Ultrasound guided site specific gene delivery system using adenoviral vectors and commercial ultrasound contrast agents

We have evaluated if ultrasound imaging (US) and various commercially available contrast microbubbles can serve as a non-invasive systemically administered delivery vehicle for site-specific adenoviral-mediated gene transfer in vitro and in vivo. The contrast agents were tested for their ability to enclose and to protect an adenoviral vector carrying the GFP marker gene (Ad-GFP) into the microbubbles. We have also evaluated the ability of the innate immune system to inactivate free adenoviruses as well as unenclosed viruses adsorbed on the surface of the contrast agents and in turn the ability of the microbubbles to enclose and to protect the viral vectors from such agents. In vitro as well as in vivo, innate components of the immune system were able to serve as inactivating agents to clear free viral particles and unenclosed adenoviruses adsorbed on the microbubbles' surface. Systemic delivery of Ad-GFP enclosed into microbubbles in the tail vein of nude mice resulted in specific targeting of the GFP transgene. Both fluorescence microscopy and GFP immunohistochemistry demonstrated US guided specific transduction in the targeted cells only, with no uptake in either heart, lungs or liver using complement-pretreated Ad-GFP microbubbles. This approach enhances target specificity of US microbubble destruction as a delivery vehicle for viral-mediated gene transfer.     

A screen for mutations in zebrafish that affect myelin gene expression in Schwann cells and oligodendrocytes

Myelin is the multi-layered glial sheath around axons in the vertebrate nervous system. Myelinating glia develop and function in intimate association with neurons and neuron-glial interactions control much of the life history of these cells. However, many of the factors that regulate key aspects of myelin development and maintenance remain unknown. To discover new molecules that are important for glial development and myelination, we undertook a screen of zebrafish mutants with previously characterized neural defects. We screened for myelin basic protein (mbp) mRNA by in situ hybridization and identified four mutants (neckless, motionless, iguana and doc) that lacked mbp expression in parts of the peripheral and central nervous systems (PNS or CNS), despite the presence of axons. In all four mutants electron microscopy revealed that myelin-forming glia were present and had formed loose wraps around axons but did not form compact myelin. We found that addition of exogenous retinoic acid (RA) rescued mbp expression in neckless mutant embryos, which lack endogenous RA synthesis. Timed application of the RA synthesis inhibitor DEAB to wild type embryos showed that RA signalling is required at least 48 h before the onset of myelin protein synthesis in both CNS and PNS.     

AMP-activated protein kinase and the regulation of glucose transport

The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by acute increases in the cellular [AMP]/[ATP] ratio. In skeletal and/or cardiac muscle, AMPK activity is increased by stimuli such as exercise, hypoxia, ischemia, and osmotic stress. There are many lines of evidence that increasing AMPK activity in skeletal muscle results in increased rates of glucose transport. Although similar to the effects of insulin to increase glucose transport in muscle, it is clear that the underlying mechanisms for AMPK-mediated glucose transport involve proximal signals that are distinct from that of insulin. Here, we discuss the evidence for AMPK regulation of glucose transport in skeletal and cardiac muscle and describe research investigating putative signaling mechanisms mediating this effect. We also discuss evidence that AMPK may play a role in enhancing muscle and whole body insulin sensitivity for glucose transport under conditions such as exercise, as well as the use of the AMPK activator AICAR to reverse insulin-resistant conditions. The identification of AMPK as a novel glucose transport mediator in skeletal muscle is providing important insights for the treatment and prevention of type 2 diabetes.     

GH receptor signaling in skeletal muscle and adipose tissue in human subjects following exposure to an intravenous GH bolus

Growth hormone (GH) regulates muscle and fat metabolism, which impacts on body composition and insulin sensitivity, but the underlying GH signaling pathways have not been studied in vivo in humans. We investigated GH signaling in biopsies from muscle and abdominal fat obtained 30 (n = 3) or 60 (n = 3) min after an intravenous bolus of GH (0.5 mg) vs. saline in conjunction with serum sampling in six healthy males after an overnight fast. Expression of the following signal proteins were assayed by Western blotting: STAT5/p-STAT5, MAPK, and Akt/PKB. IRS-1-associated PI 3-kinase activity was measured by in vitro phosphorylation of PI. STAT5 DNA binding activity was assessed with EMSA, and the expression of IGF-I and SOCS mRNA was measured by real-time RT-PCR. GH induced a 52% increase in circulating FFA levels with peak values after 155 min (P = 0.03). Tyrosine-phosphorylated STAT5 was detected in muscle and fat of all subjects after GH. Activation of MAPK was observed in several lysates but without GH dependency. Neither PKB/Akt nor PI 3-kinase activity was affected by GH. GH-induced STAT5 DNA binding and expression of IGF-I mRNA were detected in fat, whereas expression of SOCS-1 and -3 tended to increase after GH in muscle and fat, respectively. We conclude that 1) STAT5 is acutely activated in human muscle and fat after a GH bolus, but additional downstream GH signaling was significant only in fat; 2) the direct GH effects in muscle need further characterization; and 3) this human in vivo model may be used to study the mechanisms subserving the actions of GH on substrate metabolism and insulin sensitivity in muscle and fat.     

Effect of meal and propranolol on whole body and splanchnic oxygen consumption in patients with cirrhosis

Our aim was to measure whole body energy expenditure after a mixed liquid meal, with and without simultaneous propranolol infusion, in patients with cirrhosis. We also wanted to investigate the effect of propranolol on substrate fluxes and oxygen uptake in the tissues drained by the hepatic vein and azygos vein in the postprandial period in these patients. Whole-body oxygen uptake, hepatic blood flow, hepatic venous pressure gradient and net-hepatic fluxes of oxygen, lactate, glucose, glycerol, and free fatty acids (FFA) were measured in 12 patients with alcoholic cirrhosis before and for 2 h after ingestion of a mixed liquid meal (700 kcal). Half of the patients (n = 6) were randomized to a treatment group receiving intravenous infusion of propranolol in combination with the meal. The meal-induced energy expenditure was significantly lower in patients given propranolol [15.0 +/- 18.9 vs. 67.0 +/- 26.1 kJ/120 min (means +/- SD), P < 0.01]. Meal-induced whole body oxygen uptake was lower in patients receiving propranolol (19.2 +/- 38 vs. 135.7 +/- 61 mmol/120 min, P < 0.01), and the meal-induced increase in splanchnic oxygen uptake was nonexistent when propranolol was administered in combination (-13.2 +/- 34.8 vs. 110.4 +/- 34.8 mmol/120 min, P = 0.04). Postprandially, the propranolol group had a tendency toward a reduced splanchnic glucose output, and the FFA uptake was significantly reduced. Propranolol reduces meal-induced whole body oxygen uptake and energy expenditure as well as splanchnic oxygen uptake. The splanchnic reduction in oxygen consumption can explain almost the entire reduction in whole body oxygen consumption.     

Enzymatic Activation of Lysine 2,3-Aminomutase from Porphyromonas gingivalis

The development of lysine 2,3-aminomutase as a robust biocatalyst hinges on the development of an in vivo activation system to trigger catalysis. This is the first report to show that, in the absence of chemical reductants, lysine 2,3-aminomutase activity is dependent upon the presence of flavodoxin, ferredoxin, or flavodoxin-NADP⁺ reductase.     

The PP-fold solution structure of human polypeptide YY and human PYY3-36 as determined by NMR

PYY3-36 is a biopharmaceutical antiobesity agent under development as well as an endogenous satiety hormone, which is generated by dipeptidyl peptidase-IV digestion of polypetide YY (PYY), and in contrast to the parent hormone, PYY is highly selective for the Y2 versus the Y1 receptor. NMR analysis revealed a highly ordered, back-folded structure for human PYY in aqueous solution similar to the classical PP-fold structure of pancreatic polypeptide. The NMR analysis of PYY3-36 also showed a folded structure resembling a PP-fold, which however was characterized by far fewer long distance NOEs than the PP-fold observed in the full-length peptide. This suggests that either a conformational change has occurred in the N-terminal segment of PYY3-36 or that this segments is characterized by larger dynamics. The study supports the notion that the PP-fold is crucial for establishing simultaneous interactions with two subsites in the receptor for binding of, respectively, the N- and C-terminal ends of PYY. The Y2 receptor only requires recognition of the C-terminal segment of the molecule as displayed by the Y2 selective PYY3-36.