• Parasites, Hosts and Diseases
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• v.45 no.3
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• pp.165-174
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• 2007

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.

• Journal of the Korean Society of Manufacturing Process Engineers
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• v.15 no.6
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• pp.115-121
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• 2016

The aircraft flight control system controls an aircraft's direction and flying attitude, and actuators are key components of control systems. Actuators can be classified as Geared Rotary Actuator (GRA) and Ball Screw Actuator (BSA). GRA is used in mid-sized aircraft, and BSA is used in larger aircraft. A two-stage differential GRA model was suggested in this paper, and structural analysis and performance tests were performed. According to the analysis and experiment, the stiffness of the two-stage differential GRA was 17.57% higher than that of the conventional GRA, and the structural safety was improved.

• Parasites, Hosts and Diseases
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• v.52 no.4
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• pp.367-376
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• 2014

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 $GRA2_{25-105}$, $GRA3_{39-138}$, $ROP2_{324-561}$, and $MIC2_{1-284}$ domains had respectively higher value of IgG avidity. The $rGST-GRA2_{25-105}$ and $rGST-GRA3_{39-138}$ were soluble, while $rGST-ROP2_{324-561}$ and $rGST-MIC2_{1-284}$ were not. $GRA2_{31-71}$, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The $rGST-GRA2_{31-71}-ROP2_{324-561}$, a chimeric protein, appeared to be soluble. Moreover, $rGST-GRA2_{31-71}-MIC2_{1-284}$ was also soluble and had higher IgG avidity comparing to $rGST-MIC2_{1-284}$. 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.

• Parasites, Hosts and Diseases
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• v.46 no.4
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• pp.209-216
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• 2008

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.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.431-437
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• 2016

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.

• Parasites, Hosts and Diseases
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• v.44 no.4 s.140
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• pp.303-312
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• 2006

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the $X-\alpha-gal$ positive and PCR, 157 colonies of the $X-\beta-gal$ assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.

• Parasites, Hosts and Diseases
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• v.53 no.3
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• pp.345-348
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• 2015

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.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.264.1-264.1
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• 2002

We studied to compare hypoglycemic effect of GRA and MF in multiple low dose streptozotocin(STZ)-induced diabetic rats. 25 mg/kg of STZ in 100 mM citrate buffer(pH 4.5) was injected intraperitoneally for 5 consecutive days. SD rats were randomly divided into diabetic control and treatment groups. Treatment groups were administered with either 500mg/kg of GRA. 500mg/kg of MF. 250mg/kg of GRA＋250mg/kg of MF(GM 250)or 500mg/kg of GRA＋500mg/kg of MF(GM 500) for 3 weeks. (omitted)

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.1
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• pp.57-61
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• 2002

The renoprotective activities of white ginseng radix and rootlet were compared in spontaneously hypertensive rat (SHR) with diabetes. During oral administration of white ginseng radix (Ginseng Radix Alba, GRA) and white ginseng rootlet (Ginseng Radix Palva, GRP) for four weeks, arterial blood pressure and blood glucose levels were determined at every 10 days. In both GRA- and GRP-treatment groups, arterial blood pressures started to go down after 10 days of administration and maintained throughout the study period. After four weeks administrations of GRA and GRP, diastolic blood pressures were significantly decreased with 17% and 9%, respectively. GRA treatment also decreased blood glucose levels after 10 days of administration when compared with diabetic SHR group. At the end of the experiment, serum creatinine (Scr) and blood urea nitrogen (BUN) were not significantly different between the groups, except 62% higher value of BUN in diabetic SHR group when compared with SHR group. In the diabetic SHR group, the excretion of urinary albumin was increased significantly when compared with SHR. The level of urinary albumin in GRA treated group was markedly reduced when compared with diabetic SHR group $(67.8{\pm}4.7\;vs.\;131.3{\pm}13.5\;mg/24\;h).$ To examine the effects of ginseng radices on an overt diabetic nephropathy, index of kidney hypertrophy and transforming growth $factor-{\beta}1\;(TGF-{\beta}1)$ protein levels were evaluated. The glomerular and tubular cells stained positive for $TGF-{\beta}1$ seemed to be more abundant in diabetic SHR than in those with SHR, and GRA treated rats showed somewhat less $TGF-{\beta}1$ protein in glomerular and tubular cells when compared with diabetic SHR. Our results suggest that GRA might be a useful antihypertensive and antidiabetic agent with renoprotective effect.

• Parasites, Hosts and Diseases
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• v.39 no.3
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• pp.233-240
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• 2001

Although some reports have been published on the protective effect of antibodies to Toxoplasma gondii surface membrane proteins, few address the inhibitory activity of antibodies to dense granular proteins (GRA proteins) . Therefore, we performed a series of experiments to evaluate the inhibitory effects of monoclonal antibodies (mAbs) to GRA proteins (GRA2, 28 kDa; GRA6, 32 kDa) and surface membrane protein (SAGI, 30 kDa) on the invasion of T. gondii tachyzoites. Passive immunization of mice with one of three mAbs following challenge with a lethal dose of tachyzoites significantly increased survival compared with results for mice treated with control ascites. The survival times of mice challenged with tachyzoties pretreated with anti-GRA6 or anti-SAG 1 mAb were significantly increased. Mice that received tachyzoties pretreated with both mAb and complement had longer survival times than those that received tachyzoites pretreated with mAb alone. Invasion of tachyzoites into fibroblasts and macrophages was significantly inhibited in the anti-GRA2, anti-GRA6 or anti-SAG 1 mAb pretreated group. Pretreatment with mAb and complement inhibited invasion of tachyzoites in both fibroblasts and macrophages. These results suggest that specific antibodies to dense-granule molecules may be useful for controlling infection with T. gondii.