Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.     

The efficient isolation of murine splenic dendritic cells and their cytochemical features

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.     

Stimulation of histone deacetylase activity by metabolites of intermediary metabolism

Histone deacetylases (HDACs) function in a wide range of molecular processes, including gene expression, and are of significant interest as therapeutic targets. Although their native complexes, subcellular localization, and recruitment mechanisms to chromatin have been extensively studied, much less is known about whether the enzymatic activity of non-sirtuin HDACs can be regulated by natural metabolites. Here, we show that several coenzyme A (CoA) derivatives, such as acetyl-CoA, butyryl-CoA, HMG-CoA, and malonyl-CoA, as well as NADPH but not NADP(+), NADH, or NAD(+), act as allosteric activators of recombinant HDAC1 and HDAC2 in vitro following a mixed activation kinetic. In contrast, free CoA, like unconjugated butyrate, inhibits HDAC activity in vitro. Analysis of a large number of engineered HDAC1 mutants suggests that the HDAC activity can potentially be decoupled from "activatability" by the CoA derivatives. In vivo, pharmacological inhibition of glucose-6-phosphate dehydrogenase (G6PD) to decrease NADPH levels led to significant increases in global levels of histone H3 and H4 acetylation. The similarity in structures of the identified metabolites and the exquisite selectivity of NADPH over NADP(+), NADH, and NAD(+) as an HDAC activator reveal a previously unrecognized biochemical feature of the HDAC proteins with important consequences for regulation of histone acetylation as well as the development of more specific and potent HDAC inhibitors.     

Neuroprotective Effects of Methyl-3-O-methyl gallate Against Sodium Fluoride-Induced Oxidative Stress in the Brain of Rats

Methyl-3-O-methyl gallate (M3OMG) is a rare natural product that showed promising in vitro antioxidant activities. In this study, the protective role of synthetic M3OMG against sodium fluoride (NaF)-induced oxidative stress in rat brain was evaluated. Animals were treated with either M3OMG (10 and 20 mg/kg i.p.), vitamin C (10 mg/kg i.p.) as the standard antioxidant or the vehicle (5 % dimethyl sulfoxide; 1 ml/kg) for 1 week. Oxidative stress was induced in the brain by adding 600 ppm NaF in the drinking water for 7 days. At the end of the treatment period, the levels of thiobarbituric acid reactive substances (TBARS), reduced glutathione and the activities of antioxidant enzymes (superoxide dismutase and catalase) were evaluated in brain homogenates. M3OMG treatment mitigated the NaF-induced oxidative stress through normalization of the level of TBARS, reduced levels of glutathione and by the restoration of the diminished antioxidant enzyme activities. In conclusion, M3OMG could have a potential for treating neurotoxicity induced by fluoride or related environmental pollutants.     

Trace Element Level in Different Tissues of <Emphasis Type="Italic">Rutilus frisii kutum</Emphasis> Collected from Tajan River,Iran

Tajan River is among the most significant rivers of the Caspian Sea water basin. In this study, the concentration of Cr, Cu, Fe, Mn, Ni, Pb, Cd, and Zn were determined in brain, heart, liver, gill, bile, and muscle of Rutilus frisii kutum which has great economic value in the Mazandaran state. Trace element levels in fish samples were analyzed by means of atomic absorption spectrometry. Nearly all non-essential metals levels (Ni, Pb, Cd) detected in tissues were higher than limits for fish proposed by FAO/WHO, EU, and TFC. Generally, non-essential metals (Ni, Pb) were so much higher in muscle than the essential metals (Cu, Zn, and Mn) except Fe, which was higher than other metals in nearly all parts, except in gills. Fe distribution pattern in tissues was in order of heart > brain > liver > muscle > bile > gill. Distribution patterns of metal concentrations in the muscle of fish as a main edible part followed the sequence: Fe > Pb > Ni > Cu > Mn > Zn > Cd.     

Multiple potential targets of opioids in the treatment of acute respiratory distress syndrome from COVID-19

COVID-19 can present with a variety of clinical features, ranging from asymptomatic or mild respiratory symptoms to fulminant acute respiratory distress syndrome (ARDS) depending on the host's immune responses and the extent of the associated pathologies. This implies that several measures need to be taken to limit severely impairing symptoms caused by viral-induced pathology in vital organs. Opioids are most exploited for their analgesic effects but their usage in the palliation of dyspnoea, immunomodulation and lysosomotropism may represent potential usages of opioids in COVID-19. Here, we describe the mechanisms involved in each of these potential usages, highlighting the benefits of using opioids in the treatment of ARDS from SARS-CoV-2 infection.