Functional characterization of a minimal K+ channel expressed from a synthetic gene.

A gene for a slowly activating, voltage-dependent K(+) -selective channel was designed and synthesized on the basis of its known amino acid sequence. The synthetic gene was cloned into a transcription vector, and in vitro transcribed mRNA was injected into Xenopus oocytes for electrophysiological assay of the resulting ionic currents. The currents are voltage-dependent and highly selective for K+ over Na+. The selectivity among monovalent cations follows a familiar K(+)- channel sequence: K+ greater than Rb+ greater than NH4+ greater than Cs+ much greater than Na+, Li+. The currents are inhibited by Ba2+, Cs+, and tetraethylammonium (TEA), common pore blockers of K+ channels. Open-channel blockade by Cs+ (but not by Ba2+ or TEA) depends on applied voltage. The major inhibitory effect of Ba2+ is to alter channel gating by favoring the closed state; this effect is specific for Ba2+ and is relieved by external K+. The results argue that although the polypeptide expressed is very small for a eukaryotic ion channel, 130 amino acid residues in length, the ionic currents observed are indeed mediated by a genuine K(+) -channel protein. This synthetic gene is therefore well suited for a molecular analysis of the basic mechanisms of K(+) -channel function

E4orf1: A Novel Ligand That Improves Glucose Disposal in Cell Culture

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance(IR), are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase(PI3K), and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 ‘requires’ E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as ‘sufficient’ to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras–the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large(Dlg1) protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif(PBM) of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs

A Serum Factor Induces Insulin-Independent Translocation of GLUT4 to the Cell Surface which Is Maintained in Insulin Resistance

In response to insulin, glucose transporter GLUT4 translocates from intracellular compartments towards the plasma membrane where it enhances cellular glucose uptake. Here, we show that sera from various species contain a factor that dose-dependently induces GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, human adipocytes, myoblasts and myotubes. Notably, the effect of this factor on GLUT4 is fully maintained in insulin-resistant cells. Our studies demonstrate that the serum-induced increase in cell surface GLUT4 levels is not due to inhibition of its internalization and is not mediated by insulin, PDGF, IGF-1, or HGF. Similarly to insulin, serum also augments cell surface levels of GLUT1 and TfR. Remarkably, the acute effect of serum on GLUT4 is largely additive to that of insulin, while it also sensitizes the cells to insulin. In accordance with these findings, serum does not appear to activate the same repertoire of downstream signaling molecules that are implicated in insulin-induced GLUT4 translocation. We conclude that in addition to insulin, at least one other biological proteinaceous factor exists that contributes to GLUT4 regulation and still functions in insulin resistance. The challenge now is to identify this factor

Translating Birdsong: Songbirds as a Model for Basic and Applied Medical Research

Songbirds, long of interest to basic neuroscientists, have great potential as a model system for translational neuroscience. Songbirds learn their complex vocal behavior in a manner that exemplifies general processes of perceptual and motor skill learning, and more specifically resembles human speech learning. Song is subserved by circuitry that is specialized for vocal learning and production, but that has strong similarities to mammalian brain pathways. The combination of a highly quantifiable behavior and discrete neural substrates facilitates understanding links between brain and behavior, both normally and in disease. Here we highlight 1) behavioral and mechanistic parallels between birdsong and aspects of speech and social communication, including insights into mirror neurons, the function of auditory feedback, and genes underlying social communication disorders, and 2) contributions of songbirds to understanding cortical-basal ganglia circuit function and dysfunction, including the possibility of harnessing adult neurogenesis for brain repair