Regulation of p21Waf1/Cip1 expression by miR-22 in Epstein-Barr virus infected B cells

의과학과/석사Epstein-Barr virus (EBV)는 감마 허피스 바이러스로 사람의 종양 형성과 관련된 종양 바이러스이다. 바이러스 잠복기 유전자 중에서 EBNA2는 세포에 EBV가 감염되었을 때 발현되는 종양 형성에 중요한 바이러스 단백질로 세포 유전자의 전사 활성을 일으킨다. EBNA2는 세포 주기를 억제하여 종양의 증식을 감시하고 억제하는 기능을 하는 p21Waf1/Cip1의 발현도 활성화 시킨다. 종양 형성 과정에서 p21Waf1/Cip1의 단백질 발현은 감소되어야 하므로, microRNA (miRNA)에 의한 유전자 번역 억제가 일어나는 것을 조사하였다. B 세포에 EBV가 감염되었을 때 발현되는 miRNA를 miRNA microarray를 시행하여 찾았고, TargetScan을 이용하여 p21Waf1/Cip1을 표적으로 하는 miRNA를 조사한 결과, miR-22가 EBV 감염에 의해 발현이 증가하며 동시에 p21Waf1/Cip1을 표적으로 하는 것을 알아내었다. MiR-22의 표적이 p21Waf1/Cip1임을 확인하기 위하여 p21Waf1/Cip1의 3’ UTR을 가진 reporter plasmid를 이용하여 reporter assay를 시행하여 감염 초기에 발현되는 EBV miRNA인 mir-BHRF1-3와 miR-22에 의하여 p21Waf1/Cip1의 발현이 억제되는 것을 확인하였다. 또한, miRNA의 저해제인 2’-O-Methyl miR-22를 SNU719 세포에 처리해 주었을 경우에 p21Waf1/Cip1의 유전자 및 단백질 발현이 증가하는 것을 확인하였고, 세포 주기가 억제되는 것이 관찰되었다. 이는 miR-22 를 억제시키고 EBV를 감염시킨 Ramos 세포에서 p21Waf1/Cip1의 발현이 증가하는 것으로도 확인할 수 있었다.이러한 실험 결과를 통하여 EBV에 감염된 B 세포에서 EBNA2에 의하여 증가된 p21Waf1/Cip1이 miR-22 및 mir-BHRF1-3의 영향으로 단백질 발현이 억제되는 것을 확인하였다.restrictio

웹기반 건설물류 정보시스템의 설계 및 개발

學位論文(碩士)--亞州大學敎 大學院 :産業工學科,2001Maste

The effect of robot-assisted gait training on cortical activation in stroke patients: A functional near-infrared spectroscopy study

OBJECTIVE: To investigate the effects of the robot-assisted gait training on cortical activation and functional outcomes in stroke patients. METHODS: The patients were randomly assigned: training with Morning Walk (R) (Morning Walk group; n =30); conventional physiotherapy (control group; n= 30). Rehabilitation was performed five times a week for 3 weeks. The primary outcome was the cortical activation in the Morning Walk group. The secondary outcomes included gait speed, 10-Meter Walk Test (10MWT), FAC, Motricity Index-Lower (MI-Lower), Modified Barthel Index (MBI), Rivermead Mobility Index (RMI), and Berg Balance Scale (BBS). RESULTS: Thirty-six subjects were analyzed, 18 in the Morning Walk group and 18 in the control group. The cortical activation was lower in affected hemisphere than unaffected hemisphere at the beginning of robot rehabilitation. After training, the affected hemisphere achieved a higher increase in cortical activation than the unaffected hemisphere. Consequently, the cortical activation in affected hemisphere was significantly higher than that in unaffected hemisphere (P = 0.036). FAC, MBI, BBS, and RMI scores significantly improved in both groups. The Morning Walk group had significantly greater improvements than the control group in 10MWT (P = 0.017), gait speed (P = 0.043), BBS (P = 0.010), and MI-Lower (P = 0.047) scores. CONCLUSION: Robot-assisted gait training not only improved functional outcomes but also increased cortical activation in stroke patients

Neurodevelopmental Outcomes after Congenital Heart Disease Surgery in Infancy: A 2-Year Serial Follow-Up

Background: The aim of this study is to assess the neurodevelopmental status of infant patients who underwent cardiac surgery in infancy and to investigate the factors affecting the neurodevelopmental status. Methods: This retrospective study included 108 patients who underwent cardiac surgery before the age of one. We used the Bayley Scales of Infant Development II to evaluate the neurodevelopmental status. All patients were analyzed according to the presence of the syndrome. Patients without the syndrome were analyzed according to the presence of brain lesions. Results: The mean mental developmental index (MDI) and the mean psychomotor developmental index (PDI) were 76.11 PLUSMN; 20.17 and 65.95 PLUSMN; 18.34, respectively, in the first evaluation, and 73.98 PLUSMN; 22.53 and 69.48 PLUSMN; 20.86, respectively, in the second evaluation. In the subgroup analysis, no significant difference was observed between the first evaluation and the second evaluation. Conclusions: No significant difference was observed in the degree of development of the patients in the two evaluation periods. Although the presence of syndrome, brain lesion, or gestational age affected the degree of developmental delay, more than half of the patients had developmental delay in the two evaluation periods in any of the subgroup. Therefore, the necessity of early screening and early rehabilitation intervention is emphasized

Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri.

Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cellsope

Evaluation of alpha-tubulin as an antigenic and molecular probe to detect Giardia lamblia

The alpha/beta-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant alpha-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of alpha-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the alpha-tubulin gene from G. lamblia. PCR-RFLP analysis of this alpha-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that alpha-tubulin can be used as a molecular probe to detect G. lambliaope

Leukotriene B(4) receptors BLT1 and BLT2 are involved in interleukin-8 production in human neutrophils induced by Trichomonas vaginalis-derived secretory products.

OBJECTIVE AND METHOD: Trichomonas vaginalis is a flagellated protozoan parasite that causes human trichomoniasis. Although T. vaginalis itself can secrete lipid mediator leukotriene (LT) B(4) leading to neutrophil activation, information regarding the signaling mechanism involved in neutrophil activation induced by T. vaginalis-secreted LTB(4) is limited. We investigated whether LTB(4) contained in the T. vaginalis-derived secretory products (TvSP) is closely involved in interleukin (IL)-8 production in human neutrophils via LTB(4) receptors BLT1 or BLT2. RESULTS: T. vaginalis produced more than 714 pg/ml of LTB(4) per 1 × 10(7) trichomonads. The ability of trichomonads to secrete LTB(4) was inhibited by treatment of trichomonads with the 5-lipo-oxygenease inhibitor AA861, but not the cyclo-oxygenease I inhibitor FR122047. When neutrophils were incubated with TvSP obtained from 1 × 10(7) trichomonads, IL-8 protein secretion was significantly increased compared to results for cells incubated with medium alone. The stimulatory effect of TvSP on IL-8 production was strongly inhibited by pretreatment of TvSP with lipase, although pretreatment with heat or proteinase K showed little inhibitory effect. Moreover, TvSP-induced IL-8 production was efficiently inhibited when trichomonads were pretreated with AA861 or when neutrophils were pretreated with antagonists for BLT1 or BLT2. CONCLUSION: Our results suggest that LTB(4) receptors BLT1 and BLT2 are involved in IL-8 production in neutrophils induced by T. vaginalis.ope

Leukotriene B4 receptor BLT-mediated phosphorylation of NF-κB and CREB is involved in IL-8 production in human mast cells induced by Trichomonas vaginalis-derived secretory products

Trichomonas vaginalis is a protozoan parasite that causes acute tissue inflammation in vaginal trichomoniasis. In this study, we investigated the signaling mechanisms through which T. vaginalis-derived secretory products (TvSP) induce chemokine IL-8 production in human mast cells. Stimulation with TvSP induced up-regulation of IL-8 protein secretion in HMC-1 cells. In addition, TvSP induced phosphorylation of transcription factors NF-κB and CREB in HMC-1 cells. Pretreatment of TvSP with lipase, but not heat or proteinase K strongly abolished the stimulatory effect on IL-8 production. Moreover, TvSP-induced IL-8 production and phosphorylation of NF-κB or CREB were inhibited when HMC-1 cells were stimulated with modified TvSP collected from 5-lipooxygenase inhibitor-treated trichomonads. Indeed, T. vaginalis-secreted lipid mediator LTB(4) (700pg/ml) from 1×10(7) trichomonads. Furthermore, pretreatment of HMC-1 cells with antagonists for LTB(4) receptors BLT1 or BLT2 abolished the stimulatory effects of TvSP. Finally, TvSP-induced IL-8 production was inhibited by pretreatment with IκB or CREB inhibitors. These results suggest that T. vaginalis-derived secretory lipid mediator LTB(4) induces IL-8 production in mast cells via BLT-dependent activation of NF-κB and CREB.ope

NOX1 participates in ROS-dependent cell death of colon epithelial Caco2 cells induced by Entamoeba histolytica

Entamoeba histolytica, which causes amebic colitis and occasional liver abscesses in humans, can induce host cell death through apoptosis and necrosis. Recently, we have demonstrated that E. histolytica can induce cell death in neutrophils via diphenyleneiodonium-sensitive NADPH oxidase (NOX)-derived reactive oxygen species (ROS). Although there are enzyme systems similar to the phagocyte NADPH oxidase system in many non-phagocytic cell types, the signaling role of NOX-derived ROS in cell death of human colon epithelial cells induced by E. histolytica remains obscure. Incubation of colon epithelial Caco2 tumor cell lines with amebic trophozoites resulted in intracellular ROS generation and cell death in a caspase-independent manner. Pretreatment with DPI, an inhibitor of NOX, strongly decreased E. histolytica-induced cell death in Caco2 cells. As identified by RT-PCR, NOX1 transcripts were highly expressed in Caco2 cells. siRNA-mediated suppression of NOX1 protein significantly inhibited E. histolytica-induced cell death and ROS response in Caco2 cells. These results suggest that NOX1 participates in the ROS-dependent cell death of colon epithelial cells induced by amebic adhesion during the early phase of intestinal amebiasis.ope