Estimated average daily intake of antioxidants from typical vegetables consumed in Japan: a preliminary study

The hydrophilic antioxidant content of 23 vegetables commonly consumed in Japan was assessed by the hydrophilic oxygen radical absorbance capacity (H-ORAC) method to estimate the dietary intake of total antioxidants in Japan. The estimated average H-ORAC value for "typical vegetables" consumed in Japan was 594.3 μmol Trolox equivalent (TE)/100 g. Hence, 2080 μmol TE/d of hydrophilic antioxidants would be ingested when 350 g of vegetables a day are consumed.     

MUC5AC production is downregulated in NCI-H292 lung cancer cells cultured on type-IV collagen

Mucus overproduction is an important feature of bronchial asthma. MUC5AC mucin is a major component of mucus and is overproduced in patients with asthma. Although regulation of MUC5AC production has been well investigated, its regulation through the signals from extracellular matrix (ECM) is less clear. In this study, we investigated whether the signals from ECM regulate MUC5AC production in the human lung epithelial cell line NCI-H292. We found that MUC5AC production is downregulated in NCI-H292 cells cultured on type-IV collagen, a major component of ECM, but shows no obvious changes when cultured on type-I collagen or fibronectin. In contrast, MUC5AC production was upregulated on laminin and on reconstituted basement membrane (Matrigel), a complex of ECM components. Antibody-mediated inhibition of integrin β1-subunit, a major receptor involved in the adherence of cells to type-IV collagen, upregulated the MUC5AC production in NCI-H292 cells, and also in the cells cultured on type-IV collagen. Although the major signaling pathway from integrins is via Src kinase activation, treatment of cells with PP2, a Src kinase inhibitor, did not recover the downregulation of MUC5AC on type-IV collagen. In contrast, on Matrigel, the inhibition of integrin β1-subunit did not abolish the upregulation of MUC5AC production, but PP2 reduced the upregulation. These results suggest that ECM and an integrin/Src pathway play an important role in the regulation of MUC5AC production in the cell line NCI-H292. The production of MUC5AC is downregulated on type-IV collagen through a Src-independent pathway. In contrast, MUC5AC is upregulated on Matrigel through a Src-dependent pathway in NCI-H292 cells.     

Detachment-associated changes in lipid rafts of senescent human fibroblasts

We characterized the effects of in vitro cellular aging on constituents of lipid rafts in human diploid fibroblasts, TIG-1. Cholesterol recovery from lipid rafts of senescent cells was decreased by the detaching treatment, while the decrease was far less obvious in young cells. A probe that binds selectively to cholesterol in lipid rafts revealed that the amount of lipid rafts on the cell surface decreased in senescent cells upon cell detachment. Accompanying this change was the release of the raft-associated molecules caveolin and Fyn from lipid rafts upon cell detachment, suggesting a detachment-associated disorganization of lipid rafts in senescent cells. In addition, our observations showing differential sensitivities of lipid rafts from young and senescent cells to detaching treatment indicate a caution in how to detach cells. Particular attention needs to be paid to interpreting the results when lipid rafts are prepared from mechanically detached cells under detergent-free conditions.     

The internalization and metabolism of 3-deoxyglucosone in human umbilical vein endothelial cells

3-Deoxyglucosone (3-DG), a dicarbonyl compound produced by glycation, plays a role in the modification and cross-linking of long-lived proteins. We synthesized [3H]3-DG from [3H]glucose and developed an internalization assay system using HPLC to examine its cellular metabolism. When smooth muscle cells or human umbilical vein endothelial cells were incubated with [3H]3-DG, it was found that [3H]3-DG was internalized by cells in a time dependent manner. The rate of internalization was reduced when the cells were incubated at 4 degrees C or treated with phenylarsine oxide (PAO). By monitoring [3H]3-DG taken up by cells, it was confirmed that 3-DG is reduced to 3-deoxyfructose (3-DF) and that this reaction was inhibited by an aldo-keto reductase inhibitor (ARI). The presence of 3-DG led to an increase in reactive oxygen species levels in the cells and subsequent apoptosis, and the effect was enhanced by pretreatment with ARI. These results suggest that 3-DG is internalized by cells and reduced to 3-DF by aldo-keto reductases, and that the internalized 3-DG is responsible for the production of intracellular oxidative stress.     

Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities

The ubiquitin ligase Hul5 was recently identified as a component of the proteasome, a multisubunit protease that degrades ubiquitin-protein conjugates. We report here a proteasome-dependent conjugating activity of Hul5 that endows proteasomes with the capacity to extend ubiquitin chains. hul5 mutants show reduced degradation of multiple proteasome substrates in vivo, suggesting that the polyubiquitin signal that targets substrates to the proteasome can be productively amplified at the proteasome. However, the products of Hul5 conjugation are subject to disassembly by a proteasome-bound deubiquitinating enzyme, Ubp6. A hul5 null mutation suppresses a ubp6 null mutation, suggesting that a balance of chain-extending and chain-trimming activities is required for proper proteasome function. As the association of Hul5 with proteasomes was found to be strongly stabilized by Ubp6, these enzymes may be situated in proximity to one another. We propose that through dynamic remodeling of ubiquitin chains, proteasomes actively regulate substrate commitment to degradation.     

A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4

SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an alpha1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.     

Localization of sphingosine kinase-1 in mouse sperm acrosomes.

Sphingosine kinase (SPHK) catalyzes sphingosine phosphorylation to form a bioactive lipid mediator, sphingosine-1-phosphate (S1P). In the current study, we report the presence of SPHK-1 in mouse spermatozoa. SPHK-1 was localized to the acrosomes of spermatozoa, and its expression was proven by RT-PCR and Western blot analysis. SPHK activity of mouse spermatozoa was 18.1 pmol/min/mg protein. Furthermore, we identified the presence of the S1P receptors S1P1, S1P2, S1P3, and S1P5, in mouse spermatozoa by RT-PCR. These results suggest that S1P produced by SPHK-1 would play a role in the acrosomal reaction through S1P receptors.     

Early segregation of germ and somatic lineages during gonadal regeneration in the annelid Enchytraeus japonensis

Although regeneration studies are useful for understanding how organs renew, little information is available about regeneration of reproductive organs and germ cells. We here describe the behavior of germ-cell precursors during regeneration of the oligochaete annelid worm Enchytraeus japonensis, which has the remarkable feature of undergoing asexual (by fission) and sexual reproduction . We first found that the gonad can regenerate from any body fragment yielded by fission during asexual reproduction. We then examined behavior of germ-cell lineage during this regenerative process, by using a homolog of the Piwi gene (Ej-piwi) as a marker. We found that in asexually growing animals, specialized cells expressing Ej-piwi are distributed widely in the body as single cells. These cells seem to serve as a reservoir of germ-cell precursors because during asexual propagation these cells migrate into the regenerating tissue, where they ultimately settle in the prospective gonads, and give rise to germ cells upon sexualization. These cells are distinct from the neoblasts, thought to be stem cells in other animals. This is the first report to directly show that the germ and somatic lineages are segregated in asexually growing animals and behave differently during regeneration.     

Effect of cooking process on the deoxynivalenol content and its subsequent cytotoxicity in wheat products

The retention of deoxynivalenol in noodles and bread made from naturally-contaminated flour was examined by a chemical analysis (HPLC) and bioassays. The retention level of deoxynivalenol obtained from both assays was reduced by boiling process, although only the bioassays showed it to have been reduced by baking. This study is the first to estimate the exposure to deoxynivalenol from the consumption of the final products of wheat flour in Japan.