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1.
J Neurosci Methods ; 404: 110060, 2024 04.
Article in English | MEDLINE | ID: mdl-38244848

ABSTRACT

BACKGROUND: Isolation of adult Neural Stem/Progenitor Cells (NSPCs) from their neurogenic niches, is a prerequisite for studies involving culturing of NSPCs as neurospheres or attached monolayers in vitro. The currently available protocols involve the use of multiple animals and expensive reagents to establish the NSPCs culture. NEW METHOD: This unit describes a method to isolate and culture NSPCs from the two neurogenic niches in the mouse brain, the Subventricular Zone (SVZ) and Dentate gyrus (DG)/subgranular zone (SGZ), in an easy and cost-effective manner. RESULTS: NSPCs from SVZ and DG regions of adult mouse brains were isolated and cultured up to passage 15 without losing their stem/progenitor characteristics. These NSPCs could be differentiated into neurons, astrocytes, and oligodendrocytes, revealing its trilineage potential. COMPARISON WITH EXISTING METHODS: This protocol eliminates the need for multiple animals as well as the use of many expensive reagents mentioned in previous protocols, adding to the cost-effectiveness of experiments. In addition, we have effectively reduced the number of steps involved in isolation and propagation, thereby minimizing the chances of contamination. CONCLUSION: Our simplified protocol for the isolation and culturing of adult NSPCs from the SVZ and DG demonstrates a cost-effective and efficient alternative to existing methods, reducing the need for sacrificing many animals and the usage of expensive reagents. This method permits the long-term maintenance of NSPCs' stem/progenitor characteristics and their effective differentiation into the major types of cells in the brain, making it a valuable resource for researchers in the field. BASIC PROTOCOL: Isolation and Culturing of Neural Stem/Progenitor cells from the Sub ventricular Zone and the Dentate Gyrus of the adult mouse brain. SUPPORT PROTOCOL 1: Cryopreservation, and revival of frozen NSPCs. SUPPORT PROTOCOL 2: Preparation of adherent monolayer cultures of neural stem/progenitor cells for the differentiation into multiple lineages SUPPORT PROTOCOL 3: Differentiation of NSPCs to neuronal and glial lineages SUPPORT PROTOCOL 4: Characterization of differentiated cells by immunocytochemistry.


Subject(s)
Lateral Ventricles , Neural Stem Cells , Mice , Animals , Cost-Benefit Analysis , Cell Differentiation , Neurogenesis , Brain , Dentate Gyrus
2.
Metallomics ; 13(8)2021 08 23.
Article in English | MEDLINE | ID: mdl-34351413

ABSTRACT

Active surfaces with bactericidal properties are of paramount importance in health care sector as a judicious approach to confront prevalent challenges presented by disastrous pathogenic infections and antibiotic-resistant microbes. Herein, we present Bayerite underpinned Ag2O/Ag (ALD), a nanohybrid with excellent antibacterial and antibiofilm functionalities against tested standard strains and clinical isolates. The multicomponent system coexists and complement each other with respect to phase and functionalities, demonstrated by XRD, XPS, and TEM analyses. In situ reduction of Ag+ ions to Ag0 over Bayerite as a stable bound phase is favoured by pH of the reaction, yielding 60-80% bound Ag protruding outwards facilitating active surface for interaction with microbes. ALD has a minimum inhibitory concentration (MIC) of 0.068 mg/ml against clinical isolates: Pseudomonas aeruginosa RRLP1, RRLP2, Acinetobactor baumannii C78 and C80. Disc diffusion assay demonstrated excellent antibacterial activity against standard strains (positive control: standard antibiotic disc, Amikacin). ALD incorporated PMMA films (5 and 10 wt%; PALD-5 and PALD-10) exhibited significant contact killing (99.9%) of clinical isolates in drop-test besides strong antibacterial activity (disc diffusion assay) comparable to that of ALD. ALD exemplified a dose (0.034 and 0.017 mg/ml) dependent biofilm inhibition (P < 0.001) and significant eradication of pre-formed biofilms (P < 0.001) by clinical isolates. PALD 5 and PALD 10 significantly declined the number of viable biofilm associated bacteria (99.9%) compared to control. Both ALD and PALD samples are proposed as green antibacterial materials with antibiofilm properties. Results also present ample opportunity to explore PALD as antibacterial and/or antibiofilm coating formulations.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Biofilms/drug effects , Oxides/pharmacology , Silver Compounds/pharmacology , Bacteria/growth & development , Bacteria/isolation & purification , Biofilms/growth & development , Humans , Microbial Sensitivity Tests
3.
Biochemistry ; 54(24): 3739-48, 2015 Jun 23.
Article in English | MEDLINE | ID: mdl-26030260

ABSTRACT

Hypoxia inducible factor-1 (HIF1) is a stress-responsive nuclear transcription factor that is activated with a decrease in oxygen availability. HIF1 regulates the expression of genes involved in a cell's adaptation to hypoxic stress, including those with mitochondrial specific function. To gain a more comprehensive understanding of the role of HIF1 in mitochondrial homeostasis, we studied the link between hypoxia, HIF1 transactivation, and electron transport chain (ETC) function. We established immortalized mouse embryonic fibroblasts (MEFs) for HIF1α wild-type (WT) and null cells and tested whether HIF1α regulates mitochondrial respiration by modulating gene expressions of nuclear-encoded ETC components. High-throughput quantitative real-time polymerase chain reaction was performed to screen nuclear-encoded mitochondrial genes related to the ETC to identify those whose regulation was HIF1α-dependent. Our data suggest that HIF1α regulates transcription of cytochrome c oxidase (CcO) heart/muscle isoform 7a1 (Cox7a1) under hypoxia, where it is induced 1.5-2.5-fold, whereas Cox4i2 hypoxic induction was HIF1α-independent. We propose that adaptation to hypoxic stress of CcO as the main cellular oxygen consumer is mediated by induction of hypoxia-sensitive tissue-specific isoforms. We suggest that HIF1 plays a central role in maintaining homeostasis in cellular respiration during hypoxic stress via regulation of CcO activity.


Subject(s)
Electron Transport Complex IV/metabolism , Enzyme Induction , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mitochondria/metabolism , Oxygen Consumption , Animals , Cell Hypoxia , Cells, Cultured , Clone Cells , Electron Transport Complex IV/antagonists & inhibitors , Electron Transport Complex IV/genetics , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Profiling , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Mice, Transgenic , Mitochondria/drug effects , Mitochondria/enzymology , Oxidative Phosphorylation/drug effects , Oxygen Consumption/drug effects , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Transcriptional Activation/drug effects
4.
J Toxicol ; 2011: 391074, 2011.
Article in English | MEDLINE | ID: mdl-21811500

ABSTRACT

Wild-type and HIF1α -/- MEF cells were used to determine the role of HIF1α in cadmium-induced toxicity. Cadmium treatment did not affect HIF1-mediated transcription but led to caspase activation and apoptotic cell death in wild-type and HIF1α -/- cells. Cadmium-induced cell death, however, was significantly higher in HIF1α -/- cells as compared to their wild-type counterparts. Increased cell death in the HIF1α -/- cells was correlated with lower metallothionein protein, elevated levels of reactive oxygen species, and decreased superoxide dismutase enzyme activity. The total and oxidized glutathione levels, and, correspondingly, lipid peroxidation levels were elevated in the null cells compared to wild-type cells, indicating increased antioxidant demand and greater oxidative stress. Overall, the results suggest that basal levels of HIF1α play a protective role against cadmium-induced cytotoxicity in mouse embryonic fibroblasts by maintaining metallothionein and antioxidant activity levels.

5.
Toxicol Appl Pharmacol ; 254(3): 299-310, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21616089

ABSTRACT

Dioxins, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), produce a wide range of toxic effects in mammals. Most, if not all, of these toxic effects are regulated by the aryl hydrocarbon receptor (AHR). The AHR is a ligand activated transcription factor that has been shown to interact with numerous proteins capable of influencing the receptor's function. The ability of secondary proteins to alter AHR-mediated transcriptional events, a necessary step for toxicity, led us to determine whether additional interacting proteins could be identified. To this end, we have employed tandem affinity purification (TAP) of the AHR in Hepa1c1c7 cells. TAP of the AHR, followed by mass spectrometry (MS) identified ATP5α1, a subunit of the ATP synthase complex, as a strong AHR interactor in the absence of ligand. The interaction was lost upon exposure to TCDD. The association was confirmed by co-immunoprecipitation in multiple cell lines. In addition, cell fractionation experiments showed that a fraction of the AHR is found in the mitochondria. To ascribe a potential functional role to the AHR:ATP5α1 interaction, TCDD was shown to induce a hyperpolarization of the mitochondrial membrane in an AHR-dependent and transcription-independent manner. These results suggest that a fraction of the total cellular AHR pool is localized to the mitochondria and contributes to the organelle's homeostasis.


Subject(s)
Mitochondria/physiology , Mitochondrial Proteins/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Oxidative Phosphorylation Coupling Factors/metabolism , Receptors, Aryl Hydrocarbon/metabolism , ATP Synthetase Complexes/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Homeostasis/physiology , Mice , Mice, Knockout , Mitochondria/drug effects , Molecular Sequence Data , Polychlorinated Dibenzodioxins/metabolism , Polychlorinated Dibenzodioxins/toxicity , Protein Binding/physiology , Protein Subunits/metabolism
6.
Gene Expr ; 11(3-4): 181-97, 2003.
Article in English | MEDLINE | ID: mdl-14686790

ABSTRACT

Hypoxia is defined as a deficiency of oxygen reaching the tissues of the body, and it plays a critical role in development and pathological conditions, such as cancer. Once tumors outgrow their blood supply, their central portion becomes hypoxic and the tumor stimulates angiogenesis through the activation of the hypoxia-inducible factors (HIFs). HIFs are transcription factors that are regulated in an oxygen-dependent manner by a group of prolyl hydroxylases (known as PHDs or HPHs). Our understanding of hypoxia signaling is limited by our incomplete knowledge of HIF target genes. cDNA microarrays and a cell line lacking a principal HIF protein, HIF1alpha, were used to identify a more complete set of hypoxia-regulated genes. The microarrays identified a group of 286 clones that were significantly influenced by hypoxia and 54 of these were coordinately regulated by cobalt chloride. The expression profile of HIF1alpha -/- cells also identified a group of downregulated genes encoding enzymes involved in protecting cells from oxidative stress, offering an explanation for the increased sensitivity of HIF1alpha -/- cells to agents that promote this type of response. The microarray studies confirmed the hypoxia-induced expression of the HIF regulating prolyl hydroxylase, PHD2. An analysis of the members of the PHD family revealed that they are differentially regulated by cobalt chloride and hypoxia. These results suggest that HIF1alpha is the predominant isoform in fibroblasts and that it regulates a wide battery of genes critical for normal cellular function and survival under various stresses.


Subject(s)
Cell Hypoxia/physiology , Gene Expression Profiling , Gene Expression Regulation/physiology , Procollagen-Proline Dioxygenase/genetics , Signal Transduction/genetics , Transcription Factors/genetics , Animals , Cells, Cultured , Cobalt/pharmacology , Fibroblasts/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Oxygen/metabolism , Procollagen-Proline Dioxygenase/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolism , Transcription, Genetic
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