Cardiac hypertrophy can be induced in isolated human atrial and left ventricular intact myocardium by Ang II and diastolic overstretch but not by isometric afterload. The fact that the induction of cardiac growth is inhibited by the blockade of Ang II subtype 1 receptors is of scientific and clinical importance.

SOX9 is an evolutionary conserved transcription factor that is expressed in a variety of tissues, with essential functions in cartilage, testis, heart, glial cell, inner ear and neural crest development. By comparing human and pufferfish genomic sequences, we previously identified eight highly conserved sequence elements between 290 kb 5' and 450 kb 3' to human SOX9. In this study, we assayed the regulatory potential of elements E1 to E7 in transgenic mice using a lacZ reporter gene driven by a 529 bp minimal mouse Sox9 promoter. We found that three of these elements and the Sox9 promoter control distinct subsets of the tissue-specific expression pattern of Sox9. E3, located 251 kb 5' to SOX9, directs lacZ expression to cranial neural crest cells and to the inner ear. E1 is located 28 kb 5' to SOX9 and controls expression in the node, notochord, gut, bronchial epithelium and pancreas. Transgene expression in the neuroectoderm is mediated by E7, located 95 kb 3' to SOX9, which regulates expression in the telencephalon and midbrain, and by the Sox9 minimal promoter which controls expression in the ventral spinal cord and hindbrain. We show that E3-directed reporter gene expression in neural crest cells of the first but not of the second and third pharyngeal arch is dependent on beta-catenin, revealing a complex regulation of Sox9 in cranial neural crest cells. Moreover, we identify and discuss highly conserved transcription factor binding sites within enhancer E3 that are in good agreement with current models for neural crest and inner ear development. Finally, we identify enhancer E1 as a cis-regulatory element conserved between vertebrates and invertebrates, indicating that some cis-regulatory sequences that control developmental genes in vertebrates might be phylogenetically ancient.

Tyrosylprotein sulfotransferase (TPST) is a 54-to 50-kDa integral membrane glycoprotein of the transGolgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular Protein tyrosine sulfation is a widespread posttranslational modification found in all metazoan species and tissues examined (1, 2). It is catalyzed by tyrosylprotein sulfotransferase (TPST) (3), an integral membrane glycoprotein residing in the trans-Golgi network (TGN) whose catalytic site is oriented toward the TGN lumen (2). Accordingly, proteins trafficking through the TGN have been found to become tyrosinesulfated, including several identified plasma membrane and secretory proteins (2). As for its physiological role, tyrosine sulfation has been shown to promote protein-protein interaction (2, 4), be it between (i) two secretory proteins (4, 5), (ii) a secretory protein and its cell surface receptor (6, 7), or (iii) two plasma membrane proteins (8-10).Recognition by TPST requires the occurrence of certain structural features in the substrate protein; a hallmark of these is the presence of acidic amino acid residues in the vicinity of the tyrosine (11-15). The comparison of the sequence motifs of various tyrosine sulfation sites (12), the characterization of the enzymatic properties of TPST from various tissues (16), and the observation that upon SDS͞PAGE purified TPST appears as a 54-to 50-kDa doublet of two, albeit highly related, polypeptides (17), have led to the suggestion (2) that for a given organism, isoenzymes of TPST may exist. Here we report the cDNA cloning and molecular characterization of a human TPST, referred to as TPST-2, which is distinct from the human TPST (referred to as TPST-1), whose molecular cloning was reported (18) while this study was in progress. MATERIALS AND METHODS Synthesis of [35 S]PAPS. ''Carrier-free'' [ 35 S]PAPS was synthesized as described (19), purified by thin-layer electrophoresis on cellulose sheets at pH 3.5, eluted, and stored at Ϫ20°C. The [ 35 S]PAPS was adjusted to the indicated concentration by addition of unlabeled PAPS. except for the presence of one rather than two N-terminal lysine residues] was dissolved in 50% acetonitrile͞50 mM sodium borate, pH 8.5, to a final concentration of 3 mM. NHS-LC-biotin (Fluka) in acetonitrile was added from a 30-mM stock solution to a final concentration of 12 mM, and the mixture was incubated for 90 min at room temperature. The pH was maintained at a value of 8.5 by repeated addition of appropriate amounts of 1 M NaOH. After incubation, the mixture was acidified to pH Ͻ 3 by addition of trifluoroacetic acid, and ␣,-bis-(biotinyl-Ahx)-KE(EPEYGE) 3 -OH (referred to as biotinyl-SgI 3 ) was isolated by using reverse-phase chromatograph...

The purpose of the experiments reported is to provide an unambiguous demonstration that embryonie skeletal muscle contains factors that act directly on embryonie spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonie chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone bad no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Mostofthis activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).