I–II Loop Structural Determinants in the Gating and Surface Expression of Low Voltage-Activated Calcium Channels

The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3

Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2

Dengue virus (DENV) infection remains a serious health threat despite the availability of supportive care in modern medicine. Monoclonal antibodies (mAbs) of DENV would be powerful research tools for antiviral development, diagnosis and pathological investigations. Here we described generation and characterization of seventeen mAbs with high reactivity for E protein of DENV. Four of these mAbs showed high neutralizing activity against DENV-2 infection in mice. The monoclonal antibody mAb DB32-6 showed the strongest neutralizing activity against diverse DENV-2 and protected DENV-2-infected mice against mortality in therapeutic models. We identified neutralizing epitopes of DENV located at residues K310 and E311 of viral envelope protein domain III (E-DIII) through the combination of biological and molecular strategies. Comparing the strong neutralizing activity of mAbs targeting A-strand with mAbs targeting lateral ridge, we found that epitopes located in A-strand induced stronger neutralizing activity than those located on the lateral ridge. DB32-6 humanized version was successfully developed. Humanized DB32-6 variant retained neutralizing activity and prevented DENV infection. Understanding the epitope-based antibody-mediated neutralization is crucial to controlling dengue infection. Additionally, this study also introduces a novel humanized mAb as a candidate for therapy of dengue patients

FoxO and Stress Responses in the Cnidarian Hydra vulgaris

Background: In the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms. Methods/Principal Findings: We have examined the regulation of FoxO activity in members of the well-studied cnidarian genus Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases. Conclusions: These findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians an

Primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic liver disease with a slowly progressive course. Without treatment, most patients eventually develop fibrosis and cirrhosis of the liver and may need liver transplantation in the late stage of disease. PBC primarily affects women (female preponderance 9–10:1) with a prevalence of up to 1 in 1,000 women over 40 years of age. Common symptoms of the disease are fatigue and pruritus, but most patients are asymptomatic at first presentation. The diagnosis is based on sustained elevation of serum markers of cholestasis, i.e., alkaline phosphatase and gamma-glutamyl transferase, and the presence of serum antimitochondrial antibodies directed against the E2 subunit of the pyruvate dehydrogenase complex. Histologically, PBC is characterized by florid bile duct lesions with damage to biliary epithelial cells, an often dense portal inflammatory infiltrate and progressive loss of small intrahepatic bile ducts. Although the insight into pathogenetic aspects of PBC has grown enormously during the recent decade and numerous genetic, environmental, and infectious factors have been disclosed which may contribute to the development of PBC, the precise pathogenesis remains enigmatic. Ursodeoxycholic acid (UDCA) is currently the only FDA-approved medical treatment for PBC. When administered at adequate doses of 13–15 mg/kg/day, up to two out of three patients with PBC may have a normal life expectancy without additional therapeutic measures. The mode of action of UDCA is still under discussion, but stimulation of impaired hepatocellular and cholangiocellular secretion, detoxification of bile, and antiapoptotic effects may represent key mechanisms. One out of three patients does not adequately respond to UDCA therapy and may need additional medical therapy and/or liver transplantation. This review summarizes current knowledge on the clinical, diagnostic, pathogenetic, and therapeutic aspects of PBC