Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.     

Comparison between two rat sympathetic pathways activated in cold defense

In cold defense and fever, activity increases in sympathetic nerves supplying both tail vessels and interscapular brown adipose tissue (iBAT). These mediate cutaneous vasoconstrictor and thermogenic responses, respectively, and both depend upon neurons in the rostral medullary raphé. To examine the commonality of brain circuits driving these two outflows, sympathetic nerve activity (SNA) was recorded simultaneously from sympathetic fibers in the ventral tail artery (tail SNA) and the nerve to iBAT (iBAT SNA) in urethane-anesthetized rats. From a warm baseline, cold-defense responses were evoked by intermittently circulating cold water through a water jacket around the animal's shaved trunk. Repeated episodes of trunk skin cooling decreased core (rectal) temperature. The threshold skin temperature to activate iBAT SNA was 37.3 +/- 0.5 degrees C (n = 7), significantly lower than that to activate tail SNA (40.1 +/- 0.4 degrees C; P < 0.01, n = 7). A fall in core temperature always strongly activated tail SNA (threshold 38.3 +/- 0.2 degrees C, n = 7), but its effect on iBAT SNA was absent (2 of 7 rats) or weak (threshold 36.9 +/- 0.1 degrees C, n = 5). The relative sensitivity to core vs. skin cooling (K-ratio) was significantly greater for tail SNA than for iBAT SNA. Spectral analysis of paired recordings showed significant coherence between tail SNA and iBAT SNA only at 1.0 +/- 0.1 Hz. The coherence was due entirely to the modulation of both signals by the ventilatory cycle because it disappeared when the coherence spectrum was partialized with respect to airway pressure. These findings indicate that independent central pathways drive cutaneous vasoconstrictor and thermogenic sympathetic pathways during cold defense.     

Discovery of novel spiro-piperidine derivatives as highly potent and selective melanin-concentrating hormone 1 receptor antagonists

Optimization of high-throughput screening hit 1a led to the identification of a novel spiro-piperidine class of melanin-concentrating hormone 1 receptor (MCH-1R) antagonists. Compound 3c was identified as a highly potent and selective MCH-1R antagonist, which has an IC₅₀ value of 0.09 nM at hMCH-1R. The synthesis and structure–activity relationships of the novel spiro-piperidine MCH-1R antagonists are described.     

Monophyly of Lampreys and Hagfishes Supported by Nuclear DNA–Coded Genes

The phylogenetic position of hagfishes in vertebrate evolution is currently controversial. The 18S and 28S rRNA trees support the monophyly of hagfishes and lampreys. In contrast, the mitochondrial DNAs suggest the close association of lampreys and gnathostomes. To clarify this controversial issue, we have conducted cloning and sequencing of the four nuclear DNA–coded single-copy genes encoding the triose phosphate isomerase, calreticulin, and the largest subunit of RNA polymerase II and III. Based on these proteins, together with the Mn superoxide dismutase for which hagfish and lamprey sequences are available in database, phylogenetic trees have been inferred by the maximum likelihood (ML) method of protein phylogeny. It was shown that all the five proteins prefer the monophyletic tree of cyclostomes, and the total log-likelihood of the five proteins significantly supports the cyclostome monophyly at the level of ±1 SE. The ML trees of aldolase family comprising three nonallelic isoforms and the complement component group comprising C3, C4, and C5, both of which diverged during vertebrate evolution by gene duplications, also suggest the cyclostome monophyly. Received: 28 April 1999 / Accepted: 30 June 1999     

Identification of the carboxyl-terminal membrane-anchoring region of HPC-1/syntaxin 1A with the substituted-cysteine-accessibility method and monoclonal antibodies

HPC-1/syntaxin 1A is a member of the syntaxin family, and functions at the plasma membrane during membrane fusion as the target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (t-SNARE). We identified the membrane-anchoring region of HPC-1/syntaxin 1A, and examined its role in anchoring of a protein to the plasma membrane. A series of mutants was created from a cysteine-less mutant of HPC-1/syntaxin 1A by substitution of each residue at the C-terminus with cysteine. The accessibility of the thiol-groups in each mutant was analyzed in vivo. The cysteine (C145) within the N-terminal cytosolic segment was labeled, but not that at C271 or C272, or any of those introduced at the C-terminus. The addition of additional residues to the C-terminal tail of HPC-1/syntaxin 1A allowed labeling by thiol-specific reagents. A monoclonal antibody directed against the C-terminal tail peptide did not react with the protein located at the plasma membrane. In addition, subcellular fractionation and immunocytochemical analyses with various transmembrane mutants showed that the C-terminal tail comprising eight amino acids is essential for anchoring of HPC-1/syntaxin 1A to the plasma membrane. These results indicate that the C-terminal membrane-anchoring region, which comprises 23 amino acids, does not traverse the lipid-bilayer and that the C-terminal tail is essential for anchoring of HPC-1/syntaxin 1A to the plasma membrane.     

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser-15 and Ser-85 in smooth muscle of aorta in vivo. The AVP-induced phosphorylation of HSP27 at Ser-15 and Ser-85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85. The AVP-induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen-activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.