Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 39
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Phytochem Anal ; 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31512326

RESUMO

INTRODUCTION: Polygoni Orientalis Fructus (POF) is a clinically effective Chinese medicine. Raw POF (RPOF) and POF Tostus (POFT) are used separately in clinics. However, incomplete progress has been made on quality control. OBJECTIVE: To establish a comprehensive method for quality assessment of RPOF and POFT and to discriminate these two varieties. METHODOLOGY: High-performance liquid chromatography combined with the diode array detector (HPLC-DAD) methods were developed for fingerprinting and quantitative analysis of seven major compounds in RPOF and POFT, and the main components were determined by HPLC-DAD coupled with Fourier-transform ion cyclotron resonance-mass spectrometry. Chemometric approaches were performed to discriminate RPOF and POFT and to screen discriminatory components. RESULTS: Fingerprints were established and 12 common peaks were identified, cannabisin G and cannabisin E were firstly identified from POF. In quantitative analysis, all analytes showed good regression (R > 0.9996) within test ranges and the recovery of the method was in the range 96.6-104.3%. Fingerprints in conjunction with similarity analysis and hierarchical clustering analysis (HCA) demonstrated the consistent quality of RPOF and showed a clear discrimination between RPOF and POFT. Principal component analysis, partial least-squares discriminant analysis, and heatmap-HCA on quantitative data not only gave a clear differentiation between RPOF and POFT, but they also suggested that quercetin, 3,5,7-trihydroxychromone, and N-trans-feruloyltyramine acted as the main factors responsible for the sample differences. CONCLUSIONS: Chromatographic analysis in combination with chemometric analysis provides a simple and reliable method of comparing and evaluating the qualities of RPOF and POFT.

2.
Plant Physiol ; 181(4): 1441-1448, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31558579

RESUMO

The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31354847

RESUMO

Oligosaccharide esters, which are among the main active components of Polygalae Radix (PR), demonstrate significant pharmacological activities in the human nervous system. In our previous research, some other constituents in PR were able to improve the bioavailability of oligosaccharide esters such as sibiricose A5 (SA5), sibiricose A6 (SA6), and 3,6'-disinapoyl sucrose (DISS), but the related components and their underlying mechanisms remain unknown. The present study aimed to investigate the intestinal absorptive profile of SA5, SA6, and DISS and the absorptive behavior influenced by the coadministration of polygalaxanthone III and total saponins of PR (TS) using an in vitro everted rat gut sac model, along with the possible mechanisms that may influence absorption. The results showed that TS could significantly enhance the absorption of SA5, SA6, and DISS monomers. Verapamil, a P-glycoprotein inhibitor, was able to elevate the absorption of SA5 and SA6, and an absorption experiment using Rho123 led us to conclude that TS influenced the absorption of SA5 and SA6 in a manner similar to that of a P-glycoprotein inhibitor. Sodium caprate, a paracellular absorption enhancer, was found to increase the absorption of SA5, SA6, and DISS. Results showed that the absorption mechanisms of SA5 and SA6 may combine active transport with paracellular passive penetration, while DISS's absorption was dominated by paracellular passive penetration. However, the relationship between polygala saponins and the absorption of SA5, SA6, and DISS by paracellular passive penetration remain to be examined. This is the direction of our future research.

4.
Plant Mol Biol ; 96(4-5): 445-456, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29476306

RESUMO

KEY MESSAGE: We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.


Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Mutagênese/genética , Sequência de Bases , Mutagênese Insercional/genética , Mutação/genética , Reação em Cadeia da Polimerase , Edição de RNA/genética , RNA Guia/genética , RNA de Transferência/genética , Reprodutibilidade dos Testes
5.
Sci Rep ; 7: 41993, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28155921

RESUMO

Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.


Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Biologia Sintética/métodos , Arabidopsis/genética , Escherichia coli , Vetores Genéticos/genética , Plantas Geneticamente Modificadas/genética
6.
Exp Ther Med ; 12(5): 3213-3220, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27882140

RESUMO

In the present study, we investigated the effects of hydroxyethyl starch (HES) 130/0.4 on serum pro-inflammatory variables, immunologic variables, fluid balance (FB)-negative(-) rate and renal function in severe acute pancreatitis (SAP) patients. From October, 2007 to November, 2008, a total of 120 SAP patients were enrolled in this retrospective study. Fifty-nine patients in the HES group received 6% HES 130/0.4 combined with crystalloid solution for fluid resuscitation (HES group). In the control group, 61 patients received only crystalloid solution after admission. Interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-α levels in serum were measured on days 1, 2, 4 and 8. The peripheral blood CD4+CD8+ T lymphocyte rates, serum BUN and Cr values were also measured on days 1, 4 and 8. Patients with FB(-) rates were recorded from day 1 to 8. Interaction term analysis (hospital stay and fluid resuscitation methods) based on mixed-effects regression model revealed significantly lower levels of IL-1 and TNF-α in the HES group compared with the control group. The difference in curve's risk ratio was not significant for IL-6, CD4+CD8+ T lymphocyte rate, BUN and Cr values (P>0.05). In the HES group, we detected a significantly higher rate of patients with FB(-) from day 4 to 8 (P<0.05). Thus, HES 130/0.4 resuscitation could decrease the IL-1 and IL-8 levels, shorten the duration of positive FB, and preserve the patient's immune status as well as renal function during the early phase of SAP.

7.
Front Plant Sci ; 7: 863, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27379143

RESUMO

Organ abscission is an important plant developmental process that occurs in response to environmental stress or pathogens. In Arabidopsis, ligand signals, such as ethylene or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), can regulate organ abscission. Previously, we reported that overexpression of AtDOF4.7, a transcription factor gene, directly suppresses the expression of the abscission-related gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 2 (ADPG2), resulting in a deficiency of floral organ abscission. However, the relationship between AtDOF4.7 and abscission pathways still needs to be investigated. In this study, we showed that ethylene regulates the expression of AtDOF4.7, and the peptide ligand, IDA negatively regulates AtDOF4.7 at the transcriptional level. Genetic evidence indicates that AtDOF4.7 and IDA are involved in a common pathway, and a MAPK cascade can phosphorylate AtDOF4.7 in vitro. Further in vivo data suggest that AtDOF4.7 protein levels may be regulated by this phosphorylation. Collectively, our results indicate that ethylene regulates AtDOF4.7 that is involved in the IDA-mediated floral organ abscission pathway.

8.
J Huazhong Univ Sci Technolog Med Sci ; 35(6): 793-800, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26670427

RESUMO

Pancreaticoduodenectomy (PD) is the most effective treatment for patients with pancreatic head or periampullary lesions. Two major strategies exist: pylorus-preserving pancreaticoduodenectomy (PPPD) and pylorus-resecting pancreaticoduodenectomy (PRPD). However, it is yet unclear regarding the morbidity after PPPD and PRPD. This study analyzed the morbidity after PPPD and PRPD to determine the optimal surgical treatment of masses in the pancreatic head or periampullary region. A systematic search of databases identifying randomized controlled trials (RCTs) from the Cochrane Library, PubMed, EMBASE and Web of Science was performed. Outcome was compared by postoperative morbidity including overall morbidity, pancreatic fistulas, wound infections, postoperative bleeding, biliary leakage, ascites and delayed gastric emptying (DGE) rate between PPPD and PRPD. The DGE rate in the PRPD subgroups (conventional PD [CPD] and subtotal stomach-preserving PD [SSPPD], respectively) was also analyzed. The results showed that 9 RCTs including 722 participants were included for meta-analysis. Among these RCTs, 7 manuscripts described PRPD as CPD, and 2 manuscripts described PRPD as SSPPD. There were no significant differences in the overall morbidity, pancreatic fistulas, wound infections, postoperative bleeding, or biliary leakage between PPPD and PRPD. There was a lower rate of DGE with PRPD than that with PPPD (RR=2.15, P=0.03, 95% CI, 1.09-4.23). Further subgroup analysis indicated a comparable DGE rate for the CPD but a lower DGE rate for the SSPPD group than the PPPD group. However, the result did not indicate any difference between CPD and SSPPD regarding the DGE rate (P=0.92). It is suggested that PPPD is comparable to PRPD in overall morbidity, pancreatic fistulas, wound infections, postoperative bleeding and biliary leakage. The current data are not sufficient to draw a conclusion regarding which surgical procedure is associated with a lower postoperative DGE rate. Our conclusions were limited by the available data. Further evaluations of RCTs are needed.


Assuntos
Morbidade , Pancreaticoduodenectomia/efeitos adversos , Piloro/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Pancreaticoduodenectomia/métodos
9.
Genome Biol ; 16: 144, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26193878

RESUMO

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.


Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas , Genes de Plantas , Homozigoto , Mutação , Regiões Promotoras Genéticas , Arabidopsis/embriologia , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Células Germinativas Vegetais/metabolismo , Fenótipo , Regiões Terminadoras Genéticas
10.
BMC Plant Biol ; 14: 327, 2014 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-25432517

RESUMO

BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.


Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Genoma de Planta , Zea mays/genética , Agrobacterium/genética , Sequência de Bases , Vetores Genéticos/genética , Plantas Geneticamente Modificadas/genética , Protoplastos/metabolismo , Alinhamento de Sequência
11.
PLoS One ; 9(3): e90316, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24603478

RESUMO

BACKGROUND: The aim of this meta-analysis was to compare the long-term survival, mortality, morbidity and the operation-related events in patients with periampullary and pancreatic carcinoma undergoing pylorus-preserving pancreaticoduodenectomy (PPPD) and pylorus-resecting pancreaticoduodenectomy (PRPD). METHOD: A systematic search of literature databases (Cochrane Library, PubMed, EMBASE and Web of Science) was performed to identify studies. Outcome measures comparing PPPD versus PRPD for periampullary and pancreatic carcinoma were long-term survival, mortality, morbidity (overall morbidity, delayed gastric emptying [DGE], pancreatic fistula, wound infection, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage) and operation related events (hospital stays, operating time, intraoperative blood loss and red blood cell transfusions). RESULTS: Eight randomized controlled trials (RCTs) including 622 patients were identified and included in the analysis. Among these patients, it revealed no difference in long-term survival between the PPPD and PRPD groups (HR = 0.23, p = 0.11). There was a lower rate of DGE (RR = 2.35, p = 0.04, 95% CI, 1.06-5.21) with PRPD. Mortality, overall morbidity, pancreatic fistula, wound infection, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage were not significantly different between the groups. PPPDs were performed more quickly than PRPDs (WMD = 53.25 minutes, p = 0.01, 95% CI, 12.53-93.97); and there was less estimated intraoperative blood loss (WMD = 365.21 ml, p = 0.006, 95% CI, 102.71-627.71) and fewer red blood cell transfusions (WMD = 0.29 U, p = 0.003, 95% CI, 0.10-0.48) in patients undergoing PPPD. The hospital stays showed no significant difference. CONCLUSIONS: PPPD had advantages over PRPD in operating time, intraoperative blood loss and red blood cell transfusions, but had a significantly higher rate of DGE for periampullary and pancreatic carcinoma. PPPD and PRPD had comparable mortality and morbidity including pancreatic fistulas, wound infections, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage. Our conclusions were limited by the available data. Further evaluations of high-quality RCTs are needed.


Assuntos
Ampola Hepatopancreática/cirurgia , Neoplasias do Ducto Colédoco/cirurgia , Neoplasias Pancreáticas/cirurgia , Pancreaticoduodenectomia/métodos , Piloro/cirurgia , Hemorragia/etiologia , Humanos , Complicações Intraoperatórias/etiologia , Pancreaticoduodenectomia/efeitos adversos , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de Sobrevida , Fatores de Tempo , Resultado do Tratamento
12.
Artigo em Chinês | MEDLINE | ID: mdl-24812834

RESUMO

OBJECTIVE: To clone and express Plasmodium falciparum erythrocyte membrane protein 1 DBLalpha (PfEMP1-DBLalpha) and three fragments genes, and screen the strongest affinity sequence with the red blood cell surface receptors-heparin or heparin sulfate in the structure of PfEMP1-DBLalpha. METHODS: The sequence of PfEMP1-DBLalpha1245 was optimized according to the characteristics of E. coli codon, synthesized, and divided into three fragments (DBLaA, DBLalphaB, and DBLalphaC) by PCR. Full-length gene and three gene fragments were subcloned into PGEX-4T-1 vector, and transformed into E. coli BL21 and then induced with IPTG for expression. The recombinant protein was purified from bacterial lysates using glutathione-Sepharose 4B. Heparin affinity test and glycosaminoglycan (GAG) inhibition test were used to analyze the affinity between recombinant protein and heparin. RESULTS: Four recombinant proteins(DBLalpha1245, DBLalphaA, DBLalphaB, and DBLalphaC) were expressed as solubility and the relative molecular weight (M(r) 73 600, M(r) 41 600, M(r) 42 500, and M(r) 41 500) were conformed to the prediction size. Heparin affinity test and GAG inhibition test showed that the four recombinant proteins were binded to the heparin-Sepharose, but not for the GST control. DBLalphaC (Q285-Y415) had the strongest affinity to heparin. CONCLUSION: The strongest affinity sequence with heparin or heparin sulfate in the structure of PfEMP1-DBLalpha is Q285-Y415, which plays a role in binding of Plasmodium falciparum infected red blood cells to the peripheral red blood cells.


Assuntos
Eritrócitos/parasitologia , Heparina/metabolismo , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Expressão Gênica , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
J Proteomics ; 77: 423-32, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23026549

RESUMO

Toxoplasma gondii, a ubiquitous intracellular protozoan, infects one third of the world human population. It is of great medical significance, especially for pregnant women and immune-compromised patients. Accurate and early detection of T. gondii infection is crucial in the management of this disease. To obtain potential diagnostic markers, immunoproteomics was employed to identify immunodominant proteins separated by 2-D immunobloting and probed with sera collected from Toxoplasma-positive pregnant women. MALDI-TOF MS and MS/MS analyses identified a total of 18 immunoreactive proteins that were recognized by Toxoplasma-positive sera, whereas none was reactive with the negative-control sera from healthy, Toxoplasma-negative volunteers. Pregnant women showed a diverse immunoreactivity pattern with each serum recognizing one to eight identified tachyzoite proteins. The identified proteins were localized in the membrane, cytoplasm and specific organelles of T. gondii, and are involved in host cell invasion, metabolism and cell structure. Among these 18 proteins, actin, catalase, GAPDH, and three hypothetical proteins had a broad reactivity with Toxoplasma-positive sera, indicating their potential as diagnostic markers for toxoplasmosis. Each of several combinations of the identified proteins offered 100% detection of Toxoplasma infections of all 28 Toxoplasma-positive women. The study findings suggest that Toxoplasma tachyzoites are highly immunogenic and highlights the heterogeneity of host responses to Toxoplasma infection and the importance of using combinations of immunogens as diagnostic antigens. The findings have significant implications to the development of diagnostic reagents with high sensitivity and specificity.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/sangue , Imunoglobulina G/sangue , Complicações Infecciosas na Gravidez/sangue , Proteínas de Protozoários/sangue , Toxoplasma/metabolismo , Toxoplasmose/sangue , Adulto , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Proteômica/métodos , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Toxoplasmose/imunologia
14.
Food Chem ; 135(4): 2661-5, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22980855

RESUMO

Abrin is a plant toxin, which can be easily isolated from the seeds of Abrus precatorius. It may be used as a biological warfare agent. In order to detect abrin in food samples, a two-layer sandwich format enzyme-linked immunosorbent assay based on the monoclonal antibody (mAb) (as capture antibody) and rabbit polyclonal serum (as detecting antibody) was developed and applied for the determination of abrin in some food matrices. The linear range of the mAb was 1-100 µg L(-1) with a detection limit of 0.5 µg L(-1) for abrin in phosphate buffered saline (PBS). The recoveries of abrin from sausage, beer and milk samples ranged 97.5-98.6%, 95.8-98.4% and 94.8-9.6%, respectively, with a coefficient of variation (CV) of 3.7% or less. The newly developed sandwich ELISA using the mAb appears to be a reliable and useful method for detection of abrin in sausage, beer and milk.


Assuntos
Abrina/análise , Cerveja/análise , Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Produtos da Carne/análise , Leite/química , Toxinas Biológicas/análise , Animais , Anticorpos Monoclonais/análise , Bovinos , Coelhos
15.
Asian Pac J Trop Med ; 5(2): 85-90, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22221747

RESUMO

OBJECTIVE: To analyse the var gene repertoire and characterise the chondroitin sulphate A (CSA)-binding activity of the Duffy-binding like (DBL) domains encoded by the var2csa gene of a Plasmodium falciparum (P. falciparum) isolate in Hainan Province, China. METHODS: The sequences of var DBL1 regions were PCR-amplified, sequenced and the sequence characteristics was bioinformatically analysed. Recombinant proteins encoded by the var2csa genes were expressed and purified. The binding activities of the recombinant proteins to CSA receptor was detected by ELISA assays. RESULTS: Fifty six unique DBL α sequences were obtained, and the sequences represented similar diversity to the var genes of the genome parasite 3D7. There are two var2csa genes in the P. falciparum isolated from Hainan Province. Unlike in other falciparum parasites such as HB3, the two var2csa genes are more diverged. The receptor-binding capacity of DBL-5ε and DBL-6ε domains of HN var2CSA was studied. CONCLUSIONS: This work represented the diversity of var genes of a P. falciparum isolate in China.


Assuntos
Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Clonagem Molecular , Malária Falciparum/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos de Protozoários/genética , China , Sulfatos de Condroitina/genética , DNA de Protozoário/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Humanos , Malária Falciparum/genética , Dados de Sequência Molecular , Plasmodium falciparum/química , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/genética
16.
Ren Fail ; 34(4): 420-4, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22250918

RESUMO

BACKGROUND: Upper gastrointestinal (UGI) symptoms are common in hemodialysis (HD) patients, while gastric metaplasia (GM) and Helicobacter pylori infections are key causes for UGI symptoms. This study is targeted to compare GM and H. pylori infections in patients with different durations of HD. METHODS: A total of 406 subjects from Ningbo Urology and Nephrology Hospital were included. The mean age of subjects was 44.7 ± 13.5 years; 62.9% were male; and subjects were divided into four groups according to different HD durations. Upper endoscopy and lesion were performed in these patients and methylene blue staining was used in detecting H. pylori and GM. RESULTS: Erosive gastritis was the most common symptom in uremic subjects. GM was found in 139 patients. The longer the dialysis duration, the higher the incidence rate of GM (p < 0.05). H. pylori infection accounted for 24.1% in HD patients. The occurrence of H. pylori infection decreased as dialysis periods progressed within the first 4-year follow-up after the start of HD. CONCLUSIONS: Almost all patients with HD experienced gastrointestinal discomfort in the current patient cohort. The most common mucosal lesion observed in our study pool was chronic erosive gastritis. The overall incidence of GM was normal at 35.0%, since quite a part of patients are the elderly group in this study. We need not worry about this too much, unless the HD patients have registered for renal transplantation or are suffering from severe gastrointestinal discomfort.


Assuntos
Mucosa Gástrica/patologia , Infecções por Helicobacter/patologia , Helicobacter pylori/isolamento & purificação , Diálise Renal , Uremia/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , China/epidemiologia , Endoscopia Gastrointestinal , Feminino , Seguimentos , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/complicações , Infecções por Helicobacter/epidemiologia , Humanos , Incidência , Masculino , Metaplasia/epidemiologia , Metaplasia/etiologia , Metaplasia/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Uremia/complicações , Adulto Jovem
17.
Parasit Vectors ; 4: 213, 2011 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-22070984

RESUMO

BACKGROUND: Infection with the protozoan Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. Protection from acute toxoplasmosis is known to be mediated by CD8+ T cells, but the T. gondii antigens and host genes required for eliciting protective immunity have been poorly defined. The T. gondii dense granule protein 6 (GRA6), recently proved to be highly immunogenic and produces fully immune protection in T. gondii infected BALB/c mice with an H-2Ld gene. The CD8+ T cell response of H-2Ld mice infected by the T. gondii strain seemed to target entirely to a single GRA6 peptide HF10-H-2Ld complex. RESULTS: To determine whether a GRA6-based DNA vaccine can elicit protective immune responses to T. gondii in BALB/c mice, we constructed a eukaryotic expression vector pcDNA3.1-HisGRA6 and tested its immunogenicity in a mouse model. BALB/c mice were vaccinated intramuscularly with three doses of GRA6 DNA and then challenged with a lethal dose of T. gondii RH strain tachyzoites. All immunized mice developed high levels of serum anti-GRA6 IgG antibodies, and in vitro splenocyte proliferation was strongly enhanced in mice adjuvanted with levamisole (LMS). Immunization with pcDNA3.1-HisGRA6 with LMS resulted in 53.3% survival of challenged BALB/c mice as compared to 40% survival of BALB/c without LMS. Additionally, immunized Kunming mice without an allele of H-2Ld failed to survive. CONCLUSIONS: Our result supports the concept that the acquired immune response is MHC restricted. This study has a major implication for vaccine designs using a single antigen in a population with diverse MHC class I alleles.


Assuntos
Antígenos de Protozoários/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Toxoplasmose Animal/prevenção & controle , Vacinação/métodos , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Proliferação de Células , Modelos Animais de Doenças , Feminino , Imunização Secundária/métodos , Imunoglobulina G/sangue , Injeções Intramusculares , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Baço/imunologia , Análise de Sobrevida , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética
18.
Artigo em Chinês | MEDLINE | ID: mdl-21823322

RESUMO

OBJECTIVE: To clone and express three VAR2CSA duffy antigen-binding ligand (DBL) domains (DBL4/ 5/6) encoded by var2csa gene of a Hainan isolate of Plasmodium falciparum, and study the difference of chondroitin sulfate A (CSA)-binding activity among them. METHODS: Three DBL domains was amplified by PCR and cloned into the vector pMD18-T. The recombinant plasmids were identified by enzyme digestion and sequencing, and then subcloned into the prokaryotic expression vector pET-22b. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was purified with His GraciTrap kit and identified by SDS-PAGE and Western blotting. CSA-binding activity of the three recombinant DBL domains was assayed by ELISA. RESULTS: The target genes were amplified with the length of 996 bp, 859 bp and 894 bp. The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. The recombinant proteins (DBL4/5/6) were purified, the relative molecular mass of DBLfA, DBL5 and DBL6 was Mr 439 800, Mr 34,500 and Mr 36,000, respectively. The purified protein has been confirmed with immunogenicity by Western blotting. The results of adhesion experiment indicated that A405 values of DBL5 domain with different concentration were significantly higher than that of DBLA and DBL6. CONCLUSION: The three recombinant proteins (DBLA/5/6) of VAR2CSA DBL domains were expressed, and DBL5 domain has high binding affinity with CSA.


Assuntos
Antígenos de Protozoários/genética , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes/metabolismo , Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Clonagem Molecular , DNA de Protozoário/genética , Escherichia coli/metabolismo , Vetores Genéticos , Plasmodium falciparum/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética
19.
Parasit Vectors ; 4: 168, 2011 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-21871123

RESUMO

BACKGROUND: Toxoplasma gondii has been shown to trigger strong cellular immune responses to heterologous antigens expressed by the parasite in the inbred mouse model. We studied the immune response induced by T. gondii as an effective vaccine vector in chickens and rabbits. RESULTS: T. gondii RH strain was engineered to express the yellow fluorescent protein (YFP) in the cytoplasm. A subcutaneous injection of the transgenic T. gondii YFP in chickens afforded partial protection against the infection of transgenic E. tenella YFP. T. gondii YFP induced low levels of antibodies to YFP in chickens, suggesting that YFP specific cellular immune response was probably responsible for the protective immunity against E. tenella YFP infection. The measurement of T-cell response and IFN-γ production further confirmed that YFP specific Th1 mediated immune response was induced by T. gondii YFP in immunized chickens. The transgenic T. gondii stimulated significantly higher YFP specific IgG titers in rabbits than in chickens, suggesting greater immunogenicity in a T. gondii susceptible species than in a resistant species. Priming with T. gondii YFP and boosting with the recombinant YFP can induce a strong anti-YFP antibody response in both animal species. CONCLUSIONS: Our findings suggest that T. gondii can be used as an effective vaccine vector and future research should focus on exploring avirulent no cyst-forming strains of T. gondii as a live vaccine vector in animals.


Assuntos
Portadores de Fármacos , Vetores Genéticos , Vacinas Protozoárias/imunologia , Toxoplasma/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Galinhas , Imunoglobulina G/sangue , Injeções Subcutâneas , Proteínas Luminescentes/genética , Proteínas Luminescentes/imunologia , Vacinas Protozoárias/administração & dosagem , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Toxoplasma/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia
20.
Biosens Bioelectron ; 26(8): 3700-4, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21371875

RESUMO

A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA-ITCBE-Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL(-1). The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Chumbo/análise , Coloide de Ouro/química , Nanopartículas Metálicas/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA