MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.

Interactions between Growth Factors and Integrins: Latent Forms of Transforming Growth Factor-β Are Ligands for the Integrin αvβ1

The multipotential cytokine transforming growth factor-β (TGF-β) is secreted in a latent form. Latency results from the noncovalent association of TGF-β with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-β–binding protein (LTBP) produces the most common form of latent TGF-β, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-β. LTBP and the LAP propeptides of TGF-β (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-β function in the ECM, we determined whether latent TGF-β1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits αv and β1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. αvβ1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of αvβ5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was αvβ1 dependent. These results establish αvβ1 as a LAP-β1 receptor. Interactions between latent TGF-β and αvβ1 may localize latent TGF-β to the surface of specific cells and may allow the TGF-β1 gene product to initiate signals by both TGF-β receptor and integrin pathways.

Requirement of the nicotinic acetylcholine receptor β2 subunit for the anatomical and functional development of the visual system

In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, β2−/− mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the β2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.

NCX-1000, a NO-releasing derivative of ursodeoxycholic acid, selectively delivers NO to the liver and protects against development of portal hypertension

Portal hypertension resulting from increased intrahepatic resistance is a common complication of chronic liver diseases and a leading cause of death in patients with liver cirrhosis, a scarring process of the liver that includes components of both increased fibrogenesis and wound contraction. A reduced production of nitric oxide (NO) resulting from an impaired enzymatic function of endothelial NO synthase and an increased contraction of hepatic stellate cells (HSCs) have been demonstrated to contribute to high intrahepatic resistance in the cirrhotic liver. 2-(Acetyloxy) benzoic acid 3-(nitrooxymethyl) phenyl ester (NCX-1000) is a chemical entity obtained by adding an NO-releasing moiety to ursodeoxycholic acid (UDCA), a compound that is selectively metabolized by hepatocytes. In this study we have examined the effect of NCX-1000 and UDCA on liver fibrosis and portal hypertension induced by i.p. injection of carbon tetrachloride in rats. Our results demonstrated that although both treatments reduced liver collagen deposition, NCX-1000, but not UDCA, prevented ascite formation and reduced intrahepatic resistance in carbon tetrachloride-treated rats as measured by assessing portal perfusion pressure. In contrast to UDCA, NCX-1000 inhibited HSC contraction and exerted a relaxing effect similar to the NO donor S-nitroso-N-acetylpenicillamine. HSCs were able to metabolize NCX-1000 and release nitrite/nitrate in cell supernatants. In aggregate these data indicate that NCX-1000, releasing NO into the liver microcirculation, may provide a novel therapy for the treatment of patients with portal hypertension.