AAA ATPases as therapeutic targets: Structure, functions, and small-molecule inhibitors

ATPases Associated with Diverse Cellular Activity (AAA ATPase) are essential enzymes found in all organisms. They are involved in various processes such as DNA replication, protein degradation, membrane fusion, microtubule serving, peroxisome biogenesis, signal transduction, and the regulation of gene expression. Due to the importance of AAA ATPases, several researchers identified and developed small-molecule inhibitors against these enzymes. We discuss six AAA ATPases that are potential drug targets and have well-developed inhibitors. We compare available structures that suggest significant differences of the ATP binding pockets among the AAA ATPases with or without ligand. The distances from ADP to the His20 in the His-Ser-His motif and the Arg finger (Arg353 or Arg378) in both RUVBL1/2 complex structures bound with or without ADP have significant differences, suggesting dramatically different interactions of the binding site with ADP. Taken together, the inhibitors of six well-studied AAA ATPases and their structural information suggest further development of specific AAA ATPase inhibitors due to difference in their structures. Future chemical biology coupled with proteomic approaches could be employed to develop variant specific, complex specific, and pathway specific inhibitors or activators for AAA ATPase proteins

Cardiotoxicity Associated with Trastuzumab Therapy in Taiwan: A Single Medical Center's 5-Year Experience

AbstractIntroductionTrastuzumab, a recombinant humanized monoclonal antibody, targets the external domain of HER2 to improve the efficacy of HER2-positive breast cancer treatment and inhibit carcinoma cellular proliferation. The purpose of this study was to identify early changes in cardiac function and dimensional changes in heart size in patients treated with trastuzumab.Materials and MethodsSeventy three female patients with Her2/neu overexpression (IHC 3+/Fish +) in breast cancer underwent echocardiography before and after trastuzumab therapy.ResultsCardiac complications developed in 14 patients (19.2%), including asymptomatic left ventricle systolic dysfunction (n = 12), symptomatic heart failure (n = 2), new asymptomatic left bundle branch block (n = 1), new negative T waves on electrocardiogram (n = 2), pericardial effusion (n = 1), and death (n = 1). No significant deterioration in diastolic function was noted, and right heart diameters and function did not change significantly. Most patients remained in an asymptomatic stage of cardiac disease. A significant decrease in left ventricular ejection fraction (LVEF) was observed in 14 patients (19.2%), and new mitral regurgitation (≥ grade 1) was noted after 3 months of trastuzumab therapy in 7 patients (9.6%).ConclusionsTrastuzumab led to measurable decreases in LVEF (but only 2.7% was symptomatic heart failure) and new mitral regurgitation. Therefore, regular follow-up with echocardiography is essential for early detection and prevention of trastuzumab-induced cardiomyopathy

Cost-utility analysis of adjuvant goserelin (Zoladex) and adjuvant chemotherapy in premenopausal women with breast cancer

<p>Abstract</p> <p>Background</p> <p>Increased health care costs have made it incumbent on health-care facilities and physicians to demonstrate both clinical and cost efficacy when recommending treatments. Though studies have examined the cost-effectiveness of adjuvant goserelin with radiotherapy for locally advanced prostate cancer, few have compared the cost-effectiveness of adjuvant goserelin to adjuvant chemotherapy alone in premenopausal breast cancer.</p> <p>Methods</p> <p>In this retrospective study at one hospital, the records of 152 patients with stage Ia to IIIa ER + breast cancer who received goserelin or chemotherapy were reviewed. Survival analysis was assessed by the Kaplan-Meier method. Patients were interviewed to evaluate their quality of life using the European Organization for Research and Treatment Quality of Life questionnaire (EORTC-QLQ-C30, version 4.0), and to obtain the utility value by the standard gamble (SG) and visual scale (VS) methods. Total medical cost was assessed from the (National Health Insurance) NHI payer's perspective.</p> <p>Results</p> <p>Survival at 11 years was significantly better in the groserelin group (<it>P </it>< 0.0012). The lifetime lost was lower in the goserelin group (42 months vs. 66 months). The quality adjusted survival (QAS) of patients who received goserelin was longer (122.5 ± 6.3 vs. 112.2 ± 6.7 months). Total expenses of goserelin were more than cyclophosphamide, methotrexate, 5-fluorouracil (CMF) or 5-fluorouracil, epirubicin, cyclophosphamide (FEC) chemotherapy regimes, but less than docetaxel, epirubicin (TE) or docetaxel, epirubicin, cyclophosphamide (TEC) regimes. The quality-adjusted life-year was higher in the goserelin group.</p> <p>Conclusions</p> <p>Goserelin therapy results in better survival and higher utility-weighted life-years, and is more cost-effective than TC or TEC chemotherapy.</p

A Plant Kavalactone Desmethoxyyangonin PreventsInflammation and Fulminant Hepatitis in Mice

Alpinia pricei Hayata is a Formosan plant which has been popularly used as nutraceutical or folk medicine for inflammation and various disorders. An active compound of the plant rhizomes, desmethoxyyangonin (DMY), was identified in this study for its novel effect against endotoxin lipopolysaccharide (LPS)-stimulated inflammation in murine macrophages and LPS/D-galactosamine (LPS/D-GalN)-induced fulminant hepatitis in mice. DMY was observed to significantly inhibit proliferation and activation of T cells ex vivo and the activity of several pro-inflammatory mediators in vitro. DMY also protected LPS/D-GalN−induced acute hepatic damages in mice through inhibiting aminotransferases activities and infiltrations of inflammatory macrophages, neutrophils and pathogenic T cells into the liver tissues. In addition, pretreatment with DMY significantly improved the survival rate of LPS/D-GalN−treated mice to 90% (9/10), compared to LPS/D-GalN−treated group (40%, 4/10). UPLC/MS platform-based comparative metabolomics approach was used to explore the serum metabolic profile in fulminant hepatic failure (FHF) mice with or without the DMY pretreatment. The results showed that LPS/D-GalN−induced hepatic damage is likely through perturbing amino acid metabolism, which leads to decreased pyruvate formation via catalysis of aminotransferases, and DMY treatment can prevent to a certain degree of these alterations in metabolic network in mouse caused by LPS/D-GalN. Mechanistic investigation demonstrated that DMY protects LPS or LPS/D-GalN−induced damages in cell or liver tissues mainly through de-regulating IKK/NFκB and Jak2/STAT3 signaling pathways. This report provides evidence-based knowledge to support the rationale for the use of A. pricei root extract in anti-inflammation and also its new function as hepatoprotetive agent against fulminant hepatitis

The Availability of Paraffin-Embedded Cancer Tissue in Personal Identification: A Model of Breast Cancer

"鑑定"是法醫的主要工作，然而法醫學的鑑定不;是局限在死因之鑑定，在司法領;域中尚有許多和法醫有相關的鑑定工作，例;如：親子鑑定、血緣鑑定也稱人身鑑別、物證鑑定、傷痕鑑定，在面對種種來;源的證物、血斑、精;斑、唾液斑、汗斑、皮膚、毛髮、指甲，甚至牙齒、骨骼等組織檢體，大部份都;是仰賴DNA的鑑定技術。其中組織包埋在石蠟;中理;論;上其相對DNA是呈穩定狀;態被貯存的，而且從石蠟;中去萃取組織DNA來;當作鑑定已不;是困難的事，不;過，值得探討的是，假如使用的蠟;塊包埋組織來;源是癌症組織，是否有可能因有基因突變(癌症細胞增生)而造成與本來;個體之正常組織的DNA不;一樣了呢？而它能否可用來;當成作鑑定的標準？文的研究利;用在一年;期間，2007年;8月~2008年;6月搜集了50個外科切;除的乳癌病患之癌組織來;供作研究中的實驗組，並同時研究病人身體的其他正常組織如靜脈血液當對照組。在石蠟;組織的 DNA萃取，只有36個達到可分析的量;，另外有14位病人的石蠟;組織的DNA萃取的STR (short Tandem Repeats 短串;聯;重複序列;)表現不;完全，所以剔除在本分析之外，故可分析率;是72% ( 36/50) ，血液的DNA萃取則全數;成功。結果發現有92 ﹪(33/36)其癌組織STR 15 基因座與其血液是完全相匹配，而有3人的蠟;塊包埋組織發現有突變，突變率;是8.% ( 3/36), 3位突變均只發生在單一個基因座上( single locus )，在血緣鑑定中，以STR分型來;評量;，只發生單一基因座的突變並不;足以用來;排除二者非來;自於同一個體，必須有二個或以上的位點不;一樣 才能懷疑來;自不;同個體。 所以藉由本研究結果， 似乎至少證明大部份癌變組織中的DNA與未發生癌變的同一人身上其他正常組織的DNA是相同的，或許在沒有其他可比對的情況下石蠟;包埋癌症組織仍可以提供作為DNA鑑定及人身鑑別的輔助來;源或參;考。“Investigation” is the major work of Forensic Medicine. The forensic identification is not only restricted to realize the reason of death, but also involves a lot of judicial investigations, such as parentage analysis, paternity test or personal identification and biological evidence examination. Most of the forensic evidence examination, for example, blood, sperm, sweat, skin, hair, nail and even teeth and bone, relies on DNA analysis technology. Short tandem repeat (STR) system is one of the main methodologies used in criminal investigation activities. Evidences have shown that DNA in paraffin-embedded tissues is relatively stable, and it is not difficult to extract DNA from paraffin-embedded tissues. Furthermore, it has been proved that cancer is induced by accumulation of genetic mutations. Thus, it is worthy to consider that if the paraffin-embedded cancer tissues could provide a model of forensic identification.n this study, we assayed 50 paraffin-embedded breast cancer tissues, which were collected from August 2007 to June 2008, and their counterpart blood specimens as control groups. The extraction rates of DNA were 72% (36/50) in paraffin and 100% in blood specimens, respectively. We revealed that 92 % (33/36) of 15 STR loci polymorphism in cancer tissues was perfectly matched with blood samples. The rest of 3 paraffin-embedded specimens DNA were found to be mutated from control group, which the mutation rate was approximately 8% (3/36). In addition, we also discovered that these 3 tissues have the same single locus mutation. Using STR system to evaluate the parentage relationship, single locus mutation is not enough to exclude the possibility that the two specimens are from the identical source. More than two loci of different are needed. Thus, these results demonstrate that DNA in most of cancer tissues is not mutated from normal parts, indicating that in some circumstances, paraffin-embedded cancer tissues might provide usefulness source of DNA analysis and forensic personal identification.口試委員會審定書…………………………………………………………………………………...i文摘要……………………………………………………………………………………………..ii文摘要………………………………………………………………………………………….....iii錄;……………………………………………………………………………………………..…...iv目錄;………………………………………………………………………………………………..v目錄;……………………………………………………………………………………………….vi一章 …………………………………………………………………………………………….1一節 緒論; ………………………………………………………………………………….1二節 研究動機………………………………………………………… …………………...2三節 研究目的………………………………………………………… ………………….. 2四節 文獻回顧………………………………………………………………………………2二章 材料;與方法………………………………………………………………………………..9一節 檢體來;源………………………………………………………………………………9二節 實驗方法……………………………………………………………………………..10三節 儀器與設備…………………………………………………………………………..13三章 實驗結果…………………………………………………………………………………14四章 討論;………………………………………………………………………………………15五章 結論;與展望………………………………………………………………………22六;章 參;考文獻…………………………………………………………………………………24目錄;一 病例;.A.之組織病理;學H&E染色 .40X ,左下角插圖.100X………………………… 30二 病例; B之組織病理;學H&E 染色 40X ,左下角插圖.100X…………………………...30三 病例; C之組織病理;學H&E 染色 40X ,左下角插圖.100X………………………......31四 病例;A STR-15基因型 石蠟;包埋組織與血液………………………………… 32五 病例;A 第二次石蠟;包埋組織實驗結果 ………………………………………….. 33六; 病例; A 突變發生在vWA 基因座………………………………………………… 34七 病例;B STR-15基因型 石蠟;包埋組織與血液……………………………............ 35八 病例; B 突變發生在FGA基因座…………………………………………………... 36九 病例;C STR-15基因型 石蠟;包埋組織與血液………………………………….. 37十 病例;C 第二次石蠟;包埋組織實驗結果……………………………………......... 38十一 病例; C 突變發生在 D21S11 基因座…………………………………………… 39十二 體突變 (Somatic Mutation) 之 圖解說;表目錄一 本研究乳癌病人的年齡分佈………………………………………………………….. 40二 本研究病人乳癌組織型態的分佈…………………………………………………… 40三 本研究病人的乳癌期別分佈……………………………………………………………41四 本研究實驗組與對照組結果……………………………………………………………41五 本研究STR-15型實驗組與對照組比對結果……………………………………………41六 癌症組織類別與STR-15型基因座發生突變之關係……………………………………42七 乳癌病人的年齡與突變之關係…………………………………………………………42八 乳癌期別與突變關係…………………………………………………………………….43九 發生突變的乳癌案例的年齡﹑期別與突變基因座分佈……………………………….43十 病例.A.基因座分佈………………………………………………………………… 45十一 病例.B 基因座分佈……………………………………………………………….. 45十二 病例.C 基因座分佈……………………………………………………………….. 46十三 部分商品化STR檢測試劑盒之識別率…………………………………………… 46十四 FORDIS 資料庫比對病例A,B,C 結果................................................................. 47錄 人體實驗委員會 (IRB) 通過文號...................................................................... 4

The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation

The diverse modes of action of small molecule inhibitors provide versatile tools to investigate basic biology and develop therapeutics. However, it remains a challenging task to evaluate their exact mechanisms of action. We identified two classes of inhibitors for the p97 ATPase: ATP competitive and allosteric. We showed that the allosteric p97 inhibitor, UPCDC-30245, does not affect two well-known cellular functions of p97, endoplasmic-reticulum-associated protein degradation and the unfolded protein response pathway; instead, it strongly increases the lipidated form of microtubule-associated proteins 1A/1B light chain 3B (LC3-II), suggesting an alteration of autophagic pathways. To evaluate the molecular mechanism, we performed proteomic analysis of UPCDC-30245 treated cells. Our results revealed that UPCDC-30245 blocks endo-lysosomal degradation by inhibiting the formation of early endosome and reducing the acidity of the lysosome, an effect not observed with the potent p97 inhibitor CB-5083. This unique effect allows us to demonstrate UPCDC-30245 exhibits antiviral effects against coronavirus by blocking viral entry

Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication

Human coronavirus (HCoV) similar to other viruses rely on host cell machinery for both replication and to spread. The p97/VCP ATPase is associated with diverse pathways that may favor HCoV replication. In this study, we assessed the role of p97 and associated host responses in human lung cell line H1299 after HCoV-229E or HCoV-OC43 infection. Inhibition of p97 function by small molecule inhibitors shows antiviral activity, particularly at early stages of the virus life cycle, during virus uncoating and viral RNA replication. Importantly, p97 activity inhibition protects human cells against HCoV-induced cytopathic effects. The p97 knockdown also inhibits viral production in infected cells. Unbiased quantitative proteomics analyses reveal that HCoV-OC43 infection resulted in proteome changes enriched in cellular senescence and DNA repair during virus replication. Further analysis of protein changes between infected cells with control and p97 shRNA identifies cell cycle pathways for both HCoV-229E and HCoV-OC43 infection. Together, our data indicate a role for the essential host protein p97 in supporting HCoV replication, suggesting that p97 is a therapeutic target to treat HCoV infection

Flow Cytometric Detection of Mitochondrial Membrane Potential

Mitochondrial membrane potential (Δψm) is an important parameter of mitochondrial function and an indicator of cell health. Depletion of Δψm suggests the loss of mitochondrial membrane integrity reflecting the initiation of the proapoptotic signal. Recently, lipophilic cationic fluorescent dyes have been developed to detect Δψm by accumulating in the mitochondrial matrix until the Nernstian equilibrium distribution of lipophilic cations is reached. In this protocol, we applied a cell-permeant, green-fluorescent, lipophilic dye 3,3&#39;-dihexyloxacarbocyanine Iodide (DiOC6(3)) which accumulates in mitochondria due to their large negative membrane potential, it can be applied to monitor the mitochondrial membrane potential using flow cytometric detection

Flow Cytometric Detection of Reactive Oxygen Species

Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions especially exposure to environmental oxidants and certain drugs that leads to oxidative stress. Exceed ROS can cause damages in the building blocks of cells including DNA, proteins, and lipids, and eventually results in cell death. Cell-permeant 2&#39;, 7&#39;-dichlorodihydrofluorescein diacetate (H2DCFDA) is a widely used ROS indicator. The reduced non-fluorescent fluorescein H2DCFDA can be oxidized and converted into fluorescent 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry