evaluate its magnetic resonamce imaging (MRI) features in nude mice and potential diagnostic value in tumor MRI.</p><p><b>METHODS</b>Dual-targeting peptides RGD10-NGR9 were designed and synthesized. Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles were synthesized by chemical co-precipitation method and the surface was modified to be hydrophilic by coating with dextran. The dual-targeting peptides RGD10-NGR9 were conjugated to USPIO. Cell binding affinity and up-taking ability of the dual-targeting USPIO nanoparticles to integrin ανβ3-APN positive cells were subsequently tested by Prussian blue staining and phenanthroline colorimetry in vitro. The RGD10-NGR9 conjugated with USPIO was injected intravenously into xenograft mice, which were scanned by MRI at predetermined time points. The MRI and contrast-to-noise ratio (CNR) values were calculated to evaluate the ability of dual-targeting USPIO as a potential contrast agent in nude mice.</p><p><b>RESULTS</b>P-CLN-Dextran-USPIO nanoparticles with stable physical properties were successfully constructed. The average diameter of Fe3O4 nanoparticles was 8-10 nm, that of Dextran-USPIO was about 20 nm and P-CLN-Dextran-USPIO had an average diameter about 30 nm. The in vitro studies showed a better specificity of dual-targeting USPIO nanoparticles on proliferating human umbilical vein endothelia cells (HUVEC). In vivo, RGD10-NGR9-USPIO showed a significantly reduced contrast in signal intensity and 2.83-times increased the CNR in the tumor MRI in xenograft mice.</p><p><b>CONCLUSION</b>This novel synthesized RGD10-NGR9 dual-targeting USPIO is with better specific affinity in vitro and in vivo, and might be used as a molecular contrast agent for tumor angiogenesis MRI.</p>

Pathology of C3 Glomerulonephritis

C3 glomerulonephritis (C3GN) is a recently described entity that shows a glomerulonephritis on light microscopy, bright C3 staining and the absence of C1q, C4, and immunoglobulins on immunofluorescence microscopy and mesangial and/or subendothelial electron-dense deposits on electron microscopy. The term 'C3 glomerulopathy' is often used to include C3GN and dense deposit disease (DDD), CFHR5 nephropathy, those of which result from dysregulation of the alternative pathway of complement. C3GN shares some aspects of atypical hemolytic uremic syndrome, MPGN, late stage of post infectious glomerulonephritis and other glomerulonephrtis. When C3GN is considered, measurement of serum complement proteins including C3, CFH, CFI, CFB and testing for the presence of C3 nephritic factor, anti-factor H autoantibodies are necessary. To screening for mutations, genes that encode complement regulators should be evaluated. This disorder equally affected all ages, both genders, and typically presented with hematuria and proteinuria. In both the short and long term, renal function remained stable in the majority of patients.

The Impact of Marriage Migrant Women's Korean Language Ability, Self-esteem, and Acculturative Stress on Their Family Health: Focused on Mediating Effects of Marital Adjustment

PURPOSE: This study was conducted for the purpose of a structural model analysis of family health of women who came to Korea for being married to Korean men. METHODS: The data were collected from 260 immigrant women at multicultural centers located in C and B cities from May 10th to 30th, 2012. The variance analysis on the samples was conducted by using the maximum likelihood minimization function with AMOS 7.0. The fitness was evaluated by means of the SRMR, RMSEA, CFI, and TLI with a 90% confidence interval. RESULTS: First, immigrant women's self-esteem and acculturative stress were found to have significant direct effects on their family health. Second, their self-esteem and acculturative stress have direct effects on their marital adjustment. Third, their marital adjustment was found to have significant direct effects on their family health. Forth, immigrant women's Korean language ability was found not to have significant direct effects on their marital adjustment and family health. CONCLUSION: In order to enhance the family health of immigrant women, it is necessary to develop and apply nursing programs in consideration of immigrant women's self-esteem, marital adjustment and acculturative stress.

Plutella xylostella granulovirus PP31 interacts with two host proteins / 病毒学报

Protein-protein interactions between viruses and hosts are common during viral infection and replication. In this study, a cDNA library from larvae of Plutella xylostella was constructed and used for screening of genes encoding proteins interacting with Plutella xylostella granulovirus (PlxyGV) proteins. Two cDNA clones containing genes encoding proteins interacting with PlxyGV PP31 were identified by yeast two-hybrid assays. Sequence analysis showed that the genes encoded homologues of receptor for activated protein C kinase (RACK) and methionine aminopeptidase 2 (MetAP2), respectively. The P. xylostella rack gene and the PlxyGV pp31 was expressed in an E. coli strain to produce proteins fused with a 6-His or a GST tag. It was shown that the rack was expressed as a 38kD peptide as prospected. The 38kD His-tagged peptide was co-purified with GST-PP31 by GST-bind resin in GST-pulldown assays, confirming interaction between the PlxyGV PP31 and the RACK protein of P. xylostella.

Association of defective HLA-I expression with antigen processing machinery and their association with clinicopathological characteristics in Kazak patients with esophageal cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It has been confirmed that defective expression of human leukocyte antigen class I (HLA-I) molecules can contribute to the immune evasion of cancer cells in some types of cancer. The aim of this study was to examine the expression of HLA class I antigen and the antigen-processing machinery (APM) components in esophageal squamous cell carcinoma (ESCC) and their role in high risk human papillomavirus (HPV) infection, and to analyze their association with histopathological characteristics in the Kazak ethnic group.</p><p><b>METHODS</b>A total of 50 formalin-fixed, paraffin-embedded ESCC lesions were collected from the First Affiliated Hospital of Xinjiang Medical University, China. The expression levels of HLA-I antigen and APM components were determined by immunohistochemistry; the HPV DNA were detected using polymerase chain reaction (PCR).</p><p><b>RESULTS</b>A high frequency of down-regulation or loss of expression of HLA and APM components were found in esophageal cancer in Kazak people. HLA-I, TAP1, CNX, LMP7, Erp57, Tapasin and ERAP1 were down-regulated in 68%, 44%, 48%, 40%, 52%, 32% and 20% of ESCC lesions then, respectively. The loss of expression of HLA-I antigen was significantly correlated with part of the APM components and positively correlated with high risk HPV16 infection. TAP1, CNX, LMP7, Erp57 and Tapasin loss were significantly associated with tumor grading, lymph node metastasis and depth of invasion (P < 0.05).</p><p><b>CONCLUSION</b>Our results suggest that APM component defects are a mechanism underlying HLA-I antigen down-regulation in ESCC lesions, and indicate that the loss expression of HLA-I and APM components will become an important marker of ESCC and analysis of HLA-I and APM component expression can provide useful prognostic information for patients with ESCC from the Kazak ethnic group.</p>

Study on a model of street vended food choices by Korean high school students

Street vended food (SVF) includes food and beverages prepared and sold outdoors or in public areas by street merchants for consumption on the scene or later without further preparation. Due to its low price and convenience, SVF has been popular in Korea for a long time, particularly with high school students. Beyond Korea, SVF is also popular in southeast Asia and southern Africa in the form of ready-to-eat food. This study on high school students, who are main consumers of SVF in Korea, focused on the factors that affect consumer loyalty. The study was performed by questionnaire and used AMOS software to develop a structural equation model. The results of verifying the model's fidelity were chi2 = 685.989, df = 261, GFI = 0.851, AGFI = 0.814, NFI = 0.901, CFI = 0.907, RMR = 0.048, indicating a satisfying structural model. SVF quality and service, emotional response, and the physical environment had a statistically significant effect on consumer loyalty. In contrast, SVF sanitation had no statistically significant effect on consumer loyalty. Based on these results, the sanitary management of SVF needs to be addressed immediately combined with education for SVF providers to maintain a clean environment.

Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa: solving the structure - function anomaly of a reported aminopeptidase

The N-terminal amino acid sequence of an aminopeptidase from Japanese edible mushroom, Grifola frondosa, was reported to have high similarity with that of a serine proteinase from basidiomycete, Agaricus bisporous (Nishiwaki and Hayashi, 2001). The full-length cDNA and the corresponding genomic DNA of the enzyme were cloned, based on the reported N-terminal amino acid sequence. The predicted open reading frame (ORF) of the cloned cDNA, encoding a product of 379 amino acids, was expressed in E. coli using pET expression vector. The expressed pro-enzyme (40 kDa) underwent autolysis to produce the mature protein (30 kDa) and a pro-peptide (10 kDa). The mature protein and the pro-peptide remained tightly bound to each other and could not be separated by Ni-NTA metal affinity chromatography or Q-Sepharose ion-exchange chromatography. The enzyme was inactive in the bound form. Upon treatment with subtilisin, the bound pro-peptide was further hydrolyzed and a high serine proteinase activity was recovered. No aminopeptidase activity was detected at any stage of the protein processing. These results clearly indicated that the N-terminal amino acid sequence and the function of the reported aminopeptidase were not derived from the same protein entity and hence caused the structure-function anomaly.

Expression in Escherichia coli, purification and enzymatic properties of chicken aminopeptidase H / 生物工程学报

Aminopeptidase H (APH) is an universally distributed aminoendopeptidase in the tissue of many organism. However, it is hard to investigate its mechanism underlying the catalysis and the function in cell. In this paper, the full DNA sequence of this enzyme was cloned from chicken liver, then subcloned to the vector pET22 b(+). The recombined vector was transformed into E. coli Rosetta(DE3), and the APH gene was expressed by the induction of IPTG. It was found the recombinant protein exhibited same mo lecular weight as authentic APH on SDS-PAGE analysis; the expression level increased with induction time and approached maximum of 94.7 mg/L till 6 hours, which contained 16.7% of the total protein. Moreover, this recombinant protein showed similar prop erties of subunit composition, thermal stability and optimum pH with native APH, based on the enzymatic assay, purification and analysis of enzymological properties. Therefore, it is confirmed that APH was expressed in this prokaryote system with a high-level of 1636 u/L aminopeptidase activity. These results would help to elucidate the catalysis mechanism and biological function of APH by providing enough material.

Expression Change of Complement Regulator Genes of the Human Astrocytoma Cell Line after A beta1-42 and Interferon Gamma Administration

BACKGROUND: We determined the changes of complement regulator gene expression in the amyloid-beta1-42(A beta1-42) and interferon-gamma(IFN-gamma)-stimulated human astrocytoma cell line. METHODS: The human astrocytoma cell line, U373MG, was stimulated with IFN-gamma(62.5-1,000U/ml) in the presence or absence of aggregated A beta1-42(1-20micrometer) for 24 hours. Messenger RNA expression of C1 inhibitor(C1-INH), complement factor I(CFI), clusterin, vitronectin, decay accelerating factor(DAF), membrane cofactor protein(MCP), and CD59 was measured by quantitative real-time reverse transcriptase-PCR. RESULTS: IFN-gamma(final concentration, 500U/ml) markedly increased the expression of mRNA for C1-INH in a time dependent fashion. A beta1-42(final concentration, 2micrometer) induced a slight increase in the expression of C1-INH. Messenger RNAs for CFI and clusterin were minimally increased, but other regulators were unchanged or decreased by either A beta1-42 or IFN-gamma. IFN-gamma overrode A beta1-42-induced mRNA expression of C1-INH when the cells were treated with these two reagents together. CONCLUSION: Among the complement regulator genes in the human astrocytoma cell line, U373MG, only C1-INH was significantly up-regulated by IFN-gamma with or without A beta1-42 administration.

An Explanatory Model on Functional Capacity in Patients with Chronic Obstructive Pulmonary Disease / 성인간호학회지

PURPOSE: This study was conducted to develop and test an explanatory model on functional capacity in patients with chronic obstructive pulmonary disease using path analysis. METHODS: Data were collected from 149 chronic obstructive pulmonary disease patients using 6-minute walk test, measurement of oxygen saturation, pulmonary function test, and self-reported questionnaires from June to October, 2005. The collected data were analyzed using SPSS/WIN 12.0 program and AMOS/WIN 4.0 program. RESULTS: The overall fitness indices of modified model were good( chi-square = 14.324, p = .281 GFI = .981, RMSEA = .006, AGFI = .944, NFI = .927, NNFI = .999, CFI = .999, PNFI = .613, chi-square /df = 1.194). Functional capacity was influenced directly by age(beta = -.304, p = .000), dyspnea(beta = -.278, p = .000), self-efficacy(beta = .240, p = .000), social support(beta = .175, p = .004), pulmonary function(beta = .169, p = .008), and oxygen saturation(beta = .099, p = .048). These variables explained 39.3% in functional capacity. CONCLUSION: The findings of this study suggest that comprehensive nursing interventions should focus on decreasing dyspnea and increasing self-efficacy, social support, and oxygen saturation. In this perspective, pulmonary rehabilitation would be an effective strategy for improving functional capacity in patients with chronic obstructive pulmonary disease.

Identification of digestive hydrolases in the larval midgut of the camel nasal bot fly cephalopina titilla tor clark [diptera: Oestridae]

Activities of digestive hydrolases associated with midgut of the third instar larva of Cephalopina titillator were investigated. Based on the hydrolysis of synthetic substrates and optimum pH, it was found that C. titillator midgut contains trypsin-like [optimum pH, 9], chymotrypsin esterase-like [optimum pH, 8], carboxypeptidase A and B [optimum pH at 8.5 and 7 respectively], alkaline- and acidphosphatase [optimum pH at 9 and 5 respectively] and membrane bound leucine aminopeptidase [optimum pH, 8]. An acid proteinase activity was detected, by the ability to hydrolyze acid denaturated haemoglobin; and it seems to be close to pepsin than cathepsin-like enzyme. It has a maximum activity at pH 3.5. alpha-Glucosidase activity, and was also identified [optimum pH at 6] in the midgut, and seems to be membrane bound

Experimental study of the enhancement effect of aminopeptidase N inhibitor ubenimex on the differentiation induction activity of all-trans-retinoic acid in acute promyelocytic leukemia cells and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of aminopeptidase N inhibitor ubenimex on differentiation induction of all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia (APL) cells and its mechanism.</p><p><b>METHODS</b>The expression of CD11b was analyzed by flow cytometry and nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cell differentiation of APL cells. The expressions of c-Myc, ERK1/2, p38MAPK protein and the phosphorylation of ERK1/2, p38MAPK protein in NB4 cells were detected by Western blot assay.</p><p><b>RESULTS</b>Ubenimex alone induced no significant changes in NBT reduction activity and CD11b expression but potentiated the differentiation induction activity of ATRA in APL cells. 100 microg/ml of ubenimex could enhance the NBT reduction activity induced by 10 nmol/L of ATRA, intensify the down-regulation of c-Myc protein expression and inhibit the phosphorylation of p38MAPK protein induced by 10 nmol/L of ATRA in NB4 cells.</p><p><b>CONCLUSIONS</b>Ubenimex could potentiate ATRA induced differentiation in APL cells, which may be correlated with the inhibition of p38 MAPK protein phosphorylation and regulation of c-Myc protein expression.</p>

Effect of aminopeptidase inhibitor on differentiation induction activity of all-trans retinoic acid in human acute promyelocytic leukemia NB4 cells and its mechanism / 中国实验血液学杂志

This study was purposed to investigate whether aminopeptidase inhibitor, bestatin, can potentiate all-trans retinoic acid (ATRA)-inducing differentiation in NB4 cells, and to explore its mechanism. The NB4 cells were exposed to either bestatin and ATRA alone or in combination, the morphological changes of NB4 cells were observed by optical microscopy, the CD11b expression was measured by flow cytometry, the function of defferentiation cells was analyzed by nitroblue-tetrazolium (NBT) reduction assay, the mRNA expressions of c-myc and c-EBPepsilon in NB4 cells were detected by RT-PCR, the c-Myc protein expression was determined by Western blot. The results showed that treatment with bestatin alone induced no significant changes in morphology, NBT reduction activity and CD11b expression in NB4 cells. NB4 cells incubated with 10 nmol/L ATRA plus 100 microg/ml bestatin showed more morphologic feature of metamyelocyte and band neutrophil than ATRA alone treated cells. 100 microg/ml bestatin enhanced the NBT reduction activity in NB4 cells induced by various concentrations of ATRA (10, 20, 40 nmol/L). The effects of various concentrations of ATRA in combination with 100 microg/ml bestatin were statistically different from the effect of ATRA alone (P < 0.01). From 48 to 96 hours, 100 microg/ml bestatin time-dependently increased NBT reduction in NB4 cells induced by 10 nmol/L ATRA (P < 0.01). 10 nmol/L ATRA plus 100 microg/ml bestatin for 72 hours prominently elevated CD11b expression in NB4 cells as compared with ATRA alone treated NB4 cells (P < 0.01). There was a substantial decrease in c-myc mRNA levels when 100 microg/ml bestatin was added to 10 nmol/L ATRA (P < 0.05). Various concentrations (50, 75, 100 microg/ml) of bestatin combined with 10 nmol/L ATRA down-regulated the expression of c-Myc protein, which was negatively correlated with the NBT reduction activity of NB4 cells induced by 10 nmol/L ATRA alone or plus bestatin at various concentrations (r = -0.940, P = 0.017). However, 100 microg/ml bestatin plus 10 nmol/L ATRA could not induce any significant changes in the levels of c-EBPepsilon mRNA as compared with ATRA alone treated NB4 cells. It is concluded that an aminopeptidase inhibitor bestatin can potentiate ATRA-inducing differentiation of NB4 cells, possibly by down-regulating c-myc expression in synergy with ATRA.

preliminary characterization of digestive proteease in the mid-gut of the third larval instar of gasterophilus intestinalis

Protinases contained in the mid-gut of the early third instar of Gasterophilus intestinals have been tentatively identified by midgut hydrolysis of synthetic substrates. Trypsin was identified by maximal hydrolysis of benzoyl-DL-arginine-p-nitroanilide [BApNA] at pH 8 and chymotrypsin by maximal hydrolysis of benzoyl-L-tyrosine ethyl ester [BTEE] at pH 9. Carboxypeptidase A and B were identified by their maximal hydrolysis of hippuryl-DL-phenyllactic acid and hippuryl-L-arginine at pH 9 and 8 respectively. Aminopeptidase was identified by maximal hydrolysis of leucine-p-nitroanilide at pH 9. The mid-gut also showed activity of aspartic proteinase and identified it as cathepsin D.A drug [Banmith 12.5% pyrantel tartarate] used for the routine control of helminthes parasites of horses and donkies in Egypt was used in vitro to investigate its effect on the optimal activity of studied enzymes. It was found that the drug has no effect on trypsin and carboxypeptidase A while it decreases the activity of chymotrypsin, aminopeptidase and acidic proteinase and was also found to increase the activity of carboxypeptidase B greatly

Study on the properties of a novel glycine amino peptidase from Actinomucor elegans / 生物工程学报

The glycine amino peptidase of Actinomucor elegans was studied in this work. For the enzyme production Actinomucor elegans was cultured with an enzyme producing medium. Then the cells were collected and subjected to enzyme purification. The glycine aminopeptidase was purified 592 times by a DEAE-Toyopearl column, a Toyopearl HW 65-C column and a Superdex 200 column subsequently and the purified enzyme had a specific activity of 14.2 u/mg. The enzyme was estimated to have molecular mass of 320kD by gel filtration and a subunit size of 56.5kD by SDS-PAGE. It hydrolyzes glycine residue containing substrates such as glycine-betanaphthylamine more efficiently than those containing other amino acid residue. Addition to Gly-betaNA, the enzyme could also hydrolyze Ala-betaNA, Met-betaNA, Leu-betaNA, Arg-betaNA and Ser-betaNA but it had no activity on the substrates such as Trp-betaNA, Pyr-betaNA, Pro-betaNA, Asp-betaNA, Lys-betaNA, Val-betaNA. It was also observed when the glycine-betanaphthylamine concentration was higher than 2mmol/L the enzyme showed a substrate inhibition, and at the 20 mmol/L the enzyme only showed about 55% activity as it showed at the 2mmol/L. Whereas no such phenomenon was observed on the other substrate such as alanine-betanaphthylamine. The optimal temperature and pH for the reaction of this enzyme is 30 degrees C and pH 8.0, respectively. The Km and Kcat of the enzyme for glycine-betanaphthylamine is 0.24 mmol/L and 100.8 s(-1), respectively. Zn2+, Cu2+ and Cd2+ suppress almost all activities of the enzyme at the concentration of 1.0 mmol/L. Based on the study of chelating reagents, GAP belongs to the metalloenzyme. When a gelatin solution was hydrolyzed with 0.5% of alkaline proteinase together with glycine aminopeptidase at 50 degrees C for 18 hours, the glycine aminopeptidase could improve the hydrolysis degree of the protease. The total free amino acid was improved about 13% and although the enzyme mainly had the activity to hydrolyze the glycine residue, individual amino acids analysis with an amino acid analyzer showed that the contents of glycine, proline, alanine, arginine and glutamate were considerably increased. The results of this study showed that the glycine aminopeptidase would be useful in the food industry.