Polytheonamides A and B are highly cytotoxic polypeptides with 48 amino acid residues isolated from the marine sponge, Theonella swinhoei. The structure of polytheonamide B was determined by spectral and chemical methods, especially extensive 2D NMR experiments, which resulted in the unprecedented polypeptide structure; the N-terminal glycine blocked with a 5,5-dimethyl-2-oxo-hexanoyl group, the presence of eight tert-leucine, three beta-hydroxyvaline, six gamma-N-methylasparagine, two gamma-N-methyl-beta-hydroxyasparagine, and beta,beta-dimethymethionine sulfoxide residues. More significantly, it has the sequence of alternating D- and L-amino acids. Polytheonamide A is an epimer of polytheonamide B differing only in the stereochemistry of the sulfoxide of the 44(th) residue.
Synaptotagmin IV (SytIV) is an immediate early gene induced by membrane depolarization in PC12 cells and in rat brain. However, little is known about the function of SytIV or the functional relationship between SytIV and SytI, because SytIV has yet to be localized. Here we show that SytIV was localized at the Golgi and distal part of neurites in nerve growth factor-differentiated PC12 cells and cultured hippocampal neurons by immunocytochemistry using an isoform-specific antibody (antiSytIV). These SytIV signals were not colocalized well with SytI signals. Upon membrane depolarization, SytIV signals were increased at both the Golgi and distal part of neurites within several hours in both types of cells. We further show that the increase of SytIV protein levels results from protein kinase A-dependent gene up-regulation. In hippocampal neurons, SytIV was developmentally regulated. These results suggest that SytIV may play a role at the Golgi and tips of neurites during development and synaptic plasticity. Key Words: Synaptotagmin-Depolarization-Forskolin-Immediate early genesNeuronal development-Synaptic plasticity. J. Neurochem. 74, 518 -526 (2000).The synaptotagmin family consists of 11 isoforms [synaptotagmins (Syt) I-XI] in rat or mouse and shares a short amino terminus, one transmembrane region, and two C2 domains homologous to the C2 regulatory region of protein kinase C (known as the C2A and C2B domains) (Perin et al
Polytheonamide B (pTB), a highly cytotoxic polypeptide, is one of the most unusual nonribosomal peptides of sponge origin. pTB is a linear 48-residue peptide with alternating D- and L-amino acids and contains a total of eight types of nonproteinogenic amino acids. To investigate the mechanisms underlying its cytotoxic activity, we determined the three-dimensional structure of pTB by NMR spectroscopy, structure calculation, and energy minimization. pTB adopts a single right-handed β(6.3)-helical structure in a 1:1 mixture of methanol/chloroform with a length of approximately 45 A and a hydrophilic pore of ca. 4 A inner diameter. These features indicate that pTB molecules form transmembrane channels that permeate monovalent cations as gramicidin A channels do. The strong cytotoxicity of pTB can be ascribed to its ability to form single molecule channels through biological membranes.
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