Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

2008 ◽  
Vol 54 (2) ◽  
pp. 284-298 ◽  
Author(s):  
Charles R. Dietrich ◽  
Gongshe Han ◽  
Ming Chen ◽  
R. Howard Berg ◽  
Teresa M. Dunn ◽  
...  
Keyword(s):  
Cell Viability ◽  
Critical Role ◽  
Loss Of Function ◽  
Inducible Rnai

scholarly journals Effects of Titanium Surfaces on the Developmental Profile of Monocytes/Macrophages

2014 ◽  
Vol 25 (2) ◽  
pp. 96-103 ◽  
Author(s):  
Camilla Christian Gomes Moura ◽  
Darceny Zanetta-Barbosa ◽  
Paula Dechichi ◽  
Valessa Florindo Carvalho ◽  
Priscilla Barbosa Ferreira Soares
Keyword(s):  
Cell Viability ◽  
Colorimetric Assay ◽  
Initial Period ◽  
Critical Role ◽  
Surface Characteristics ◽  
Th2 Cytokine ◽  
Titanium Surfaces ◽  
Control Surfaces ◽  
Immunoenzymatic Assays

Due to the critical role of monocytes/macrophages (Mϕ) in bone healing, this study evaluated the effects of bio-anodized, acid-etched, and machined titanium surfaces (Ti) on Mϕ behavior. Cells were separated from whole human blood from 10 patients, plated on Ti or polystyrene (control) surfaces, and cultured for 72 h. At 24, 48 and 72 h, cell viability, levels of IL1β, IL10, TNFα, TGFβ1 inflammatory mediators, and nitric oxide (NO) release were analyzed by mitochondrial colorimetric assay (MTT assay) and immunoenzymatic assays, respectively. Real-time PCR was used to verify the expression of TNFα and IL10 at 72 h. The data were subjected to a Kruskal-Wallis analysis. IL1β, TNFα and TGFβ1 release were not significantly different between the Ti surfaces (p>0.05). The presence of NO and IL10 was not detected in the samples. Cell viability did not differ between the samples cultivated on Ti and those cultivated on control surfaces, except at 24 h (p=0.0033). With respect to the mediators evaluated, the surface characteristics did not induce a typical Th1 or Th2 cytokine profile, although the cell morphology and topography were influenced by the Ti surface during the initial period.

Role of EGFR/ErbB2 and PI3K/AKT/e-NOS in Lycium barbarum polysaccharides Ameliorating Endothelial Dysfunction Induced by Oxidative Stress

2019 ◽  
Vol 47 (07) ◽  
pp. 1523-1539 ◽  
Author(s):  
Wenjuan Zhang ◽  
Huifang Yang ◽  
Lingqin Zhu ◽  
Yan Luo ◽  
Lihong Nie ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Dysfunction ◽  
Cell Viability ◽  
Therapeutic Option ◽  
Critical Role ◽  
Ang Ii ◽  
Lycium Barbarum ◽  
Underlying Mechanisms ◽  
Lycium Barbarum Polysaccharides

Lycium barbarum polysaccharides (LBP) are the major ingredients of wolfberry. In this study, we investigated the role of LBP in endothelial dysfunction induced by oxidative stress and the underlying mechanisms using thoracic aortic endothelial cells of rat (RAECs) as a model. We found that Ang II inhibits cell viability of RAECs with 10[Formula: see text][Formula: see text]mol/L of Ang II treatment for 24[Formula: see text]h most potential ([Formula: see text]), the level of reactive oxygen species (ROS) is increased by Ang II treatment ([Formula: see text]), and the expression of Occludin and Zonula occludens-1 (ZO-1) is decreased by Ang II treatment ([Formula: see text]). However, preincubation of cells with LBP could inhibit the changes caused by Ang II, LBP increased cell viability ([Formula: see text]), decreased the level of ROS ([Formula: see text]), and up-regulated the expression of Occludin ([Formula: see text]) and ZO-1. In addition, Ang II treatment increased the expression of EGFR and p-EGFR (Try1172) and which can be inhibited by LBP. On the contrary, expression of ErbB2, p-ErbB2 (Try1248), PI3K, p-e-NOS (Ser1177) ([Formula: see text]), and p-AKT (Ser473) ([Formula: see text]) was inhibited by Ang II treatment and which can be increased by LBP. Treatment of the cells with inhibitors showed that the regulation of p-e-NOS and p-AKT expression by Ang II and LBP can be blocked by PI3K inhibitor wortmannin but not EGFR and ErbB2 inhibitor AC480. Taken together, our results suggested that LBP plays a critical role in maintaining the integrality of blood vessel endothelium through reduced production of ROS via regulating the activity of EGFR, ErbB2, PI3K/AKT/e-NOS, and which may offer a novel therapeutic option in the management of endothelial dysfunction.

scholarly journals Critical role of caveolin-1 in aflatoxin B1-induced hepatotoxicity via the regulation of oxidation and autophagy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Qingqiang Xu ◽  
Wenwen Shi ◽  
Pan Lv ◽  
Wenqi Meng ◽  
Guanchao Mao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Aflatoxin B1 ◽  
Cell Apoptosis ◽  
Critical Role ◽  
Hepatic Cell ◽  
Hepatocellular Injury ◽  
Caveolin 1 ◽  
Induced Apoptosis

AbstractAflatoxin B1 (AFB1) is a potent hepatocarcinogen in humans and exposure to AFB1 is known to cause both acute and chronic hepatocellular injury. As the liver is known to be the main target organ of aflatoxin, it is important to identify the key molecules that participate in AFB1-induced hepatotoxicity and to investigate their underlying mechanisms. In this study, the critical role of caveolin-1 in AFB1-induced hepatic cell apoptosis was examined. We found a decrease in cell viability and an increase in oxidation and apoptosis in human hepatocyte L02 cells after AFB1 exposure. In addition, the intracellular expression of caveolin-1 was increased in response to AFB1 treatment. Downregulation of caveolin-1 significantly alleviated AFB1-induced apoptosis and decreased cell viability, whereas overexpression of caveolin-1 reversed these effects. Further functional analysis showed that caveolin-1 participates in AFB1-induced oxidative stress through its interaction with Nrf2, leading to the downregulation of cellular antioxidant enzymes and the promotion of oxidative stress-induced apoptosis. In addition, caveolin-1 was found to regulate AFB1-induced autophagy. This finding was supported by the effect that caveolin-1 deficiency promoted autophagy after AFB1 treatment, leading to the inhibition of apoptosis, whereas overexpression of caveolin-1 inhibited autophagy and accelerated apoptosis. Interestingly, further investigation showed that caveolin-1 participates in AFB1-induced autophagy by regulating the EGFR/PI3K-AKT/mTOR signaling pathway. Taken together, our data reveal that caveolin-1 plays a crucial role in AFB1-induced hepatic cell apoptosis via the regulation of oxidation and autophagy, which provides a potential target for the development of novel treatments to combat AFB1 hepatotoxicity.

scholarly journals Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans

2016 ◽  
Vol 310 (3) ◽  
pp. C233-C242 ◽  
Author(s):  
Erik Allman ◽  
Qian Wang ◽  
Rachel L. Walker ◽  
Molly Austen ◽  
Maureen A. Peters ◽  
...  
Keyword(s):  
Genetic Model ◽  
Expression Patterns ◽  
Critical Role ◽  
Model Organism ◽  
Loss Of Function ◽  
Homologous Proteins ◽  
Relative Significance ◽  
C Elegans ◽  
Fluorescent Tags

Calcineurin B homologous proteins (CHP) are N-myristoylated, EF-hand Ca2+-binding proteins that bind to and regulate Na+/H+ exchangers, which occurs through a variety of mechanisms whose relative significance is incompletely understood. Like mammals, Caenorhabditis elegans has three CHP paralogs, but unlike mammals, worms can survive CHP loss-of-function. However, mutants for the CHP ortholog PBO-1 are unfit, and PBO-1 has been shown to be required for proton signaling by the basolateral Na+/H+ exchanger NHX-7 and for proton-coupled intestinal nutrient uptake by the apical Na+/H+ exchanger NHX-2. Here, we have used this genetic model organism to interrogate PBO-1's mechanism of action. Using fluorescent tags to monitor Na+/H+ exchanger trafficking and localization, we found that loss of either PBO-1 binding or activity caused NHX-7 to accumulate in late endosomes/lysosomes. In contrast, NHX-2 was stabilized at the apical membrane by a nonfunctional PBO-1 protein and was only internalized following its complete loss. Additionally, two pbo-1 paralogs were identified, and their expression patterns were analyzed. One of these contributed to the function of the excretory cell, which acts like a kidney in worms, establishing an alternative model for testing the role of this protein in membrane transporter trafficking and regulation. These results lead us to conclude that the role of CHP in Na+/H+ exchanger regulation differs between apical and basolateral transporters. This further emphasizes the importance of proper targeting of Na+/H+ exchangers and the critical role of CHP family proteins in this process.

scholarly journals Heterozygous variants in KCNC2 cause a broad spectrum of epilepsy phenotypes associated with characteristic functional alterations

2021 ◽  
Author(s):  
Niklas Schwarz ◽  
Simone Seiffert ◽  
Manuela Pendziwiat ◽  
Annika Rademacher ◽  
Tobias Bruenger ◽  
...  
Keyword(s):  
Functional Analysis ◽  
De Novo ◽  
Critical Role ◽  
Specific Response ◽  
Loss Of Function ◽  
Causative Gene ◽  
Characteristic Functional ◽  
Research Collaborations ◽  
The Brain

Background KCNC2 encodes a member of the shaw-related voltage-gated potassium channel family (KV3.2), which are important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain. Methods Individuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic and functional analysis. The cases were referred through clinical and research collaborations in our study. Four de novo variants were examined electrophysiologically in Xenopus laevis oocytes. Results We identified novel KCNC2 variants in 27 patients with various forms of epilepsy. Functional analysis demonstrated gain-of-function in severe and loss-of-function in milder phenotypes as the underlying pathomechanisms with specific response to valproic acid. Conclusion These findings implicate KCNC2 as a novel causative gene for epilepsy emphasizing the critical role of KV3.2 in the regulation of brain excitability with an interesting genotype-phenotype correlation and a potential concept for precision medicine.

scholarly journals DUSP12 regulates the tumorigenesis and prognosis of hepatocellular carcinoma

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11929
Author(s):  
Gaoda Ju ◽  
Tianhao Zhou ◽  
Rui Zhang ◽  
Xiaozao Pan ◽  
Bing Xue ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Immune Cells ◽  
Malignant Tumors ◽  
Critical Role ◽  
Normal Liver ◽  
Functional Enrichment ◽  
Loss Of Function ◽  
Liver Tissues

Background Dual specificity protein phosphatase (DUSP)12 is an atypical member of the protein tyrosine phosphatase family, which are overexpressed in multiple types of malignant tumors. This protein family protect cells from apoptosis and promotes the proliferation and motility of cells. However, the pathological role of DUSP12 in hepatocellular carcinoma (HCC) is incompletely understood. Methods We analyzed mRNA expression of DUSP12 between HCC and normal liver tissues using multiple online databases, and explored the status of DUSP12 mutants using the cBioPortal database. The correlation between DUSP12 expression and tumor-infiltrating immune cells was demonstrated using the Tumor Immune Estimation Resource database and the Tumor and Immune System Interaction Database. Loss of function assay was utilized to evaluate the role of DUSP12 in HCC progression. Results DUSP12 had higher expression along with mRNA amplification in HCC tissues compared with those in normal liver tissues, which suggested that higher DUSP12 expression predicted shorter overall survival. Analyses of functional enrichment of differentially expressed genes suggested that DUSP12 regulated HCC tumorigenesis, and that knockdown of DUSP12 expression by short hairpin (sh)RNA decreased the proliferation and migration of HCC cells. Besides, DUSP12 expression was positively associated with the infiltration of cluster of differentiation (CD)4+ T cells (especially CD4+ regulatory T cells), macrophages, neutrophils and dendritic cells. DUSP12 expression was positively associated with immune-checkpoint moieties, and was downregulated in a C3 immune-subgroup of HCC (which had the longest survival). Conclusion These data suggest that DUSP12 may have a critical role in the tumorigenesis, infiltration of immune cells, and prognosis of HCC.

scholarly journals Critical Role of E1623 Residue in S3-S4 Loop of Nav1.1 Channel and Correlation Between Nature of Substitution and Functional Alteration

2022 ◽  
Vol 14 ◽  
Author(s):  
Tao Su ◽  
Meng-Long Chen ◽  
Li-Hong Liu ◽  
Hen Meng ◽  
Bin Tang ◽  
...  
Keyword(s):  
Current Density ◽  
Genetic Disorders ◽  
Critical Role ◽  
Missense Variant ◽  
Loss Of Function ◽  
Channel Conductance ◽  
Peak Current Density ◽  
Functional Changes ◽  
Functional Alteration

Objective: An overwhelming majority of the genetic variants associated with genetic disorders are missense. The association between the nature of substitution and the functional alteration, which is critical in determining the pathogenicity of variants, remains largely unknown. With a novel missense variant (E1623A) identified from two epileptic cases, which occurs in the extracellular S3-S4 loop of Nav1.1, we studied functional changes of all latent mutations at residue E1623, aiming to understand the relationship between substitution nature and functional alteration.Methods: Six latent mutants with amino acid substitutions at E1623 were generated, followed by measurements of their electrophysiological alterations. Different computational analyses were used to parameterize the residue alterations.Results: Structural modeling indicated that the E1623 was located in the peripheral region far from the central pore, and contributed to the tight turn of the S3-S4 loop. The E1623 residue exhibited low functional tolerance to the substitutions with the most remarkable loss-of-function found in E1623A, including reduced current density, less steady-state availability of activation and inactivation, and slower recovery from fast inactivation. Correlation analysis between electrophysiological parameters and the parameterized physicochemical properties of different residues suggested that hydrophilicity of side-chain at E1623 might be a crucial contributor for voltage-dependent kinetics. However, none of the established algorithms on the physicochemical variations of residues could well predict changes in the channel conductance property indicated by peak current density.Significance: The results established the important role of the extracellular S3-S4 loop in Nav1.1 channel gating and proposed a possible effect of local conformational loop flexibility on channel conductance and kinetics. Site-specific knowledge of protein will be a fundamental task for future bioinformatics.

Determinating the role of the E3 Ubiquitin Ligase Ariadne 2 in mammalian systemss

2007 ◽  
Vol 30 (4) ◽  
pp. 87
Author(s):  
A. E. Lin ◽  
A. Wakeham ◽  
A. You-Ten ◽  
G. Wood ◽  
T. W. Mak
Keyword(s):  
Function Analysis ◽  
Critical Role ◽  
Ring Finger ◽  
E3 Ligase ◽  
Signalling Pathways ◽  
Loss Of Function ◽  
In Vivo Models

Ubiquitination is a eukaryotic process of selective proteolysis, where a highly conserved ubiquitin protein is selectively added as a chain to the targeted to a protein for degradation. In recent years, the process of ubiquitination has been shown to be a critical mechanism that can affect essential signalling pathways, including apoptosis, cell cycle arrest and induction of the inflammatory response. Thus, alterations in the ubiquitination process can alter signalling pathways pivotal to numerous disease pathologies. This is clearly demonstrated in perturbations of ubiquitination in the NFκB giving rise to cancer and other immunological disease processes. To gain insight into pathways that require regulation by ubiquitination, our lab has directed focus on the highly conserved E3 ligase, Ariadne 2. Ariadne 2 is characterized as a putative RING finger E3 ligase and is part of the family of highly conserved RBR (RING-B-Box-RING) superfamily. The role of Ariadne 2 has been well studied in Drosophila melanogaster, however, little is known of the function of Ariadne 2 in mammalian systems. Therefore, the main objectives of the project are as follows: To determine the biological role of Ariadne 2, the role of Ariadne 2 in development and differentiation, and the consequences of in vivo loss of Ariadne 2 expression. We are currently investigating the role of Ariadne 2 as an E3 ligase and its involvement in the immune response. To date, we have shown that Ariadne 2 is ubiquitously expressed, especially in the brain, heart, spleen and thymus. For in vivo loss of function analysis, mice were generated by homologous recombination to be deficient for Ariadne 2. These deficient mice die prematurely soon after birth, suggesting a critical role for Ariadne 2 in development and survival. We are currently focusing on the role of Ariadne 2 in development and it’s role in immune pathologies, in particular, spontaneous autoimmunity, using both in vitro studies and in vivo models.

scholarly journals MAB21L1 loss of function causes a syndromic neurodevelopmental disorder with distinctive cerebellar, ocular, craniofacial and genital features (COFG syndrome)

2018 ◽  
Vol 56 (5) ◽  
pp. 332-339 ◽  
Author(s):  
Abolfazl Rad ◽  
Umut Altunoglu ◽  
Rebecca Miller ◽  
Reza Maroofian ◽  
Kiely N James ◽  
...  
Keyword(s):  
Neurodevelopmental Disorder ◽  
Critical Role ◽  
Missense Variant ◽  
Corneal Dystrophy ◽  
Reproductive Organs ◽  
Homozygosity Mapping ◽  
Facial Dysmorphism ◽  
Cerebellar Hypoplasia ◽  
Loss Of Function

BackgroundPutative nucleotidyltransferase MAB21L1 is a member of an evolutionarily well-conserved family of the male abnormal 21 (MAB21)-like proteins. Little is known about the biochemical function of the protein; however, prior studies have shown essential roles for several aspects of embryonic development including the eye, midbrain, neural tube and reproductive organs.ObjectiveA homozygous truncating variant in MAB21L1 has recently been described in a male affected by intellectual disability, scrotal agenesis, ophthalmological anomalies, cerebellar hypoplasia and facial dysmorphism. We employed a combination of exome sequencing and homozygosity mapping to identify the underlying genetic cause in subjects with similar phenotypic features descending from five unrelated consanguineous families.ResultsWe identified four homozygous MAB21L1 loss of function variants (p.Glu281fs*20, p.Arg287Glufs*14 p.Tyr280* and p.Ser93Serfs*48) and one missense variant (p.Gln233Pro) in 10 affected individuals from 5 consanguineous families with a distinctive autosomal recessive neurodevelopmental syndrome. Cardinal features of this syndrome include a characteristic facial gestalt, corneal dystrophy, hairy nipples, underdeveloped labioscrotal folds and scrotum/scrotal agenesis as well as cerebellar hypoplasia with ataxia and variable microcephaly.ConclusionThis report defines an ultrarare but clinically recognisable Cerebello-Oculo-Facio-Genital syndrome associated with recessive MAB21L1 variants. Additionally, our findings further support the critical role of MAB21L1 in cerebellum, lens, genitalia and as craniofacial morphogenesis.

scholarly journals Domain-Specific Response of Imprinted Genes to Reduced DNMT1

2010 ◽  
Vol 30 (16) ◽  
pp. 3916-3928 ◽  
Author(s):  
Jamie R. Weaver ◽  
Garnik Sarkisian ◽  
Christopher Krapp ◽  
Jesse Mager ◽  
Mellissa R. W. Mann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Critical Role ◽  
Imprinted Genes ◽  
Specific Response ◽  
Loss Of Function ◽  
Partial Loss ◽  
Domain Specific ◽  
Parent Of Origin

ABSTRACT Imprinted genes are expressed in a monoallelic, parent-of-origin-specific manner. Clusters of imprinted genes are regulated by imprinting control regions (ICRs) characterized by DNA methylation of one allele. This methylation is critical for imprinting; a reduction in the DNA methyltransferase DNMT1 causes a widespread loss of imprinting. To better understand the role of DNA methylation in the regulation of imprinting, we characterized the effects of Dnmt1 mutations on the expression of a panel of imprinted genes in the embryo and placenta. We found striking differences among imprinted domains. The Igf2 and Peg3 domains showed imprinting perturbations with both null and partial loss-of-function mutations, and both domains had pairs of coordinately regulated genes with opposite responses to loss of DNMT1 function, suggesting these domains employ similar regulatory mechanisms. Genes in the Kcnq1 domain were less sensitive to the absence of DNMT1. Cdkn1c exhibited imprinting perturbations only in null mutants, while Kcnq1 and Ascl2 were largely unaffected by a loss of DNMT1 function. These results emphasize the critical role for DNA methylation in imprinting and reveal the different ways it controls gene expression.

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