Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium

Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, Chenopodium album WSCP (CaWSCP) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding Chenopodium ficifolium Class I WSCPs, CfWSCP1, and CfWSCP2. Sequence analyses revealed that the open reading frames of CfWSCP1 and CfWSCP2 were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in Escherichia coli as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.     

DNase II and the Chk2 DNA damage pathway form a genetic barrier blocking replication of horizontally transferred DNA

We have previously shown that DNA from dying tumor cells may be transferred to living cells via the uptake of apoptotic bodies and may contribute to tumor progression. DNA encoding H-ras(V12) and c-myc oncogenes may be transferred to the nucleus of the phagocyte but will only integrate and propagate in p53- and p21-deficient mouse embryonic fibroblasts, whereas normal cells are resistant to transformation. Here, we show that this protective mechanism (activation of p53 and p21 after uptake of apoptotic bodies) is dependent on DNA fragmentation, where inhibition of the caspase-activated DNase in the apoptotic cells, in conjunction with genetic ablation of lysosomal DNase II in the phagocytes, completely blocks p53 activation and consequently allows DNA replication of transferred DNA. We, therefore, suggest that there is a causal relationship between DNA degradation during apoptosis and p53 activation. In addition, we could further show that Chk2-/- cells were capable of replicating the hyg(R) gene taken up from engulfed apoptotic cells, suggesting involvement of the DNA damage response. These data show that the phagocytosing cell is sensing the degraded DNA within the apoptotic cell, hence preventing these genes from being replicated, probably through activation of the DNA damage response. We, therefore, hypothesize that DNase II together with the Chk2, p53, and p21 pathway form a genetic barrier blocking the replication of potentially harmful DNA introduced via apoptotic bodies, thereby preventing transformation and malignant development.     

The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.     

IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.     

Relationship between eyed-egg percentage and levels of cortisol and thyroid hormone in masu salmon Oncorhynchus masou

A possible cause of the low eyed-egg percentage that afflicts pond-reared masu salmon Oncorhynchus masou at a fish hatchery (Kumaishi Fish Hatchery, Hokkaido, Japan) was investigated. Serum cortisol levels of masu salmon during the spawning period were compared between individuals from Kumaishi and those from another station (Mori). Cortisol, thyroxine (T₄) and triiodothyronine (T₃) hormones were also measured in eyed eggs and their levels were compared to the eyed-egg percentage. Serum cortisol levels were significantly higher at Kumaishi than at Mori in May and July of the breeding season. At the Kumaishi station, there was a linear positive relationship between serum cortisol and fertilized-egg cortisol levels of female masu salmon. As cortisol levels in both ovulating females and eyed eggs increased, the eyed-egg percentage decreased, indicating a highly significant negative relationship between cortisol levels and eyed-egg percentage. In contrast, as T₃ and T₄ levels in eyed eggs increased, the eyed-egg percentage increased. The strong positive correlation between high cortisol levels in serum and in eyed eggs at Kumaishi indicates that the frequently higher cortisol levels may have caused the lowering of the eyed-egg percentage.     

Occurrence of a specific protein in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304 inductively with a cell-wall preparation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation (CWP) of Schizophyllum commune, has been reported to have an activity to form protoplasts from S. commune mycelia. The SDS-polyacrylamide gel electrophoreses described here demonstrated that a specific proteinous component (molecular weight: 150,000) occurred in KA-prep. The protein (P150T) was also formed in culture filtrates with CWP of several basidiomycetes, which could release the protoplasts, suggesting that the component was an indispensable factor for protoplast formation. P150T, isolated from an ammonium sulfate fraction of KA-prep (0-30% saturation), did not have any protoplast-forming activity. Results were obtained indicating that P150T participates in protoplast formation together with chitinase(s) and beta-glucanase(s) in KA-prep. The N-terminal amino acid sequence indicated an analogy of P150T to mutanase (alpha-1,3-glucanase) from Bacillus sp. RM1, and actually P150T hydrolyzed mutan as well as S-(alpha-1,3) glucan from S. commune.     

CYP3A induction aggravates endotoxemic liver injury via reactive oxygen species in male rats

We carried out this experiment to evaluate the relationship between isoforms of cytochrome P450 (P450) and liver injury in lipopolysaccharide (LPS)-induced endotoxemic rats. Male rats were intraperitoneally administered phenobarbital (PB), a P450 inducer, for 3 days, and 1 day later, they were intravenously given LPS. PB significantly increased P450 levels (200% of control levels) and the activities (300-400% of control) of the specific isoforms (CYP), CYP3A2 and CYP2B1, in male rats. Plasma AST and ALT increased slightly more in PB-treated rats than in PB-nontreated (control) rats with LPS treatment. Furthermore, either troleandomycin or ketoconazole, specific CYP3A inhibitors, significantly inhibited LPS-induced liver injury in control and PB-treated male rats. To evaluate the oxidative stress in LPS-treated rats, in situ superoxide radical detection using dihydroethidium (DHE), hydroxy-2-nonenal (HNE)-modified proteins in liver microsomes and 8-hydroxydeoxyguanosine (8-OHdG) in liver nuclei were measured in control and PB-treated rats. DHE signal intensity, levels of HNE-modified proteins, and 8-OHdG increased significantly in PB-treated rats. LPS further increased DHE intensity, HNE-modified proteins, and 8-OHdG levels in normal and PB-treated groups. CYP3A inhibitors also inhibited the increases in these items. Our results indicate that the induction or preservation of CYP isoforms further promotes LPS-induced liver injury through mechanisms related to oxidative stress. In particular, CYP3A2 of P450 isoforms made an important contribution to this LPS-induced liver injury.     

The canine mast cell activation via CRP

We report here canine mastocytoma-derived cell (CMMC) activation via two pentraxin, limulus- and human-CRP. Mast cell chemotaxis was measured by Boyden's blindwell chamber. To confirm that the cell migration was chemotactic, "checkerboard" analysis was performed. We used Fura-2 to investigate CRP-mediated cytosolic calcium elevation. To examine whether CRP-induced stimulation is mediated through G-proteins, CMMC were incubated with pertussis toxin (PTx) before use in chemotaxis assay and Ca(2+) mobilization. CMMC migration in response to CRP was both chemokinetic and chemotactic. Limulus-CRP induced a transient Ca(2+)-mobilization dose-dependently. Preincubation of the cells with PTx inhibited CRP chemotaxis and Ca(2+)-mobilization, suggesting that G-proteins of the Gi-class are involved in the chemotaxis. We suggest that CRP may participate in the migration of mast cells to inflamed tissues during an acute-phase response. CRP-mediated recruitment of mast cells might play an important role in hypersensitivity and inflammatory processes.