HSP20, low-molecular-weight heat shock-related protein,acts extracellularly as a regulator of platelet functions: a novel defense mechanism

We previously showed that a dissociated form of a low-molecular-weight heat shock-related protein 20 (HSP20) but not an aggregated form of HSP20 suppresses platelet aggregation. In the present study, we investigated the behavior of HSP20 in response to endothelial injury and the possible mechanism of HSP20 in platelet functions. The levels of HSP20 in vessel wall after endothelial injury were markedly reduced. This observation was supported by the results of Western blotting analysis and immunohistochemical analysis. Additionally, the plasma levels of HSP20 in cardiomyopathic hamsters were markedly elevated. Centrifugation on sucrose density gradients allowed detection mainly of the dissociated form of plasma HSP20 in these hamsters. Human platelets showed specific binding sites for HSP20. Moreover, HSP20 markedly reduced thrombin-induced phosphoinositide hydrolysis by phospholipase C in human platelets. Taken together, our results strongly suggest that HSP20, which immediately responds to pathological events, acts extracellularly as a regulator of platelet functions.     

Coevolution of the S-locus genes SRK,SLG and SP11/SCR in Brassica oleracea and B. rapa

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (pi(S) and pi(N)) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of pi(N):pi(S) for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.     

Sphingomyelinase amplifies BMP-4-induced osteocalcin synthesis in osteoblasts: role of ceramide

We previously reported that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblast-like MC3T3-E1 cells and that mitogen-activated protein (MAP) kinases are involved in bone morphogenetic protein (BMP)-4-stimulated osteocalcin synthesis in these cells. In the present study, we investigated whether sphingomyelinase affects BMP-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. Sphingomyelinase significantly enhanced the BMP-4-stimulated osteocalcin synthesis. Among sphingomyelin metabolites, C(2)-ceramide enhanced the BMP-4-stimulated osteocalcin synthesis while sphingosine and sphingosine 1-phosphate had little effect on the synthesis. D-erythro-MAPP, an inhibitor of ceramidase, amplified the sphingomyelinase-effect on the osteocalcin synthesis. C(2)-ceramide suppressed the BMP-4-induced phosphorylation of p44/p42 MAP kinase, while having little effect on the phosphorylation of Smad1 and p38 MAP kinase. Taken together, our results strongly suggest that extracellular sphingomyelinase enhances the BMP-stimulated osteocalcin synthesis via ceramide in osteoblasts and that the effect of ceramide is exerted at a point upstream from p44/p42 MAP kinase.     

Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata

CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.     

Effects of stereochemistry of sugars on protein stabilities

We investigated thermal stabilities of four proteins in the presence of four kinds of sugars to analyze the mechanism of stabilization of proteins by additives. These proteins were stabilized by the addition of sugars, and the degree of stabilization correlated to the partial molar isentropic compressibility of the sugar.     

Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability

The association of the p27(Kip1) protein with cyclin and cyclin-dependent kinase complexes inhibits their kinase activities and contributes to the control of cell proliferation. The p27(Kip1) protein has now been shown to be phosphorylated in vivo, and this phosphorylation reduces the electrophoretic mobility of the protein. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser(10) accounted for approximately 70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was approximately 25- and 75-fold greater than that at Ser(178) and Thr(187), respectively. The phosphorylation of p27(Kip1) was markedly reduced when the positions of Ser(10) and Pro(11) were reversed, suggesting that a proline-directed kinase is responsible for the phosphorylation of Ser(10). The extent of Ser(10) phosphorylation was markedly increased in cells in the G(0)-G(1) phase of the cell cycle compared with that apparent for cells in S or M phase. The p27(Kip1) protein phosphorylated at Ser(10) was significantly more stable than the unphosphorylated form. Furthermore, a mutant p27(Kip1) in which Ser(10) was replaced with glutamic acid in order to mimic the effect of Ser(10) phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser(10) is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr(187), contributes to regulation of p27(Kip1) stability.     

Participation of testicular spermatids in development upon intracytoplasmic injection into eggs of the sawfly, Athalia rosae (Insecta, hymenoptera)

Fertilization by intracytoplasmic injection of mature sperm into mature eggs has previously been achieved in the sawfly, Athalia rosae (Insecta, Hymenoptera). In the present study, we examined the potential of spermatids, premature male gametes, for participating in development. When round spermatids and elongating spermatids from pupal testes were injected into the anterior end of mature eggs, about 5% of the total injected eggs developed into chimeric embryos (independent participation in development of the egg and spermatid nuclei). Some of them developed further, hatched, and pupated, with 1-2% of the total injected eggs becoming haploid chimeric male adults in which both the egg-derived and injected spermatid-derived nuclei contributed to the germline. No fertilized embryos were obtained by these injections. Elongated spermatids (immature sperm) from newly eclosed adult male testes upon injection did produce fertilized embryos that developed into normal diploid females (about 7% of the total injected). These results indicate that insect spermatids (round and elongating) have the potential to participate in development, but only independently of the egg nucleus. J. Exp. Zool. 286:181-192, 2000.     

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.     

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.