• Parasites, Hosts and Diseases
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• v.52 no.6
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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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• v.13 no.2
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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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• v.21 no.6
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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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• v.26 no.2
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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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• v.13 no.6
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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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.3
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• pp.253-258
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• 2002

The androgenic gland secretes a hormone, androgenic gland hormone, which is believed to act on the differentiation of the primary, secondary, and behavioral sex characteristics in most malacostracan crustaceans. Based on the changes in gonado-somatic indexe and histological observation of testis and androgenic gland cell, testicular maturation and spermatogenesis of M. nipponense occurred early in summer (May to July), and generally spermatogenesis was absent in August. It could be, concluded that May to July is the period when adult males of this species are sexually active, and androgenic gland showed signs of increased secretory activity.

• The Korean Journal of Zoology
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• v.32 no.3
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• pp.221-231
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• 1989

The histochemical characteristics of the cutaneous mucous glands and its fine structure of the Korean fire bellied toad, Bombina orientolis Boulenger, were studied with light and electron microscopes. The mucous glands of the toad are simple alveolar gland, and are composed of inner glandular epithelial cells and outermost myoepithelial cells. Histochemical studies suggest that main secretory materials released from the mucous glands might be acid mucopolysaccharides which is more abundant in the ventral skin than in the dorsal skin. The scanning electron microscopic observation showed that the morphology of the opening sites of the gland duct is similar to the morphology of stomata on the plant leaves. Glandular epithelial layers of the mucous glands are composed of two cell types, the mucous secreting cells and the mitochondria rich cells. The mucous secreting cells could be classified according to the morphology and inner textures of cytoplasm and those characteristics were seemed to be related to the degree of maturation of the cells. Furthermore the mucous within the mucous secreting cells appears to be synthesized in the rough endoplasmic reticulum.

• Parasites, Hosts and Diseases
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• v.53 no.6
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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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• v.42 no.4
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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.

• Journal of KIISE:Software and Applications
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• v.34 no.7
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• pp.595-602
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• 2007

This paper proposes a method that discriminates signal peptide and predicts the cleavage site of the secretory proteins cleaved by the signal peptidase I. The preprocessing stage uses hydrophobicity scales of amino acids in order to predict the presence of signal sequence and the cleavage site. The preprocessing enhances the performance of the prediction method by eliminating the non-secretory proteins in the early stage of prediction. for the effective use of support vector machine for the signal sequence prediction, the biologically relevant distance between the amino acid sequences is defined by using the hydrophobicity and substitution matrix; the hydrophobicity can be used to Predict the location of amino acid in a cell and the substitution matrix represents the evolutionary relationships of amino acids. The proposed method showed 98.9% discrimination rates from signal sequences and 88% correct rate of the cleavage site prediction on Swiss-Prot release 50 protein database using the 5-fold-cross-validation. In the comparison tests, the proposed method has performed significantly better than other prediction methods.