뼈 발생 과정에서 Ninjurin1 단백질의 기능 및 분자적 특성 연구

학위논문 (박사)-- 서울대학교 대학원 : 약학과, 2017. 2. 김규원.Osteoclasts (OCs) are bone-resorbing cells that originate from hematopoietic stem cells and develop through the fusion of mononuclear myeloid precursors. Dysregulation of OC development causes skeletal disorders such as osteopetrosis, osteoporosis, and rheumatoid arthritis. Although the molecular mechanisms underlying osteoclastogenesis have been well established, the means by which OCs maintain their survival during OC development remain unknown. In the present study, it was found that Ninjurin1 (Ninj1), a cell surface protein, supports OC development by enhancing the survival of prefusion OCs (preOCs). Ninj1 expression was dynamically regulated during osteoclastogenesis. In addition, Ninj1-/- mice exhibit mild osteopetrosis owing to impaired OC development. However, typical markers of OC differentiation such as Nfatc1, c-Fos, integrinβ3, Oscar, and Calcr were unaffected by Ninj1 deficiency. Furthermore, other important events of OC development such as transmigration, fusion, and actin ring formation were also not impaired by the absence of Ninj1. Instead, Ninj1 deficiency increased Caspase-9-dependent intrinsic apoptotic cell death in preOCs. Furthermore, overexpression of Ninj1 enhanced the survival of mouse macrophage/preOC RAW264.7 cells in osteoclastogenic culture, which suggests that Ninj1 is important for the survival of preOCs. Finally, analysis of publicly available microarray datasets revealed a potent correlation between high NINJ1 expression and bone disorders in humans. Regarding to Ninj1 molecular property, it was revealed that Ninj1 assembles into a homomeric protein complex composed of two to six monomeric Ninj1 molecules, and the intracellular region of Ninj1 encompassing Leu101 to Ala110 is important for Ninj1 assembly. Furthermore, Ninj1 is an N-glycosylated protein, and the N-glycosylation enhances homomer formation and regulates protein stability and plasma membrane sorting. These findings suggest that Ninj1 plays an important role in bone homeostasis by enhancing the survival of preOCs and might represent a potent therapeutic target for destructive bone disorders. Moreover, targeting of N-glycosylation in Ninj1 might be a regulatory strategy for modulation of Ninj1 function(s).INTRODUCTION 1 1. Bone and osteoblast 1 2. Osteoclast 4 3. Ninjurin1 8 4. Homomeric protein complex 11 PURPOSE OF THIS STUDY 12 MATERIALS AND METHODS 14 1. Ethics statement 14 2. Animals 14 3. Radiologic analysis 15 4. Plasmid construction, transfection and retroviral infection 15 5. Reagents 17 6. In vitro osteoclastogenesis 18 7. Cell culture 19 8. TRAP activity staining and measurement of TRAP activity 20 9. FACS analysis 21 10. Confocal microscopy 23 11. Proliferation assay 25 12. Protein cross-linking with formaldehyde 25 13. Immunoblot assay 26 14. RNA isolation and qRT-PCR 26 15. Microarray analysis 27 16. Statistical analysis 28 RESULTS 32 1. OCs express high amount of Ninj1 and deletion of Ninj1 induces bone abnormality in mice 32 2. Ninj1 deficiency induces mild osteopetrosis in mice 35 3. Ninj1 is expressed in OCs, not in OBs 40 4. Ninj1 expression is dynamically regulated during osteoclastogenesis 42 5. Ninj1 deficiency reduces multinucleated OCs 45 6. Ninj1 is dispensable for OC differentiation 50 7. Ninj1 deletion augments development of OPCs 54 8. Ninj1-deficient OC precursor cells develop normally from OPCs. 56 9. Ninj1 is expendable for preOC migration 59 10. Macrophage fusion is enhanced by Ninj1 deficiency 61 11. Ninj1 deletion reduces mature OC area 67 12. Lack of Ninj1 attenuates TRAP activity in cultured media 69 13. Ninj1 is important for maintenance of cell population during preOC stage 72 14. Ninj1 enhances preOC survival during osteoclastogenesis 74 15. Ninj1 deficiency induces Caspase-9-dependent intrinsic apoptosis in preOCs 78 16. A high level of Ninj1 improves preOC survival 83 17. High NINJ1 expression correlates with human bone disorders 86 18. Ninj1 might be a co-stimulatory molecule and/or co-receptor for ITAMmediated signals 89 19. Ninj1 possesses homophilic binding affinity 92 20. Ninj1 is a cis-interacting protein 95 21. Ninj1 assembles into a homomeric protein complex 99 22. Intracellular region of Ninj1 from Leu101 to Ala110 is required for Ninj1 assembly 103 23. Ninj1 is an N-linked glycosylated protein 107 24. N-glycosylation stabilizes the Ninj1 homomeric complex 112 25. N-glycosylation is important for Ninj1 trafficking and stability 116 DISCUSSION 126 REFERENCES 138 요약 (국문초록) 150Docto

Formation of Warped Disks by Galactic Fly-by Encounters. I. Stellar Disks

Warped disks are almost ubiquitous among spiral galaxies. Here we revisit and test the `fly-by scenario' of warp formation, in which impulsive encounters between galaxies are responsible for warped disks. Based on N-body simulations, we investigate the morphological and kinematical evolution of the stellar component of disks when galaxies undergo fly-by interactions with adjacent dark matter halos. We find that the so-called `S'-shaped warps can be excited by fly-bys and sustained for even up to a few billion years, and that this scenario provides a cohesive explanation for several key observations. We show that disk warp properties are governed primarily by the following three parameters; (1) the impact parameter, i.e., the minimum distance between two halos, (2) the mass ratio between two halos, and (3) the incident angle of the fly-by perturber. The warp angle is tied up with all three parameters, yet the warp lifetime is particularly sensitive to the incident angle of the perturber. Interestingly, the modeled S-shaped warps are often non-symmetric depending on the incident angle. We speculate that the puzzling U- and L-shaped warps are geometrically superimposed S-types produced by successive fly-bys with different incident angles, including multiple interactions with a satellite on a highly elongated orbit.Comment: 16 pages, 13 figures, 3 tables. Accepted for publication in Ap

Učinci prenatalne i postnatalne izloženosti perfluorooktanskoj kiselini na ekspresiju glavnih gena povezanih s reprodukcijom u mišjem hipotalamusu i spolnim žlijezdama

Perfluorooctanoic acid (PFOA), a ubiquitous environmental pollutant reported to be an endocrine disruptor, is used in many industrial and consumer products. Although the adverse effects of PFOA on the reproductive health of animals and humans have been widely reported, most studies have focused on assessing the anatomical features and conventional histology of adult gonads. Therefore, the molecular mechanisms activated in the hypothalamus and gonads following PFOA exposure during the pre- and postnatal periods are not clear. This study used a mouse model to evaluate the effects of PFOA exposure on the alteration of molecular mechanisms in the hypothalamus and gonads during the prenatal and postpartum periods. Changes in gene and protein expression following PFOA exposure were evaluated by quantitative polymerase chain reaction and Western blotting, respectively. Kisspeptin 1 and gonadotropin-releasing hormone expression in the hypothalamus of female and male mouse pups was significantly decreased. Additionally, Cyp17a1 expression was upregulated in male offspring testes, while Cyp17a1 and Cyp19a1 expression was downregulated in female offspring ovaries. Changes at the molecular level due to PFOA exposure in the early stages of development did not show sex-related differences in the hypothalamus; however, such differences were confirmed in the gonads. These results could be used as basic data to study the molecular mechanisms underlying the reproductive dysfunction caused by PFOA exposure in the early stages of embryonic development.Perfluorooktanska kiselina (PFOA) sveprisutna je onečišćujuća tvar za okoliš, za koju je zabilježeno da uzrokuje i endokrinopatije, a upotrebljava se u mnogim industrijskim proizvodima. Bez obzira na poznate i zabilježene nuspojave PFOA-e na reproduktivno zdravlje životinja i ljudi, većina se istraživanja usredotočuje na procjenu anatomskih histoloških značajki u spolnim žlijezdama odraslih jedinki. Molekularni mehanizmi koji se aktiviraju u hipotalamusu i spolnim žlijezdama nakon izlaganja PFOA-i stoga nisu razjašnjeni. U ovom je istraživanju upotrijebljen mišji model za procjenu učinaka izlaganja PFOA-i na promjenu molekularnih mehanizama u hipotalamusu i spolnim žlijezdama za vrijeme prijeporođajnog i poslijeporođajnog razdoblja. Promjene u ekspresiji gena i proteina nakon izloženosti PFOA-i analizirane su kvantitativnom PCR metodom i metodom Western blotting. Ekspresija kispeptina 1 i hormona koji oslobađa gonadotropin u hipotalamusu ženske i muške mladunčadi bila je znakovito smanjena. Osim toga ekpresija Cyp17a1 bila je pojačana u testisima muških potomaka, dok je ekspresija Cyp17a1 i Cyp19a1 u jajnicima ženskih potomaka bila smanjena. Kod promjena na molekularnoj razini u hipotalamusu u ranim razvojnim stadijima nije bilo razlike među spolovima, dok je kod promjena u spolnim žlijezdama povrđena razlika među spolovima. Rezultati ovog istraživanja mogli bi biti korisni kao osnovni podaci u proučavanju molekularnih mehanizama podležeće reproduktivne disfunkcije uzrokovane izloženošću PFOA-i u ranim stadijima embrionalnog razvoja

Effects of Lowering Dialysate Calcium Concentrations on Arterial Stiffness in Patients Undergoing Hemodialysis

BACKGROUND/AIMS: We assessed changes in hemodynamic and arterial stiffness parameters following reductions of dialysate calcium concentrations in patients undergoing hemodialysis. METHODS: In this prospective study, 20 patients on maintenance hemodialysis (10 females, 10 males) with dialysate calcium concentrations of 1.75 mmol/L were enrolled. At the start of the study, the dialysate calcium level was lowered to 1.50 mmol/L. Serial changes in biochemical, hemodynamic, and arterial stiffness parameters, including pulse wave velocity (PWV) and augmentation index (AIx), were assessed every 2 months for 6 months. We also examined changes in the calcification-inhibitory protein, serum fetuin-A. RESULTS: During the 6-month study period, serum total calcium and ionized calcium decreased consistently (9.5 ± 1.0 to 9.0 ± 0.7, p = 0.002 vs. 1.3 ± 0.1 to 1.1 ± 0.1, p = 0.035). Although no apparent changes in blood pressure were observed, heart-femoral PWW (hf-PWV) and AIx showed significant improvement (p = 0.012, 0.043, respectively). Repeated-measures ANOVA indicated a significant effect of lowering dialysate calcium on hf-PWV (F = 4.58, p = 0.004) and AIx (F = 2.55, p = 0.049). Accompanying the change in serum calcium, serum fetuin-A levels significantly increased (95.8 ± 45.8 pmol/mL at baseline to 124.9 ± 82.2 pmol/mL at 6 months, p = 0.043). CONCLUSIONS: Lowering dialysate calcium concentration significantly improved arterial stiffness parameters, which may have been associated with upregulation of serum fetuin-A.ope

Generation of subspecies level-specific microbial diagnostic microarrays using genes amplified from subtractive suppression hybridization as microarray probes

The generation of microarray probes with specificity below the species level is an ongoing challenge, not least because the high-throughput detection of microorganisms would be an efficient means of identifying environmentally relevant microbes. Here, we describe how suppression subtractive hybridization (SSH) can be applied to the production of microarray probes that are useful for microbial differentiation at the subspecies level. SSH was used to initially isolate unique genomic sequences of nine Salmonella strains, and these were validated in quadruplicate by microarray analysis. The results obtained indicate that a large group of genes subtracted by SSH could serve together, as one probe, for detecting a microbial subspecies. Similarly, the whole microbial genome (not subjected to SSH) can be used as a species-specific probe. The detailed methods described herein could be used and adapted for the estimation of any cultivable bacteria from different environments

EFFECTS OF MO, CR, AND V ADDITIONS ON TENSILE AND CHARPY IMPACT PROPERTIES OF API X80 PIPELINE STEELS

In this study, four API X80 pipeline steels were fabricated by varying Mo, Cr, and V additions, and their microstructures and crystallographic orientations were analyzed to investigate the effects of their alloying compositions on tensile properties and Charpy impact properties. Because additions of Mo and V promoted the formation of fine acicular ferrite (AF) and granular bainite (GB) while prohibiting the formation of coarse GB, they increased the strength and upper-shelf energy (USE) and decreased the energy transition temperature (ETT). The addition of Cr promoted the formation of coarse GB and hard secondary phases, thereby leading to an increased effective grain size, ETT, and strength, and a decreased USE. The addition of V resulted in a higher strength, a higher USE, a smaller effective grain size, and a lower ETT, because it promoted the formation of fine and homogeneous of AF and GB. The steel that contains 0.3 wt pct Mo and 0.06 wt pct V without Cr had the highest USE and the lowest ETT, because its microstructure was composed of fine AF and GB while its maintained excellent tensile properties.X1126sciescopu

Use of a Tunneling Technique to Achieve a Lower Defibrillation Threshold during Implantable Cardioverter Defibrillator Implantation via the Right Subclavian Vein

A 56-yr-old man with aborted sudden cardiac death underwent implantable cardioverter defibrillator (ICD) implantation. While the ICD was being implanted, a left subclavian venogram failed to visualize the left subclavian vein, which was attributed to likely prolonged indwelling of the left subclavian sheath for venous access. Accordingly, the right subclavian vein was punctured and the ICD lead was diverted from the right side area to the active Can in the left pectoral area by tunneling over the sternum for high defibrillation threshold. The approach used in this case may be considered in patients who had difficult left subclavicular venous access and it may be prudent to save the left subclavian vein for ICD implantation in patients with fatal tachyarrhythmia

mtDNAmanager: a Web-based tool for the management and quality analysis of mitochondrial DNA control-region sequences

BACKGROUND: For the past few years, scientific controversy has surrounded the large number of errors in forensic and literature mitochondrial DNA (mtDNA) data. However, recent research has shown that using mtDNA phylogeny and referring to known mtDNA haplotypes can be useful for checking the quality of sequence data. RESULTS: We developed a Web-based bioinformatics resource "mtDNAmanager" that offers a convenient interface supporting the management and quality analysis of mtDNA sequence data. The mtDNAmanager performs computations on mtDNA control-region sequences to estimate the most-probable mtDNA haplogroups and retrieves similar sequences from a selected database. By the phased designation of the most-probable haplogroups (both expected and estimated haplogroups), mtDNAmanager enables users to systematically detect errors whilst allowing for confirmation of the presence of clear key diagnostic mutations and accompanying mutations. The query tools of mtDNAmanager also facilitate database screening with two options of "match" and "include the queried nucleotide polymorphism". In addition, mtDNAmanager provides Web interfaces for users to manage and analyse their own data in batch mode. CONCLUSION: The mtDNAmanager will provide systematic routines for mtDNA sequence data management and analysis via easily accessible Web interfaces, and thus should be very useful for population, medical and forensic studies that employ mtDNA analysis. mtDNAmanager can be accessed at http://mtmanager.yonsei.ac.krope