scholarly journals Exploring the Regulatory Mechanism of Stress Responses in the Paraventricular Nucleus of the Hypothalamus: Backgrounds and Future Perspectives of Corticotropin-Releasing Factor-Modified Yellow Fluorescent Protein-Knock-In Mouse

2015 ◽  
Vol 21 (3) ◽  
pp. 213-224
Author(s):  
Keiichi ITOI
Keyword(s):  
Paraventricular Nucleus ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Regulatory Mechanism ◽  
Yellow Fluorescent Protein ◽  
Corticotropin Releasing Factor ◽  
Future Perspectives

scholarly journals Clp and Lon Proteases Occupy Distinct Subcellular Positions in Bacillus subtilis

2008 ◽  
Vol 190 (20) ◽  
pp. 6758-6768 ◽  
Author(s):  
Lyle A. Simmons ◽  
Alan D. Grossman ◽  
Graham C. Walker
Keyword(s):  
Bacillus Subtilis ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Normal Growth ◽  
Temporal Organization ◽  
Hexameric Atpase ◽  
Fluorescent Moiety ◽  
Green Fluorescent

ABSTRACT Among other functions, ATP-dependent proteases degrade misfolded proteins and remove several key regulatory proteins necessary to activate stress responses. In Bacillus subtilis, ClpX, ClpE, and ClpC form homohexameric ATPases that couple to the ClpP peptidase. To understand where these peptidases and ATPases localize in living cells, each protein was fused to a fluorescent moiety. We found that ClpX-GFP (green fluorescent protein) and ClpP-GFP localized as focal assemblies in areas that were not occupied by the nucleoid. We found that the percentage of cells with ClpP-GFP foci increased following heat shock independently of protein synthesis. We determined that ClpE-YFP (yellow fluorescent protein) and ClpC-YFP formed foci coincident with nucleoid edges, usually near cell poles. Furthermore, we found that ClpQ-YFP (HslV) localized as small foci, usually positioned near the cell membrane. We found that ClpQ-YFP foci were dependent on the presence of the cognate hexameric ATPase ClpY (HslU). Moreover, we found that LonA-GFP is coincident with the nucleoid during normal growth and that LonA-GFP also localized to the forespore during development. We also investigated LonB-GFP and found that this protein localized to the forespore membrane early in development, followed by localization throughout the forespore later in development. Our comprehensive study has shown that in B. subtilis several ATP-fueled proteases occupy distinct subcellular locations. With these data, we suggest that substrate specificity could be determined, in part, by the spatial and temporal organization of proteases in vivo.

scholarly journals Golgi anti-apoptotic proteins are evolutionarily conserved ion channels that regulate cell death in plants

2019 ◽  
Author(s):  
Maija Sierla ◽  
David L. Prole ◽  
Nuno Saraiva ◽  
Guia Carrara ◽  
Natalia Dinischiotu ◽  
...  
Keyword(s):  
Cell Death ◽  
Ion Channels ◽  
Programmed Cell Death ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Lesion Mimic ◽  
Evolutionarily Conserved ◽  
Apoptotic Proteins ◽  
Regulate Cell Death

ABSTRACTProgrammed cell death regulates developmental and stress responses in eukaryotes. Golgi anti-apoptotic proteins (GAAPs) are evolutionarily conserved cell death regulators. Human and viral GAAPs inhibit apoptosis and modulate intracellular Ca2+fluxes, and viral GAAPs form cation-selective channels. Although most mammalian cell death regulators are not conserved at the sequence level in plants, the GAAP gene family shows expansion, with five paralogues (AtGAAP1-5) in the Arabidopsis genome. We pursued molecular and physiological characterization of AtGAAPs making use of the advanced knowledge of their human and viral counterparts. Structural modeling of AtGAAPs predicted the presence of a channel-like pore, and electrophysiological recordings from purified AtGAAP3 reconstituted into lipid bilayers confirmed that plant GAAPs can function as ion channels. AtGAAP1 and AtGAAP4 localized exclusively to the Golgi within the plant cell, while AtGAAP2, AtGAAP3 and AtGAAP5 also showed tonoplastic localization. Gene expression analysis revealed differential spatial expression and abundance of transcript forAtGAAPparalogues in Arabidopsis tissues. We demonstrate that AtGAAP1-5 inhibit Bax-induced cell death in yeast. However, overexpression of AtGAAP1 induces cell death inNicotiana benthamianaleaves and lesion mimic phenotype in Arabidopsis. We propose that AtGAAPs function as Golgi-localized ion channels that regulate cell death by affecting ionic homeostasis within the cell.HighlightArabidopsis Golgi anti-apoptotic proteins (GAAPs) share functional conservation with their human and viral counterparts in cell death regulation and ion channel activityAbbreviationsAtGAAP,Arabidopsis thalianaGAAP; BI-1, Bax inhibitor-1; CFP, cyan fluorescent protein; CMLV, camelpox virus; ER, Endoplasmic reticulum; GAAP, Golgi anti-apoptotic protein; GFP, green fluorescent protein; hGAAP, human GAAP; LFG, Lifeguard; LMM, lesion mimic mutant; PCD, programmed cell death; TMBIM, transmembrane Bax inhibitor-1 motif-containing; TMDs, transmembrane domains; vGAAP, viral GAAP; YFP, yellow fluorescent protein

scholarly journals Function and Mechanism of WRKY Transcription Factors in Abiotic Stress Responses of Plants

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1515
Author(s):  
Weixing Li ◽  
Siyu Pang ◽  
Zhaogeng Lu ◽  
Biao Jin
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Abiotic Stress ◽  
Stress Responses ◽  
Regulatory Mechanism ◽  
Structural Characteristics ◽  
Wrky Transcription Factor ◽  
Future Perspectives ◽  
Wrky Transcription Factors ◽  
Different Response

The WRKY gene family is a plant-specific transcription factor (TF) group, playing important roles in many different response pathways of diverse abiotic stresses (drought, saline, alkali, temperature, and ultraviolet radiation, and so forth). In recent years, many studies have explored the role and mechanism of WRKY family members from model plants to agricultural crops and other species. Abiotic stress adversely affects the growth and development of plants. Thus, a review of WRKY with stress responses is important to increase our understanding of abiotic stress responses in plants. Here, we summarize the structural characteristics and regulatory mechanism of WRKY transcription factors and their responses to abiotic stress. We also discuss current issues and future perspectives of WRKY transcription factor research.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

109 Chemogenetic silencing of corticotropin releasing factor neurons in the paraventricular nucleus: the effect on post-stress sleep

SLEEP ◽  
2021 ◽  
Vol 44 (Supplement_2) ◽  
pp. A45-A45
Author(s):  
Irma Gvilia ◽  
Sunil Kumar ◽  
Dennis McGinty ◽  
Ronald Szymusiak
Keyword(s):  
Paraventricular Nucleus ◽  
Nrem Sleep ◽  
Corticotropin Releasing Factor ◽  
Male Rats ◽  
Male Rat ◽  
Sleep Onset Latency ◽  
Post Surgery ◽  
Crf Neurons ◽  
Emg Electrodes ◽  
Post Stress

Abstract Introduction We have previously shown that pharmacological elevation of corticotropin releasing factor (CRF) signaling in the brain results in exacerbation of sleep disturbances evoked by the exposure of rats to an acute stressor, the dirty cage of a male rat. In the present study we (1) assessed wake-sleep behavior of mice after the exposure to the dirty cage stress paradigm, and (2) examined the effect of chemogenetic silencing of CRF neurons in the hypothalamic paraventricular nucleus (PVN) on sleep occurring following the exposure to this stressor. Methods First, a group of mice (n=12) was implanted with EEG/EMG electrodes. In two weeks, post-surgery, six mice were transferred to dirty cages of male rats and recorded for 24 hours. Control mice were transferred to clean cages. In the second study, a group of CRF-ires-cre mice (n=8) received bilateral injections of AAV-hSyn-DIO-hM4Di-mCherry targeting the PVN. The other group of CRF-ires-cre mice (n=8) was injected AAV-hSyn-DIO-mCherry (control vector). All mice were implanted with EEG/EMG electrodes. Dirty cage experiments were started following a 4-week postsurgical period to allow gene recombination and expression. Mice were subjected to intraperitoneal (IP) administration of clozapine-n-oxide (CNO; 3 mg/kg) at ZT1, placed into dirty cages, and recorded for post-stress sleep. Results: Results In mice expressing hM4Di inhibitory DREADDs (designer receptors activated by designer drugs) versus mice injected with control AAV, IP CNO (3 mg/kg) resulted in a significant decrease of post-stress sleep onset latency, decrease of time spent in wakefulness (first hour, 74±5.3 vs. 89±11.0, second hour, 37.2±10.3% vs. 81.3±9.3%; third hour, 40.1±3.3% vs. 47.1±14.3%; fourth hour, 44.4±6.0 vs. 55.5±9.9), and increase in non-rapid eye movement (NREM) sleep time (26.0±5.4% vs. 11.0±11.1%; 62.8%±9.8 vs. 18.7 ± 9.6%; 59.9±3.2% vs. 52.9±14.5%; 55.6±6.2 vs. 44.5±10.0). The hM4Di expressing mice exhibited longer episodes of NREM sleep, compared to mice injected with control AAV (first hour, 133.3±80.1sec vs. 21±1.7sec; second hour, 43256±83.4sec vs. 73.5±44.1sec; third hour, 459.2±139.8sec vs. 139±80.6sec; fourth hour, 233.1±82.6sec vs. 190±72.3sec). Conclusion Chemogenetic silencing of CRF neurons in the PVN attenuates acute stress-induced sleep disturbance in mice. Support (if any) Supported by Department of Veterans Affairs Merit Review Grant # BX00155605 and SRNSF (Georgia) grant FR-18-12533

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