Green tea polyphenol suppresses tumor invasion and angiogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-induced bladder cancer

Background: Green tea polyphenol (GTP) suppresses malignancy in bladder cancer cell lines. However, the detail of its anti-carcinogenic effect in vivo is not fully understood. This study investigated the effect of GTP on bladder tumor size and angiogenesis in mice given N-butyl-(-4-hydroxybutyl) nitrosamine (BBN), with and without GTP. Methods: Eight-week-old female C3H/He mice were treated with and without 0.05% BBN solution for 14 or 24 weeks. In addition, they were also treated with and without 0.5% GTP solution for the same periods. Histopathological diagnosis was established using hematoxylin and eosin staining, and microvessel density (MVD) was estimated by counting CD34- and von Willebrand factor-positive vessels in the tumor area. Results: At 14 weeks, cancer cells were detected in BBN and BBN + GTP mice [5/14 (35.7%) and 3/14 (21.4%), respectively, p = 0.678]. At 24 weeks, the incidence of cancer cells was also similar between the groups (BBN + GTP: 61.9% vs. BBN: 82.6%; p = 0.179). However, the frequency of invasive tumors in BBN + GTP mice was significantly lower (23.8%; p = 0.030) than in those given BBN alone (65.2%). Tumor volume and MVD of intratumoral and stromal region in the BBN + GTP group were also significantly lower than in BBN mice. Conclusion: The results showed that GTP had no anti-carcinogenic effect, but inhibited tumor growth and invasion in mice with established bladder cancer, at least in part through the regulation of angiogenesis. Our data suggest that GTP seems to suppress tumor development in bladder cancer.     

Probing phenylalanine environments in oligomeric structures with pentafluorophenylalanine and cyclohexylalanine

Stabilization of protein structures and protein-protein interactions are critical in the engineering of industrially useful enzymes and in the design of pharmaceutically valuable ligands. Hydrophobic interactions involving phenylalanine residues play crucial roles in protein stability and protein-protein/peptide interactions. To establish an effective method to explore the hydrophobic environments of phenylalanine residues, we present a strategy that uses pentafluorophenylalanine (F5Phe) and cyclohexylalanine (Cha). In this study, substitution of F5Phe or Cha for three Phe residues at positions 328, 338, and 341 in the tetramerization domain of the tumor suppressor protein p53 was performed. These residues are located at the interfaces of p53-p53 interactions and are important in the stabilization of the tetrameric structure. The stability of the p53 tetrameric structure did not change significantly when F5Phe-containing peptides at positions Phe328 or Phe338 were used. In contrast, the substitution of Cha for Phe341 in the hydrophobic core enhanced the stability of the tetrameric structure with a T(m) value of 100 degrees C. Phe328 and Phe338 interact with each other through pi-interactions, whereas Phe341 is buried in the surrounding alkyl side-chains of the hydrophobic core of the p53 tetramerization domain. Furthermore, high pressure-assisted denaturation analysis indicated improvement in the occupancy of the hydrophobic core. Considerable stabilization of the p53 tetramer was achieved by filling the identified cavity in the hydrophobic core of the p53 tetramer. The results indicate the status of the Phe residues, indicating that the "pair substitution" of Cha and F5Phe is highly suitable for probing the environments of Phe residues.     

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion–shallot monosomic additions and allotriploid-bunching onion single alien deletions

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion–shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST–SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261 cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.     

Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.     

Mucosal allergic sensitization to cockroach allergens is dependent on proteinase activity and proteinase-activated receptor-2 activation

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization.     

Binding and selectivity of the marine toxin neodysiherbaine A and its synthetic analogues to GluK1 and GluK2 kainate receptors

Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C₉ hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors.     

Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells.

Lysyl oxidase, an extracellular amine oxidase, controls the maturation of collagen and elastin. We examined the regulation of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium (RPE) cells in relation to the changes in subretinal fluid transport and phenotype of RPE cells. The level of the mRNA in cells grown on microporous membranes was markedly increased by application of hyperosmotic mannitol solution on the apical side (191% of control), implying that RPE cells express more lysyl oxidase in the condition which may cause the accumulation of subretinal fluid. Platelet-derived growth factor increased the mRNA level in subconfluent cells in culture (137% of control) and basic fibroblast growth factor decreased it (79% of control). In addition, exposure of cells to retinoic acid alone or in combination with dibutyryl cAMP for 22 days markedly decreased the level of lysyl oxidase mRNA (52 or 35% of control) while increasing the level of mRNA of N-acetylglucosaminidase (NAG), a marker enzyme for lysosomes (162 or 142% of control). Moreover, the level of lysyl oxidase mRNA in cells grown on microporous membranes was lower than that in cells grown on plastic dishes, while the level of NAG mRNA in the former cells was higher than that in the latter. Taken together, the expression of lysyl oxidase seemed to increase during proliferation of RPE cells and decrease toward differentiation. beta-Aminopropionitrile, an inhibitor of lysyl oxidase, significantly inhibited the contraction of collagen gels by fetal calf serum, suggesting that lysyl oxidase may be involved in pathogenesis caused by RPE cells.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.