• Archives of Pharmacal Research
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• v.25 no.6
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• pp.770-780
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• 2002

The airway surface liquid (ASL), often referred to as mucus, is a thin layer of fluid covering the luminal surface of the airway. The major function of mucus is to protect the lung through mucociliary clearance against foreign particles and chemicals entering the lung. The mucus is comprised of water, ions, and various kinds of macromolecules some of which possess the protective functions such as anti-microbial, anti-protease, and anti-oxidant activity. Mucus glycoproteins or mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for the effective mucociliary clearance. There are at least eight mucin genes identified in the human airways, which will potentially generate various kinds of mucin molecules. At present, neither the exact structures of mucin proteins nor their regulation are understood although it seems likely that different types of mucins are involved in different functions and might also be associated with certain airway diseases. The fact that mucins are tightly associated with various macromolecules present in ASL seems to suggest that the defensive role of ASL is determined not only by these individual components but rather by a combination of these components. Collectively, mucins in ASL may be compared to aircraft carriers carrying various types of weapons in defense of airborne enemies.

• Journal of Periodontal and Implant Science
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• v.39 no.sup2
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• pp.231-237
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• 2009

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.

• Parasites, Hosts and Diseases
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• v.49 no.1
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• pp.79-83
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• 2011

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage ${\lambda}$ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, ${\alpha$-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

• IMMUNE NETWORK
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• v.2 no.1
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• pp.35-40
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• 2002

Background: CTLA-4 (Cytotoxic T Lymphocyte associated Antigen 4, CD152) has been known as a homologue of CD28, an accessory molecule providing a key costimulatory signal for successful antigen-driven activations of T lymphocyte. Most of biochemical and cell biological characteristics of the CD152 protein remain unknown while those of CD28 have been characterized in detail. Methods: In this study CD152 expression in both $CD4^+$ and $CD8^+$ PBLs was studied by using flow cytometry. And intracellular CD152 multiprotein complex was purified and used for generating antibodies recognizing proteins composing of intracellular CTLA-4 multi protein complex. Results: Level of surface expression of this molecule was peaked at 2 days of PHA stimulation in flow cytometric analysis. 40~45% of PHA blast cells were $CD152^+$ in both of two subsets at this stage and the level of expression were equivalent in both two subsets. Contrary to this surface expression, intracellular expression was peaked at day 3 and it was preferentially induced in $CD8^+$ cells and about 60% of $CD8^+$ cells were $CD152^+$ at this stage. High molecular weight (>350 kD) intacellular CD152 protein complex purified by using preparative electrophoresis were immunized into rabbits and then 3 different anti-P34PC4, anti-P34PC7 and anti-P34PC8 antibodies were obtained. Using these 3 antibodies two unknown antigens associated with intracellular CD152 multiprotein complex were found and their molecular weights were 54 kD and 75 kD, respectively. Among these, the former was present as 110 kD homodimer in non-reducing condition. Conclusion: It seemed that 34 kD intracellular CD152 molecule forms high molecular weight multiprotein complex at least with 2 proteins of 75 kD monomer and 110 kD homodimer.

• Journal of Animal Science and Technology
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• v.65 no.2
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• pp.401-411
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• 2023

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

• Molecules and Cells
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• v.28 no.5
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• pp.441-446
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• 2009

During epididymal transit, mammalian sperm acquire selected proteins secreted by the epididymis. We previously showed that a disintegrin and metalloprotease (ADAM) 7 is expressed specifically in the epididymis and transferred to the sperm surface during epididymal transit. Here, we show that mouse ADAM7 secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Furthermore, we found that ADAM7 can be transferred directly from epididymal vesicles to sperm and that it is an integral plasma membrane protein in sperm. Thus, our study provides new information regarding the unique mode of secretion and interaction of ADAM7 during the epididymis-to-sperm transfer process.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.421-431
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• 2016

Recently, poly-γ-glutamic acid synthetase A (pgsA) has been applied to display exogenous proteins on the surface of Lactobacillus casei or Lactococcus lactis, which results in a surface-displayed component of bacteria. However, the ability of carrying genes encoded by plasmids and the expression efficiency of recombinant bacteria can be somewhat affected by the longer gene length of pgsA (1,143 bp); therefore, a truncated gene, pgsA, was generated based on the characteristics of pgsA by computational analysis. Using murine IL-10 as an exogenous gene, recombinant Lactobacillus plantarum was constructed and the capacity of the surface-displayed protein and functional differences between exogenous proteins expressed by these strains were evaluated. Surface expression of IL-10 on both recombinant bacteria with anchorins and the higher expression levels in L. plantarum-pgsA'-IL-10 were confirmed by western blot assay. Most importantly, up-regulation of IL-1β, IL-6, TNF-α, IFN-γ, and the nuclear transcription factor NF-κB p65 in RAW264.7 cells after stimulation with Poly(I:C) or LPS was exacerbated after co-culture with L. plantarum-pgsA. By contrast, IL-10 expressed by these recombinant strains could reduce these factors, and the expression of these factors was associated with recombinant strains that expressed anchorin (especially in L. plantarum-pgsA'-IL-10) and was significantly lower compared with the anchorin-free strains. These findings indicated that exogenous proteins could be successfully displayed on the surface of L. plantarum by pgsA or pgsA', and the expression of recombinant bacteria with pgsA' was superior compared with bacteria with pgsA.

• Korean Journal of Veterinary Service
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• v.44 no.4
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• pp.185-194
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• 2021

Polyglutamine extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyglutamine aggregates in Huntington's disease (HD). Mutant huntingtin can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. To better understand the mechanism by which an elongated polyglutamine causes aggregates, we have developed an in vitro binding assay system of polyglutamine tract from truncated huntingtin. We made GST-HD exon1 fusion proteins which have expanded polyglutamine epitopes (e.g., 17, 23, 32, 46, 60, 78, 81, and 94 CAG repeats). In the present emergence of new study adjusted nanotechnology on protein chip such as surface plasmon resonance strategy which used to determine the substance which protein binds in drug discovery platform is worth to understand better neurodegenerative diseases (i.e., Alzheimer disease, Parkinson disease and Huntington disease) and its pathogenesis along with development of therapeutic measures. Hence, we used strengths of surface plasmon resonance (SPR) technology which is enabled to examine binding specificity and explore targeted molecular epitope using its electron charged wave pattern in HD pathogenesis utilize conjugated mutant epitope of HD protein and its interaction whether wild type GST-HD interacts with mutant GST-HD with maximum binding affinity at pH 6.85. We found that the maximum binding affinity of GST-HD17 with GST-HD81 was higher than the binding affinities of GST-HD17 with other mutant GST-HD constructs. Furthermore, our finding illustrated that the mutant form of GST-HD60 showed a stronger binding to GST-HD23 or GST-HD17 than GST-HD60 or GST-HD81. These results indicate that the binding affinity of mutant huntingtin does not correlate with the length of polyglutamine. It suggests that the aggregation of an expanded polyglutamine might have easily occurred in the presence of wild type form of huntingtin.

• BMB Reports
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• v.31 no.2
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• pp.196-200
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• 1998

Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the subcellular targeting process without the ER retention signal. Circumstantial data indicate that the 27K zein is an ER transmembrane protein. The potential transmembrane domain may permit the 27K zein to remain in the ER. This study investigated the potential transmembrane feature by employing alkaline extraction, proteinase K digestion, and surface biotinylation on isolated intact protein bodies. These assays consistently support the possibility of the 27K zein as a transmembrane protein. The 27K zein polypeptide was shown to be associated with alkali-stripped membranes. The polypeptide was digested by proteinase K to a smaller fragment. According to surface biotinylation, the 27K zeins was labeled to the exclusion of other classes of zeins. This study, therefore, concludes that the 27K zein has an ER transmembrane domain, which may serve as an anchor for zeins' ER retention.

• Proceedings of the Korean Institute of Surface Engineering Conference
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• 2016.11a
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• pp.122.2-122.2
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• 2016

Surface topographical cues has been highlighted to control the fate of neural stem cells (NSCs). Herein we developed a hierarchically patterned substrate (HPS) platform for regulating NSC differentiation. The HPS induced cytoskeleton alignment and highly activated focal adhesion in hNSCs as indicated by enhanced expression of focal adhesion proteins such as focal adhesion kinase (FAK) and vinculin. hNSCs cultured on HPS exhibited enhanced neuronal differentiation compared to flat group. We also developed a graphene oxide (GO)-based hierarchically patterned substrates (GPS) that promote focal adhesion formation and neuronal differentiation of hNSCs. Enhanced focal adhesion and differentiation of hNSCs on the HPS was reversed by blocking the ${\beta}1$ integrin binding and mechanotransduction-associated signals including Rho-associated protein kinase (ROCK) and extracellular-regulated kinase (ERK) pathway, which may suggest a potential mechanism of beneficial effects of HPS. In addition, hNSCs on the HPS differentiated into functional neurons exhibiting sodium currents and action potentials as confirmed by whole cell patch-clamping analysis. The hierarchical topography can direct differentiation of NSCs towards functional neurons, and therefore would be an important element for the design of functional biomaterials for neural tissue regeneration applications.