• 대한수의학회지
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• 제44권3호
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• pp.455-462
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• 2004

Change in ${\beta}$-tubulin nucleic acid and protein sequences was the only known difference between Haemonchus contortus fenbendazole (FBZ)-resistant and -susceptible isolates. This change was sufficient to determine the pathologic effect induced by FBZ treatment. This research was initiated to investigate further differences from these two isolates. Since ${\beta}$-tubulin is involved in formation of microtubule, which has functions in secretory vesicle transport, DNase activities from excretory/secretory products (ESP) of the two isolates were compared, based on pH, sensitivity to DNase inhibitors, molecular masses and production of 3'-OH. The most significant difference detected was that a 38.5 kDa DNase activity was identified from ESP of H. contortus FBZ-susceptible isolates but not from those of H. contortus FBZ-resistant isolates. However, it was shown that the 38.5 kDa DNase is expressed with similar level of activity in intestine and whole worm of H. contortus FBZ-resistant and -susceptible isolates. This result demonstrated that the secretory transport pathway of the 38.5 kDa DNase was inhibited by unknown mechanisms, which may be related with ${\beta}$-tubulin sequence change in FBZ-resistant isolates. Other DNases of 34, 36 and 37 kDa were detected from ESP of both H. contortus FBZ-resistant and -susceptible isolates. Overall DNase activities found from ESP of these two isolates were not inhibited by 10 mM EDTA at pH 5.0, but largely inhibited by pH 7.0. In addition, DNase activities in two isolates produced DNA fragments with mixtures of 3'- hydroxyls (OH) and 3'-phosphates (P) at each pH although the 3'-end labeling ratios at pH 5.0 and 7.0 were shown different. Identification of inhibition of the 38.5 kDa DNase secretion in FBZ-resistant isolates suggests existence of further differences, in addition to ${\beta}$-tubulin sequence change, in two isolates. This shows complex effect of FBZ on H. contortus biological mechanisms.

• Parasites, Hosts and Diseases
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• 제49권3호
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• pp.291-294
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• 2011

Trichomonas vaginalis is a flagellated lumen-dwelling extracellular protozoan parasite that causes human trichomoniasis via sexual intercourse. Human neutrophils play a crucial role in acute tissue inflammatory responses in T. vaginalis infection. In this study, we investigated the signaling mechanism of neutrophil responses when stimulated with T. vaginalis-derived secretory products (TvSP), which were collected from $1{\times}10^7$ live trichomonads. Incubation of human neutrophils isolated from peripheral blood with TvSP induced up-regulation of IL-8 protein secretion. In addition, stimulation with TvSP induced phosphorylation of NF-${\kappa}B$ and CREB in neutrophils. Moreover, TvSP-induced IL-8 production was also significantly inhibited by pretreatment of neutrophils with $i{\kappa}B$ inhibitor or CREB inhibitor. These results suggest that transcription factors NF-${\kappa}B$ and CREB are involved in IL-8 production in human neutrophils induced by stimulation with T. vaginalis infection.

• Applied Microscopy
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• 제50권
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• pp.16.1-16.11
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• 2020

Silk is produced by a variety of insects, but only silk made by terrestrial arthropods has been examined in detail. To fill the gap, this study was designed to understand the silk spinning system of aquatic insect. The larvae of caddis flies, Hydatophylax nigrovittatus produce silk through a pair of labial silk glands and use raw silk to protect themselves in the aquatic environment. The result of this study clearly shows that although silk fibers are made under aquatic conditions, the cellular silk production system is quite similar to that of terrestrial arthropods. Typically, silk production in caddisworm has been achieved by two independent processes in the silk glands. This includes the synthesis of silk fibroin in the posterior region, the production of adhesive glycoproteins in the anterior region, which are ultimately accumulated into functional silk dope and converted to a silk ribbon coated with gluey substances. At the cellular level, each substance of fibroin and glycoprotein is specifically synthesized at different locations, and then transported from the rough ER to the Golgi apparatus as transport vesicles, respectively. Thereafter, the secretory vesicles gradually increase in size by vesicular fusion, forming larger secretory granules containing specific proteins. It was found that these granules eventually migrate to the apical membrane and are exocytosed into the lumen by a mechanism of merocrine secretion.

• Parasites, Hosts and Diseases
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• 제62권3호
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• pp.281-293
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• 2024

We previously reported that leukotriene B₄ (LTB₄) contained in Trichomonas vaginalis-derived secretory products (TvSP) play an essential role in interleukin-8 (IL-8) production in human mast cell line (HMC-1 cells) via LTB₄ receptor (BLT)-mediated Nuclear Factor-kappa B (NF-κB) activation. Dynamin, a GTPase, has been known to be involved in endocytosis of receptors for signaling of production of cytokine or chemokines. In the present study, we investigated the role of dynamin-mediated BLT1 endocytosis in TvSP-induced IL-8 production. When HMC-1 cells were transfected with BLT1 or BLT2 siRNA, TvSP-induced IL-8 production was significantly inhibited compared with that in cells transfected with control siRNA. In addition, pretreatment of HMC-1 cells with a dynamin inhibitor (Dynasore) reduced IL-8 production induced by TvSP or LTB₄. TvSP- or LTB₄-induced phosphorylation of NF-κB was also attenuated by pretreatment with Dynasore. After exposing HMC-1 cells to TvSP or LTB₄, BLT1 was translocated from the intracellular compartments to the plasma membrane within 30 min. At 60 min after stimulation with TvSP or LTB₄, BLT1 remigrated from the cell surface to intracellular areas. Pretreatment of HMC-1 cells with dynamin-2 siRNA blocked internalization of BLT1 induced by TvSP or LTB₄. Co-immunoprecipitation experiments revealed that dynamin-2 strongly interacted with BLT1 60 min after stimulation with TvSP or LTB₄. These results suggest that T. vaginalis-secreted LTB₄ induces IL-8 production in HMC-1 cells via dynamin 2-mediated endocytosis of BLT1 and phosphorylation of NF-κB.

• Parasites, Hosts and Diseases
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• 제62권3호
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• pp.270-280
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• 2024

Trichomoniasis is caused by a sexually transmitted flagellate protozoan parasite Trichomonas vaginalis. T. vaginalis-derived secretory products (TvSP) contain lipid mediators such as leukotriene B₄ (LTB₄) and various cysteinyl leukotrienes (CysLTs) which included LTC₄, LTD₄, and LTE₄. However, the signaling mechanisms by which T. vaginalis-induced CysLTs stimulate interleukin (IL)-8 production in human mast cells remain unclear. In this study, we investigated these mechanisms in human mast cells (HMC-1). Stimulation with TvSP resulted in increased intracellular reactive oxygen species (ROS) generation and NADPH oxidase 2 (NOX2) activation compared to unstimulated cells. Pre-treatment with NOX2 inhibitors such as diphenyleneiodonium chloride (DPI) or apocynin significantly reduced ROS production in TvSP-stimulated HMC-1 cells. Additionally, TvSP stimulation increased NOX2 protein expression and the translocation of p47^phox from the cytosol to the membrane. Pretreatment of HMC-1 cells with PI3K or PKC inhibitors reduced TvSP-induced p47^phox translocation and ROS generation. Furthermore, NOX2 inhibitors or NOX2 siRNA prevented CREB phosphorylation and IL-8 gene expression or protein secretion induced by TvSP. Pretreatment with a CysLTR antagonist significantly inhibited TvSP-induced ROS production, CREB phosphorylation, and IL-8 production. These results indicate that CysLT-mediated activation of NOX2 plays a crucial role in ROS-dependent IL-8 production in human mast cells stimulated by T. vaginalis-secreted CysLTs. These findings enhance our understanding of the inflammatory response in trichomoniasis and may inform the development of targeted therapies to mitigate this response.

• Applied Microscopy
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• 제18권2호
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• pp.91-101
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• 1988

The ultrastructure of the sac and tail portion of the large ampullate gland and production of the silk materials in the orb web spider, Nephila clavata L. Koch, are studied with electron microscope. Large ampullate glands, the largest glands among the seven kinds of silk glands in this species, are composed of three parts which are the excretory duct, the storage sac and the convoluted tail. The wall of the sac is composed of a single layer of columnar epithelial cells. In the cytoplasm of these cells several kinds of the secretory granules, which are commonly associated with the rough endoplasmic reticula and had characteristic crystalloid, are seen. According to the morphology and internal textures of these granules, the sac portion is subdivided into proximal(connected with the convoluted tail) and distal(connected with excretory duct) portion. Between these two portions, the proximal portion is longer than the distal by four times. Silk materials, being synthesized to the shape of secretory granules, within the glandular epithelial cell of the tail portion are released to the inner cavity by the mechanism of the eccrine secretion. These secretory granules are originated from the rough endoplasmic reticula of the glandular epithelial cells, whereas no Golgi complexes has been found in any of the cells which have been examined.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1264-1269
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• 2011

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular functions and immunity of animals. In this study, the recombinant duck IL-2 (rduIL-2) was secretory expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks and then scaled up in a 5.0-l bioreactor. The result showed that the maximal fresh-cell-weight of 594.1 g/l and the maximal $OD_{600}$ of 408 were achieved in the bioreactor. The rduIL-2 was purified by two steps of purification procedures, and approximately 311 mg of rduIL-2/L fermentation supernatant was obtained. SDS-PAGE showed that the purified rduIL-2 constituted a homogeneous band of ~16 kDa or ~14 kDa corresponding to the glycosylated or non-glycosylated duIL-2 protein in size, respectively. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay. The result indicated that the rduIL-2 greatly promoted the proliferation of ConA-stimulated lymphocytes in vitro. The P. pastoris expression system described here could provide promising, inexpensive, and large-scale production of the rduIL-2, which lays the foundation for development of novel immunoadjuvants to enhance both the immunity of ducks against various infectious pathogens and vaccine efficacy.

• IMMUNE NETWORK
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• 제21권5호
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• pp.36.1-36.16
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• 2021

Peroxiredoxins (Prxs) are ubiquitously expressed peroxidases that reduce hydrogen peroxide or alkyl peroxide production in cells. Prxs are released from cells in response to various stress conditions, and they function as damage-associated molecular pattern molecules. However, the secretory mechanism of Prxs and their roles have not been elucidated. Thus, we aimed to determine whether inflammasome activation is a secretory mechanism of Prxs and subsequently identify the effect of the secreted Prxs on activation of the classical complement pathway. Using J774A.1, a murine macrophage cell line, we demonstrated that NLRP3 inflammasome activation induces Prx1, Prx2, Prx5, and Prx6 secretion in a caspase-1 dependent manner. Using HEK293T cells with a transfection system, we revealed that the release of Prx1 and Prx2 relies on gasdermin-D (GSDMD)-mediated secretion. Next, we confirmed the binding of both Prx1 and Prx2 to C1q; however, only Prx2 could induce the C1q-mediated classical complement pathway activation. Collectively, our results suggest that inflammasome activation is a secretory mechanism of Prxs and that GSDMD is a mediator of their secretion. Moreover, secreted Prx1 and Prx2 bind with C1q, but only Prx2 mediates the classical complement pathway activation.

• The Korean Journal of Physiology and Pharmacology
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• 제19권6호
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• pp.549-555
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• 2015

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors /PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2005년도 2005 Annual Meeting & International Symposium
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• pp.131-136
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• 2005