UV and IR matrix-assisted laser desorption/ionization mass spectrometry (UV-and IR-MALDI) have demonstrated their potential for the accurate and sensitive mass determination of oligonucleotides. Metastable molecule ion fragmentation was found to he the main limitation in both desorption schemes for the analysis of larger nucleic acid sequences. Fragment ions from additional prompt decays, observed only in IR-MALDI, offer structural data, which allow sequence information to be derived for low-picomok amounts of oligodeoxynucleotides with up to 21 bases from a single mass spectrum. Two examples demonstrating the feasibility of this new sequencing technique are given. A model is introduced and discussed, which proposes a reaction scheme for the observed fragment ion patterns of nucleic acids and differentiates prompt and metastable fragmentation mechanisms. The role of fragmentation in direct mass spectrometric sequencing schemes and as a limitation for the accessible mass range in nucleic acid MALDI mass spectrometry is discussed.

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Abstract. Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the ~-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an l 1-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.ROWTH development, and differentiation of epithelial tissues are multistage processes that are driven by a combination of paracrine and autocrine signaling factors and interactions of a cell with its extracellular matrix (reviewed in 51, 60, 62). In the mammary gland, these complex interactions are regulated by various steroid and peptide hormones (29,32,46,60). Development of the mouse mammary gland at puberty is characterized by sparsely branching ducts which invade the stroma, followed by the development of lobuloalveolar structures and functional differentiation, i.e., synthesis of milk constituents at pregnancy (1,17,32). By use of endocrine ablation (44), organ culture systems (1,35,54,65) and mammary cells (MEC) ~ growing on a

The three‐dimensional structure of the holo form of recombinant cellular bovine heart fatty‐acid‐binding protein (H‐FABPc), a polypeptide of 133 amino acid residues with a molecular mass of 15 kDa, has been determined by multidimensional homonuclear and heteronuclear NMR spectroscopy applied to uniformly 15N‐labeled and unlabeled protein. A nearly complete set of 1H and 15N chemical shift assignments was obtained. A total of 2329 intramolecular distance constraints and 42 side‐chain χi dihedral‐angle constraints were derived from cross‐relaxation and J coupling information. 3D nuclear Overhauser enhancement and exchange spectroscopy combined with heteronuclear multiple‐quantum coherence (NOESY‐HMQC) experiments, performed on a sample of uniformly 13C‐labeled palmitic acid bound to unlabeled cellular heart fatty‐acid‐binding protein revealed 10 intermolecular contacts that determine the orientation of the bound fatty acid. An ensemble of protein conformations was calculated with the distance‐geometry algorithm for NMR applications (DIANA) using the redundant dihedral‐angle constraint (REDAC) strategy. After docking the fatty acid into the protein, the protein‐ligand arrangement was subject to distance‐restrained energy minimization. The overall conformation of the protein is a β‐barrel consisting of 10 antiparallel β‐strands which form two nearly orthogonal β‐sheets of five strands each. Two short helices form a helix‐turn‐helix motif in the N‐terminal region of the polypeptide chain. The palmitic acid is bound within the protein in a U‐shaped conformation close to the two helices. The obtained solution structure of the protein is consistent with a number of fatty‐acid‐binding‐protein crystal structures.

High resolution nuclear magnetic resonance (NMR) and ethidium bromide binding studies are used to demonstrate that poly d(G-T) forms an ordered double helical structure at low temperatures (below 24 degrees C in 0.3 M NaCl) in which G and T are hydrogen bonded together in a wobble base pair hydrogen bonding scheme as proposed earlier by Lezius and Domin. Alternative hydrogen bonding schemes involving the tautomeric form of either T or G, such as have been proposed to account for mutation rates in DNA synthesis, are eliminated.