Mechanism of graphene-induced cytotoxicity: Role of endonucleases.

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.

Cell-based high-throughput screening identifies galactocerebrosidase enhancers as potential small-molecule therapies for Krabbe's disease.

Krabbe's disease, also known as globoid cell leukodystrophy (GLD), is a lysosomal storage disease caused by the deficiency of the lysosomal enzyme ß-galactocerebrosidase (GALC), resulting in severe neurological manifestations related to demyelination secondary to elevated galactosylsphingosine (psychosine) with its subsequent cytotoxicity. The only available treatment is hematopoietic stem cell transplantation, which delays disease onset but does not prevent long-term neurological manifestations. This article describes the identification of small molecules that enhance mutant GALC activity, identified by quantitative cell-based high-throughput screening (qHTS). Using a specific neurologically relevant murine cell line (145M-Twi) modified to express common human hGALC-G270D mutant, we were able to detect GALC activity in a 1,536-well microplate format. The qHTS of approximately 46,000 compounds identified three small molecules that showed significant enhancements of residual mutant GALC activity in primary cell lines from GLD patients. These compounds were shown to increase the levels of GALC-G270D mutant in the lysosomal compartment. In kinetic assessments, these small molecules failed to disturb the GALC kinetic profile under acidic conditions, which is highly desirable for folding-assisting molecules operating in the endoplasmic reticulum and not affecting GALC catalytic properties in the lysosomal compartment. In addition, these small molecules rescued the decreased GALC activity at neutral pH and partially stabilized GALC under heat-denaturating conditions. These drug-like compounds can be used as the starting point to develop novel small-molecule agents to treat the progressive neurodegenerative course of GLD. © 2016 Wiley Periodicals, Inc.

Beta1-adrenergic receptor-mediated dilation of rat cerebral artery requires Shaker-type KV1 channels on PSD95 scaffold.

Postsynaptic density-95 (PSD95) is a scaffolding protein in cerebral vascular smooth muscle cells (cVSMCs), which binds to Shaker-type K(+) (KV1) channels and facilitates channel opening through phosphorylation by protein kinase A. ß1-Adrenergic receptors (ß1ARs) also have a binding motif for PSD95. Functional association of ß1AR with KV1 channels through PSD95 may represent a novel vasodilator complex in cerebral arteries (CA). We explored whether a ß1AR-PSD95-KV1 complex is a determinant of rat CA dilation. RT-PCR and western blots revealed expression of ß1AR in CA. Isoproterenol induced a concentration-dependent dilation of isolated, pressurized rat CA that was blocked by the ß1AR blocker CGP20712. Cranial window imaging of middle cerebral arterioles in situ showed isoproterenol- and norepinephrine-induced dilation that was blunted by ß1AR blockade. Isoproterenol-induced hyperpolarization of cVSMCs in pressurized CA was blocked by CGP20712. Confocal images of cVSMCs immunostained with antibodies against ß1AR and PSD95 indicated strong colocalization, and PSD95 co-immunoprecipitated with ß1AR in CA lysate. Blockade of KV1 channels, ß1AR or disruption of PSD95-KV1 interaction produced similar blunting of isoproterenol-induced dilation in pressurized CA. These findings suggest that PSD95 mediates a vasodilator complex with ß1AR and KV1 channels in cVSMCs. This complex may be critical for proper vasodilation in rat CA.

Novel cytoprotective inhibitors for apoptotic endonuclease G.

Apoptotic endonuclease G (EndoG) is responsible for DNA fragmentation both during and after cell death. Previous studies demonstrated that genetic inactivation of EndoG is cytoprotective against various pro-apoptotic stimuli; however, specific inhibitors for EndoG are not available. In this study, we have developed a high-throughput screening assay for EndoG and have used it to screen a chemical library. The screening resulted in the identification of two potent EndoG inhibitors, PNR-3-80 and PNR-3-82, which are thiobarbiturate analogs. As determined by their IC50s, the inhibitors are more potent than ZnCl2 or EDTA. They inhibit EndoG at one or two orders of magnitude greater than another apoptotic endonuclease, DNase I, and do not inhibit the other five tested cell death-related enzymes: DNase II, RNase A, proteinase, lactate dehydrogenase, and superoxide dismutase 1. Exposure of natural EndoG-expressing 22Rv1 or EndoG-overexpressing PC3 cells rendered them significantly resistant to Cisplatin and Docetaxel, respectively. These novel EndoG inhibitors have the potential to be utilized for amelioration of cell injuries in which participation of EndoG is essential.

Novel high-throughput deoxyribonuclease 1 assay.

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z' ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

Anti-platelet aggregating and anti-oxidative activities of 11-O-(4'-O-methylgalloyl)-bergenin, a new compound isolated from Crassula cv. 'Himaturi'.

A new compound, 11- O-(4'- O-methylgalloyl)-bergenin was isolated from the MeOH extract of Crassula cv. 'Himaturi'. The structure of the compound was determined on the basis of chemical and spectroscopic data. It inhibited arachidonic acid-induced platelet aggregation more efficiently than acetylsalicylic acid and showed an anti-oxidative effect (EC (50) = 23.9 microM) equivalent to that of L-ascorbic acid or quercetin.