Abstract 13585: Up Regulation of Autophagy in Hsp60 Mutant Heart is an Adaptive Response to Increased Oxidative Stress and Causes Cardiomyopathy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Hirokazu Enomoto ◽  
Shinji Makino ◽  
Nishant Mittal ◽  
Akinori Kimura ◽  
Takuro Arimura ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Condition ◽  
Heat Shock Protein 60 ◽  
Loss Of Function ◽  
Lower Survival Rate ◽  
Protein Levels ◽  
Fish Model ◽  
Electron Microscopy Analysis ◽  
Pericardial Edema ◽  
Microscopy Analysis

Introduction: Despite the recent advance of genetic studies, genetic causes of hereditary dilated cardiomyopathy (DCM) are still unknown in most cases. Heat shock protein 60 (Hsp60) is a well-known chaperonin, responsible for correct folding and transportation of cytoplasmic protein to mitochondria. This study is aimed to investigate whether dysfunction of Hsp60 leads to cardiomyopathy in a fish model. Methods: We previously developed a zebrafish mutant, nbl, which has a missense mutation in hsp60, leading to the loss of function. To evaluate the phenotype of cardiomyopathy in nbl, we performed RT-PCR, western blot and immunohistochemistry of the hearts. Results: Homozygous nbl embryos showed lower survival rate (65%), compared to 81% in wild-type (WT) embryos, when subjected to 33°C (stress condition). We observed pericardial edema in 92% of nbl homozygous mutants. Also, nbl homozygotes showed sudden death at around 8 months post fertilization (mpf), when grown in non-stress condition. At 8 mpf, nbl mutants showed dilated heart and high expression of reactive oxygen species (ROS). Both mRNA and protein levels of Hsp60 were similar in nbl homozygotes and WT, at 3 mpf but, much higher expression of Hsp60 in nbl homozygotes was observed at 6 mpf, beginning of death of nbl homozygous mutants. Electron microscopy analysis showed dark mitochondria, disrupted sarcomeric structure and higher number of autophagosomes in nbl homozygote hearts at 8 mpf. We, then, analyzed autophagy related genes and found that atg5, atg3 and gabarap mRNAs were increased in nbl homozygotes, suggesting the increased autophagy might underlie the pathogenesis of DCM. Furthermore, analysis of genetically unrelated patients with familial DCM, who had no mutations in the known DCM-causing genes, identified an hsp60 mutation in one DCM family in which two of four mutation prone individuals died suddenly. Over expression of nbl mutation or DCM-associated hsp60 mutation, but not normal hsp60, increased autophagosomes in Hela cells carrying GFP-LC3. Conclusions: Functional loss of Hsp60 increased oxidative stress in the heart, which leads to increased autophagy and confer the susceptibility to cardiomyopathy.

scholarly journals PACLOBUTRAZOL-RESISTANCE Gene Family Regulates Floral Organ Growth with Unequal Genetic Redundancy in Arabidopsis thaliana

2019 ◽  
Vol 20 (4) ◽  
pp. 869 ◽  
Author(s):  
Kihye Shin ◽  
Inhye Lee ◽  
Eunsun Kim ◽  
Soon Park ◽  
Moon-Soo Soh ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Family ◽  
Floral Organ ◽  
High Order ◽  
Loss Of Function ◽  
Genetic Redundancy ◽  
Organ Growth ◽  
Helix Loop Helix ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis

A PACLOBUTRAZOL-RESISTANCE (PRE) gene family, consisting of six genes in Arabidopsis thaliana, encodes a group of helix-loop-helix proteins that act in the growth-promoting transcriptional network. To delineate the specific role of each of the PRE genes in organ growth, we took a reverse genetic approach by constructing high order pre loss-of-function mutants of Arabidopsis thaliana. In addition to dwarf vegetative growth, some double or high order pre mutants exhibited defective floral development, resulting in reduced fertility. While pre2pre5 is normally fertile, both pre2pre6 and pre5pre6 showed reduced fertility. Further, the reduced fertility was exacerbated in the pre2pre5pre6 mutant, indicative of the redundant and critical roles of these PREs. Self-pollination assay and scanning electron microscopy analysis showed that the sterility of pre2pre5pre6 was mainly ascribed to the reduced cell elongation of anther filament, limiting access of pollens to stigma. We found that the expression of a subset of flower-development related genes including ARGOS, IAA19, ACS8, and MYB24 was downregulated in the pre2pre5pre6 flowers. Given these results, we propose that PREs, with unequal functional redundancy, take part in the coordinated growth of floral organs, contributing to successful autogamous reproduction in Arabidopsis thaliana.

scholarly journals Hypertrophic adipocyte-derived exosomal miR-802-5p contributes to insulin resistance in cardiac myocytes through targeting HSP60

2020 ◽  
Author(s):  
Zhongyuan Wen ◽  
Junfeng Li ◽  
Yalin Fu ◽  
Yuyang Zheng ◽  
Mingke Ma ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Heat Shock Protein 60 ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Reporter Assays ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Epicardial adipose tissue (EAT) is implicated in insulin resistance, which has been recognized as a strongest predictor of the development of diabetic cardiomyopathy and subsequent heart failure. However, the underlying mechanism remains incompletely understood. Herein, we investigated the effect of hypertrophic adipocytes on cardiac insulin resistance. We found that hypertrophic adipocyte-derived exosomes (h-Exo) induced insulin resistance in NRVMs. Furthermore, h-Exo high-expressed miR-802-5p. Insulin sensitivity of NRVMs was impaired by miR-802-5p mimic but improved by its inhibitor. TargetScan and luciferase reporter assays revealed that heat shock protein 60 (HSP60) was a direct target of miR-802-5p. Both h-Exo and miR-802-5p mimic could downregulate HSP60 protein levels. In addition, HSP60 silencing induced insulin resistance and mitigated the insulin-sensitizing effects of adiponectin. HSP60 depletion also significantly increased the expression levels of CHOP, a marker of the unfolded protein response (UPR), and enhanced oxidative stress, accompanied by the increased phosphorylation of JNK and IRS-1 Ser307. Inhibition of both miR-802-5p and endocytosis abolished the impacts of HSP60 knockdown on the UPR and oxidative stress. In summary, hypertrophic adipocyte-derived exosomal miR-802-5p caused cardiac insulin resistance in NRVMs through downregulating HSP60. These findings provide a novel mechanism by which EAT impairs cardiac function.

scholarly journals Hypertrophic adipocyte-derived exosomal miR-802-5p contributes to insulin resistance in cardiac myocytes through targeting HSP60

2020 ◽  
Author(s):  
Zhongyuan Wen ◽  
Junfeng Li ◽  
Yalin Fu ◽  
Yuyang Zheng ◽  
Mingke Ma ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Insulin Sensitivity ◽  
Ventricular Myocytes ◽  
Underlying Mechanism ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Heat Shock Protein 60 ◽  
Akt Phosphorylation ◽  
Protein Levels

Abstract Background : Epicardial adipose tissue (EAT) is implicated in insulin resistance, which has been recognized as a strongest predictor of the development of diabetic cardiomyopathy and subsequent heart failure. However, the underlying mechanism remains incompletely understood. Herein, we investigated the effect of hypertrophic adipocytes on cardiac insulin resistance. Methods : Palmitate was used to induce hypertrophic 3T3-L1 adipocytes. Exosomes were purified from normal control or hypertrophic 3T3-L1 adipocyte-associated conditioned medium. Exosome-exposed neonatal rat ventricular myocytes (NRVMs) were treated with insulin to investigate the effects of exosomes on insulin signaling. Insulin sensitivity was evaluated by measuring insulin-stimulated Akt phosphorylation and glucose uptake. SiRNA techniques were used to downregulate protein levels and its efficiency was evaluated by western blot.Results : Hypertrophic adipocyte-derived exosomes (h-Exo) induced insulin resistance in NRVMs. Furthermore, h-Exo high-expressed miR-802-5p. Insulin sensitivity of NRVMs was impaired by miR-802-5p mimic but improved by its inhibitor. TargetScan and luciferase reporter assays revealed that heat shock protein 60 (HSP60) was a direct target of miR-802-5p. Both h-Exo and miR-802-5p mimic could downregulate HSP60 protein levels. In addition, HSP60 silencing induced insulin resistance and mitigated the insulin-sensitizing effects of adiponectin. HSP60 depletion also significantly increased the expression levels of CHOP, a marker of the unfolded protein response (UPR), and enhanced oxidative stress, accompanied by the increased phosphorylation of JNK and IRS-1 Ser307. Inhibition of both miR-802-5p and endocytosis abolished the impacts of HSP60 knockdown on the UPR and oxidative stress. Conclusion : Hypertrophic adipocyte-derived exosomal miR-802-5p caused cardiac insulin resistance in NRVMs through downregulating HSP60. These findings provide a novel mechanism by which EAT impairs cardiac function.

Material analysis techniques using the ion mill

1996 ◽  
Vol 54 ◽  
pp. 1034-1035
Author(s):  
J. P. Benedict ◽  
R. M. Anderson ◽  
S. J. Klepeis
Keyword(s):  
Polished Surface ◽  
Surface Material ◽  
Analysis Techniques ◽  
Material Analysis ◽  
Optical Inspection ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Argon Ions ◽  
The Impact ◽  
Scanning Electron

Ion mills equipped with flood guns can perform two important functions in material analysis; they can either remove material or deposit material. The ion mill holder shown in Fig. 1 is used to remove material from the polished surface of a sample for further optical inspection or SEM ( Scanning Electron Microscopy ) analysis. The sample is attached to a pohshing stud type SEM mount and placed in the ion mill holder with the polished surface of the sample pointing straight up, as shown in Fig 2. As the holder is rotating in the ion mill, Argon ions from the flood gun are directed down at the top of the sample. The impact of Argon ions against the surface of the sample causes some of the surface material to leave the sample at a material dependent, nonuniform rate. As a result, the polished surface will begin to develop topography during milling as fast sputtering materials leave behind depressions in the polished surface.

scholarly journals LOXL1 confers antiapoptosis and promotes gliomagenesis through stabilizing BAG2

2020 ◽  
Vol 27 (11) ◽  
pp. 3021-3036 ◽  
Author(s):  
Hua Yu ◽  
Jun Ding ◽  
Hongwen Zhu ◽  
Yao Jing ◽  
Hu Zhou ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Glioma Cell ◽  
Lysyl Oxidase ◽  
The Cancer Genome Atlas ◽  
Loss Of Function ◽  
Protein Levels ◽  
Important Mediator ◽  
Cancer Genome Atlas ◽  
Potential Strategy ◽  
Glioma Progression

Abstract The lysyl oxidase (LOX) family is closely related to the progression of glioma. To ensure the clinical significance of LOX family in glioma, The Cancer Genome Atlas (TCGA) database was mined and the analysis indicated that higher LOXL1 expression was correlated with more malignant glioma progression. The functions of LOXL1 in promoting glioma cell survival and inhibiting apoptosis were studied by gain- and loss-of-function experiments in cells and animals. LOXL1 was found to exhibit antiapoptotic activity by interacting with multiple antiapoptosis modulators, especially BAG family molecular chaperone regulator 2 (BAG2). LOXL1-D515 interacted with BAG2-K186 through a hydrogen bond, and its lysyl oxidase activity prevented BAG2 degradation by competing with K186 ubiquitylation. Then, we discovered that LOXL1 expression was specifically upregulated through the VEGFR-Src-CEBPA axis. Clinically, the patients with higher LOXL1 levels in their blood had much more abundant BAG2 protein levels in glioma tissues. Conclusively, LOXL1 functions as an important mediator that increases the antiapoptotic capacity of tumor cells, and approaches targeting LOXL1 represent a potential strategy for treating glioma. In addition, blood LOXL1 levels can be used as a biomarker to monitor glioma progression.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

Effects of Various Antioxidant Pretreatment Modalities on Adhesion to Sound and Caries-Affected Dentin: An In Vitro Study

2021 ◽  
pp. 232020682199798
Author(s):  
Beyza Unalan Degirmenci ◽  
Alperen Degirmenci ◽  
Emine Kara
Keyword(s):  
Bond Strength ◽  
In Vitro Study ◽  
Natural Antioxidants ◽  
Microtensile Bond Strength ◽  
Mandibular Molar ◽  
Electron Microscopy Analysis ◽  
Molar Teeth ◽  
Microscopy Analysis ◽  
Two Parameters

Aim: Natural antioxidants were offered as the answer of dentin adhesion issue. The aim of this study is to investigate the effects of proanthocyanidin and lycopene as pretreatment agents on the sound and caries-affected dentin surface on microtensile bond strength and microleakage. Materials and Methods: This study was designed as in vitro because of that 84 mandibular molar teeth were collected. Forty-two of the included teeth were carious teeth, while the other 42 were without caries. Sixty of them were used for microleakage and 24 for microtensile bond strength testing and scanning electron microscopy analysis. The samples were divided into six subgroups randomly according to dentin pretreatments: 5% proanthocyanidin, 5% lycopene, and no antioxidant application. After the restorative procedures, samples were attached to the microtensile tester. Samples were subjected to tensile stress in the load cell until they broke at a speed of 0.5 mm per min. Microtensile bond strength (µTBS) and microleakage test data were analyzed with two-way analysis of variance, Bonferroni correction, and Tamhane’s T2 tests. Results: Two-way variance analysis showed that dentin pretreatment applications, dentin substrate, and the interaction between these two parameters had statistically significant effects on µTBS values ( P < .001). There was no difference between dentin pretreatment applications in terms of microleakage scores ( P > .05). Conclusion: The application of dentin pretreatment with proanthocyanidin is a successful procedure that increases the bond strength in both dentin substrate, while pretreatment with lycopene in caries-affected dentin reduces it.

scholarly journals Antiparasitic Properties of Cardiovascular Agents against Human Intravascular Parasite Schistosoma mansoni

2021 ◽  
Vol 14 (7) ◽  
pp. 686
Author(s):  
Raquel Porto ◽  
Ana C. Mengarda ◽  
Rayssa A. Cajas ◽  
Maria C. Salvadori ◽  
Fernanda S. Teixeira ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Screening Strategy ◽  
Early Infection ◽  
Global Public Health ◽  
Parasitic Worm ◽  
Electron Microscopy Analysis ◽  
Health Significance ◽  
Microscopy Analysis

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Praziquantel is the only drug available to treat schistosomiasis and there is an urgent demand for new anthelmintic agents. Adopting a phenotypic drug screening strategy, here, we evaluated the antiparasitic properties of 46 commercially available cardiovascular drugs against S. mansoni. From these screenings, we found that amiodarone, telmisartan, propafenone, methyldopa, and doxazosin affected the viability of schistosomes in vitro, with effective concentrations of 50% (EC50) and 90% (EC90) values ranging from 8 to 50 µM. These results were further supported by scanning electron microscopy analysis. Subsequently, the most effective drug (amiodarone) was further tested in a murine model of schistosomiasis for both early and chronic S. mansoni infections using a single oral dose of 400 mg/kg or 100 mg/kg daily for five consecutive days. Amiodarone had a low efficacy in chronic infection, with the worm and egg burden reduction ranging from 10 to 30%. In contrast, amiodarone caused a significant reduction in worm and egg burden in early infection (>50%). Comparatively, treatment with amiodarone is more effective in early infection than praziquantel, demonstrating the potential role of this cardiovascular drug as an antischistosomal agent.

Sign in / Sign up

Export Citation Format

Share Document