scholarly journals An alcoholic extract of Thuja orientalis L. leaves inhibits autophagy by specifically targeting pro-autophagy PIK3C3/VPS34 complex

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Juneyoung Jung ◽  
Yoomi Chun ◽  
Young Pyo Jang ◽  
Myung Sook Oh ◽  
Jeong Hee Kim ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Beclin 1 ◽  
Specific Factor ◽  
Alcoholic Extract ◽  
Cellular Functions ◽  
Complex Activity ◽  
Protein Protein Interaction ◽  
Complex Core ◽  
Thuja Orientalis

AbstractAutophagy is a lysosome-dependent degradation program to maintain cellular homeostasis in response to a variety of stressful conditions, such as long-lived or non-functional subcellular organelles, protein aggregates, nutrient limitation, and virus/bacteria infection. Accordingly, dysregulation of autophagy is closely associated with many human pathophysiological conditions, such as neurodegenerative diseases, aging, and cancer, and autophagy is highlighted as an important therapeutic target for these human diseases. In autophagy process, PIK3C3/VPS34 complex plays important roles in autophagosome biogenesis. Accumulating evidences that inhibition of PIK3C3/VPS34 complex successfully blocks autophagy make the complex as an attractive target for the development of autophagy-specific inhibitors. However, considering that various forms of PIK3C3/VPS34 complex exist and they are involved in many different cellular functions, the targeting of the pro-autophagy PIK3C3/VPS34 complex is required to specifically inhibit autophagy. To identify autophagy inhibitors targeting the pro-autophagy complex, we have performed the screening of a customized natural product library consisting of 35 herbal extracts which are widely used in the oriental medicine as anti-inflammation and/or anti-tumor reagents. We discovered that an alcoholic extract of Thuja orientalis L. leaves inhibits pro-autophagy complex formation by disrupting the interaction between autophagy-specific factor, ATG14L, and the complex core unit Vps34-Beclin 1 in vitro. Also, it inhibits the nutrient starvation induced autophagy and diminished pro-autophagy PIK3C3/VPS34 complex containing either ATG14L or UVRAG in several cell lines. Our results strongly suggest that Thuja orientalis L. leave extract functions as an autophagy-specific inhibitor not decreasing the complex activity nor the protein level, but preventing protein–protein interaction between autophagy-specific factor (ATG14L and UVRAG) and PIK3C3/VPS34 complex core unit, Vps34-Beclin 1, thereby specifically depleting the pro-autophagy complex to inhibit autophagy.

scholarly journals Complex Activity and Short-term Memories in Reciprocally Connected Cerebral Organoids

2021 ◽  
Author(s):  
Tatsuya Osaki ◽  
Yoshiho Ikeuchi
Keyword(s):  
Human Brain ◽  
Three Dimensional ◽  
Oscillatory Activity ◽  
Cellular Functions ◽  
Complex Activity ◽  
Axonal Connections ◽  
Brain Organoid ◽  
External Stimuli ◽  
The Brain

AbstractMacroscopic axonal connections in the human brain distribute information and neuronal activity across the brain. Although this complexity previously hindered elucidation of functional connectivity mechanisms, brain organoid technologies have recently provided novel avenues to investigate human brain function by constructing small segments of the brain in vitro. Here, we describe the neural activity of human cerebral organoids reciprocally connected by a bundle of axons. Compared to conventional organoids, connected organoids produced significantly more intense and complex oscillatory activity. Optogenetic manipulations revealed that the connected organoids could re-play and recapitulate over time temporal patterns found in external stimuli, indicating that the connected organoids were able to form and retain temporal memories. Our findings suggest that connected organoids may serve as powerful tools for investigating the roles of macroscopic circuits in the human brain – allowing researchers to dissect cellular functions in three-dimensional in vitro nervous system models in unprecedented ways.

scholarly journals NLRP3 Inflammasome Activation Attenuates Neuronal Apoptosis by Upregulating Autophagy through AMPK/Beclin-1 Pathway after Intracerebral Hemorrhage with Ventricular Extension in Rats

2021 ◽  
Author(s):  
Zhaoqi Zhang ◽  
Peiwen Guo ◽  
Zhengcai Jia ◽  
Tunan Chen ◽  
Hua Feng
Keyword(s):  
Intracerebral Hemorrhage ◽  
Nlrp3 Inflammasome ◽  
Neuronal Apoptosis ◽  
Specific Inhibitor ◽  
Beclin 1 ◽  
Tunel Staining ◽  
Inflammasome Activation ◽  
Sprague Dawley

Abstract BackgroundIn brain, NLRP3 inflammasome, mainly derived from macrophage/microglia, is involved in proinflammatory and neurodeficits after hemorrhage, and autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggested that NLRP3 inflammasome and autophagy played an important role in intracerebral hemorrhage (ICH). Thus, this study was designed to further explore the pathogenesis of neurodeficits after in posthemorrhagic hydrocephalus.MethodsAutologous blood injection model was induced to mimic ICH with ventricular extension (ICH-IVH) in Sprague-Dawley rats. To elucidate the underlying mechanism, the NLRP3 inflammasome inhibitor MCC950 was administered abdominally at 1 h after ICH-IVH. Magnetic resonance imaging, neurobehavioral tests, immunofluorescence, western blotting, Fluoro-Jade C- staining, Tunel staining, and Quantitative RNA Sequencing were performed.ResultsIn the acute phase of ICH-IVH, both the expression of NLRP3 inflammasome and the autophagy of neurons were upregulated. The activated NLRP3 in macrophage/microglia promoted the release of IL-1β to extracellular, which contributed to excessive autophagy, leading to neurons apoptosis both in vivo and in vitro. AMPK/Beclin-1 pathway played an important role in NLRP3-related neurons autophagy. Using MCC950(NLRP3 inflammasome specific inhibitor) treatment after ICH-IVH significantly reduced ventricles dilation, improved neurofunction, down-regulated the release of IL-1β, and alleviated neuroinflammation and excessive autophagy.ConclusionsOur finding demonstrated that NLRP3 inflammasome activated in microglia/macrophage aggravated neurological outcomes and neuronal apoptosis by upregulating autophagy after ICH-IVH, which was partly mediated by the AMPK/Beclin-1 pathway. Therefore, inhibiting the activation of NLRP3 may be a potential therapeutic strategy for the neurodeficits of ICH-IVH patients.

scholarly journals Cyclophilin A plays an important role in embryo implantation through activating Stat3

Reproduction ◽  
2020 ◽  
Vol 160 (3) ◽  
pp. 343-351
Author(s):  
Tao Yu ◽  
Shuai Lin ◽  
Rui Xu ◽  
Tian-Xi Du ◽  
Yang Li ◽  
...  
Keyword(s):  
Embryo Implantation ◽  
Specific Inhibitor ◽  
Cyclophilin A ◽  
Cellular Functions ◽  
Ovariectomized Mice ◽  
Inflammatory Stimuli ◽  
Implantation Sites ◽  
Cypa Expression

Embryo implantation is a crucial step for the successful establishment of mammalian pregnancy. Cyclophilin A (CYPA) is a ubiquitously expressed intracellular protein and is secreted in response to inflammatory stimuli to regulate diverse cellular functions. However, there are currently no reports about the role of CYPA in embryo implantation. Here, we examine the expression pattern of CYPA during mouse early pregnancy and explore the potential role of CYPA during implantation. CYPA is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy, but not at inter-implantation sites. In ovariectomized mice, estrogen and progesterone significantly stimulate CYPA expression. When pregnant mice are injected intraperitoneally with CYPA inhibitor, the numbers of implantation sites are significantly reduced. Using an in vitro stromal cell culture system, Ppia siRNA knockdown of CYPA and CYPA-specific inhibitor treatment partially inhibits levels of CD147, MMP3 and MMP9. Decreased CYPA expression also significantly inhibits Stat3 activity and expands estrogen responsiveness. Taken together, CYPA may play an important role during mouse embryo implantation.

Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Hideki Tatemoto ◽  
Norio Muto
Keyword(s):  
Protein Kinase ◽  
Time Course ◽  
Polar Body ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Cellular Functions ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Second Polar Body

The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.

scholarly journals Interferon regulatory factors and TFIIB cooperatively regulate interferon-responsive promoter activity in vivo and in vitro.

1996 ◽  
Vol 16 (11) ◽  
pp. 6313-6324 ◽  
Author(s):  
I M Wang ◽  
J C Blanco ◽  
S Y Tsai ◽  
M J Tsai ◽  
K Ozato
Keyword(s):  
In Vitro Transcription ◽  
Specific Factor ◽  
Dependent Manner ◽  
Promoter Activation ◽  
Functional Interaction ◽  
Regulatory Factors ◽  
Protein Protein Interaction ◽  
Interferon Regulatory Factors

Interferon regulatory factors (IRFs) bind to the interferon-stimulated response element (ISRE) and regulate interferon- and virus-mediated gene expression. IRF-1 acts as a transcriptional activator, while IRF-2 acts as a repressor. Here we show that IRF-1 and IRF-2 bind to both cellular TFIIB, a component of the basal transcription machinery, and recombinant TFIIB (rTFIIB) and that this protein-protein interaction facilitates binding of IRFs to the ISRE. A functional interaction between TFIIB and IRF was assessed by a newly established in vitro transcription assay in which recombinant IRF-1 (rIRF-1) stimulated transcription specifically from an ISRE-containing template. With this assay we show that rIRF-1 and rTFIIB cooperatively enhance the ISRE promoter in vitro. We found that the activity of an ISRE-containing promoter was cooperatively enhanced upon cotransfection of TFIIB and IRF-1 cDNAs into P19 embryonal carcinoma cells, further demonstrating functional interactions in vivo. The cooperative enhancement by TFIIB and IRF-1 was independent of the TATA sequence in the ISRE promoter but dependent on the initiator sequence (Inr) and was abolished when P19 cells were induced to differentiate by retinoic acid treatment. In contrast, cotransfection of TFIIB and IRF-1 into NIH 3T3 cells resulted in a dose-dependent repression of promoter activation which occurred in a TATA-dependent manner. Our results indicate the presence of a cell type-specific factor that mediates the functional interaction between IRFs and TFIIB and that acts in conjunction with the requirement of TATA and Inr for promoter activation.

Illuminating Cellular Physiology: Recent Developments

2007 ◽  
Vol 90 (2-3) ◽  
pp. 129-160 ◽  
Author(s):  
Lubov Y. Brovko ◽  
Mansel W. Griffiths
Keyword(s):  
Protein Trafficking ◽  
Cell Biology ◽  
Translational Regulation ◽  
Cellular Functions ◽  
Protein Protein Interaction ◽  
Target Drug ◽  
Signal Transduction Protein ◽  
Recent Developments

Bioluminescent methods are gaining more and more attention among scientists due to their sensitivity, selectivity and simplicity; coupled with the fact that the bioluminescence can be monitored both in vitro and in vivo. Since the discovery of bioluminescence in the 19th century, enzymes involved in the bioluminescent process have been isolated and cloned. The bioluminescent reactions in several different organisms have also been fully characterized and used as reporters in a wide variety of biochemical assays. From the 1990s it became clear that bioluminescence can be detected and quantified directly from inside a living cell. This gave rise to numerous possibilities for the in vivo monitoring of intracellular processes non-invasively using bioluminescent molecules as reporters. This review describes recent developments in the area of bioluminescent imaging for cell biology. Newly developed imaging methods allow transcriptional/translational regulation, signal transduction, protein–protein interaction, oncogenic transformation, cell and protein trafficking, and target drug action to be monitored in vivo in real-time with high temporal and spatial resolution; thus providing researchers with priceless information on cellular functions. Advantages and limitations of these novel bioluminescent methods are discussed and possible future developments identified.

scholarly journals Arginine Methyltransferase PRMT5 Methylates and Destabilizes Mxi1 to Confer Radioresistance in Lung Cancer

2021 ◽  
Author(s):  
Xijie Yang ◽  
Zhen Zeng ◽  
Xiaohua Jie ◽  
Ye Wang ◽  
Jun Han ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Biological Effects ◽  
Specific Inhibitor ◽  
Arginine Methylation ◽  
Arginine Methyltransferase ◽  
Protein Protein Interaction ◽  
Underlying Mechanisms ◽  
The Stability

Abstract BackgroundRadioresistance is regarded as the main cause of local recurrence and distant metastasis in lung cancer. However, the underlying mechanisms of radioresistance remain incompletely understood. This study investigates the roles and regulatory mechanisms of arginine methyltransferase PRMT5 in lung cancer radioresistance.MethodsImmunoprecipitation assay and GST pulldown were used to detect the protein-protein interaction. The methylation of Mxi1 was determined by in vivo and in vitro arginine methylation assays. In vivo ubiquitination and CHX chase assays were performed to examine the stability of Mxi1. The biological effects of PRMT5 and its specific inhibitor EPZ015666 in lung cancer were evaluated both in vitro and in vivo.ResultsWe show that the arginine methyltransferase PRMT5 interacts with and methylates Mxi1, which promotes the binding of the β-Trcp ligase to Mxi1, facilitating the ubiquitination and degradation of Mxi1 in lung cancer. Furthermore, genetic blockade of PRMT5 impairs DNA damage repair and enhances lung cancer radiosensitivity in vitro and in vivo, and these phenotypes are partially reversed by Mxi1 silencing. More importantly, pharmacological inhibition of PRMT5 with the specific inhibitor EPZ015666 leads to extraordinary radiosensitization in vitro and in vivo in lung cancer.ConclusionsOur data indicate that PRMT5 methylates and destabilizes Mxi1 to confer radioresistance, suggesting that PRMT5 may be a promising radiosensitization target in lung cancer.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

A Hexagonal (II) Phase Phospholipid Neutralization Assay for Lupus Anticoagulant Identification

1993 ◽  
Vol 70 (05) ◽  
pp. 787-793 ◽  
Author(s):  
Douglas A Triplett ◽  
Linda K Barna ◽  
Gail A Unger
Keyword(s):  
Hemophilia A ◽  
Clinical Laboratory ◽  
Neutralization Assay ◽  
Confirmatory Test ◽  
Specific Factor ◽  
Clinical Settings ◽  
Drug Induced ◽  
Variable Screening ◽  
Routine Clinical Laboratory

SummaryLupus anticoagulants (LAs) are immunoglobulins (IgG, IgM, or both) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e.g. APTT, dilute PT, dilute Russell Viper Venom Time). These antibodies may be identified in a wide variety of clinical settings. With the exception of heparinized patient samples, the presence of LAs is often the most common cause of an unexplained APTT in a routine clinical laboratory. The diagnosis of LAs is difficult due to variable screening reagent sensitivity and intrinsic heterogeneity of LAs. Recently, Rauch and colleagues have shown human monoclonal hybridoma LAs were inhibited by hexagonal (II) phase PLs. In contrast, lamellar phase PLs had no effect. We have evaluated a new assay system, Staclot LA®, which utilizes a hexagonal (II) phase PL (egg phosphatidylethanolamine [EPE]) as a confirmatory test for LAs. Plasma samples from the following patient populations were studied: LA positive, heparinized, oral anticoagulated, hemophilia A and B, and specific factor inhibitors (factors V, VIII, IX). Unlike previous studies, the LA positive patients were a mixed population including: autoimmune diseases, drug-induced, and post-infection. Our findings confirm the specificity of hexagonal (II) phase PL neutralization of LAs.

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