• Parasites, Hosts and Diseases
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• 제52권6호
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• pp.595-603
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• 2014

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time-and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.

• IMMUNE NETWORK
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• 제13권2호
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• pp.63-69
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• 2013

IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membranebound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-${\alpha}$. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the $CD8^+$ T cell-depleted mice than in $CD4^+$ T cell-depleted or normal mice. These results suggest that $CD8^+$ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and $CD4^+$ helper T cells.

• Journal of Microbiology and Biotechnology
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• 제21권6호
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• pp.637-645
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• 2011

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.

• Molecules and Cells
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• 제26권2호
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• pp.113-120
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• 2008

Laser light microscopy enables observation of various simultaneously occurring events in living cells. This capability is important for monitoring the spatiotemporal patterns of the molecular interactions underlying such events. Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multiphoton excitation, is one of the most promising candidates for such imaging. The advantages of two-photon microscopy have spurred wider adoption of the method, especially in neurological studies. Multicolor excitation capability, one advantage of two-photon microscopy, has enabled the quantification of spatiotemporal patterns of $[Ca^{2+}]_i$ and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocytosis" for the first time, a process which has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Our newly developed method, the two-photon extracellular polar-tracer imaging-based quantification (TEPIQ) method, can be used for determining fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Furthermore, two-photon microscopy has the demonstrated capability of obtaining cross-sectional images from deep layers within nearly intact tissue samples over long observation times with excellent spatial resolution. Recently, we have successfully observed a neuron located deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. This microscopy also enables the monitoring of long-term changes in neural or glial cells in a living mouse. This minireview describes both the current and anticipated capabilities of two-photon microscopy, based on a discussion of previous publications and recently obtained data.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.892-896
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• 2003

High-level expression of the lipase B26 gene from Bacillus pumilus was achieved using Bacillus subtilis Chungkookjang isolated from the Korean traditional fermented bean paste, Chungkookjang. For the secretory production of recombinant lipase B26 in a Bacillus host system, pLipB26 was constructed by ligating the lipase B26 gene into the recently designed Escherichia coli-Bacillus shuttle vector, pLipSM, and that was then transformed into B. subtilis Chungkookjang. Among the various vector, medium, and host combinations, B. subtilis Chungkookjang harboring the pLipB26 exhibited the highest lipase activity in PY medium, and B. subtilis Chungkookjang secreted two times more enzymes than B. subtilis DB 104 under the same condition. When B. subtilis Chungkookjang harboring the pLipB26 was cultured in a 5-1 jar-fermentor containing 21 of a PY medium, the maximum lipase activity (140 U/ml) and production yield (0.68 g/l) were obtained during the late exponential phase from a cell-free culture broth. Although B. subtilis Chungkookjang also secreted extracellular proteases at the late exponential phase, these results suggested the potential of B. subtilis Chungkookjang as a host for the secretory production of foreign proteins.

• 한국수산과학회지
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• 제35권3호
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• pp.253-258
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• 2002

Androgenic gland는 대부분의 수컷 연갑류의 제1, 2차 성징의 분화와 성적행동을 유발시키는 호르몬을 분비하는 것으로 알려져 있다. 생식소지수와 정소 및 AC의 조직학적 조사결과, 본 종에서 정자형성이 활발히 일어나는 시기는 5$\~$7월이며 8월부터는 대부분의 개체에서 정자형성은 관찰되지 않았다. 따라서 본 종의 수컷은 5월에서 7월이 성적으로 가장 활발하며 이 시기에 AG는 increased secretory activity를 나타내는 여러 징후를 보인다.

• 한국동물학회지
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• 제32권3호
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• pp.221-231
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• 1989

피부선이 잘 발달되어 있는 Bombina속 중에서 유일하게 국내에 서식하는 무당개구리를 재료로 하여 피부 점액선의 미세구조와 점액분비물질의 조직화학적 성질을 광학 및 전자현미경으로 관찰하였다. 무당개구리의 점액선은 진피에 위치한 단포상선으로서 선분비부는 외부의 근상피세포층과 내부의 선상피세포층으로 이루어져 있었다. 조직화학반응의 결과, 점액선에서 생성되는 물질의 주성분이 산성다당류임을 확인하였으며, 배피에 비해 복피에서 강한 양성반응을 나타내었다. 또한 주사전자현미경을 이용한 피부표면의 관찰 결과, 점액선의 개구부는 피부의 표면에 돌출된 식물의 기공과 비슷한 모양을 하고 있으며, 과립선의 개구부에 비해 분포밀도는 높았으나 크기는 소형인 것으로 관찰되었다. 점액선의 선상피세포층에는 점액분비세포와 미토콘드리아가 잘 발달되어 있는 세포의 2종류로 이루어져 있었고, 선의 대부분을 차지하고 있는 점액분비세포는 세포의 분화정도에 따라서 성숙 또는 미성숙된 세포로 구분되었으며, 세포질 내에 함유된 점액분비물질의 주변에서는 조면소포체가 잘 발달되어 있었다.

• Parasites, Hosts and Diseases
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• 제53권6호
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• pp.675-682
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• 2015

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

• Applied Microscopy
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• 제42권4호
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• pp.186-193
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• 2012

The ultrastructure of the secretory granules of the submandibular gland was examined in the Korean spider shrew, Sorex caecutiens, lesser white-toothed shrew, Crocidura suaveolens and big white-toothed shrew, C. lasiura. The mucous and serous acinar granules of S. caecutiens with a border of the lucid corona differed from those of C. suaveolens and C. lasiurar with a dense lateral border. The mucous acinar granules of C. lasiura with several bands producing a variety of patterns in the matrix were similar to those of C. suaveolens. The serous acinar granules of C. lasiura had a homogenous pale center surrounded by minute dense specks or had an unusual substructure showing a geometric pattern according to the inclusions in the pale matrix of the granules. This is the first report of an unusual substructure showing a geometric pattern of the serous acinar granules of C. lasiura. The myelin-like body was observed in the granular duct cell of the three species of shrew. The myelin-like body of S. caecutiens with layers of unit membranes was different from that of C. suaveolens and C. lasiura with paired membranes. Therefore, the layers composing of the paired membranes of the myelin-like body might be one of the characteristics of Crocidura.

• 한국정보과학회논문지:소프트웨어및응용
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• 제34권7호
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• pp.595-602
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• 2007

본 논문에서는 미지의 아미노산 서열이 신호 펩티다제 I에 의해 절단되는 분비성 단백질인지를 판별하고, 분비성 단백질일 경우에는 절단 위치를 예측하는 방법을 제안한다. 아미노산의 소수성을 이용한 전처리를 수행하여 분비성 단백질의 선도서열인 신호서열의 존재와 절단 위치를 추정한다. 전처리를 통해서 신호서열 아닌 서열을 초기에 제외함으로써 신호서열 예측의 정확도를 높인다. 지지벡터기계를 신호서열의 예측에 효과적으로 적용하기 위해서, 생물학적 정보와 관련된 아미노산 서열간의 거리를 제안한다. 아미노산의 세포내 위치를 예측할 수 있는 소수성 척도와 아미노산의 진화적인 관계를 나타낼 수 있는 치환행렬을 이용하여 아미노산 서열간의 거리를 정의한다. Swiss-Prot release 50 단백질 자료에 대하여 교차타당성 기법을 사용하여 실험한 결과 제안한 방법은 신호서열중에 98.9%를 신호서열로 판별하였고, 88%의 절단위치 예측정확도를 보였다. 기존의 방법과의 비교실험을 통해서 제안한 방법이 신호서열의 예측에 더욱 효과적임을 확인하였다.