HuB and HuD repress telomerase activity by dissociating HuR from TERC.

The ubiquitous RNA-binding protein HuR (ELAVL1) promotes telomerase activity by associating with the telomerase noncoding RNA TERC. However, the role of the neural-specific members HuB, HuC, and HuD (ELAVL2-4) in telomerase activity is unknown. Here, we report that HuB and HuD, but not HuC, repress telomerase activity in human neuroblastoma cells. By associating with AU-rich sequences in TERC, HuB and HuD repressed the assembly of the TERT-TERC core complex. Furthermore, HuB and HuD competed with HuR for binding to TERC and antagonized the function of HuR that was previously shown to enhance telomerase activity to promote cell growth. Our findings reveal a novel mechanism controlling telomerase activity in human neuroblastoma cells that involves a competition between HuR and the related, neural-specific proteins HuB and HuD.

Safety Studies for a 44-Channel Suprachoroidal Retinal Prosthesis: A Chronic Passive Study.

Purpose: Following successful clinical outcomes of the prototype suprachoroidal retinal prosthesis, Bionic Vision Australia has developed an upgraded 44-channel suprachoroidal retinal prosthesis to provide a wider field of view and more phosphenes. The aim was to evaluate the preclinical passive safety characteristics of the upgraded electrode array. Methods: Ten normal-sighted felines were unilaterally implanted with an array containing platinum electrodes (44 stimulating and 2 returns) on a silicone carrier near the area centralis. Clinical assessments (color fundus photos, optical coherence tomography, full-field electroretinography, intraocular pressure) were performed under anesthesia prior to surgery, and longitudinally for up to 20 weeks. Histopathology grading of fibrosis and inflammation was performed in two animals at 13 to 15 weeks. Results: Eight animals showed safe electrode array insertion (good retinal health) and good conformability of the array to the retinal curvature. Eight animals demonstrated good mechanical stability of the array with only minor (<2 disc diameters) lateral movement. Four cases of surgical or stability complications occurred due to (1) bulged choroid during surgery, (2) hemorrhage from a systemic bleeding disorder, (3) infection, and (4) partial erosion of thin posterior sclera. There was no change in retinal structure or function (other than that seen at surgery) at endpoint. Histopathology showed a mild foreign body response. Electrodes were intact on electrode array removal. Conclusions: The 44-channel suprachoroidal electrode array has an acceptable passive safety profile to proceed to clinical trial. The safety profile is expected to improve in human studies, as the complications seen are specific to limitations (anatomic differences) with the feline model.

Chronic electrical stimulation with a suprachoroidal retinal prosthesis: a preclinical safety and efficacy study.

PURPOSE: To assess the safety and efficacy of chronic electrical stimulation of the retina with a suprachoroidal visual prosthesis. METHODS: Seven normally-sighted feline subjects were implanted for 96-143 days with a suprachoroidal electrode array and six were chronically stimulated for 70-105 days at levels that activated the visual cortex. Charge balanced, biphasic, current pulses were delivered to platinum electrodes in a monopolar stimulation mode. Retinal integrity/function and the mechanical stability of the implant were assessed monthly using electroretinography (ERG), optical coherence tomography (OCT) and fundus photography. Electrode impedances were measured weekly and electrically-evoked visual cortex potentials (eEVCPs) were measured monthly to verify that chronic stimuli were suprathreshold. At the end of the chronic stimulation period, thresholds were confirmed with multi-unit recordings from the visual cortex. Randomized, blinded histological assessments were performed by two pathologists to compare the stimulated and non-stimulated retina and adjacent tissue. RESULTS: All subjects tolerated the surgical and stimulation procedure with no evidence of discomfort or unexpected adverse outcomes. After an initial post-operative settling period, electrode arrays were mechanically stable. Mean electrode impedances were stable between 11-15 kΩ during the implantation period. Visually-evoked ERGs & OCT were normal, and mean eEVCP thresholds did not substantially differ over time. In 81 of 84 electrode-adjacent tissue samples examined, there were no discernible histopathological differences between stimulated and unstimulated tissue. In the remaining three tissue samples there were minor focal fibroblastic and acute inflammatory responses. CONCLUSIONS: Chronic suprathreshold electrical stimulation of the retina using a suprachoroidal electrode array evoked a minimal tissue response and no adverse clinical or histological findings. Moreover, thresholds and electrode impedance remained stable for stimulation durations of up to 15 weeks. This study has demonstrated the safety and efficacy of suprachoroidal stimulation with charge balanced stimulus currents.

Secretion of the glucose-regulated selenoprotein SEPS1 from hepatoma cells.

SEPS1 (also called selenoprotein S, SelS, Tanis or VIMP) is a selenoprotein, localized predominantly in the ER membrane and also on the cell surface. In this report, we demonstrate that SEPS1 protein is also secreted from hepatoma cells but not from five other types of cells examined. The secretion can be abolished by the ER-Golgi transport inhibitor Brefeldin A and by the protein synthesis inhibitor cycloheximide. Using a sandwich ELISA, SEPS1 was detected in the sera of 65 out of 209 human subjects (31.1%, average=15.7+/-1.1 ng/mL). Fractionation of human serum indicated that SEPS1 was associated with LDL and possibly with VLDL. The function of plasma SEPS1 is unclear but may be related to lipoprotein metabolism.

Activation of the selenoprotein SEPS1 gene expression by pro-inflammatory cytokines in HepG2 cells.

SEPS1 (also called selenoprotein S, SelS) plays an important role in the production of inflammatory cytokines and its expression is activated by endoplasmic reticulum (ER) stress. In this report, we have identified two binding sites for the nuclear factor kappa B in the human SEPS1 promoter. SEPS1 gene expression, protein levels and promoter activity were all increased 2-3-fold by TNF-alpha and IL-1beta in HepG2 cells. We have also confirmed that the previously proposed ER stress response element GGATTTCTCCCCCGCCACG in the SEPS1 proximate promoter is fully functional and responsive to ER stress. However, concurrent treatment of HepG2 cells with IL-1beta and ER stress produced no additive effect on SEPS1 gene expression. We conclude that SEPS1 is a new target gene of NF-kappaB. Together with our previous findings that SEPS1 may regulate cytokine production in macrophage cells, we propose a regulatory loop between cytokines and SEPS1 that plays a key role in control of the inflammatory response.

Regulation of the selenoprotein SelS by glucose deprivation and endoplasmic reticulum stress - SelS is a novel glucose-regulated protein.

SelS is a newly identified selenoprotein and its gene expression is up-regulated in the liver of Psammomys obesus after fasting. We have examined whether SelS is regulated by glucose deprivation and endoplasmic reticulum (ER) stress in HepG2 cells. Glucose deprivation and the ER stress inducers tunicamycin and thapsigargin increased SelS gene expression and protein content several-fold in parallel with glucose-regulated protein 78. The overexpression of SelS increased Min6 cell resistance to oxidative stress-induced toxicity. These results indicate that SelS is a novel member of the glucose-regulated protein family and its function is related to the regulation of cellular redox balance.

Bis(pyridine)-based bromonium ions. Molecular structures of bis(2,4,6-collidine)bromonium perchlorate and bis(pyridine)bromonium triflate and the mechanism of the reactions of 1,2-bis(2'-pyridylethynyl)benzenebrominum triflate and bis(pyridine)bromonium triflate with acceptor olefins.

1,2-Bis(2'-pyridylethynyl)benzenebromonium triflate (4) and bis(pyridine)bromonium triflate (5) have been prepared and the mechanism of their reaction with various acceptors including eight alkenes of various structure, collidine, and Br(-) are reported. The reaction of 4 with neutral acceptors is second-order overall and involves a preequilibrium dissociation of the bidentate-bound Br(+) to form an unstable monodentate open form (4-op), which reacts with all neutral acceptors at or near the diffusion limit. Br(-) reacts with 4 by a different mechanism involving a direct nucleophilic attack on the Br(+). The reaction of 5 with acceptors proceeds by a dissociative mechanism to reversibly form an unstable intermediate (pyr-Br(+)), which reacts with 4-penten-1-ol, 4-pentenoic acid adamantylidineadamantane and cyclohexene with nearly the same selectivity. The crystal and molecular structures of bis(2,4,6-collidine)bromonium perchlorate (2-ClO(4)) and 5 were determined by X-ray crystallography.

Elevation in Tanis expression alters glucose metabolism and insulin sensitivity in H4IIE cells.

Increased hepatic glucose output and decreased glucose utilization are implicated in the development of type 2 diabetes. We previously reported that the expression of a novel gene, Tanis, was upregulated in the liver during fasting in the obese/diabetic animal model Psammomys obesus. Here, we have further studied the protein and its function. Cell fractionation indicated that Tanis was localized in the plasma membrane and microsomes but not in the nucleus, mitochondria, or soluble protein fraction. Consistent with previous gene expression data, hepatic Tanis protein levels increased more significantly in diabetic P. obesus than in nondiabetic controls after fasting. We used a recombinant adenovirus to increase Tanis expression in hepatoma H4IIE cells and investigated its role in metabolism. Tanis overexpression reduced glucose uptake, basal and insulin-stimulated glycogen synthesis, and glycogen content and attenuated the suppression of PEPCK gene expression by insulin, but it did not affect insulin-stimulated insulin receptor phosphorylation or triglyceride synthesis. These results suggest that Tanis may be involved in the regulation of glucose metabolism, and increased expression of Tanis could contribute to insulin resistance in the liver.