• Tuberculosis and Respiratory Diseases
• /
• 제43권2호
• /
• pp.210-220
• /
• 1996

식세포인 호중구나 단핵세포는 생체외실험에 사용하기 위한 세포분리법인 플라스틱 표면부착만으로도 세포활성화가 일어나 이후의 실험결과에 영향을 주고 이 과정에 부착분자가 연관되어 있는 것으로 알려져 있다. 폐의 주된 면역세포인 폐포대식세포도 대부분 플라스틱 표면부착에 의해 세포를 분리하므로 사람의 폐포대식세포가 표면부착 자체에 의해 활성화되는지 알아보고 세포활성회에 부착분자와 같은 기전이 관여하는지 밝히기 위해 적어도 한 쪽 폐가 정상인 사람에서 기관지폐포세척술을 통해 얻은 폐포대식세포를 대상으로 표면 부착이 미치는 영향을 과산화수소 분비량 측정으로 분석하여 다음과 같은 결과를 얻었다. 1) 폐포대식세포는 플라스틱 표면에 부착되면 부착 자체에 의해 과산화수소 분비능이 증가하고 이런 상태에서는 PMA나 fMLP와 같은 추가적인 화학자극물질에 의해 과산화수소 분비가 증가되지 않았다. 2) 여러가지 표면중 A549세포단층에 부착될 경우에만 이후의 PMA와 fMLP자극 모두에 의해 과산회수소 분비가 증가하였다. 3) PMA는 세포 부착여부에 관계없이 과산화수소 분비를 자극하지만 fMLP는 폐포대식세포가 표면에 부착된 상태에서만 자극효과가 나타났고 이런 부착세포에서의 fMLP에 의한 과산회수소 분비 효과는 단백합성억제제인 cycloheximide, G단백차 단제인 일해독소와 $\beta_2$ integrin 부착분자에 대한 항체인 항CD18 단세포항체 3가지 모두의 의해 차단되었다. 이상의 결과로 사람의 폐포대식세포는 플라스틱 부착자체에 의해 활성화되므로 부착 이후의 자극물질에 대한 효과가 반감되지만 폐포상피세포와 같은 생물학적 표면에 부착될 경우에는 이후의 세포자극에 민감하게 반응한다는 것을 알 수 있었고, PMA는 세포 부착여부에 관계없이 세포를 자극하는 반면 fMLP는 세포 부착상태에서만 자극효과가 나타나며 이런 부착세포에서의 fMLP에 의한 산소유리기 자극효과는 G단백결합 수용체를 통한 새로운 단백합성 과정으로 이루워지면서 $\beta_2$ integrin을 통한 폐포대식세포와 폐포상피세포의 부착에 의존하는 것으로 사료된다.

• Parasites, Hosts and Diseases
• /
• 제39권2호
• /
• pp.171-176
• /
• 2001

Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), meroxoite surface protein (MSP-1), apical merozoite antigen (AMA- 1), serine repeat antigen (SERA), and exported antigen (EXP- 1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using S antigens (91.9%) rather than using each antigen solely (55.6 - 80%), a combination of 2 (76.3 - 87.5%), 3 (85.6 - 90.6%), or 4 antigens (89.4 - 91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.

• BMB Reports
• /
• 제30권6호
• /
• pp.431-437
• /
• 1997

Since the role of CD40 on the interleukin-4(IL-4) -induced B cell activation has been strongly implicated in the agumentation of IgE production and response, we have investigated the intracelluar signaling pathways utilized by IL-4 and CD40 for type II IgE receptor (CD23) expression. IL-4 and anti-CD40 antibody treatment of human B cells, independently caused a rapid induction of CD23 gene activation within 2 h. There was a noticeable synergism between the action of the two agents inducing CD23 expression: the addition of anti-CD40 to the IL-4-treated culture significantly agumented the IL-4-induced CD23 on both mRNA and surface protein levels, and the inclusion of IL-4 in the anti-CD40-treated cells caused a further increase of CD23 expression far above the maximal level induced by anti-CD40. Protein tyrosine kinase (PTK) inhibitors effectively suppressed the both IL-4- and anti -CD40-induced CD23 expression. whereas protein kinase C (PKC) inhibitors had no effects. Electrophoretic mobility shift assays (EMSA) have shown that IL-4 and anti-CD40 induce the activation of NF-IL-4 and $NF-_{K}B$, respectively, binding to the CD23 promoter, both in a PKC-independent and PTK-dependent manner. These data suggest that the synergistic activation of CD23 gene expression by IL-4 and anti-CD40 is mediated by co-operative action of distinct nuclear factors. each of which is rapidly activated via PKC-independent and PTK-dependent process.

• Journal of Periodontal and Implant Science
• /
• 제39권sup2호
• /
• pp.231-237
• /
• 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.

• The Plant Pathology Journal
• /
• 제35권6호
• /
• pp.564-574
• /
• 2019

Like many fungal pathogens, the conidium and appressorium play key roles during polycyclic dissemination and infection of Magnaporthe oryzae. Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein. In animalia, RanBPM has been implicated in apoptosis, cell morphology, and transcription. However, the functional roles of RanBPM, encoded by MGG_00753 (named MoRBP9) in M. oryzae, have not been elucidated. Here, the deletion mutant ΔMorbp9 for MoRBP9 was generated via homologous recombination to investigate the functions of this gene. The ΔMorbp9 exhibited normal conidial germination and vegetative growth but dramatically reduced conidiation compared with the wild type, suggesting that MoRBP9 is involved in conidial production. ΔMorbp9 conidia failed to produce appressoria on hydrophobic surfaces, whereas ΔMorbp9 still developed aberrantly shaped appressorium-like structures at hyphal tips on the same surface, suggesting that MoRBP9 is involved in the morphology of appressorium-like structures from hyphal tips and is critical for development of appressorium from germ tubes. Taken together, our results indicated that MoRBP9 played a pleiotropic role in polycyclic dissemination and infection-related morphogenesis of M. oryzae.

• Archives of Pharmacal Research
• /
• 제26권4호
• /
• pp.338-343
• /
• 2003

The purpose of the present study was to investigate the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was saturable, with a $K_m of 29.1\pm3.2 \mu M and V_{max} of 2.9\pm0.1$ mmol/min/mg protein. Subsequently, the initial efflux rate of ANS from isolated hepatocytes was determined by resuspending preloaded cells to 3.0% (w/v) BSA buffer. The efflux process for total ANS revealed a little saturability. The mean value of the efflux clearance was $2.2\pm0.1 \mu$ L/min/mg protein. The efflux rate of ANS from hepatocytes was markedly decreased at $4^{\circ}C$, indicating that the apparent efflux of ANS might not be attributed to the release of ANS bound to the cell surface, but to the efflux of ANS from intracellular space. The efflux clearance was furthermore corrected for the unbound intracellular ANS concentration on the basis of its binding parameters to cytosol. The relation between efflux rate and unbound ANS concentration was fitted well to the Michaelis-Menten equation with a saturable and a nonsaturable components. The $V_{max} and K_m$ values were 0.54 mmol/min/mg protein, and 10.0 $\mu$ M, respectively. Based on the comparison of the ratios of $V_{max} to K_m (V_{max}/K_m)$ corresponding to the transport clearance, the influx clearance was two times higher than the efflux clearance. Together with our preliminary studies that ATP suppression in hepatocytes substantially inhibited ANS influx rate, we concluded that the hepatic uptake of ANS is actively taken up into hepatocytes via the carrier mediated transport system.

• 한국수정란이식학회지
• /
• 제28권3호
• /
• pp.289-295
• /
• 2013

Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of ${\alpha}1$,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human $EF1{\alpha}$ promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human $EF1{\alpha}$ promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

• The Korean Journal of Physiology and Pharmacology
• /
• 제4권6호
• /
• pp.487-496
• /
• 2000

Insulin stimulates glucose transport in muscle and fat cells by promoting the translocation of glucose transporter (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3-kinase) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt and $PKC-{\zeta}$, those are known as the downstream target of PI3-kinase in regulation of GLUT4 translocation, is not known yet. An interesting possibility is that these protein kinases phosphorylate GLUT4 directly in this process. In the present study, $PKB-{\alpha}$ and $PKC-{\zeta}$ were added exogenously to GLUT4-containing vesicles purified from low density microsome (LDM) of the rat adipocytes by immunoadsorption and immunoprecipitation for direct phosphorylation of GLUT4. Interestingly GLUT4 was phosphorylated by $PKC-{\zeta}$ and its phosphorylation was increased in insulin stimulated state but GLUT4 was not phosphorylated by $PKB-{\alpha}.$ However, the GST-fusion proteins, GLUT4 C-terminal cytoplasmic domain (GLUT4C) and the entire major GLUT4 cytoplasmic domain corresponding to N-terminus, central loop and C-terminus in tandem (GLUT4NLC) were phosphorylated by both $PKB-{\alpha}$ and $PKC-{\zeta}.$ The immunoblots of $PKC-{\zeta}$ and $PKB-{\alpha}$ antibodies with GLUT4-containing vesicles preparation showed that $PKC-{\zeta}$ was co-localized with the vesicles but not $PKB-{\alpha}.$ From the above results, it is clear that $PKC-{\zeta}$ interacts with GLUT4-containing vesicles and it phosphorylates GLUT4 protein directly but $PKB-{\alpha}$ does not interact with GLUT4, suggesting that insulin-elicited signals that pass through PI3-kinase subsequently diverge into two independent pathways, an Akt pathway and a $PKC-{\zeta}$ pathway, and that later pathway contributes, at least in part, insulin stimulation of GLUT4 translocation in adipocytes via a direct GLUT4 phosphorylation.

• 한국식품과학회지
• /
• 제21권6호
• /
• pp.780-787
• /
• 1989

쌀과 분리대두단백 혼합에 따른 조직화 특성에서 쌀 혼합량 30%까지 증가할수록 토출구온도, 수용성 질소지수 및 조직잔사지수가 떨어지며 씹힘성과 검성이 감하여 부드러운 조직감이 형성되었다. 또한 쌀혼합량의 증가에 따라 최종 제품의 수분함량은 증가되었으나 밀도에는 변화가 없었고 재흡수력은 감소하는 경향이었다. 한편 복원 후에는 제품의 명도가 밝아졌다. 압출성형 준의 압력분포는 $15{\sim}100kg/cm^3$의 범위였다. 미세구조에서는 쌀혼합량이 증가할수록 기공의 크기가 증대하였으며 쌀 전분질이 호화된 표면구조를 지니고 있었다.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.19-19
• /
• 2001

Mammalian urothelium undergoes unique membrane specialization by making the asymmetric unit membrane (AUM) that is covered with the apical cell surface during terminal differentiation. The AUM contains several major integral membrane proteins including uroplakin Ia, Ib, II and III. The genes for uroplakins have been cloned from humans and mice, but not from porcine. In this study, we report the cloning of the UPII genomic DNA, which codes for the full length open reading frame for the uroplakin II protein. The deduced amino acid sequence encodes of a hydrophobic NH$_2$-terminal peptide, a prosequence, and a mature protein. The prosequence contains three potential N-glycosylation sites and a RGRR cleavage site that may be involved in uroplakin II processing and maturation. Northern and immunohistochemistry analyses showed that the porcine UPII gene is only expressed in urothelium and that the protein was specifically localized in urothelial superficial cells. A 2kb of upstream in the promoter sequence contains multiple transcription factor binding sites, including GC-box, SPI, AP2, and GATA-box sites, but not for TATA or CAAT-box sequences. Comparison of the porcine UPII promoter sequence with that of the murine by MEME system presented two conserved motifs, suggesting a cis-acting regulatory role for the conserved sequences. Sequence homology between two species in motif A and B was 79% and 80% respectively, although their relative locations were different. During the gestation, mouse bladder at estrus stages and day 10 after parturition showed higher UPII expression, while showed lower expression at peri-implantation stage. Taken together, our results showed that the porcine UPII gene was expressed highly and specifically in the bladder urothelium and that steroid hormones for implantation changed the expression of UPII in the bladder, although the biological significance of UPII remains to be not determined.