scholarly journals Gentiopicroside, a Secoiridoid Glycoside from Gentiana rigescens Franch, Extends the Lifespan of Yeast via Inducing Mitophagy and Antioxidative Stress

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Qian Liu ◽  
Lihong Cheng ◽  
Akira Matsuura ◽  
Lan Xiang ◽  
Jianhua Qi
Keyword(s):  
Stress Condition ◽  
Fluorescent Protein ◽  
Treated Group ◽  
Protein Levels ◽  
Antioxidative Stress ◽  
Green Fluorescent ◽  
Yeast Mutants ◽  
Mitotracker Red ◽  
Gentiana Rigescens ◽  
Secoiridoid Glycoside

Gentiopicroside (GPS), an antiaging secoiridoid glycoside, was isolated from Gentiana rigescens Franch, a traditional Chinese medicine. It prolonged the replicative and chronological lifespans of yeast. Autophagy, especially mitophagy, and antioxidative stress were examined to clarify the mechanism of action of this compound. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 and the colocation signal of MitoTracker Red CMXRos and GFP were increased upon the treatment of GPS. The free GFP in the cytoplasm and free GFP and ubiquitin of mitochondria were significantly increased at the protein levels in the GPS-treated group. GPS increased the expression of an essential autophagy gene, ATG32 gene, but failed to extend the replicative and chronological lifespans of ATG32 yeast mutants. GPS increased the survival rate of yeast under oxidative stress condition; enhanced the activities of catalase, superoxide dismutase, and glutathione peroxidase; and decreased the levels of reactive oxygen species and malondialdehyde. The replicative lifespans of Δsod1, Δsod2, Δuth1, and Δskn7 were not affected by GPS. These results indicated that autophagy, especially mitophagy, and antioxidative stress are involved in the antiaging effect of GPS.

scholarly journals A New Geniposidic Acid Derivative Exerts Antiaging Effects through Antioxidative Stress and Autophagy Induction

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 987
Author(s):  
Ying Wang ◽  
Yanjun Pan ◽  
Yanan Liu ◽  
Dejene Disasa ◽  
Matsuura Akira ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Replicative Lifespan ◽  
Gardenia Jasminoides ◽  
Autophagy Induction ◽  
Antioxidative Stress ◽  
Green Fluorescent ◽  
Yeast Mutants ◽  
Acid Compound ◽  
Gene Expression Levels ◽  
Geniposidic Acid

Two compounds that can prolong the replicative lifespan of yeast, geniposidic acid (Compound 1) and geniposide (Compound 2), were isolated from Gardenia jasminoides Ellis. Compared with Compound 1, Compound 2 was different at C11 and showed better bioactivity. On this basis, seven new geniposidic derivatives (3–9) were synthesized. Geniposidic 4-isoamyl ester (8, GENI), which remarkably prolonged the replicative and chronological lifespans of K6001 yeast at 1 µM, was used as the lead compound. Autophagy and antioxidative stress were examined to clarify the antiaging mechanism of GENI. GENI increased the enzymes activities and gene expression levels of superoxide dismutase (SOD) and reduced the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) to improve the survival rate of yeast under oxidative stress. In addition, GENI did not extend the replicative lifespan of ∆sod1, ∆sod2, ∆uth1, ∆skn7, ∆cat, and ∆gpx mutants with K6001 background. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 was increased by GENI. The protein level of free GFP showed a considerable increase and was time-dependent. Furthermore, GENI failed to extend the replicative lifespans of ∆atg32 and ∆atg2 yeast mutants. These results indicated that antioxidative stress and autophagy induction were involved in the antiaging effect of GENI.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals Molecular signatures of cell migration in C. elegans Q neuroblasts

2009 ◽  
Vol 185 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Guangshuo Ou ◽  
Ronald D. Vale
Keyword(s):  
Cell Migration ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Live Animal ◽  
Α Subunit ◽  
C Elegans ◽  
Protein Levels ◽  
Neuroblast Migration ◽  
Green Fluorescent

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin α subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans.

scholarly journals Using Morpholinos for Gene Knockdown in Giardia intestinalis

2009 ◽  
Vol 8 (6) ◽  
pp. 916-919 ◽  
Author(s):  
Meredith L. Carpenter ◽  
W. Zacheus Cande
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Giardia Intestinalis ◽  
Gene Knockdown ◽  
Specific Antibodies ◽  
Protein Levels ◽  
Green Fluorescent

ABSTRACT We used translation-blocking morpholinos to reduce protein levels in Giardia intestinalis. Twenty-four hours after electroporation with morpholinos targeting either green fluorescent protein or kinesin-2b, levels of these proteins were reduced by 60%. An epitope-tagged transgene can also be used as a reporter for morpholino efficacy with targets lacking specific antibodies.

Accelerated urokinase-receptor protein turnover triggered by interference with the addition of the glycolipid anchor

2011 ◽  
Vol 434 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Hector Avila ◽  
Heng Wang ◽  
Santosh Chauhan ◽  
Sean Hartig ◽  
Douglas D. Boyd
Keyword(s):  
Fluorescent Protein ◽  
Tumour Progression ◽  
Receptor Protein ◽  
Protein Levels ◽  
Type Plasminogen Activator ◽  
Endogenous Protein ◽  
Class B ◽  
Glycolipid Synthesis ◽  
Green Fluorescent

u-PAR (urokinase-type plasminogen activator receptor), anchored to the cell surface via a glycolipid moiety, drives tumour progression. We previously reported that colon cancer cells (RKO clone 2 FS2), attenuated for in vivo tumorigenicity, are diminished >15-fold for u-PAR display when compared with their tumorigenic isogenic counterparts (RKO clone 2), this disparity not reflecting altered transcription/mRNA stability. FACS, confocal microscopy and Western blotting using a fused u-PAR–EGFP (enhanced green fluorescent protein) cDNA revealed a >14-fold differential in the u-PAR–EGFP signal between the isogenic cells, ruling out alternate splicing as a mechanism. Although metabolic labelling indicated similar synthesis rates, pulse–chase revealed accelerated u-PAR–EGFP turnover in the RKO clone 2 FS2 cells. Expression in RKO clone 2 cells of a u-PAR–EGFP protein unable to accept the glycolipid moiety yielded diminished protein amounts, thus mirroring the low endogenous protein levels evident with RKO clone 2 FS2 cells. Transcript levels for the phosphatidylglycan anchor biosynthesis class B gene required for glycolipid synthesis were reduced by 65% in RKO clone 2 FS2 cells, and forced overexpression in these cells partially restored endogenous u-PAR. Thus attenuated u-PAR levels probably reflects accelerated turnover triggered by inefficient addition of the glycolipid moiety.

scholarly journals Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6 β-Cells

2005 ◽  
Vol 16 (6) ◽  
pp. 2670-2680 ◽  
Author(s):  
Aniko Varadi ◽  
Takashi Tsuboi ◽  
Guy A. Rutter
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Neuroendocrine Cells ◽  
Secretory Vesicles ◽  
Tail Domain ◽  
Cortical Actin ◽  
Β Cells ◽  
Protein Levels ◽  
Myosin Va ◽  
Green Fluorescent

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic β-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative–acting globular tail domain of MyoVa decreased by ∼50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in β-cells.

scholarly journals SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

2017 ◽  
Vol 38 (2) ◽  
Author(s):  
Venkatesh Kota ◽  
Gunhild Sommer ◽  
E. Starr Hazard ◽  
Gary Hardiman ◽  
Jeffery L. Twiss ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Hek293 Cells ◽  
Sequencing Data ◽  
Protein Levels ◽  
Specific Mrnas ◽  
Green Fluorescent

ABSTRACTThe cancer-associated RNA-binding protein La is posttranslationally modified by phosphorylation and sumoylation. Sumoylation of La regulates not only the trafficking of La in neuronal axons but also its association with specific mRNAs. Depletion of La in various types of cancer cell lines impairs cell proliferation; however, the molecular mechanism whereby La supports cell proliferation is not clearly understood. In this study, we address the question of whether sumoylation of La contributes to cell proliferation of HEK293 cells. We show that HEK293 cells stably expressing green fluorescent protein (GFP)-tagged wild-type La (GFP-LaWT) grow faster than cells expressing a sumoylation-deficient mutant La (GFP-LaSD), suggesting a proproliferative function of La in HEK293 cells. Further, we found that STAT3 protein levels were reduced in GFP-LaSDcells due to an increase in STAT3 ubiquitination and that overexpression of STAT3 partially restored cell proliferation. Finally, we present RNA sequencing data from RNA immunoprecipitations (RIPs) and report that mRNAs associated with the cell cycle and ubiquitination are preferentially bound by GFP-LaWTand are less enriched in GFP-LaSDRIPs. Taken together, results of our study support a novel mechanism whereby sumoylation of La promotes cell proliferation by averting ubiquitination-mediated degradation of the STAT3 protein.

scholarly journals Expressional and functional interactions of two Apis cerana cerana olfactory receptors

2018 ◽  
Author(s):  
Lina Guo ◽  
Huiting Zhao ◽  
Yusuo Jiang
Keyword(s):  
Fluorescent Protein ◽  
Apis Cerana ◽  
Olfactory Receptors ◽  
Surface Expression ◽  
Apis Cerana Cerana ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Green Fluorescent ◽  
Insight Into

Apis cerana cerana relies on the sensitive olfactory system to perform the foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile with olfactory receptor in Apis cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of Apis cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda Sf9 cells. The results showed that both mRNA and protein levels of AcerOr1 and AcerOr2 were drastically reduced when treated with their respective double stranded (ds) RNA compared to those in the control and double-stranded green fluorescent protein (dsGFP)-treated cells. The response to Ca2+ using 33 volatile odorants indicated that the molecular receptive range of AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1, 2, 4- triazol-3-yl) thio) acetamide (VUAA1) whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol, and it revealed distinct changes in the dose-response curve. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them using an electroantennography (EAG) assay. The response increased with the concentration of the odorant. Further, both AcerOr1 and AcerOr2 knockdowns exhibited significantly reduced intracellular Ca2+ levels in response to the corresponding ligands in vitro. Overall, the present study provides insight into the mechanism of olfactory discrimination in Apis cerana cerana.

scholarly journals Expressional and functional interactions of two Apis cerana cerana olfactory receptors

2018 ◽  
Author(s):  
Lina Guo ◽  
Huiting Zhao ◽  
Yusuo Jiang
Keyword(s):  
Fluorescent Protein ◽  
Apis Cerana ◽  
Olfactory Receptors ◽  
Surface Expression ◽  
Apis Cerana Cerana ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Green Fluorescent ◽  
Insight Into

Apis cerana cerana relies on the sensitive olfactory system to perform the foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile with olfactory receptor in Apis cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of Apis cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda Sf9 cells. The results showed that both mRNA and protein levels of AcerOr1 and AcerOr2 were drastically reduced when treated with their respective double stranded (ds) RNA compared to those in the control and double-stranded green fluorescent protein (dsGFP)-treated cells. The response to Ca2+ using 33 volatile odorants indicated that the molecular receptive range of AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1, 2, 4- triazol-3-yl) thio) acetamide (VUAA1) whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol, and it revealed distinct changes in the dose-response curve. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them using an electroantennography (EAG) assay. The response increased with the concentration of the odorant. Further, both AcerOr1 and AcerOr2 knockdowns exhibited significantly reduced intracellular Ca2+ levels in response to the corresponding ligands in vitro. Overall, the present study provides insight into the mechanism of olfactory discrimination in Apis cerana cerana.

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