Comparison of biological activities of endothelin-1, -2 and -3 in murine and human fibroblast cell lines

The effects of three forms of endothelin (ET), ET-1, -2 and -3, on intracellular free Ca2+ concentration ([Ca2+]i) and their receptor binding activities have been compared in murine fibroblast cell line Swiss 3T3 as well as diploid human fibroblast cell line FS-4. In both cell lines, ET-1 and -2 induced a profound increase in [Ca2+]i with a similar dose-response relationship; ET-3 was far less potent. ET-1 and -2 showed specific binding to both cell lines with similar dissociation constants and numbers of binding sites. Negligible specific binding of ET-3 was detected. These findings indicate that these two fibroblast populations possess high affinity receptors for ET-1 and -2 and practically no ET-3 receptors for ET-3, and binding of ET-1 and -2 increases [Ca2+]i in these cell lines.     

Glutamate Acts as a Partial Inverse Agonist to Metabotropic Glutamate Receptor with a Single Amino Acid Mutation in the Transmembrane Domain

Metabotropic glutamate receptor (mGluR), a prototypical family 3 G protein-coupled receptor (GPCR), has served as a model for studying GPCR dimerization, and growing evidence has revealed that a glutamate-induced dimeric rearrangement promotes activation of the receptor. However, structural information of the seven-transmembrane domain is severely limited, in contrast to the well studied family 1 GPCRs including rhodopsins and adrenergic receptors. Homology modeling of mGluR8 transmembrane domain with rhodopsin as a template suggested the presence of a conserved water-mediated hydrogen-bonding network between helices VI and VII, which presumably constrains the receptor in an inactive conformation. We therefore conducted a mutational analysis to assess structural similarities between mGluR and family 1 GPCRs. Mutational experiments confirmed that the disruption of the hydrogen-bonding network by T789Y^6.43 mutation induced high constitutive activity. Unexpectedly, this high constitutive activity was suppressed by glutamate, the natural agonist ligand, indicating that glutamate acts as a partial inverse agonist to this mutant. Fluorescence energy transfer analysis of T789Y^6.43 suggested that the glutamate-induced reduction of the activity originated not from the dimeric rearrangement but from conformational changes within each protomer. Double mutational analysis showed that the specific interaction between Tyr-789^6.43 and Gly-831^7.45 in T789Y^6.43 mutant was important for this phenotype. Therefore, the present study is consistent with the notion that the metabotropic glutamate receptor shares a common activation mechanism with family 1 GPCRs, where rearrangement between helices VI and VII causes the active state formation.     

A Crucial Role for CDC42 in Senescence-Associated Inflammation and Atherosclerosis

Risk factors for atherosclerosis accelerate the senescence of vascular endothelial cells and promote atherogenesis by inducing vascular inflammation. A hallmark of endothelial senescence is the persistent up-regulation of pro-inflammatory genes. We identified CDC42 signaling as a mediator of chronic inflammation associated with endothelial senescence. Inhibition of CDC42 or NF-κB signaling attenuated the sustained up-regulation of pro-inflammatory genes in senescent human endothelial cells. Endothelium-specific activation of the p53/p21 pathway, a key mediator of senescence, also resulted in up-regulation of pro-inflammatory molecules in mice, which was reversed by Cdc42 deletion in endothelial cells. Likewise, endothelial-specific deletion of Cdc42 significantly attenuated chronic inflammation and plaque formation in atherosclerotic mice. While inhibition of NF-κB suppressed the pro-inflammatory responses in acute inflammation, the influence of Cdc42 deletion was less marked. Knockdown of cdc-42 significantly down-regulated pro-inflammatory gene expression and restored the shortened lifespan to normal in mutant worms with enhanced inflammation. These findings indicate that the CDC42 pathway is critically involved in senescence-associated inflammation and could be a therapeutic target for chronic inflammation in patients with age-related diseases without compromising host defenses.     

Peptide ligand screening of α-synuclein aggregation modulators by <Emphasis Type="Italic">in silico</Emphasis> panning

Background  

α-Synuclein is a Parkinson's-disease-related protein. It forms aggregates in vivo, and these aggregates cause cell cytotoxicity. Aggregation inhibitors are expected to reduce α-synuclein cytotoxicity, and an aggregation accelerator has recently been reported to reduce α-synuclein cytotoxicity. Therefore, amyloid aggregation modulating ligands are expected to serve as therapeutic medicines.     

Winner and loser effects of juvenile cricket Gryllus bimaculatus

Journal of Ethology - Agonistic encounters of juvenile male crickets were analyzed behaviourally. In a pairing between adult and juvenile male crickets, the juvenile crickets were usually beaten by...     

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.     

Decay-accelerating factor functions as a signal transducing molecule for human monocytes.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that protects cells from damage by autologous complement activation. Of the four mAb against DAF prepared in our laboratory, 1C6 completely blocked DAF function, whereas 5B2 partially blocked it. Using these mAb, we investigated whether human monocytes were activated via DAF molecules. When monocytes were incubated with 1C6 alone, glucose was consumed in significant amounts and phagocytosis of latex beads was enhanced, indicating that the monocytes had been activated. However, 1C6 did not enhance the production of monokines, TNF-alpha, and IL-1 alpha and -beta. The F(ab')2 fragment of 1C6 also activated monocytes, whereas 5B2 and the Fab fragment of 1C6 could not. To further examine monocyte activation, these cells were treated with phosphatidylinositol-specific phospholipase C. Increased glucose consumption and enhanced phagocytic activity by 1C6 were considerably reduced in monocytes treated with phosphatidylinositol-specific phospholipase C. In addition, we found that 1C6 stimulated the generation of inositol trisphosphate. These results demonstrate that the signal transmitted via the DAF molecule is capable of stimulating monocytes.