Binding of YC-1/BAY 41-2272 to soluble guanylate cyclase: A new perspective to the mechanism of activation

Soluble guanylate cyclase (sGC), a heterodimeric heme protein, catalyses the conversion of GTP in to cyclic GMP, which acts as a second messenger in cellular signaling. Nitric oxide activates this enzyme several hundred folds over its basal level. Carbon monoxide, along with some activator molecules like YC-1 and BAY, also synergistically activate sGC. Mechanism of this synergistic activation is a matter of debate. Here we review the existing literature to identify the possible binding site for YC-1 and BAY on bovine lung sGC and its mechanism of activation. These two exogenous compounds bind sGC on α subunit inside a pocket and thus exert allosteric effect via subunit interface, which is relayed to the catalytic site. We used docking studies to further validate this hypothesis. We propose that the binding of YC-1/BAY inside the sensory domain of the α subunit modulates the interactions on the subunit interface resulting in rearrangements in the catalytic site into active conformation and this partly induces the cleavage of Fe-His bond.     

Design and synthesis of 6-fluoro-2-naphthyl derivatives as novel CCR3 antagonists with reduced CYP2D6 inhibition

In our previous study on discovering novel types of CCR3 antagonists, we found a fluoronaphthalene derivative (1) that exhibited potent CCR3 inhibitory activity with an IC(50) value of 20 nM. However, compound 1 also inhibited human cytochrome P450 2D6 (CYP2D6) with an IC(50) value of 400 nM. In order to reduce its CYP2D6 inhibitory activity, we performed further systematic structural modifications on 1. In particular, we focused on reducing the number of lipophilic moieties in the biphenyl part of 1, using ClogD(7.4) values as the reference index of lipophilicity. This research led to the identification of N-{(3-exo)-8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3-yl}-3-(piperidin-1-ylcarbonyl)isonicotinamide 1-oxide (30) which showed comparable CCR3 inhibitory activity (IC(50)=23 nM) with much reduced CYP2D6 inhibitory activity (IC(50)=29,000 nM) compared with 1.     

A role of kinase inactive ZAP-70 in altered peptide ligand stimulated T cell activation

T cell activation signals induced by altered peptide ligands (APLs) are different from those induced by the original agonistic peptide. The characteristics of the former are partial phosphorylation of TCR-zeta and no tyrosine-phosphorylation of zeta-associated protein-70 (ZAP-70). To analyze further those signaling pathways, we introduced a dominant negative (DN) form of ZAP-70 into a human CD4(+) T cell clone in which fully and partially agonistic peptide ligands have been well characterized. We found that some over-expressed partially agonistic ligands (OPALs) induced T cell responses without tyrosine-phosphorylation and kinase activation of ZAP-70. However, those responses were inhibited in T cells expressing DN ZAP-70, which could associate with partially phosphorylated TCR-zeta. In OPAL-stimulated T cells, PLC-gamma1 was phosphorylated and it was suppressed by DN ZAP-70 expression, suggesting that the ZAP-70-TCR-zeta association mediates the activation of PLC-gamma1 leading to T cell responses even in the absence of kinase activation of ZAP-70.     

Molecular cloning and characterization of novel ficolins from<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins and are thought to play crucial roles in host defense through opsonization and complement activation. To elucidate the evolution of ficolins and the primordial complement lectin pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its lectin activity.     

Studies on the active sites ofBacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis

Summary Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites ofBacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced inBacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G towardp-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP andp-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities.     

Expression of carboxylesterase and lipase genes in rat liver cell-types

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.     

rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.     

Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers

This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.