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.     

In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice

The skin micronucleus test combined with irradiation due to a sunlight simulator having a spectrum almost identical to solar irradiation was used as a novel in vivo testing method for detecting or comparing the photochemical chromosome damage of quinolone antibacterial agents (quinolones). Eight-week-old male SKH1 hairless mice were orally administered once lomefloxacin (LFLX), a strong in vitro photochemical clastogen, at 25 or 50 mg/kg, followed by light irradiation at 7.9-9.4J/cm2 of ultraviolet A (UVA). Animals were killed on Days 2, 3, 4, 5 or 8 (the dosing day was designated as Day 1), and the incidence of micronucleus in the epidermis was determined. As results, LFLX at either dose caused significant increases in the micronucleus frequency, which peaked on Day 4. These changes tended to return to the control level on Day 8. Then, the micronucleus induction potential of the quinolone derivatives levofloxacin (LVFX) and clinafloxacin (CLFX) at 10, 20 or 40 mg/kg was assessed on Day 4 under the same experimental conditions as for LFLX. Although LVFX was negative even at 40 mg/kg, CFLX dose-dependently induced significant increases in micronucleus frequency at all doses. The correlation of magnitude among the three quinolones in the skin micronucleus test with light irradiation was similar to that in our previous in vitro photochemical clastogenicity study. No significant increase in micronucleus frequency was observed in any of three quinolones employed without light irradiation. In conclusion, the experimental method presented here would be a useful tool for detecting in vivo photochemical chromosome damage and for research on photochemical carcinogenesis of chemicals.     

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.     

Roles for the two-hybrid system in exploration of the yeast protein interactome

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.     

A method for estimating torque-vector directions of shoulder muscles using surface EMGs

In this study, a new method is proposed to estimate the torque-vector directions of each shoulder muscle. The method is based on a multiple regression model that reconstructs shoulder torque, which is calculated from the hand force and posture, from the surface EMG of many muscles recorded simultaneously. The torque-vector directions of eleven shoulder muscles of four subjects were obtained at up to 30 different arm postures with this method. The mean confidence interval (p < 0.05) of the estimated torque-vector direction of each subject was 7.7-10.6 degrees. The correlation coefficient between the measured shoulder torque and reconstructed shoulder torque was between 0.76-0.84. The results for majority of the muscles were in accordance with previous studies, and reasonable from the viewpoint of anatomy. The torque-vector directions of a muscle, which are estimated with this method, have more of a functional meaning than a pure anatomical or mechanical one. These indicate the direction of the shoulder torque accompanying the muscle activation for a normal shoulder action that involves the cooperative contraction of many muscles.