Trehalose augments osteoprotegerin production in the FHs74Int human intestinal epithelial cell line

The disaccharide trehalose has been shown to inhibit both bone loss in ovariectomized mice and excessive osteoclastogenesis in lipopolysaccharide-injected mice. However, the mechanism of osteoclastogenesis inhibition by oral administration of trehalose is still unclear. We report here for the first time that a human intestinal epithelial cell line, FHs74Int, also produces osteoprotegerin (OPG) and that trehalose augments OPG production by this cell line. Thus, these results suggest that trehalose promotes the production of OPG by intestinal epithelial cells, which then acts on bone marrow cells, resulting in the suppression of osteoclastogenesis.     

Transport of fluorescein methotrexate by multidrug resistance-associated protein 3 in IEC-6 cells

The transport characteristics of fluorescein methotrexate (F-MTX) were studied by using the rat intestinal crypt cell line IEC-6. Enhanced accumulation of F-MTX at 4 degrees C suggests the existence of an active efflux system. MK-571, an inhibitor of the multidrug resistance-associated protein/ATP binding cassette C (MRP/ABCC) family, also enhanced the accumulation of F-MTX. Transcellular transport of F-MTX from the apical to the basolateral compartment was 2.5 times higher than the opposite direction. This vectorial transport was also reduced by MK-571, indicating the presence of Mrp-type transporter(s) on the basolateral membrane. Mrp3 mRNA was readily detectable, and the protein was localized on the basolateral membrane. Uptake of FMTX into membrane vesicles from IEC-6 cells and Spodoptera frugiperda-9 cells expressing rat Mrp3 were both ATP dependent and saturable as a function of the F-MTX concentration. Similar Km values (11.0 +/- 1.8 and 4.5 +/- 1.1 microM) and inhibition profiles by MK-571, estradiol-17beta-d-glucuronide, and taurocholate for the ATP-dependent transport of F-MTX into these vesicles were obtained. These findings suggest that the efflux of F-MTX is mediated by Mrp3 on the basolateral membrane of IEC-6 cells.     

Electron transfer of site-specifically cross-linked complexes between ferredoxin and ferredoxin-NADP(+) reductase

Ferredoxin (Fd) and Fd-NADP(+) reductase (FNR) are redox partners responsible for the conversion between NADP(+) and NADPH in the plastids of photosynthetic organisms. Introduction of specific disulfide bonds between Fd and FNR by engineering cysteines into the two proteins resulted in 13 different Fd-FNR cross-linked complexes displaying a broad range of activity to catalyze the NADPH-dependent cytochrome c reduction. This variability in activity was thought to be mainly due to different levels of intramolecular electron transfer activity between the FNR and Fd domains. Stopped-flow analysis revealed such differences in the rate of electron transfer from the FNR to Fd domains in some of the cross-linked complexes. A group of the cross-linked complexes with high cytochrome c reduction activity comparable to dissociable wild-type Fd/FNR was shown to assume a similar Fd-FNR interaction mode as in the native Fd:FNR complex by analyses of NMR chemical shift perturbation and absorption spectroscopy. However, the intermolecular electron transfer of these cross-linked complexes with two Fd-binding proteins, nitrite reductase and photosystem I, was largely inhibited, most probably due to steric hindrance by the FNR moiety linked near the redox center of the Fd domain. In contrast, another group of the cross-linked complexes with low cytochrome c reduction activity tends to mediate higher intermolecular electron transfer activity. Therefore, reciprocal relationship of intramolecular and intermolecular electron transfer abilities was conferred by the linkage of Fd and FNR, which may explain the physiological significance of the separate forms of Fd and FNR in chloroplasts.     

Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis in Japanese population

Autoantibodies to myeloperoxidase (MPO) are a subset of anti-neutrophil cytoplasmic antibody (ANCA, MPO-ANCA) detected in the sera of some patients with primary systemic vasculitis. The titer of MPO-ANCA does not always reflect disease activity and this inconsistency may be attributable to differences in epitopic specificity by MPO-ANCA among various patients with vasculitis. Epitope analysis may also explain the occurrence of MPO-ANCA in different vasculitic syndromes. We screened the sera of 148 MPO-ANCA positive patients from six vasculitic syndromes: rapidly progressive gromerulonephritis (RPGN), microscopic polyangiitis (MPA), idiopathic crescentic glomerulonephritis (I-CrGN), classic polyangiitis nodosa (cPAN), Churg-Strauss syndrome (CSS), Kawasaki disease (KD); and from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The sera were collected by the Intractable Vasculitis Research Project Group in Japan. No serum showed epitopes La and Lb of light chain of MPO, and sera with 68.6% of patients showed a positive reaction to one or more epitopes in heavy chain of MPO. Analysis of binding level showed that RPGN, I-CrGN and MPA sera mainly reacted to the Ha epitope at the N-termimus of the MPO heavy chain, CSS sera reacted to Ha and the Hf epitope close to the C-terminus of the MPO heavy chain, KD reacted mainly to Hf, while SLE and RA sera reacted to all epitopes. These results suggest that MPO-ANCA recognizing specific regions of the N-terminus of the MPO H-chain confer an increased risk of vasculitis RPGN, I-CrGN, MPA and CSS. Furthermore, the epitopic specificity of MPO-ANCA differentiates vasculitic from non-vasculitic syndromes associated with MPO-ANCA positivity and differentiates in the cirtain type of vasculitis from various vasculitic syndromes. In particular, vasculitic syndromes associated with kidney involvement had similar epitopic reactivity which suggests that this pattern confers an increased risk of vasculitis.     

Escherichia coli phage-shock protein A (PspA) binds to membrane phospholipids and repairs proton leakage of the damaged membranes

Escherichia coli phage-shock protein A (PspA), a 25.3 kDa peripheral membrane protein, is induced under the membrane stress conditions and is assumed to help maintain membrane potential. Here, we report that purified PspA, existing as a large oligomer, is really able to suppress proton leakage of the membranes. This was demonstrated for membrane vesicles prepared from the PspA-lacking E. coli mutants, and for membrane vesicles damaged by ethanol and Triton X-100 prepared from the mutant and the wild-type cells. PspA also suppressed proton leakage of damaged liposomes made from E. coli total lipids. Furthermore, we found that PspA bound preferentially to liposomes containing phosphatidylserine and phosphatidylglycerol. All these effects were not observed for monomer PspA that was prepared by refolding of urea-denatured PspA. These results indicate that oligomers of PspA bind to membrane phospholipids and suppress proton leakage.     

Chemical identification and ethological function of soldier-specific secretion in Japanese subterranean termite Reticulitermes speratus (Rhinotermitidae)

We identified the soldier-specific compounds in the Japanese subterranean termite, Reticulitermes speratus, to clarify their ethological roles. Silica gel column chromatography separated one major soldier-specific compound in the hexane fraction accounting for 70-80% of the total amount of the fraction, while cuticular hydrocarbons constituted the rest. We identified the compound as β-selinene by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Comparative GC analyses of the major exocrine glands detected the compound in the soldier's frontal gland. Both soldiers and workers made aggregation to the hexane fraction, as well as to the crushed heads and head extract of the soldiers. They did not aggregate to cuticular hydrocarbons, making it likely that β-selinene was the aggregation pheromone in this species. The opportunistic predator of this termite, Lasius japonicus, was also attracted to the compounds. The ant workers, therefore, would use the termite aggregation pheromone as a kairomone for hunting them.     

Amino acid sequence of a ferredoxin from Rhodymenia palmata, a red alga in the florideophyceae

The amino acid sequence of a [2Fe-2S] ferredoxin from a red alga, Rhodymenia palmata in the family Florideophyceae, was determined by conventional methods. The ferredoxin is composed of 97 amino acid residues having five cysteines, but lacking methionine and tryptophan. It possesses a number of structural features of particular interest. The amino acid sequence is compared with those previously determined for ferredoxins from two red algae in the family Bangiophyceae. Conclusions from a comparison of the structures, by noting features such as the presence of gaps in the sequences and by constructing a phylogenetic tree, were consistent with the proposed taxonomic relationship among these algae.     

Low temperature protects mammalian cells from apoptosis initiated by various stimuli in vitro

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we examined the effects of low temperature (32 degrees C) on cells exposed to a variety of stress in vitro. We found that 32 degrees C suppressed induction of apoptosis by cytotoxic stimuli such as adriamycin, etoposide, thapsigargin, NaCl, H(2)O(2), and anti-Fas antibody. In adriamycin-treated BALB/3T3 cells, the down-shift in temperature from 37 degrees C to 32 degrees C increased the Bcl-xL protein level and decreased the mRNA level of Puma and mitochondrial translocation of Bax, suppressing caspase-9-mediated apoptosis. Furthermore, the protein level and stability of p53 were decreased, and its nuclear export was increased concomitant with Mdm2 mRNA upregulation. The low temperature effect was not observed in p53(-/-)/Mdm2(-/-) mouse embryonic fibroblasts, suggesting that the effect is mediated by suppression of the p53 pathway. In contrast, while thapsigargin-induced apoptosis was suppressed by the low temperature, no effect on the p53 protein level was observed. Furthermore, the survival rate of p53(-/-)/Mdm2(-/-) cells exposed to thapsigargin was increased when cultured at 32 degrees C compared with 37 degrees C. In conclusion, mild hypothermia protects cells from a variety of stress by p53-dependent and p53-independent mechanisms.     

Role of 14-3-3gamma in FE65-dependent gene transactivation mediated by the amyloid beta-protein precursor cytoplasmic fragment

The amyloid beta-protein precursor intracellular domain fragment (AICD) is generated from amyloid beta-protein precursor by consecutive cleavages. AICD is thought to activate FE65-dependent gene expression, but the molecular mechanism remains under consideration. We found that dimeric 14-3-3gamma bound both AICD and FE65 simultaneously, and this binding facilitated FE65-dependent gene transactivation by enhancing the association of AICD with FE65. 14-3-3gamma bound to the 667VTPEER672 motif of AICD and, most interestingly, the phosphorylation of AICD at Thr-668 in this motif inhibited the interaction with 14-3-3gamma and blocked gene transactivation. 14-3-3gamma required a sequence between the WW domain and the first phosphotyrosine interaction domain of FE65 for association with FE65. Deletion of this region blocked 14-3-3gamma binding to FE65 and suppressed AICD-mediated FE65-dependent gene transactivation, although the deletion mutant FE65 was still able to bind Tip60, a histone acetyltransferase that forms a complex with FE65 in the nucleus. Taken together, these data demonstrate that 14-3-3gamma facilitates FE65-dependent gene transactivation by forming a complex containing AICD and FE65, and phosphorylation of AICD down-regulates FE65-dependent gene transactivation through the dissociation of 14-3-3gamma and/or FE65 from AICD. Our findings suggest that multiple interactions of AICD with FE65 and 14-3-3gamma modulate FE65-dependent gene transactivation.