Study on the Spectrum-effect Relationship of Different Solvent Extracts from Trollius chinensis against Hypoxia/ reoxygenation Injury of Cardiomyocyte / 中国药房

OBJECTIVE：To study spectrum-effect relationship of 11 different solvent extracts from Trollius chinensis against hypoxia/reoxygenation injury of cardiomyocyte . METHODS ：HPLC-MS/MS method was used to establish the fingerprints of 11 different solvent extracts from T. chinensis ，the compounds corresponding to the common peaks were identified by comparing with the substance control and literature information. MTT assay was used to detect the effects of 11 different solvent extracts from T. chinensis on the survival rate of rat myocardial H 9c2 cells injured by hypoxia/reoxygenation. The MDA content ，ROS level in cells and LDH content in the supernatant were detected by ELISA. GRA and PLS method were used to analyze the spectrum-effect relationship between the compounds corresponding to common peak and anti-hypoxia/reoxygenation injury of cardiomyocyte （drug effect）. RESULTS ：There were 22 common peaks in 11 different solvent extracts from T. chinensis ，and 22 compounds were identified. Compared with hypoxia/reoxygenation injury group ，survival rate of hypoxia/reoxygenation injury+S 1-S6，S9 and S 10 groups were increased significantly ，while MDA content ，ROS level and LDH content were decreased significantly （P＜0.05）； ROS level and LDH content of hypoxia/reoxygenation injury+S 8 group w ere decreased significantly （P＜0.05）. The r of GRA analysis of 22 compounds with drug effects were all higher than 0.8. Except for peaks 1，2，7，13，14 and 21，r of PLS analysis of rest peaks with drug effects were higher than 0 发。电话：0431-86058683。E-mial：nml2000@163.com （being positive correlation ）. Top 9 common peaks in the list of contribution rate were peak 6＞11＞4＞5＞8＞9＞12＞10＞15. CONCLUSIONS ：Orientin（peak 6），vitexin（peak 11）， orientin-2″-O-β-L-galacto- pyranosl （peak 4），orientin-2″-O-β-D-Pyrine xylosides （peak 5），quercetin-3-O-glucopyranoside（peak 8），vitexin-2″-O-β-L-galactoside（peak 9），hyperoside（peak 12），vitexin-2″-O-β-D-pyrine xylosides （peak 10），2″-O-（2″′- methylbutyry-loxy）-orientin（peak 15）may be the main components of anti-hypoxia/reoxygenation injury of cardiomyocytes.

Grey Correlation Analysis on Rate of Embryo, Endogenous Hormone and Enzyme Activity of Acanthopanax senticosus Seeds / 中国实验方剂学杂志

Objective::To study the correlation between endogenous hormone content, related enzyme activity and embryogenesis during the stratification of Acanthopanax senticosus seeds, and to provide theoretical basis for application of endogenous hormones and related physiological indicators in regulating germination of A. senticosus seeds in production and scientific research. Method::Endophytic fungi as well as different concentrations of nitric oxide and hydrogen peroxide solution were used for soaking the seeds of A. senticosus, and then thermophilic stratification was conducted for the seeds. The content of endogenous hormones such as gibberellin (GA3), abscisic acid (ABA), indole acetic acid (IAA), indole butyric acid (IBA) and salicylic acid (SA) in seeds of A. senticosus were tested by high performance liquid chromatography (HPLC), and the activity change of enzymes in vivo such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and malondialdehyde (MDA) were tested. The correlation between the embryo rate and the hormones and their enzyme activities in the seeds of A. senticosus was studied by the grey correlation method. Result::The embryo rate was significantly positively correlated with IAA, GA3, and CAT, with correlation coefficient of 1.086, 0.935 and 1.067, respectively. There was a significant positive correlation between IAA, GA3, CAT, IBA, and SA, but with a significant negative correlation with ABA, MDA, and POD. The correlation degree was as follows: IAA>CAT>GA3>IBA>SA>SOD>ABA>POD>MDA. Conclusion::IAA, GA3 and CAT have significant promoting effects on embryo rate, and there is a significant correlation between hormone content and enzyme activity, which provides a basis for exploring the seed germination mechanism of A. senticosus.

Extended thymectomy by subxiphoid approach video-assisted thoracoscopic surgery(VATS) for myasthenia gravis:clinical analysis of 64 cases / 中华胸心血管外科杂志

To investigate the clinical efficacy of extended thymectomy by subxiphoid approach video-assisted thoracoscopic surgery(VATS) for myasthenia gravis. Methods We retrospectively analyzed the clinical date of 64 cases of myasthenia gravis treated by subxiphoid approach VATS in the same surgical team from September 2015 to April 2018. The patients were equally divided into 4 groups(A, B, C and D) according to the date of operation. Comparisons were made among the four groups in operation time, blood loss during operation, rate of conversion to thoracotomy, postoperative complications, postoperative hospital stay, duration and amount of postoperative chest tube drainage, frequenlly of surgery. The operative effect of different stage was analyzed. Results There were no intraoperative deaths. 1 patient(group A) was converted to thoracotomy. 3 patients(2 cases of group A; 1 case of group D) had lung infection. 1 patient(group B) developed myasthenia crisis after surgery, and the rest patients showed obvious improvement in postoperative myasthenia symptoms. No significant differences were found in postoperative complications, rate of conversion to thoracotomy, postoperative hospital stay, duration and amount of postoperative chest tube drainage among the 4 groups(P >0. 05). The operation time was significantly longer in group A(186. 25 ± 25. 79) min than the other 3 groups [B(128. 75 ± 16. 28) min, C(135. 00 ± 21. 29) min, D(128. 75 ± 19. 62)min], P <0. 05. The blood loss in surgery was significsntly more in group A(110. 00 ±38. 82)ml than that in the other 3 groups[B(63. 75 ±28. 26)ml, C(58. 13 ±27. 86)ml, D(58. 75 ±25. 00)ml], P <0. 05, while no statistical difference was found among group B, C and D. The frequency of surgery was increased from 1. 6 cases in group A to 2. 3, 2. 7 and 2. 7 cases one month in B, C and D, respectively. Conclusion The results of the present study have shown that subxiphoid approach VATS thymectomy is safe and feasible for the treatment of MG patients. For thoracic surgeons with certain experience in thoracoscopic technique, a plateau of the surgical skill of the subxiphoid opproach can be reached after learning curve procedures.

Some thoughts on deepening the classification training of postgraduates of clinical medicine under the background of collaboration of health system and education system / 中华医学教育探索杂志

Under the collaborative background of health system and education system,the classified cultivation of clinical medicine master of professional degree and academic degree is facing some problems and challenges,including different ideas,lacking awareness,unequal policies and unbalanced development of two degrees.Therefore,we recommended clarifying the cultivation goal and target;actively reforming the medical education,systematically tracing and evaluating the reform effectiveness,and completing the policy;learning the advanced foreign knowledge,focusing on China's condition and accelerating the reform of medical degree system.

Evaluation of Protective Immune Response Induced by a DNA Vaccine Encoding GRA8 against Acute Toxoplasmosis in a Murine Model

Toxoplasma gondii is an apicomplexan zoonotic protozoan parasite that infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicate a need for the development of an effective vaccine. T. gondii GRA8 is a member of the dense granules protein family and is used as a marker of acute infection. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pDsRed2-GRA8, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with the pDsRed2-GRA8 plasmid and then challenged by infection with the highly virulent GFP-RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii of this vaccine were analyzed by measuring cytokine and serum antibody titers, splenocyte proliferation assays, and the survival times of mice after challenge. Our results showed that mice immunized with pDsRed2-GRA8 demonstrated specific humoral and cellular responses, induced higher IgG antibody titers with predominant IgG2a production; increased levels of IL-10, IL-12 (p70), IFN-γ, TNF-α, and splenocyte proliferation; and prolonged survival times compared to those of control mice. The present study showed that DNA immunization with pDsRed2-GRA8 induced humoral and cellular immune responses, and all immunized mice showed greater Th1-type immune responses and longer survival times than those of control mice. These results indicated that T. gondii GRA8 DNA immunization induces a partial protective effect against acute toxoplasmosis.

Grã-parentalidade: revisão integrativa da literatura / Grandparenthood: integrative review of literature / Gran-paternidad: revisión integrativa de la literatura

O nascimento de uma criança implica a transição para a parentalidade que ocorre, simultaneamente, com a transição para a grã-parentalidade. Neste artigo, procedeu-se a uma revisão integrativa da literatura existente em bases eletrônicas de dados, com vista a responder à questão: "Como é vivida a transição para a grã-parentalidade?" A grã-parentalidade pode ser identificada como: uma transição; um processo adaptativo; a procura do sentido de vida; uma oportunidade de crescimento pessoal; um evento normativo.

The birth of a child brings the transition to parenthood occurs and, simultaneously, the grandparents start on a new transition, the transition to grandparenthood. To systematize the state of art, it was done a integrative review of literature, in electronic databases in order to answer the question "How is experienced the transition to the Grand-parenting?". The grandparenthood can be seen as a transition or as an adaptive process; the search for the meaning of life; opportunity for personal growth; normative event.

El nacimiento de un niño implica la transición a la paternidad que se produce simultáneamente con la transición a la Gran-paternidad. En este artículo, se procedió a una revisión integradora de la literatura, en bases de datos electrónicas, con el fin de responder a la pregunta: "¿Cómo vivió la transición a la Gran-paternidad?". La Gran-paternidad puede ser vista como una transición o como un proceso de adaptación; la búsqueda del sentido de la vida; una oportunidad para el crecimiento personal; un evento normativo.

Study of recombinant GRA5 protein onimmuno-diagnosis of Toxoplasma infection / 安徽医科大学学报

Objective To investigate the immunodiagnostic valueofrecombinant granulocvte protein 5(rGRA5) protein in prokaryotic Toxoplasma gondii.Methods The PCR was used to ampliify GRA5 gene,then insert the target gene into the prokaryotic expression vector pET28a,and identified by double digestion and sequencing, then transfecte the correct targert gene into BL21 competent cells, SDS-PAGE and Western blot were used to detect the expression of the recombinant protein in BL21.ELISA was used to detect the serum of 100 cases of serologically Toxoplasma-positive individuals.Results The products of GRA5 gene of 363 bp in length was successfully amplified by PCR,the prokaryotic expression vector pET28a-GRA5 was constructed successfully, the result of double digestion showed that the insert fragment size was correct, and DNA sequencing results showed that the homology of GRA5 gene with GenBank was 100%.Also the expression of GRA5 protein was successfully detected in BL21 by Western blot(about 14 ku).ELISA method was used to detect 100 cases of patients with 73 cases showed positive results, the positive diagnosis rate was 73.0%.Among them, the positive detection rate of IgG positive samples was 72.5% in 40 cases,the positive detection rate of IgM positive samples was 53.3% in 30 cases,the positive detection rate of IgG and IgM positive samples was 93.3% in 30 cases.Conclusion The prokaryotic expression vector pET28a-GRA5 is constructed successfully, and the recombinant protein has potential for immunological diagnosis of toxoplasmosis.

Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6

The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents.

Determination of Loteprednol Etabonate and the Related Substances in Loteprednol Etabonate and Tobra-mycin Compound Ophthalmic Suspension by HPLC with Gradient Elution / 中国药师

Objective:To develop an HPLC with gradient elution method for the determination of loteprednol etabonate and the re-lated substances in loteprednol etabonate and tobramycin compound ophthalmic suspension. Methods:HPLC with gradient elution was performed with Inertsil ph phenyl column (250 mm × 4. 6 mm, 5 μm). The mobile phase A and B was 0. 25% acetic acid solution-acetonitrile (80 ∶20) and acetonitrile, respectively. The flow rate was 2. 0 ml·min-1 and the detection wavelength was 244 nm. The column temperature was 30 ℃ and the injection volume was 20 μl. Results:The main ingredient and the related substances could be well separated. Loteprednol etabonate had a good linear relationship within the range of 0. 001-1. 02 mg·ml-1(r=0. 999 9), and the average recovery was 99. 9%(RSD=0. 9, n=9). Conclusion:The assay method is sensitive, accurate and convenient with good res-olution. It can be applied to control the quality of loteprednol etabonate and tobramycin compound ophthalmic suspension.

Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.

Comparation of Toxoplasma gondii separated from HIV-positive people and RH strain GRA6 gene / 中国血吸虫病防治杂志

Objective To comparatively analyze Toxoplasma gondii separated from HIV-positive people and RH strain GRA6 gene. Methods By using the nested PCR the amplification of Dali HIV-positive blood samples and RH strains of Toxo-plasma GRA6 genome was performed. The GRA6 gene amplification positive product was selected and the electrophoresis imag-ing was performed by being digested with the Mse I endonuclease and the gene sequences were measured and analyzed. Re-sults The GRA6 gene fragment 800 bp was successfully amplified and about 600 bp and 200 bp bands were got by Mse I. The sequencing results showed that T. gondii GRA6 gene positive samples had 2 nucleotide variation compared with T. gondii strain RH namely 447 base pair at C becoming G and 623 base pair at G becoming T. At 146 bp and 690 bp the Mse I restric-tion sites TTAA were found. Conclusion The preliminary judgment shows that the Dali HIV-positive T. gondii genotype is consistent with RH strain belonging to genotype I.

Entre Londres e Paris, 1945-1975: formação da psicanálise contemporânea / Between London and Paris, 1945-1975: the formation of contemporary psychoanalysis / Entre Londres y Paris, 1945-1975: formación del psicoanálisis contemporáneo

Traçando um panorama da Psicanálise entre 1945 e 1980, este artigo busca situar nele as obras de Jean Laplanche, Jean-Bertrand Pontalis, André Green, Joyce McDougall e Betty Joseph.

Dibujando un panorama del Psicoanálisis entre 1945 y 1980, este artículo trata de localizar en el mismo las obras de Jean Laplanche, Jean-Bertrand Pontalis, André Green, Joyce McDougall y Betty Joseph.

Sketching a broad view of the development of Psychoanalysis between 1945 and 1980, this paper offers some suggestions about the place occupied in it by the works of Jean Laplanche, Jean-Bertrand Pontalis, André Green, Joyce McDougall and Betty Joseph.

A Rapid Diagnostic Test for Toxoplasmosis using Recombinant Antigenic N-terminal Half of SAG1 Linked with Intrinsically Unstructured Domain of GRA2 Protein

Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis.

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.

Analysis of water quality factors influencing the speciation of inorganic nitrogen using GRA.

Based on the water quality data from 2006 to 2008, grey relational analysis (GRA) is used to analyze factors that may have influence on the speciation of inorganic nitrogen in the Chengdu section of middle Min river. The results show that water temperature, changing from 20.2±2.7, 13.4±5.7 and 16.8±5.6oC, is the first restrictive factor for the speciation of inorganic nitrogen; it is negatively correlated with the ratio of total ammonia nitrogen to total inorganic nitrogen contents [m(AN)/m(TIN)] in three different periods of wet season, dry season and year-round. The average pH values for years, in wet and dry periods are 7.6±0.4, 7.3±0.3 and 7.8±0.2, respectively, and have different influences in different seasons. It is the second restrictive factor and positive correlation between pH and m(AN)/m(TIN) in wet season and through the year yet it is the fourth factor in dry seasons. The values of dissolved oxygen (DO), which are 4.6±1.4, 4.6±2.4, 4.6±2.0 respectively, is the third factor and negatively correlates with m(AN)/m(TIN) in third different periods. The chemical oxygen demand (COD) indirectly inhibits the nitrifying bacteria because the DO is depleted in the decomposition of organic matter by heterotrophic bacteria, showing the positive correlation. As the alkalinity can meet the requirement of nitrification in wet season and through the year, it is not restrictive factor. However, it is the second restrictive factor in dry season because of low content of alkalinity inhibiting the growth of nitrifying bacteria.

GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane

The dense granule of Toxoplasma gondii is a secretory vesicular organelle of which the proteins participate in the modification of the parasitophorous vacuole (PV) and PV membrane for the maintenance of intracellular parasitism in almost all nucleated host cells. In this review, the archives on the research of GRA proteins are reviewed on the foci of finding GRA proteins, characterizing molecular aspects, usefulness in diagnostic antigen, and vaccine trials in addition to some functions in host-parasite interactions.

Interaction between Parasitophorous Vacuolar Membraneassociated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.