Tumor-Associated Macrophage-Induced Invasion and Angiogenesis of Human Basal Cell Carcinoma Cells by Cyclooxygenase-2 Induction

Tumor-associated macrophages (TAMs) and cyclooxygenase-2 (COX-2) are associated with invasion, angiogenesis, and poor prognosis in many human cancers. However, the role of TAMs in human basal cell carcinoma (BCC) remains elusive. We found that the number of TAMs infiltrating the tumor is correlated with the depth of invasion, microvessel density, and COX-2 expression in human BCC cells. TAMs also aggregate near COX-2 expressing BCC tumor nests. We hypothesize that TAMs might activate COX-2 in BCC cells and subsequently increase their invasion and angiogenesis. TAMs are a kind of M2 macrophage derived from macrophages exposed to Th2 cytokines. M2-polarized macrophages derived from peripheral blood monocytes were cocultured with BCC cells without direct contact. Coculture with the M2 macrophages induced COX-2-dependent invasion and angiogenesis of BCC cells. Human THP-1 cell line cells, after treated with phorbol myristate acetate (PMA), differentiated to macrophages with M2 functional profiles. Coculture with PMA-treated THP-1 macrophages induced COX-2-dependent release of matrix metalloproteinase-9 and subsequent increased invasion of BCC cells. Macrophages also induced COX-2-dependent secretion of basic fibroblast growth factor and vascular endothelial growth factor-A, and increased angiogenesis in BCC cells

Cardiac Myosin Binding Protein C and MAP-Kinase Activating Death Domain-Containing Gene Polymorphisms and Diastolic Heart Failure

OBJECTIVE: Myosin binding protein C (MYBPC3) plays a role in ventricular relaxation. The aim of the study was to investigate the association between cardiac myosin binding protein C (MYBPC3) gene polymorphisms and diastolic heart failure (DHF) in a human case-control study. METHODS: A total of 352 participants of 1752 consecutive patients from the National Taiwan University Hospital and its affiliated hospital were enrolled. 176 patients diagnosed with DHF confirmed by echocardiography were recruited. Controls were matched 1-to-1 by age, sex, hypertension, diabetes, renal function and medication use. We genotyped 12 single nucleotide polymorphisms (SNPs) according to HapMap Han Chinese Beijing databank across a 40 kb genetic region containing the MYBPC3 gene and the neighboring DNA sequences to capture 100% of haplotype variance in all SNPs with minor allele frequencies ≥ 5%. We also analyzed associations of these tagging SNPs and haplotypes with DHF and linkage disequilibrium (LD) structure of the MYBPC3 gene. RESULTS: In a single locus analysis, SNP rs2290149 was associated with DHF (allele-specific p = 0.004; permuted p = 0.031). The SNP with a minor allele frequency of 9.4%, had an odds ratio 2.14 (95% CI 1.25-3.66; p = 0.004) for the additive model and 2.06 for the autosomal dominant model (GG+GA : AA, 95% CI 1.17-3.63; p = 0.013), corresponding to a population attributable risk fraction of 12.02%. The haplotypes in a LD block of rs2290149 (C-C-G-C) was also significantly associated with DHF (odds ratio 2.10 (1.53-2.89); permuted p = 0.029). CONCLUSIONS: We identified a SNP (rs2290149) among the tagging SNP set that was significantly associated with early DHF in a Chinese population

The Study on the Model of the Unfolding of Cytochrome C and its Thermodynamic Properties

蛋白質結構與其功能的完整性息息相關，不正常的折疊會造成蛋白質失去其功能並且處於較不穩定的狀態，一些疾病如阿茲海默症與帕金森氏症等等，就是導因於蛋白質的折疊錯誤，了解蛋白質摺疊-展開的機制，將使我們對這些疾病的形成有進一步地了解，甚至發現預防或治療此種疾病的方法。在本研究中，我們企圖用本實驗室所提出的Ising model來描述蛋白質展開的行為，在我們的模型裡，我們將蛋白質視為由單元σi(unit)所組成，單元可以是肽鍵、胺基酸、摺疊子或任何蛋白質摺疊的單位，每個單元只有兩個狀態：自然折疊狀態σi=1或展開狀態σi=-1，數個單元組成一個蛋白質。在實驗上，我們選用細胞色素C當做研究對象，藉由改變尿素濃度或提高溫度，讓細胞色素C逐漸展開，並利用吸收光譜、螢光光譜還有旋光光譜對細胞色素C做偵測，藉以得知細胞色素C展開時局部結構的變化，我們發現Ising model成功地擬合了我們的實驗數據，我們發現這三種光譜所偵測到的過渡曲線皆為一個標準的兩態過渡曲線，故我們將這三種光譜所測到的局部位置各視為一個單元，我們稱之為α-螺旋局部位置(α-helix local site)、色氨酸-59局部位置(Trp-59 local site)，原血紅素局部位置(heme local site)，我們發現這三個局部位置的熱力學穩定度由高至低排列是α-螺旋局部位置、色胺酸-59局部位置，原血紅素局部位置，與傳統的兩態模型不同的是：藉由Ising model的擬合，我們不僅得知其局部位置的熱力學穩定度而且還得知其局部位置與其他單元的作用力 J。藉由比較小角度散射的實驗，我們得知細胞色素C在展開時整個構型的改變，和光譜偵測的結果比較下，我們發現原血紅素局部位置展開對細胞色素C的構型影響不大，其展開的構型改變主要來自於α-螺旋局部位置和色胺酸-59局部位置展開的貢獻。The function of protein is dependent of its folding process. For studying thermodynamic properties of folding-unfolding of protein, cytochrome c is chosen to be the model. Equilibrium unfolding behaviors of cytochrome c induced by presence of urea or changes in temperature are examined via spectroscopic techniques, including absorption、fluorescence and circular dichroism spectrometer. The local structure change can be detected by these spectrometers. The experimental data are fitted by the modified Ising model successfully, and some thermodynamic parameters of the unfolding of cytochrome c are derived, for example, the denature free energy of the local site. Results indicate that the thermodynamic stability of three local sites in cytochrome c ,from stable to unstable, are α-helix local site、Trp-59 local site and heme local site. By comparing with the data of small angle X-ray scattering, we find that native cytochrome c is close to a globular envelope. The envelope elongates gradually to an ellipsoidal shape at high urea concentration (9M) and high temperature (338K). We find the unfolding of heme local site just influences the global volume a little bit. But with the unfolding of α-helix local site and Trp-59 local site, the semi-major axis of cytochrome c elongates dramatically, whereas the semi-minor axis stays roughly the same.口試委員會審定書 i謝 ii文摘要 iii文摘要 iv一章、介紹 1二章、樣品準備與實驗方法 13-1. 偵測原理 13-1-1. 吸收光譜對細胞色素C的偵測 13-1-2. 螢光光譜對細胞色素C的偵測 15-1-3. 旋光光譜對細胞色素C的偵測 17-2. 樣品準備 18-2-1. 偵測色素蛋白C溫度效應的樣品準備 18-2-1-1. 吸收光譜的樣品準備 18-2-1-2. 旋光光譜的樣品準備 19-2-2. 偵測色素蛋白C變性劑效應的樣品準備 19-3. 偵測色素蛋白C展開的實驗方法 20-3-1. 溫度效應的偵測 20-3-1-1. 吸收光譜偵測蛋白質展開溫度效應 20-3-1-2. 旋光光譜偵測蛋白質展開溫度效應 20 2-3-2. 變性劑效應的偵測 21-4. 數據處理 21 2-4-1. 對變性劑效應實驗數據的處理 21-4-2.溫度效應實驗數據的處理 22 2-5. 升溫容槽的設計 24三章、描述蛋白質展開的模型 26 3-1.兩態模型描述蛋白質展開行為 26-2. Ising model描述蛋白質展開的原理 34 3-3. Regular protein描述蛋白質展開的原理 41四章、結果與討論 44-1. 變性劑效應結果與討論 44-2. 溫度效應結果與討論 53五章、結論 60六章、參考文獻 61七章、附錄 65 7-1. 附錄二. 螢光共振能量轉移的原理簡介 65-2. 附錄三.誘導偶極矩的簡介 6

<i>MYBPC3</i> and <i>MADD</i> gene haplotype/DHF association analysis.

*<p>Permutation 10,000 times.</p

Baseline characteristics of patients and controls.

<p>DHF, diastolic heart failure; BMI, body mass index; BP, blood pressure; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin II type I receptor blocker.</p

Global and Local Structural Changes of Cytochrome c and Lysozyme Characterized by a Multigroup Unfolding Process

Equilibrium unfolding behaviors of cytochrome c and lysozyme induced by the presence of urea (0–10 M) as well as changes in temperature (295–363 K) or pH (1.8–7) are examined via small-angle x-ray scattering and spectroscopic techniques, including circular dichroism and optical absorption. Denaturant and temperature effects are incorporated into the free energy expression for a general multigroup unfolding process. Results indicate that there are at least four unfolding groups in the temperature-, urea-, or pH-induced unfolding of cytochrome c: two of these are related to the prosthetic heme group, and the other two correspond, respectively, to the unfolding of α-helices and global changes in protein morphology that are largely unaccounted for by the first two groups. In contrast, the unfolding of lysozyme approximately follows a simple one-group process. A modified mean-field Ising model is adopted for a coherent description of the unfolding behaviors observed. Thermodynamic parameters extracted from simple denaturing processes, on the basis of the Ising model, can closely predict unfolding behaviors of the proteins in compounded denaturing environments

Surface vimentin is critical for the cell entry of SARS-CoV

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. RESULTS: A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. CONCLUSIONS: A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection

ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs.

The coronavirus SARS-CoV-2 is the causative agent of the ongoing severe acute respiratory disease pandemic COVID-19. Tissue and cellular tropism is one key to understanding the pathogenesis of SARS-CoV-2. We investigate the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of human donors using a diverse panel of banked tissues. Here, we report our discovery that the ACE2 receptor protein robustly localizes within the motile cilia of airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during host respiratory transmission. We further determine whether ciliary ACE2 expression in the upper airway is influenced by patient demographics, clinical characteristics, comorbidities, or medication use, and show the first mechanistic evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) does not increase susceptibility to SARS-CoV-2 infection through enhancing the expression of ciliary ACE2 receptor. These findings are crucial to our understanding of the transmission of SARS-CoV-2 for prevention and control of this virulent pathogen