• Parasites, Hosts and Diseases
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• 제44권4호
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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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• 제35권2호
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• pp.79-86
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• 1997

톡소포자충의 숙주-기생충 상호관계 연구의 일환으로 마우스 비장에서 분리한 T 림프구. B 림프 구 및 과립구(대부분 호중구로 구성)에 톡소포자층의 tachyzoites를 감염시킨 후 감염된 림프구와 호중구의 미세형태 변화를 관찰하는 한편 각 세포의 충체 감염에 대한 감수성을 동위원소 흡수시 험법을 이용하여 정량화하였다. 충체는 병원성이 강한 RH 주를 샤용하였고 각 세포는 BALB/c와 CBA 마우스의 비장에서 분리하여 사용하였다. 감염 후 24시간에 관찰한 결과, T 림프구, B 림프 구 및 호중구는 마우스 주에 상관없이 세포질 내에 tachyzoites가 한 개, 두 개 또는 7-8개까지 관찰되었다. 감염된 T 림프구는 충체 주변에 형성죈 parasitophorous vacuole로 인해 핵이 한 쪽으로 밀리며. 미토콘드리아의 수가 증가하였다 감염된 B 림프구는 조내형질세망(RER)이 대조군에 비해 발달하지 않았으며 감염된 호중구는 과립의 수가 현저히 감소하였다 림프구와 호중구의 톡소포자충 감염에 대한 감수성을 3H-uracil 흡수량으로 정량화한 결과. 마우스 주에 따른 차이는 없었고 모든 종류의 세포 내에서 충체가 활발히 증식함이 확인되었다. 이상의 결과로 볼 때, BALB/c와 CBA 마우스의 비장 T 림프구, B 림프구 및 호중구는 모두 톡소포자충의 tachyzoites 감염에 대해 감수성이 높음을 알 수 있었고, 감염된 면역세포는 그 기능이 저하될 것으로 추측된다.

• 대한수의학회지
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• 제37권4호
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• pp.817-823
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• 1997

Suckling piglets and weaned pigs showed anorexia, dehydration, severe abdominal breathing, emaciation and paresis from Oct. 1993. to Nov. 1993. Five 2-week-old piglets were submitted for diagnosis in Kangwon National University. At necropsy, the pin-point well demarcated yellowish white foci were scattered on the surface of the lung, heart, liver, spleen and kidney. Histologically, multifocal areas of necrosis with mononuclear cells infiltration were found in the lung, heart, liver, lymph node, spleen, kidney and small intestine. These lesions tended to be associated with blood vessels. Variable round to ovoid tachyzoites were located at the periphery of the lesions. The organisms were demonstrated as Toxoplasma gondii by immunohistochemical staining method. Ultrastructurally, this parasite was surrounded with parasitophorous vacuole in alveolar macrophage. The parasite was crescent-shaped and $6{\sim}8{\times}1{\sim}2{\mu}m$ in size. It was enclosed by an thick outer membrane and an underlying thin inner membrane. Several club-shaped paired organelles and conoids lay in the cytoplasm at the anterior. Numerous round body and one to several mitochondria were presented in the cytoplasm. Based on the gross findings, histopathology, immunohistochemical and electron microscopic findings, this case was diagnosed as toxoplasmosis in piglets.

• Parasites, Hosts and Diseases
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• 제41권2호
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• pp.89-96
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• 2003

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5’-RACE as 1,592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.

• 대한수의학회지
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• 제35권2호
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• pp.317-326
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• 1995

The purpose of this study was to establish a method for in vitro culture of C parvum isolated in Korea by determination of suitable cell model to complete development of this parasite. The result obtained were summerized as follows: 1. To determine the most suitable cell line, six types of cell line were examined by microscopy. All cell lines were infected with C parvum and showed the highest infection score in HmLu cells. 2. The staining methods including DMSO-modified acid-fast(A-F) stain, hematoxylin-eosin(H & E) stain and immunofluorescence antibody(IFA) stain were applied to examine the infection of C parvum in cell culture. These staining methods were possible to examine the infection of C parvum in cell culture. The most sensitive one was IFA staining technique. 3. Developmental stages of C parvum in HmLu cell were observed. After the initial 8 hour incubation period, some trophozoites were observed. The meronts and gametes were appeared at 24-48 hour post inoculation(PI), and oocysts were observed firstly at 48-72 hour PI. 4. In H & E stain, the parasite appeared as basophilic within parasitophorous vacuole membrane(PVM) and lying in cytoplasm at near the nucleus of the host cells. It was able to distinguish the type I, type II meronts and gametes. 5. In DMSO-modified acid-fast stain, specific stained parasites were appeared firstly after 48 hour PI. The parasites were showed with different degrees of staining bright red color within PVM. 6. The endogenous stages of parasites in HmLu cell recovered at 48, 96, 120 and 144 hour after inoculation were reacted with rabbit immunized serum in immunofluorescence antibody and avidin-biotin complex peroxidase staining technique.

• Parasites, Hosts and Diseases
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• 제33권3호
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• pp.155-164
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• 1995

Cyptosporidium parvum의 발달 단계별 actin과 myosin의 분포 위치를 면역황금염색법을 이용하여 관찰하였다. $Depomedrol^{\circledR}$을 ICR마우스에 피하주사하여 면역억제시킨 후 C. parvum이 발현된 마우스 회장을 잘라 LR gold로 포매하여 초박절편을 떴다. 일차항체로는 chickenbackmuscle actin과 bovine uterus myosin에 대한 rabbit polyclonal antibody를 사용하였고 이차항체로는 10 mm 크기의 황금입자가 결합된 goatanti-rabbit lgG를 반응시켰다 Uranylacetate와 leadcitrate로 염색한 후 투과전자현미경으로 관찰하였다 Trophozoite에서는 세포막에서 주로 actin과 myosin이 관찰되었고 feederorganelle 주위 세포질에는 actin이 분포하였다. Meront와 같이 활발히 분열하고 있는 단계에서는 세포막과 세포질 전체에 actin이 분포되어있었으며 myosin은 세포막에서만 소량 관찰되었다. 핵과 anlage of rhoptries 등은 두 단백질에 모두 염색되지 않았다. Macrogametocyte 에서는 amylopectin-lile bodies에서 actin과 myosin이 모두 관찰되었으나 wall forming bodies에서는 관찰되지 않았고 feederorganelle 주위 세포질 부분에서는 actin이 관찰되었다. Sporozoite를 포함하는 oocyst와 merozoite를 포함하는 meront에서는 세포막과 세포막사이에서 actin이 다수 관찰 되었으며 myosin은 소량 관찰되었다. Merozoites가 빠져나가 속이 비어있는 parasitophorous vacuole중에는 microspike를 형성한 것들이 종종 관찰되었고 이것이 좀더 길어져 마치 microvilli와 같이 보이는 경우도 있었으며 이러한 구조물에서도 actin이 다수 관찰되었다. 이상의 결과로 미루어 actin과 myosin은 세포막에 주로 분포하면서 C. parvum의 형태를 유지시키며 또한 세포막의 움직임을 조절하는 cytoskeletalproteiA으로서의 역할이 주된 작용일 것으로 생각되었다.

• Parasites, Hosts and Diseases
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• 제52권6호
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• pp.613-619
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• 2014

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.