Optimization of enzymatic digestion conditions of shrimp allergens in mass spectrometry by response surface methodology / 预防医学

Objective@#To optimize the enzymatic digestion conditions of trypsin, so as to improve the testing capacity of mass spectrometry for shrimp allergens.@*Methods@#The enzymatic digestion test was carried out by response surface methodology for optimizing pH, temperature and time. After enzymatic hydrolysis, the peptides were separated by chromatography and determined by high-resolution mass spectrometry with Q-orbitrap. The allergen protein was identified and quantified by UniProt database and MaxQuant software.@*Results@#Two allergen proteins, tropomyosin and arginine kinase, were isolated from shrimp, and their intensities ranged from 100.2×106 to 436.5×106. Response surface analysis showed that when the digestion time was 4.29 hours, the temperature was 44.15 ℃ and pH value was 6.55, the maximal intensity of the allergen proteins was 457.48×106. The experiment was validated with the digestion time of 4.2 h, pH value of 6.5, and temperature of 44 ℃, then resulted in the average intensity of 448.1×106. The deviation from the predicted value was 2.1%.@*Conclusions@#The conditions of enzymatic digestion can be optimized by response surface methodology. The enzyme may have the best performance with the pH value of 6.5, temperature of 44 ℃ and digestion time of 4.2 hours.

Vaccine pretreatment for quantification of 146S antigen in foot-and-mouth disease vaccines by high performance size exclusion chromatography / 生物工程学报

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 μg/mL by ultracentrifugation, 9.7 and 10.4 μg/mL by PEG precipitation, and 10.5 and 10.4 μg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 μL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.

Expression, purification and activity verification of human norovirus NS6 protein / 中华实验和临床病毒学杂志

Objective@#To express the NS6 nonstructural protein of human norovirus (NoV) in Escherichia coli, and to detect its enzymatic activity after purification.@*Methods@#Human NoV NS6 gene was cloned into the prokaryotic expression vector pDE1 and then was transformed into E. coli BL21 for expression. NS6 protein was purified by Ni-NTA affinity chromatography and Superdex 200 pg column. The activity of NS6 protein was determined by digestion of fusion protein 15VP1-6P at 37 ℃.@*Results@#Human NoV NS6 protein was stably and highly expressed in E. coli. After purification, the expressed product reached a purity of more than 95%, and the relative molecular weight of NS6 protein was about 23×103 Da. NS6 protein could cleave the fusion protein containing rhinovirus 3C cleavage site.@*Conclusions@#The nonstructural protein NS6 of human NoV was successfully expressed in Escherichia coli, which laid a foundation for further study on the pathogenesis of human NoV.

Análisis genético de la resistencia a isoniacida en cepas de Mycobacterium tuberculosis / Genetic analysis of isoniazid resistance in strains of Mycobacterium tuberculosis

Objetivo: Analizar genéticamente la resistencia a Isoniacida en cepas de Mycobacterium tuberculosis mediante PCR-RFLP de la región S315T del gen katG. Materiales y métodos: El estudio se realizó a partir de cultivos positivos de Mycobacterium tuberculosis receptados en el Centro de Referencia Nacional de Micobacterias, durante el período 2013 ­ 2014. El ADN extraído fue cuantificado y evaluada su pureza, por espectrofotometría. Para determinar el polimorfismo en la región 315 del gen katG a partir de un producto de amplificación de 630 pb se realizó digestiones con las enzimas de restricción MspI y SatI. Resultados: Del total de 498 cepas analizadas, 215 cepas presentaron características fenotípicas de resistencia a isoniacida (32,6 % monorresistencia, 19,5 % MDR y 47,9 % polirresistencia), 283 cepas eran sensibles. 251 cepas correspondieron a pacientes vírgenes al tratamiento (VT); 174 fueron pacientes antes tratados (AT) y 73 fueron pacientes se encontraban con tratamiento (CT). La mayoría de los casos provenía de la provincia del Guayas (77,2 %). La PCR-RFLP-SatI presentó alto porcentaje de sensibilidad (98,6 %) y especificidad (98,2 %), mientras que con la enzima MspI el porcentaje de sensibilidad fue 88,8 % y 7,4 % de especificidad. Conclusión: La PCR-RFLP-SatI demostró ser específica y económica para la detección de resistencia a isoniacida, proporcionando resultados de forma rápida, la aplicación de esta técnica como apoyo para el diagnóstico permitiría al paciente acceder a un tratamiento más oportuno.

Objective: Genetically analyze the Isoniazid resistance in Mycobacterium tuberculosis cultures by PCR-RFLP of the S315T region of the atG. Materials and methods: The study was carried out in positive cultures of Mycobacterium tuberculosis, received at the National Reference Center of Mycobacteria, during the period 2013-2014. The DNA extracted was quantified and its purity was evaluated by spectrophotometry. To determine the polymorphism in the 315 region of the at G gene, digestions were made with the restriction enzymes MspI and SatI from a 630 bp amplification product. Results: Of 498 culture strains analyzed, 215 strains showed phenotypic characteristics of resistance to Isoniazid (32,6 % monoresistance, 19,5 % MDR and 47,9 % polyresistance) and 283 strains were sensitive. 251 strains corresponded to virgin patients to treatment (VT); 174 were patients before treated (AT) and 73 were patients treated (CT). The majority of cases came from the province of Guayas (77,2 %). The PCR-RFLP- SatI presented a high percentage of sensitivity (98,6 %) and specificity (98,2 %), while with the MspI enzyme the sensitivity percentage was 88,8 % and 7,4 % specificity. Conclusion: The PCR-RFLP SatI proved to be specific and economical for the detection of resistance to isoniazid, providing results quickly, the application of this technique as a support for the diagnosis would allow the patient to access a more timely treatment.

An improved method for stem cell derivation from human great saphenous vein / 生物工程学报

The objectiue was to explore how to improve stem cell derivation from human great saphenous vein. After the saphenous vein was cut into small pieces, the cells of the vessel wall were obtained by tissue adherent method and digestion with type Ⅱ collagenase. The morphological changes of blood vessel wall were observed under inverted microscope. The survival of vascular wall cells was assessed by trypan blue staining. Stem cells doubly positive for CD34 and CD117 were sorted out by immunofluorescent staining and flow cytometry. The cells obtained by tissue adherence method exhibited signs of fibrotic changes and aging at the third passage (P3), while the cells extracted by enzymatic digestion still showed colony-like growth. Survival rates of these two groups of cells were (91.7±1.2)% and (97.2±0.7)%, (P=0.005). The results of flow cytometry showed that the positive rates of CD34 and CD117 double positive cells in these two groups were (0.16± 0.05)% and (0.44±0.07)%, respectively, with statistical significance (P=0.005). Immunofluorescent staining showed that the positive rates of double positive stem cells in the two groups were (89.41±2.06)% and (94.03±1.83)%, P<0.05 one week after the sorted stem cells were cultured. The positive rates of CD31, VEGF2 and SMA in the stem cells determined by flow cytometry were (0.12±0.01)%, (0.19±0.02)% and (0.45±0.01)%, respectively, which were not statistically different from those of the control groups. This could rule out substantial inclusion of mature endothelial cells and smooth muscle cells. Tube forming experiment confirmed that these vascular stem cells had developmental plasticity. More viable and morphologically healthy vascular stem cells can be derived by enzymatic digestion. These cells can be widely used in clinical and basic research.

Diatom Detection Using Enzyme Combined with Strong Acid Digestion Method / 法医学杂志

OBJECTIVES@#To detect diatom in the organs of drowners by enzyme combined with strong acid digestion method, and evaluate its application value.@*METHODS@#A total of 40 cases which have been identified as drowning in local region were collected. Samples of the lung, liver, kidney, and the water of the scene were also gathered from each case. Strong acid digestion method, enzyme combined with strong acid digestion method, and enzymic digestion method were respectively performed to detect the diatom in the samples. The comparative analysis was made on digestion time, digestive power and detection rate of diatom, etc.@*RESULTS@#Enzyme combined with strong acid digestion method was significantly better than enzymic digestion method on digestion time and digestive power; enzyme combined with strong acid digestion method were obviously superior to strong acid digestion method on the detection rate of diatom.@*CONCLUSIONS@#Enzyme combined with strong acid digestion method combines the advantages of strong acid digestion method and enzymic digestion method. It has the characters of operation safety with little pollution to environment, which is worthy of further popularization and practice.

Antimicrobial Activity of Protein Hydrolysate from Marine Molluscs Babylonia spirata (Linnaeus, 1758).

The present study is to investigate the antimicrobial activity of protein hydrolysate of marine water mollusks Babylonia spirata (Linnaeus, 1758). Protein hydrolysate was prepared from tissue of Babylonia spirata by enzymatic hydrolysis. Enzyme digestion were carried out with the enzyme Trypsin. The protein concentration was estimated by Bradford’s method and the protein quantification was done by using SDS PAGE analysis. Antibacterial assay was carried out against four bacterial pathogens by agar well diffusion method and antifungal activity was performed against three human pathogenic fungal strains. 2.6mg/ml protein concentration was estimated by Bradford’s method and 40 to 200 kDa protein bands were resulted in SDS PAGE analysis. In antimicrobial activity, the maximum zone of inhibition was observed against Staphylococcus aureus22.16 +1.04mm at 1000μg/ml concentration and the maximum zone of inhibition was observed in Aspergillus fumigatus13.5+0.5 in 1000μg/ml concentration. These results are signify that the protein hydrolysate of marine molluscs Babylonia spirata express remarkable antimicrobial activity.

Application of modified enzyme digestion method in rapid primary culture of human glioma cells / 解放军医学杂志

Objective To explore the applied value of modified enzyme digestion method in primary culture of human glioma cells. Methods A traditional enzyme digestion method was modified based on literatures and our work experience. The glioma cells from 32 glioma patients with different grades were primarily cultured by the modified enzyme digestion method. The morphological features of these cells were observed under an inverted phase contrast microscope. The primary cells were purified by differential adhesion during passage. The primary cells were identified by immunofluorescence technique, and the growth curves were drawn by cell proliferation assays (CCK-8 method) for investigating the proliferation of the cells cultured in vitro. Results The primary human glioma cells were successfully cultured and transferred by the new method, with a success rate of 87.5%. The cells cultured successfully in vitro showed good adherent growth, stable morphologies, thus can be passaged. Fluoroimmunoassay showed positive expression of glial fibrillary acidic protein, which confirms the cultured cells were glioma cells. Cell proliferation assays revealed active cell proliferation in vitro, the higher the tumor grade, the higher the proliferative capacity. Conclusion The modified enzyme digestion method is simpler and more efficient for primary culture of human glioma cells, and the success rate is also higher, thus being able to provide a good guarantee for fundamental research of glioma.

Progress in the isolation and purification methods of adipose-derived stem cells / 中国比较医学杂志

Adipose-derived stem cells ( ASCs ) as potential seeded cells have been widely used in tissue engineering.Thus to obtain enough, high activity, high purity adipose-derived stem cells is the particular important premise of the application in tissue engineering.In this paper, the isolation and purification methods of ASCs were reviewed and the merit and demerit of different methods were compared in order to provide theoretical basis for safe and high-effective isolation and purification of ASCs.

Identification and characterization of Legionella busanensis strains isolated from environmental and air conditioning cooling water in Guangzhou / 中华微生物学和免疫学杂志

Objective To identify two suspected Legionella pneumophila (L.pneumophila) strains isolated from environmental water and air conditioning cooling water systems in Guangzhou city.Methods The two strains were identified by their cultural characteristics,biochemical test,Legionella-specific primer PCR identification,PCR-enzymatic digestion analysis,16S rRNA,mip and rpoB gene sequencing analysis.Results The two suspected L.pneumophila isolates were identified as gram-negative bacillus appeared as white colonies on BCYEα-agar after incubation for 48 hours at 36℃.Both isolates were positive for oxidase,gelatinase and hydrolysis of hippurate,and negative for urease activity and nitrate reduction.Their phenotypic characteristics were similar to those of L.pneumophila strains.Results of PCR identification by using Legionella-specific primer were positive.Enzymatic digestion analysis showed that the 226 bp PCR products of two isolates were not digested by Taa Ⅰ.The two strains were classified as Legionella busanensis as indicated by gene sequencing analysis of 16S rRNA,mip and rpoB gene.Conclusion Two L.busanensis strains were first isolated from environmental and air conditioning cooling water systems in China.Due to their biochemical characteristics,L.busanensis strains were commonly misidentified as L.pneumophila,but could be effectively identified by PCR-enzymatic digestion analysis and multiple genes identification.

Produção e regeneração de protoplastos de Colletotrichum gloeosporioides em diferentes condições de cultivo / Production and regeneration of protoplasts from Colletotrichum gloeosporioides in different conditions

A obtenção de protoplastos de fungos, utilizando-se enzimas degradadoras de parede celular, tem sido o método mais utilizado em processos de transformação genética. Foram testados dois tipos de estruturas fúngicas (micélio e conídios), diferentes concentrações enzimáticas (5, 10, 20 mg), estabilizadores osmóticos (NaCl 0,7 mol.L-1 pH 5,7; (NH4)2SO4 1,2 mol.L-1 pH 5,8; KCl 0,7 mol.L-1 pH 5,8; MgSO4 0,7 mol.L-1 pH 5,5; Sacarose 0,5 mol.L-1 pH 5,7; SorbitoL 0,6 mol.L-1 pH 5,7) e seis tempos de exposição dos protoplastos ao sistema lítico, para estabelecer condições otimizadas de obtenção e regeneração de protoplastos de Colletotrichum gloeosporioides, agente relacionado a mancha manteigosa em cafeeiros. Protoplastos de C. gloeosporiodes foram obtidos em maior quantidade quando o micélio foi exposto durante 4 horas, com 10 mg.mL-1 de Lysing Enzime em KCl 0,7 mol.L-1 que se apresentou como melhor estabilizador osmótico, com frequência de regeneração de 11,64%.

The isolation and regeneration of protoplasts from fungal cells, using several cell wall degrading enzymes, has been the most common method to prepare competent cells for genetic studies of filamentous fungi. In this work two types of fungal structures, different enzyme concentrations (5, 10, 20 mg), osmotic stabilizers (NaCl 0,7 mol.L-1 pH 5,7; (NH4)2SO4 1,2 mol.L-1 pH 5,8; KCl 0,7 mol.L-1 pH 5,8; MgSO4 0,7 mol.L-1 pH 5,5; Sacarose 0,5 mol.L-1 pH 5,7; Sorbitol 0,6 mol.L-1 pH 5,7), and six exposure times to establish optimum conditions of isolation and regeneration of protoplasts of Colletotrichum gloeosporioides, agent of blister spot on coffee, were tested. Protoplast of C. gloeosporioides were obtained in greater quantities when the mycelium was exposed for 5 hours with 15mg.mL-1 of Lysing Enzyme in 0.7 mol.L -1 of KCl as osmotic stabilizer, presenting a regeneration rate of 11.64%.

Application of Tissue-explant Technique in Culturing Rat Submandibular Gland Cells / 中国医科大学学报

Objective To explore the application of tissue-explant technique in culturing rat submandibular gland cell(RSGC)and the characteristics of RSGC.Methods RSGCs were cultured using tissue-explant technique.The cells were purified by enzymatic digestion and differential adhesion.The cell phenotype was identified by cytokeratin-8 immunohistochemical staining.Cellular morphology was observed and photographed under inverted microscope.The cell viability and growth were determined by a double-staining procedure using FDA-PI and MTT assay,respectively.Results Cytokeratin-8 was positive stained in the immunohistochemical staining.The cell viability was more than 95%.The cell growth curve showed that RSGCs were in logarithmic phase since day 5.Conclusion Tissue-explant technique is an easy way to purify plentiful RSGC with normal functions,and it can be used in further research of tissue engineering of submandibular gland.

Characterization of dengue virus serotype 1 in epidemics in Porto Velho, Rondônia, in 2001-2003

The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universal primers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98 percent similarity to sequences from Southeast Asia that were obtained from GenBank.

A primeira epidemia de febre do dengue no Estado de Rondônia, Região Ocidental do Brasil foi registrado em 1997, sem confirmação laboratorial. Em seguida, houve uma epidemia descrita em 1998, em Manaus, no vizinho Estado do Amazonas, onde os vírus DENV-1 e DENV-2 foram isolados de pacientes. No presente artigo, foi descrito a caracterização do sorotipo do vírus dengue isolado de pacientes com suspeitas clinicas de dengue em Porto Velho, Rondônia, entre 2001 a 2003. Foram coletadas 150 amostras de sangue, entre primeiro e quinto dia de sintomas. Setenta amostras de vírus isolados foram submetidas a identificação do dengue pela RT-PCR usando primers universais para gene da NS1 do DENV que amplifica um fragmento de 419pb. O amplicon obtidos foram submetidos a digestão enzimática para caracterização do sorotipo viral. Todas as amostras analisadas foram DENV-1. A seqüência nucleotídica de um dos amplicons aleatoriamente selecionada também DENV-1 demonstrou 98 por cento similaridade com as seqüências do Sudeste Asiático obtidas no GenBank.

ULTRASTRUCTURAL OBSERVATIONS OF THE ENZYMATICALLY DIGESTED HUMAN AMNIOTIC BASEMENT MEMBRANE / 解剖学报

Using enzymatic digestions on human amniotic basement membrane (HABM), it was observed ultrastructurally that heparinase Ⅱ digestion resulted in formation of microvilli on the surface of HABM and thinning and vesiculation of lamina densa and lamina lucida layers. This suggested that the heparinase Ⅱ-digested HABM had increased its surface area for more efficient cell attachment. The main action of heparinase Ⅱ is to digest heparan sulphate on HABM surface, which leads to exposure of more sites of laminin for cell attachment and neurite outgrowth; therefore heparinase Ⅱ-digested HABM can increase neuronal growth to 150%. For those HABM digested by heparinase Ⅰ, collagenase Ⅰ, and chondroitinase AC, they lost most of the sites for attachment and neurite outgrowth and as a consequence, the growth of neurons decreased. This morphological study supports heparinase Ⅱ digested HABM as an ideal substratum for neuronal attachment and growth.

COMPONENT ANALYSIS OF ENZYMATICALLY DIGESTED HUMAN AMNIOTIC BASEMENT MEMBRANE WITH REFERENCE TO ITS ROLE IN SUPPORTING GROWTH OF CHICK EMBRYO FOREBRAIN NEURONS / 解剖学报

Cell culture and SDS-polyacrylamide gel electrophoresis (PAGE) methods were employed to analyse the role of human amniotic basement membrane (HABM) in supporting the growth of forebrain neurons and the components of HABM after enzymatic digestion. It was found that HABM treated with heparinase Ⅱ supported the growth of forebrain neurons 150% of that of the untreated HABM control. The results of SDS-PAGE analysis showed that HABM treated with heparinase Ⅱ had collagen-like polypeptides 2?_1 (260kD) and 2?_2 (225kD) reduced and lamininlike peptides (212kD and 200kD) increased; and that HABM treated with heparinase I or collagenase I had laminin reduced. This indicated that laminin is the major component in HABM to promote the growth of forebrain neurons.

EFFECT OF ENZYMATICALLY DIGESTED HUMAN AMNIOTIC BASEMENT MEMBRANE ON THE GROWTH OF CHICK EMBRYONIC FOREBRAIN NEURONS / 解剖学报

Enzymatic digestion of the human amniotic basement membrane (HABM) using heparinase Ⅰ, heparinase Ⅱ, collagenase and chondroitinase ABC enabled us to study the supportiveness of HABM for forebrain neuronal growth. HABM-coated 24-well plates were enzymatically digested for 1 hr. at 37℃ before dissociated neurons from the forebrain of E8 chick embryo were seeded. The neurons were cultured for 20hr. in RPMI 1640 medium containing 20mmol/L HEPES and 60％ fetal calf serum before being quantified for neuronal growth by an automatic colorimetric microassay. The assay, utilizing a tetrazolium derivative named MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] which can be converted by viable cells to form a formazan product, can be measured with an Elisa reader. The results revealed that heparinase Ⅰ, collagenase, and chondroitinase ABC had significantly removed from the HABM the attachment sites for neurons, and that heparinase Ⅱ had significantly increased the sites for attachment and/or enhanced the survival growth of neurons.