Changes in responses of UVB irradiated skin of brownish guinea pigs with aging

It is known that skin often shows irregular pigmentation during aging, which is frequently associated with hyperpigmentation. Many studies have utilized brownish A1 guinea pigs to investigate the pathogenesis of ultraviolet B (UVB)-induced skin pigmentation, however, responses associated with aging following UVB irradiation have not been elucidated. To characterize those responses, dorsal skin of A1 guinea pigs from 14-weeks to 5-yr old were investigated. The minimal erythema dose was found to increase with aging. Further, in pigmentation induced by UVB radiation, skin brightness (DeltaL*-value) decreased equally in both the 14-week old (young) group and in the 3-yr old (old) group of guinea pigs. The DeltaL*-value recovered in the young group from 21 d after UVB irradiation, whereas no such recovery was seen in the old group. In addition, the amount of melanin and the number of melanocytes returned near pre-irradiation levels in the young group, while they remained high in the old group. Our results therefore demonstrate for the first time that skin responses following UVB irradiation change with aging in A1 guinea pigs.     

Novel antifungal compounds produced by Sterile Dark, an unidentified wheat rhizosphere fungus

Two novel antifungal compounds were isolated from a culture broth of Sterile Dark, an unidentified fungus isolated from the rhizosphere of wheat grown in a continuous cropping field. These compounds were elucidated to be phthalide-based compounds by spectroscopic analyses.     

Dynamics of methane in mesotrophic Lake Biwa,Japan

As a part of a core project of IGBP (International Geosphere-Biosphere Programme), distribution, production, oxidation and transport processes of methane in bottom sediments and lake water in a mesotrophic lake (Lake Biwa) have been studied with special reference to the spatial heterogeneity of each process. In this study, we attempted to synthesize previously reported results with newly obtained ones to depict the methane dynamics in the entire lake. The pelagic water column exhibited subsurface maxima of dissolved methane during a stratified period. Transect observation at the littoral zone suggested that horizontal transportation may be a reason for the high methane concentration in epilimnion and thermocline at the offshore area. Tributary rivers and littoral sediments were suggested to be the source. Observations also showed that the internal wave caused resuspension of the bottom sediment and release of methane from the sediment into the lake water. The impact of the internal waves was pronounced in the late stage of a stratified period. The littoral sediment showed much higher methanogenic activity than the profundal sediments, and the bottom water of the littoral sediments had little methanotrophic activity. In the profundal sediment, most of the methane that diffused up from the deeper part was oxidized when it passed through the oxic layer. Active methane oxidation was also observed in the hypolimnetic water, while the lake water in the epilimnion and thermocline showed very low methane oxidation, probably due to the inhibitory effect of light. These results mean a longer residence time for methane in the epilimnion than in the hypolimnion. Horizontal inflow of dissolved methane from the river and/or littoral sediment, together with the longer residence time in the surface water, may cause the subsurface maxima, which have also been observed in other lakes and in the ocean.     

Vialinin A, a novel 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenger from an edible mushroom in China

While screening for bioactive compounds from edible mushrooms, a new potent antioxidant, vialinin A (1), together with a known compound, ganbajunin B (2), and a mixture of ganbajunins D (3) and E (4), were isolated from the dry fruiting bodies of Thelephora vialis. The structure of 1, 5',6'-bis(phenylacetoxy)-1,1':4',1'-terphenyl-2',3',4,4'-tetraol, was elucidated by spectroscopic and chemical methods. This compound had strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity with an EC(50) value of 14.0 microM, nearly equal to that of butylated hydroxytoluene (BHT; EC(50) = 10.0 microM). A radical scavenging experiment using 1 and DPPH radicals indicated that 1 donated two hydrogen atoms to two molecules of the DPPH radical under hydrophobic conditions.     

O(2)- and H(2)O(2)-dependent verdoheme degradation by heme oxygenase: reaction mechanisms and potential physiological roles of the dual pathway degradation

Heme oxygenase (HO) catalyzes the catabolism of heme to biliverdin, CO, and a free iron through three successive oxygenation steps. The third oxygenation, oxidative degradation of verdoheme to biliverdin, has been the least understood step despite its importance in regulating HO activity. We have examined in detail the degradation of a synthetic verdoheme IXalpha complexed with rat HO-1. Our findings include: 1) HO degrades verdoheme through a dual pathway using either O(2) or H(2)O(2); 2) the verdoheme reactivity with O(2) is the lowest among the three O(2) reactions in the HO catalysis, and the newly found H(2)O(2) pathway is approximately 40-fold faster than the O(2)-dependent verdoheme degradation; 3) both reactions are initiated by the binding of O(2) or H(2)O(2) to allow the first direct observation of degradation intermediates of verdoheme; and 4) Asp(140) in HO-1 is critical for the verdoheme degradation regardless of the oxygen source. On the basis of these findings, we propose that the HO enzyme activates O(2) and H(2)O(2) on the verdoheme iron with the aid of a nearby water molecule linked with Asp(140). These mechanisms are similar to the well established mechanism of the first oxygenation, meso-hydroxylation of heme, and thus, HO can utilize a common architecture to promote the first and third oxygenation steps of the heme catabolism. In addition, our results infer the possible involvement of the H(2)O(2)-dependent verdoheme degradation in vivo, and potential roles of the dual pathway reaction of HO against oxidative stress are proposed.     

Genetic Basis of Sexual Isolation in Drosophila melanogaster

Sexual isolation between Zimbabwe (abbreviated as Z) and cosmopolitan (abbreviated as M) races exists in Drosophila melanogaster. Typically, when given a choice, the females from the Zimbabwe race mate only with males from the same race, whereas females from the cosmopolitan race mate readily with males from both races non-discriminatorily. Genetic tools available in this experimental organism permit the detail genetic analyses of the sexual isolation behavior. On the other hand, the search for the actual signaling systems involved in the mate recognition is still limited in this system. In this paper, we review the studies, which attempt to dissect the genetic basis of the sexual isolation system, and document the complex features of the genetic architecture and the behavioral traits that evolved at an incipient stage of speciation. The evolution and the maintenance of this behavioral polymorphism are also discussed.     

A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.     

Marrow stromal cells and osteoclast precursors differentially contribute to TNF-alpha-induced osteoclastogenesis in vivo

The marrow stromal cell is the principal source of the key osteoclastogenic cytokine receptor activator of NF-kappaB (RANK) ligand (RANKL). To individualize the role of marrow stromal cells in varying states of TNF-alpha-driven osteoclast formation in vivo, we generated chimeric mice in which wild-type (WT) marrow, immunodepleted of T cells and stromal cells, is transplanted into lethally irradiated mice deleted of both the p55 and p75 TNFR. As control, similarly treated WT marrow was transplanted into WT mice. Each group was administered increasing doses of TNF-alpha. Exposure to high-dose cytokine ex vivo induces exuberant osteoclastogenesis irrespective of in vivo TNF-alpha treatment or whether the recipient animals possess TNF-alpha-responsive stromal cells. In contrast, the osteoclastogenic capacity of marrow treated with lower-dose TNF-alpha requires priming by TNFR-bearing stromal cells in vivo. Importantly, the osteoclastogenic contribution of cytokine responsive stromal cells in vivo diminishes as the dose of TNF-alpha increases. In keeping with this conclusion, mice with severe inflammatory arthritis develop profound osteoclastogenesis and bone erosion independent of stromal cell expression of TNFR. The direct induction of osteoclast recruitment by TNF-alpha is characterized by enhanced RANK expression and sensitization of precursor cells to RANKL. Thus, osteolysis attending relatively modest elevations in ambient TNF-alpha depends upon responsive stromal cells. Alternatively, in states of severe periarticular inflammation, TNF-alpha may fully exert its bone erosive effects by directly promoting the differentiation of osteoclast precursors independent of cytokine-responsive stromal cells and T lymphocytes.     

Genes associated with the formation of germ cells from embryonic stem cells in cultures containing different glucose concentrations

In a previous study, we established a system for visualizing the development of germ cells from mouse embryonic stem (ES) cells in culture using knock-in ES clones in which visual reporter genes were expressed from the mouse vasa homolog, Mvh. While assessing various culture conditions, we found that germ-cell formation was markedly depressed in low glucose medium. Using a repeated polymerase chain reaction (PCR) subtraction method, we identified genes that were differentially expressed in low versus high glucose media. Three genes that were predominantly expressed in high glucose medium, thioredoxin-interacting protein (Txnip), pituitary tumor-transforming gene 1 (Pttg), and RuvB-like protein 2 (RuvBl2), were further investigated. These genes were also found to be highly expressed in adult and embryonic gonads, and RuvBl2 in particular, which encodes an ATP-dependent DNA helicase, was specifically detected in the spermatocytes and spermatids of the adult testis as well as in primordial germ cells. Furthermore, using a green fluorescent protein (GFP) fusion construct, we found that RuvBl2 was expressed in both the nucleus and cytoplasm of testicular germ cells. These findings suggest a possible relationship between glucose metabolism and germ-cell development.     

Analysis of total N-glycans in cell membrane fractions of cancer cells using a combination of serotonin affinity chromatography and normal phase chromatography

Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.