MicroRNA-423 may regulate diabetic vasculopathy.

To test the hypothesis that microRNAs may play a role in diabetic retinopathy, we measured the levels of different markers [microRNAs, vascular endothelial growth factor (VEGF), nitric oxide (NO), and total antioxidant capacity (TAO)] in patients with type 2 diabetes mellitus (T2DM) and microvascular complications. Sixty-nine patients were recruited: 22 healthy subjects, ten T2DM patients without retinopathy, 22 with nonproliferative diabetic retinopathy, and 15 with proliferative diabetic retinopathy (PDR). Serum levels of NO, VEGF, TAO and 16 candidate microRNAs were measured. Additionally, the mRNA levels of endothelial nitric oxide synthase (eNOS), induced NOS (iNOS), C reactive protein (CRP), VEGF, tumor necrosis factor α (TNFα), PON2, p22, and SOD2 were measured in human vascular endothelial cells cultured in the presence of pooled sera from the subject groups. Plasma miR-423 levels showed a significant ~ twofold decrease in patients with PDR compared to controls. P lasma NO levels were significantly higher in retinopathy, VEGF levels were significantly lower, and TAO was significantly decreased. eNOS mRNA levels were lower in the cells of T2DM patients without retinopathy, but higher in PDR. PON2, p22, and SOD2 mRNA levels were all significantly lower in PDR. CRP, TNFα, iNOS, and VEGF mRNA levels showed no significant association with disease status. Lowered miR-423 levels in diabetic patients showed a correlation with VEGF and an inverse correlation between NO and eNOS expression. Our findings suggest a cross talk between miR-423 and VEGF signaling, affecting eNOS function. miR-423 may be involved in the regulation of diabetic vascular retinal proliferation.

Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

Implication of Galpha i proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production.

Previous studies have suggested that heterotrimeric G proteins and tyrosine kinases may be involved in lipopolysacchaide (LPS) signaling events. Signal transduction pathways activated by LPS we examined in human pomonocytic THP-l cells. We hypothesized that Gi proteins and Src tyrosine kinase differentially affect mitogen-activated protein (MAP) kinases (MAPK) and nuclear factor kappa(NF-kappaB) activation. Post-receptor coupling to Ga, proteins were examined using pertussis toxin (PTx),which inhibits Galpha i receptor-coupling. The involvement of the Src family of tyrosine kinases was examined using the selective Src tyrosine kinase inhibitor pyrazolopyrimidine-2 (PP2). Pretreatment of THP-1 cells with PTx attenuated LPS-induced activation of c-Jun-N-terminal kinase (JNK) and p38 kinase, and production of tumor necrosis factor-alpha (TN-alpha) and thromboxane B2 (TXB2). Pretreatment with PP2 inhibited TNF-alpha and TxB2 production, but had no effect on p38 kinase or JNK signaling. Therefore, the Ga i-coupled signaling pathways and Src tyrosine kinase-coupled signaling pathways are necessary for LPS-induced TNF-alpha and TxB2 production, but differ in their effects on MAPK activation. Neither PTx nor PP2 inhibited LPS-induced activation of interleukin receptor activated kinase (IRAK) or inhibited translocation of NF-kappaB. However, PP2 inhibited LPS-induced NF-kappaB transactivation of a luciferase reporter gene construct in a concentration-dependent manner. Thus, LPS induction of Src tyrosine kinases may be essential in downstream NF-kappaB tansactivation of genes following DNA binding. PTx had no effect on NF-kaapaB activation of the reporter construct. These data suggest upstream divergence in signaling through Galpha i,pathways leading to MAPK activation and other signaling events leading to IkappaBalpha degradation and NF-kaapaB DNA binding.