Trafficking of Connexin36 (Cx36) in the early secretory pathway.

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing activities or network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif "SAYV" mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system.

Intralumenal docking of connexin 36 channels in the ER isolates mistrafficked protein.

The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ-binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ-binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ-binding motif is critical for proper endoplasmic reticulum (ER) export of Cx36. Removing the PDZ-binding motif of Cx36 results in ER retention and the formation of multimembrane vesicles containing gap junction-like connexin aggregates. Using a combination of site-directed mutagenesis and electron micrographs, we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.

Effect of cyanocobalamin (vitamin B12) on paraquat-induced brain injury in mice.

Objectives: The goal of this study was to evaluate the neuroprotective effects of vit B12 on paraquat-induced neurotoxicity. Materials and Methods: Thirty-six male mice were randomly divided into six groups. Three groups were treated intraperitoneally with paraquat (10 mg/kg) twice a week (with a 3-day interval) for 3 weeks. Normal saline, vit B12 (1 mg /kg), or vit C (50 mg/kg) was injected 30 min before paraquat administration. Other groups only received normal saline (control), vit B12, or vit C in the same protocol. Motor performance and coordination were assayed by challenging beam traversal, pole, open field, and rotarod tests. The hippocampus and serum samples were isolated to evaluate the oxidative stress (GSH and ROS), apoptosis (caspase 3), and inflammatory markers (TNF-α and IL-1ß). Results: Administration of paraquat leads to induction of motor deficits, which were improved by treatment with vit B12. In addition, vit B12 could prevent oxidative damage, apoptosis, and inflammation caused by paraquat. Conclusion: It seems that vit B12 could be a novel therapeutic agent in the management of paraquat induced-neurotoxicity.

Metformin inhibits polyphosphate-induced hyper-permeability and inflammation.

Circulating inflammatory factor inorganic polyphosphate (polyP) released from activated platelets could enhance factor XII and bradykinin resulted in increased capillary leakage and vascular permeability. PolyP induce inflammatory responses through mTOR pathway in endothelial cells, which is being reported in several diseases including atherosclerosis, thrombosis, sepsis, and cancer. Systems and molecular biology approaches were used to explore the regulatory role of the AMPK activator, metformin, on polyP-induced hyper-permeability in different organs in three different models of polyP-induced hyper-permeability including local, systemic short- and systemic long-term approaches in murine models. Our results showed that polyP disrupts endothelial barrier integrity in skin, liver, kidney, brain, heart, and lung in all three study models and metformin abrogates the disruptive effect of polyP. We also showed that activation of AMPK signaling pathway, regulation of oxidant/anti-oxidant balance, as well as decrease in inflammatory cell infiltration constitute a set of molecular mechanisms through which metformin elicits it's protective responses against polyP-induced hyper-permeability. These results support the clinical values of AMPK activators including the FDA-approved metformin in attenuating vascular damage in polyP-associated inflammatory diseases.

The relationship between neuropsychological function and responsiveness to vitamin D supplementation using artificial neural networks.

BACKGROUND: Vitamin D has recently attracted interest for its pleiotropic effects. Vitamin D supplements are a potentially important public health intervention, but the response to supplementation varies between individuals. AIM: We aimed to assess the association between several neuropsychological parameters and the magnitude of response to vitamin D supplementation using an artificial neural network method. METHODS: Neuropsychological function was assessed in 619 participants using standard questionnaires. The study participants received vitamin D capsules containing 50,000 IU vitamin D per week over 9 weeks. To assess the relationship between responsiveness to vitamin D supplements and the impact on these neuropsychological parameters, the best-performing artificial neural network algorithms were selected from a combination of different transfer functions in hidden and output layers and variable numbers of hidden layers (between two and 50). The performance of the artificial neural network algorithm was assessed by receiver operating characteristic analysis and variables of importance were identified. RESULTS: The artificial neural network algorithm with sigmoid transfer function in both hidden and output layers could predict responsiveness to vitamin D supplementation effectively. The sensitivity and specificity were between 0.60 and 0.70 and 0.66 and 0.70, respectively. Cognitive abilities (42.5%), basal vitamin D (21.3%), body mass index (9.5%), and daytime sleepiness (8%) are the most widely used variables to predict changes in serum vitamin D levels. CONCLUSIONS: Cognitive abilities status and baseline 25-hydroxyvitamin D are important novel modifiers of the enhancement in circulating 25-hydroxyvitamin D after vitamin D supplementation.

Curcumin Loaded in Niosomal Nanoparticles Improved the Anti-tumor Effects of Free Curcumin on Glioblastoma Stem-like Cells: an In Vitro Study.

Using a novel curcumin-loaded niosome nanoparticle (CM-NP), the present study was designed to evaluate the effect of curcumin on human glioblastoma stem-like cells (GSCs). CM-NP has a diameter of ~ 60 nm and a zeta potential of ~ - 35 mV with a constant physicochemical stability. The cytotoxic effects of free curcumin (CM) and CM-NP were investigated on GSCs obtained during the removal of a brain tumor. Both CM and CM-NP caused a dose-dependent decrease in cell proliferation and viability of GSCs. The IC50 values of CM and CM-NP on GSCs were 50 and 137 µg/ml after 24 h, respectively. CM-NP exerted significantly higher effects on GSC viability, apoptosis, cell cycle arrest, and the expression of Bax, a pro-apoptotic marker, compared with CM. In addition, the migration of GSCs was significantly impaired following the administration of CM-NP compared with CM. Furthermore, CM-NP significantly increased the values of reactive oxygen species and decreased the mRNA expressions of NF-κB and IL-6 of GSCs compared with CM. Our data also revealed that CM-NP could significantly reduce the invasiveness of GSCs compared with CM, possibly via MCP-1-mediated pathways. In addition, CM-NP exhibited a significantly greater inhibitory effect on colony formation of GSCs compared with CM. These data indicate that CM-NP exhibited stronger anti-tumor effects on GSCs than CM. Although further in vivo investigations are warranted, our results suggest that CM-NP could be an ideal carrier to deliver curcumin for potential therapeutic approaches into glioblastoma.

Crocin as a novel therapeutic agent against colitis.

Ulcerative colitis is a chronic inflammatory bowel disease with high incidence and prevalence worldwide. To investigate the therapeutic potency of crocin, as a pharmacologically active component of saffron, in dextran sodium sulfate (DSS)-induced colitis mice model. Experimental colitis was induced by 7-day administration of DSS dissolved in water at a concentration of 1.5% (w/v). The animals were randomly divided into four groups (n»6 for each group). (1) Control group received regular drinking water for four weeks, (2) the second group of mice received regular drinking water for three weeks and then received DSS for one week, (3) and (4) the other two groups received 50-ppm or 200-ppm crocin for three weeks, respectively, and then treated with DSS for one week. Our results showed that Crocin attenuates colitis disease activity index including body weight loss, diarrhea, rectal bleeding, and colon shortening in crocin pre-tread mice. Comparison of histology of colon tissues between groups showed that crocin significantly decreases colon histopathological score, at least partially, by eliciting anti-inflammatory responses in DSS-induced colitis mice. These results clearly showed that crocin is a novel therapeutic agent with low toxicity as well as great clinical significance in treatment of colitis.

Phytosomal curcumin inhibits tumor growth in colitis-associated colorectal cancer.

Colorectal-cancer (CRC) is the third leading cause of death due to cancer, supporting the need for identification of novel anticancer drug to improve the efficacy of current-therapy. There is growing bodies of data showing the antitumor-activity of curcumin, although it is associated with low absorption. The aim of current study was explored the therapeutic-potential of novel phytosomal curcumin as well as its application in combination with 5-Flurouracil (5-FU) in a mouse-model of colitis-associated colon-cancer. The anti-proliferative-activity of phytosomal curcumin was assessed in 2- and 3-dimensional cell-culture-models as well as in a mouse-model of colitis-associated colon-cancer. The expression-levels of CyclinD1, beclin, E-cadherin, and p-GSK3a/b were investigated by qRT-PCR and/or Western-blotting. We evaluated the anti-inflammatory of this agent by pathological-evaluation and disease-activity-index (DAI). Moreover, oxidant/antioxidant activity was examined by malondialdehyde (MDA), total-thiols (T-SH), superoxide-dismutase (SOD), and catalase (CAT) activity parameters. Our data showed that phytosomal curcumin and its combination with 5-FU inhibited cell growth and invasive behavior of CRC cells through modulation of Wnt-pathway and E-cadherin. Combination of curcumin with 5-FU dramatically reduced the tumor-number and tumor-size in both distal and middle parts of colon in colitis-associated colon cancer followed by reduction in DAI. Also, curcumin suppressed the colonic inflammation and notably recovered the increased levels of MDA, decreased thiol level and reduced activity of CAT. We demonstrated the antitumor-activity of novel form of curcumin in CRC, supporting further investigations on the therapeutic-potential of this approach in colorectal-cancer.