Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide

The novel chemical structure and immunobiological activities of Prevotella intermedia ATCC 25611 lipid A were investigated. A lipopolysaccharide (LPS) preparation of P. intermedia was extracted using a phenol-chloroform-petroleum ether method, after which its purified lipid A was prepared by weak acid hydrolysis followed by chromatographic separations. The lipid A structure was determined by mass spectrometry and nuclear magnetic resonance to be a diglucosamine backbone with a phosphate at the 4-position of the non-reducing side sugar, as well as five fatty acids containing branched long chains. It was similar to that of Bacteroides fragilis and Porphyromonas gingivalis, except for the phosphorylation site. P. intermedia lipid A induced weaker cytokine production and NF-kappaB activation in murine cells via Toll-like receptor (TLR) 4 as compared to Escherichia coli synthetic lipid A (compound 506). Our results indicate that P. intermedia lipid A activates cells through a TLR4-dependent pathway similar to E. coli-type lipid A, even though these have structural differences.     

Adjustment function among antioxidant substances in acatalasemic mouse brain and its enhancement by low-dose X-ray irradiation

The catalase activities in blood and organs of the acatalasemic (C3H/AnLCsbCsb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCsaCsa) mouse. We conducted a study to examine changes in the activities of antioxidant enzymes, such as catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX), the total gluathione content, and the lipid peroxide level in the brain, which is more sensitive to oxidative stress than other organs, at 3, 6, or 24 hr following X-ray irradiation at doses of 0.25, 0.5, or 5.0 Gy to the acatalasemic and the normal mice. No significant change in the lipid peroxide level in the acatalasemic mouse brain was seen under non-irradiation conditions. However, the acatalasemic mouse brain was more damaged than the normal mouse brain by excessive oxygen stress, such as a high-dose (5.0 Gy) X-ray. On the other hand, we found that, unlike 5.0 Gy X-ray, a relatively low-dose (0.5 Gy) irradiation specifically increased the activities of both catalase and GPX in the acatalasemic mouse brain making the activities closer to those in the normal mouse brain. These findings may indicate that the free radical reaction induced by the lack of catalase is more properly neutralized by low dose irradiation.     

NADPH-diaphorase and calcium binding proteins in the trigeminal nucleus oralis of rats

We have examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the calcium binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV), in the trigeminal nucleus oralis (Sp5O). NADPH-d was detected by histochemistry while CBP was detected by immunohistochemistry. NADPH-d-positive neurons were distributed in the medial rostro-dorsomedial part (RDMsp5O) and dorsomedial part (DMsp5O) of Sp5O, and the rostrolateral part of the nucleus of the solitary tract (NTS). CB- and CR-positive neurons were mainly distributed in the dorsal part of Sp5O. In contrast, PV-positive neurons were mainly distributed in the ventral part of Sp5O. NADPH-d colocalized with CB (40%) and CR (20%) but not with PV in neurons of DMsp5O/ NTS. The mean cell sizes of neurons in RDMsp5O were larger than those in DMsp5O/NTS. PV-positive neurons were larger than NADPH-d-positive neurons. NADPH-d-, CB- and CR-positive neurons were generally small in RDMsp5O and DMsp5O/NTS. Few neurons were retrogradely labeled in RDMsp5O and DMsp5O from the thalamus, when numerous labeled neurons were in the principal and interpolar nuclei. These data indicate that NADPH-d histochemistry and CB, CR and PV immunohistochemistry identify a discrete cell population in Sp5O. Those labeled neurons in RDMsp5O and DMsp5O/NTS were considered to be involved in sensorimotor reflexive function of the intra-oral structures.     

Identification of a toxic mechanism of the plasticizers,phtahlic acid esters,which are putative endocrine disrupters: time-dependent increase in quinolinic acid and its metabolites in rats fed di(2-ethylhexyl)phthalate

We have reported that the administration of di(2-ethylhexyl)phthalate (DEHP) increased the formations of quinolinic acid (QA) and its lower metabolites on the tryptophan-niacin pathway. To discover the mechanism involved in disruption of the tryptophan-niacin pathway by DEHP, we assessed the daily urinary excretion of QA and its lower metabolites, and enzyme activities on the tryptophan-niacin pathway. Rats were fed with a niacin-free, 20% casein diet or the same diet supplemented with 0.1% DEHP or 0.043% phthalic acid and 0.067% 2-ethylhexanol added for 21 days. Feeding of DEHP increased the urinary excretions of QA and its lower metabolites in a time-dependent manner, and the increase of these excretions reached a peak at 11 days, but feeding of phthalic acid and 2-ethylhexanol had no effect. Feeding of DEHP, however, did not affect any enzyme activity including alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), affecting the formation of QA, on the tryptophan-niacin pathway.