Externally applied pressure activates pancreatic stellate cells through the generation of intracellular reactive oxygen species

Local tissue pressure is higher in chronic pancreatitis than in the normal pancreas. We reported recently that pressure application induces synthesis of extracellular matrix (ECM) and cytokines in pancreatic stellate cells (PSCs) and that epigallocatechin gallate (EGCG), a potent antioxidant, inhibits the transformation of PSCs from quiescent to activated phenotype and ethanol-induced synthesis of ECM and cytokines in PSCs. These results suggest that oxidative stress and reactive oxygen species (ROS) are important in PSC activation. The aim of this study was to clarify the effects of ROS on activation and functions of pressure-stimulated PSCs. We used freshly isolated rat PSCs and culture-activated PSCs. Pressure was applied on rat cultured PSCs by adding compressed helium gas into a pressure-loading apparatus. PSCs were cultured with or without antioxidants (EGCG and N-acetyl cysteine) under normal or elevated pressure. Externally applied high pressure (80 mmHg) resulted in a gradual decrease of superoxide dismutase activity in PSCs and increased intracellular ROS generation as early as 30 s, reaching a peak level at 1 h. Antioxidants significantly inhibited ROS generation. Pressure increased the expression levels of alpha-smooth muscle actin, alpha(1)(I)-procollagen, and TGF-beta1 in PSCs. EGCG suppressed these alterations, abolished pressure-induced phosphorylation of p38 MAPK, and suppressed pressure-induced PSC transformation to activated phenotype. Our results indicated that ROS is a key player in pressure-induced PSC activation and ECM synthesis. Antioxidants could be potentially effective against the development of pancreatic fibrosis in patients with chronic pancreatitis.     

Mechanisms for asporin function and regulation in articular cartilage

Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-beta (TGF-beta), a key cytokine in OA pathogenesis, and inhibits TGF-beta-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-beta1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-beta1; in turn, TGF-beta1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-beta1 form a regulatory feedback loop. Asporin inhibits TGF-beta/Smad signaling upstream of TGF-beta type I receptor activation in vivo by co-localizing with TGF-beta1 on the cell surface and blocking its interaction with the TGF-beta type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA.     

Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats

An herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51+/-0.22 mg/dl (mean+/-SE) from 0.28+/-0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86+/-0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8+/-13.0% and 220.8+/-39.3% (mean+/-SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity.     

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.     

A recurrent mutation in type II collagen gene causes Legg-Calvé-Perthes disease in a Japanese family

Legg-Calvé-Perthes disease (LCPD) is a common childhood hip disorder characterized by sequential stages of involvement of the capital femoral epiphyses, including subchondral fracture, fragmentation, re-ossification and healing with residual deformity. Most cases are sporadic, but familial cases have been described, with some families having multiple affected members. Genetic factors have been implicated in the etiology of LCPD, but the causal gene has not been identified. We have located a missense mutation (p.G1170S) in the type II collagen gene (COL2A1) in a Japanese family with an autosomal dominant hip disorder manifesting as LCPD and showing considerable intra-familial phenotypic variation. This is the first report of a mutation in hereditary LCPD. COL2A1 mutations may be more common in LCPD patients than currently thought, particularly in familial and/or bilateral cases.     

Synthesis and antibacterial activities of new quinolone derivatives utilizing 1-azabicyclo[1.1.0]butane

The ring-opening reactions of 1-azabicyclo[1.1.0]butane 3 with thiols 6a-f gave 3-sulfenylazetidine derivatives 7a-f in 50-92% yields. Treatment of 3 with aromatic amines 11a-e and dibenzylamine 11f in the presence of Mg(ClO(4))(2) afforded the corresponding 3-aminoazetidine derivatives 12a-f in 24-53% yields. These azetidine derivatives were introduced into the C7 position of a quinolone nucleus 8 to afford the corresponding fluoroquinolones 9a-f and 13a-f in 21-83% yields. Some of them exhibited superior antibacterial activity against quinolone-susceptible MRSA in comparison with clinically used fluoroquinolones, such as levofloxacin, ciprofloxacin, and gatifloxacin.     

Synthesis and matrix metalloproteinase (MMP)-12 inhibitory activity of ageladine A and its analogs

Ageladine A (1) and its analogs 2-10 were expeditiously synthesized by featuring the biosynthetic route proposed for 1 (for 1-10) and by employing 2-(N-t-butoxycarbonylamino)imidazol-4-carbaldehyde as the starting material (for 1-8). From MMP-12 inhibitory activity assay, it appeared evident that the two bromine atoms and the three NH groups (1-NH, 14-NH, and 15-NH2) were indispensable for 1 to exhibit excellent activity.     

Mice lacking alpha1,3-fucosyltransferase IX demonstrate disappearance of Lewis x structure in brain and increased anxiety-like behaviors

The 3-fucosyl-N-acetyllactosamine [Lewis x (Le(x)), CD15, SSEA-1] carbohydrate structure is expressed on several glycolipids, glycoproteins, and proteoglycans of the nervous system and has been implicated in cell-cell recognition, neurite outgrowth, and neuronal migration during development. To characterize the functional role of Le(x) carbohydrate structure in vivo, we have generated mutant mice that lack alpha1,3-fucosyltransferase IX (Fut9(-/-)). Fut9(-/-) mice were unable to synthesize the Le(x) structure carried on glycoproteins and glycolipids in embryonic and adult brain. However, no obvious pathological differences between wild-type and Fut9(-/-) mice were found in brain. In behavioral tests, Fut9(-/-) mice exhibited increased anxiety-like responses in dark-light preference and in elevated plus maze tests. Immunohistochemical analysis showed that the number of calbindin-positive neurons was decreased in the basolateral amygdala in Fut9(-/-) mice. These observations indicated that the carbohydrates synthesized by Fut9 play critical roles in functional regulations of interneurons in the amygdalar subdivisions and suggested a role for the Le(x) structure in some aspects of emotional behavior in mice.     

Spirodiketopiperazine-based CCR5 antagonists: Lead optimization from biologically active metabolite

Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from beta-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1alpha to human CCR5, compared with the non-hydroxylated derivatives and the other isomers.