GABA (gamma-amino-butyric acid), the principal inhibitory neurotransmitter in the brain, signals through ionotropic (GABA(A)/ GABA(c)) and metabotropic (GABA(B)) receptor systems. Here we report the cloning of GABA(B) receptors. Photoaffinity labelling experiments suggest that the cloned receptors correspond to two highly conserved GABA(B) receptor forms present in the vertebrate nervous system. The cloned receptors negatively couple to adenylyl cyclase and show sequence similarity to the metabotropic receptors for the excitatory neurotransmitter L-glutamate.
We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 500. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double-and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel. The electrophoretic mobility of nucleic acids in polyacrylamide or agarose gels depends on both molecular weight and conformation (1, 2). Removing secondary and tertiary structure should make the electrophoretic mobility a simple function of molecular weight. Gels containing the denaturing agents formaldehyde (3), formamide (4,5), methylmercuric hydroxide (6), and urea (7) have all been used for molecular weight determinations. Here we present a simple and convenient method that we feel offers a number of advantages over those previously described. This method employs denaturation of nucleic acids and reaction with glyoxal, followed by electrophoresis in a slab gel.
Gene fusions prevalent in prostate cancer (CaP) lead to the elevated expression of the ERG proto-oncogene. ERG activation present in 50–70% of prostate tumors underscores one of the most common oncogenic alterations in CaP. Despite numerous reports of gene fusions and mRNA expression, ERG oncoprotein status in CaP still remains to be defined. Furthermore, development of ERG protein-based assays may provide a new dimension to evaluation of gene fusions involving diverse androgen-regulated promoters and the ERG protein-coding sequence. Through exhaustive evaluations of 132 whole-mount prostates (261 tumor foci and over 200 000 benign glands) for the ERG oncoprotein nuclear expression, we demonstrated 99.9% specificity for detecting prostate tumor cells using a highly specific anti-ERG monoclonal antibody. The ERG oncoprotein expression correlated well with fusion transcript or gene fusion in randomly selected specimens. Strong concordance of ERG-positive foci of prostatic intraepithelial neoplasia (PIN) with ERG-positive carcinoma (82 out of 85 sections with PIN, 96.5%) affirms the biological role of ERG in clonal selection of prostate tumors in 65% (86 out of 132) of patients. Conversely, ERG negative PINs were associated with ERG-negative carcinoma. Taken together, the homogeneous and strong ERG expression detected in individual tumors establishes the potential for ERG oncoprotein-based stratification of CaP.
RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.
Transforming growth factors /? belong to a group of cytokines that control cellular proliferation and differentiation. Five isoforms are known that share approximately 75% sequence identity, but exert different biological activities. The structure of TGF-/?3 was solved by X-ray crystallography and refined to a final R-factor of 17.5% at 2.0 A resolution. Comp~ison with the structure of TGF-02 (Schlunegger MP, Grutter MG, 1992, Nature 358430-434; Daopin S, Piez KA, Ogawa Y, Davies DR, 1992, Science 252369-373) reveals a virtually identical central core. Differences exist in the conformations of the N-terminal a-helix and in the P-sheet loops. In TGF-03, the N-terminal cu-helix has moved = 1 A away from the central core. This movement can be correlated with the mutation of Leu 17 to Val and Ala 47. to Pro in TGF-63. The /?-sheet loops rotate as a rigid body 9" around an axis that runs approximately parallel to the dimer axis. If these differences are recognized by the TGF-0 receptors, they might account for the individual cellular responses. A molecule of the precipitating agent dioxane is bound in a crystal contact, forming a hydrogen bond with Trp 32. This dioxane may occupy a carbohydrate-binding site, because dioxane possesses some structural similarity with a carbohydrate. The dioxane is in contact with two tryptophans, which are often involved in carbohydrate recognition.
Exogenously applied transforming growth factor-beta (TGF-beta) isoforms enhance wound healing processes in animal models; however, little is known about the expression of endogenous TGF-beta s and TGF-beta receptors in intact human skin or during wound healing. The present study has revealed several unexpected findings by means of in situ hybridization and immunohistology techniques. In humans, TGF-beta 3 is constitutively expressed in the epidermis of intact skin and in that of acute and chronic wounds--a pattern of expression closely mirrored by the TGF-beta type II receptor. Although not detected in intact skin, TGF-beta 1 mRNA expression was observed in the regenerating epidermis of acute (thermal) wounds but was not found in chronic decubital (pressure) wounds. TGF-beta 2 mRNA expression was not detected in the epidermis of any human skin or wound biopsies. From these findings we suggest that constitutive expression of TGF-beta 3 is important for maintenance of epidermal differentiation and that an induction of TGF-beta 1 expression is essential for re-epithelialization of human skin wounds. Lack of TGF-beta 1 expression in chronic pressure wounds may be associated with their protracted healing tendencies.
Monoclonal antibodies directed against rabbit reticulocyte protein synthesis initiation factor 4A (eIF-4A) were used to isolate mouse cDNA clones expressing eIF-4A protein sequences in E. coli. The identity of cDNA clones encoding eIF-4A sequences was confirmed by hybrid-selected translation and peptide mapping of the translation product. Analysis of the mRNA coding for eIF-4A from mouse liver and HeLa cells by Northern hybridization revealed two discrete mRNA species of approximately 2000 and 1600 nucleotides in length. The existence of two mRNAs in mouse and HeLa cells encoding eIF-4A was confirmed by cDNA sequencing.
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