Mouse mast cell protease-1 cleaves angiotensin I to form angiotensin II

The ability to convert angiotensin (Ang) I to Ang II was compared between human alpha-chymase and two mouse beta-chymases, mouse mast cell protease (mMCP)-1 and mMCP-4. Human chymase hydrolyzed Ang I to produce Ang II without further degradation. mMCP-1 similarly generated Ang II from Ang I in a time-dependent manner and the formation of the fragment other than Ang II was marginal. In contrast, mMCP-4 hydrolyzed Ang I at two sites, Tyr(4)-Ile(5) and Phe(8)-His(9), with Ang II formation being tentative. Consistently, mMCP-4 but not human chymase hydrolyzed Ang II and mMCP-1 showed little hydrolytic activity against Ang II. These data suggest that not only human chymase but also mMCP-1 might possess a physiological role in Ang II formation. Our findings also imply that the Ang-converting activity of chymase may not be related to the categorization of chymase into alpha- or beta-type based on their primary structure.     

Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function

Soy isoflavones have been extensively studied because of their possible benefits to human health. Genistein, the major isoflavone aglycone, has received most attention; however, it undergoes extensive metabolism (e.g. conjugation with sulfuric acid) in the gut and liver, which may affect its biological properties. This study investigated the antioxidant activity and free radical-scavenging properties of genistein, genistein-4'-sulfate and genistein-4'-7-disulfate as well as their effect on platelet aggregation and monocyte and endothelial function. Electron spin resonance spectroscopy (ESR) and spin trapping data and other standard antioxidant assays indicated that genistein is a relatively weak antioxidant compared to quercetin and that its sulfated metabolites are even less effective. Furthermore, genistein-4'-sulfate was less potent than genistein, and genistein-4'-7-disulfate even less potent, at inhibiting collagen-induced platelet aggregation, nitric oxide (NO) production by macrophages, and secretion by primary human endothelial cells of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). The current data suggest that sulfation of genistein, with the associated loss of hydroxyl groups, decreases its antioxidant activity and its effect on platelet aggregation, inflammation, cell adhesion and chemotaxis.     

Cooperation of syndecan-2 and syndecan-4 among cell surface heparan sulfate proteoglycans in the actin cytoskeletal organization of Lewis lung carcinoma cells

Syndecan-2 cooperates with integrin alpha 5 beta 1 in cell adhesion to a fibronectin substratum and regulates actin cytoskeletal organization in an expression level-dependent manner; Lewis lung carcinoma-derived P29 cells with high expression form stress fibers, whereas the same tumor-derived low expressers, LM66-H11 cells, form cortex actin [Munesue, S., Kusano, Y., Oguri, K., Itano, N., Yoshitomi, Y., Nakanishi, H., Yamashina, I., and Okayama, M. (2002) BIOCHEM: J. 363, 201-209]. In this study we examined the participation of other cell surface heparan sulfate proteoglycans in this signaling. The two clones expressed syndecan-1, -2 and -4, and glypican-1 at similar levels except for syndecan-2. Treatment of cells with phosphatidylinositol-specific phospholipase C or immobilized anti-syndecan-1 antibodies demonstrated that neither glypican-1 nor syndecan-1 was involved in this signaling, indicating that individual cell surface heparan sulfate proteoglycans have functional specificity. Stimulation with immobilized anti-syndecan-2 or -4 antibodies induced stress fiber formation in P29 cells but not in LM66-H11 cells, despite the similar levels of syndecan-4 expression, suggesting that stress fiber formation required a threshold expression level of syndecan-2 acting downstream of syndecan-4. This was confirmed by cells in which syndecan-2 expression was artificially suppressed by antisense mRNA oligonucleotide treatment or elevated by cDNA transfection. This is the first report demonstrating that syndecan-2 and -4 cooperate in situ in actin cytoskeletal organization.     

Crisscross CTL induction by SYT-SSX junction peptide and its HLA-A*2402 anchor substitute

To investigate the effects of anchor substitutions in SYT-SSX junction peptide, an HLA-A24 anchor residue (position 9) of the SYT-SSX B peptide (GYDQIMPKK) was substituted to more favorable residues according to the HLA-A24-binding motif. Among four substitutes constructed, a substitute with isoleucine (termed K9I peptide) most apparently enhanced the affinity for HLA-A24 molecule. Subsequent in vitro CTL induction analysis using PBMCs of 15 HLA-A24(+) synovial sarcoma patients revealed that the original B peptide allowed to induce synovial sarcoma-specific CTLs from 7 patients (47%), whereas such CTLs were inducible from 12 patients (80%) with K9I peptide. Moreover, the extent of cytotoxicity against HLA-A24(+) synovial sarcoma cell lines was higher in K9I peptide-induced CTLs than B peptide-induced CTLs. Influence of anchor substitution on peptide/TCR interaction was evaluated by cytotoxicity assays against autologous cells and tetramer analysis. CTLs induced from a synovial sarcoma patient using K9I peptide did not lyse autologous PHA blasts or EBV-infected B cells. In vitro stimulations of PBMCs from 5 HLA-A24(+) synovial sarcoma patients with K9I peptide increased the frequency of T cells reacting with both HLA-A24/K9I peptide tetramer and HLA-A24/B peptide tetramer. In contrast, the frequency of T cells reacting with HLA/HIV-derived peptide tetramer remained low. These findings support the validity in design of anchor residue substitution in SYT-SSX fusion gene-derived peptide, and provide a potential clue to the current stagnation in vaccination trials of fusion gene-derived natural junction peptides.     

Change from a hard to soft diet alters the expression of insulin-like growth factors,their receptors,and binding proteins in association with atrophy in adult mouse masseter muscle

To study the role of insulin-like growth factors (IGFs) in the atrophy of mouse masseter muscle in response to a change from a hard to a soft diet, we analyzed the amounts of mRNA and the immunolocalization for IGF-I, IGF-II, their receptors (IGFRs), and binding proteins (IGFBPs). Sixteen male ICR mice were fed a hard diet after weaning; they were divided into two groups at 6 months of age and fed a hard or a soft diet for 1 week. The soft diet treatment decreased masseter weight by 19% (P<0.01) and the minimal diameter of masseter myofibers by 19% (P<0.01), verifying that a soft diet led to atrophy of mouse masseter muscle. The soft diet treatment induced a 30% reduction in the amount of IGF-I mRNA (P<0.05) in preparations of whole masseter tissues. Immunohistochemical findings suggested that a reduction in the expression of IGF-I protein took place in the neural tissues, not in the masseter myofibers. The soft diet treatment induced a 56% decrease in IGF-II mRNA (P<0.05), a 21% increase in IGFR2 mRNA (P<0.01), and a 38% decrease in IGFBP5 mRNA (P<0.01). Immunohistochemical results suggested that these changes at the protein level occurred in the masseter myofibers. No significant or marked difference in the mRNA amount or immunostaining pattern for IGFR1, IGFBP3, IGFBP4, or IGFBP6 was found between the soft and hard diet groups. No IGFBP1 or IGFBP2 mRNA was detected. Thus, IGF-I, IGF-II, IGFR2, and IGFBP5 seem to play a role in the atrophy of mouse masseter muscle in response to the change from a hard to a soft diet in an autocrine and/or paracrine manner.Part of the present study was supported by a grant-in-aid for funding scientific research (no. 13671955), Bio-ventures and High-Technology Research Center, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan     

Dietary resistant starch alters the characteristics of colonic mucosa and exerts a protective effect on trinitrobenzene sulfonic acid-induced colitis in rats

The protective effect of a dietary high-amylose cornstarch (HAS) against trinitrobenzene sulfonic acid (TNBS)-induced colitis was examined in rats. Rats were fed a HAS-free basal diet or, a 15% or 30% HAS supplemented diet for 10 d, and then received intracolonic TNBS to induce colitis and fed the respective diets for a further 8 d. HAS ingestion significantly protected colonic injuries as evidenced by lower colonic myeloperoxidase activity. Rats fed the HAS diet showed greater cecal short-chain fatty acid (SCFA) production than those fed the basal diet. Further, just before TNBS administration, HAS ingestion dose-dependently increased fecal and cecal mucin contents, and protein and nucleic acid contents in the colonic mucosa. HAS ingestion also reduced colonic permeability. The protective effect of HAS ingestion on TNBS-induced colitis is perhaps exerted through alterations in colonic mucosa, possibly due to cecal SCFA production.     

Characterization of 298 ESTs from porcine back fat tissue and their assignment to the SSRH radiation hybrid map

Following several criteria, we collected, clustered, and functionally categorized 653 expressed sequence tags (ESTs) of 5 ends from porcine back fat libraries from the >15,000 porcine ESTs collected to date. By searching the LocusLink and Mapviewer database, we knew the positions of these 653 ESTs on human chromosomes (HSAs). Sus scrofa radiation hybrid (SSRH) mapping revealed that 298 porcine EST clusters out of 653 were localized near microsatellite (MS) markers. Among these EST clusters, we could assign 182 to their porcine chromosomes (SSCs) on the SSRH map.