Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.     

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.     

Dietary diacylglycerol suppresses high fat diet-induced hepatic fat accumulation and microsomal triacylglycerol transfer protein activity in rats

We have recently shown that the long-term ingestion of dietary diacylglycerol (DAG) mainly containing 1,3-isoform reduces body fat accumulation in humans as compared to triacylglycerol (TAG) with the same fatty acid composition. The fat reduction in this human experiment was most pronounced in visceral fat and hepatic fat. Recent animal studies have also indicated that dietary DAG induces alteration of lipid metabolism in the rat liver. In the present study, the dietary effects of DAG on high fat diet-induced hepatic fat accumulation and hepatic microsomal triglyceride transfer protein (MTP) activity were examined in comparison with those of TAG diet in rats. When the TAG oil content was increased from 10 to 30 g/100 g diet, hepatic TAG concentration, hepatic MTP activity and MTP large subunit mRNA levels were significantly increased after 21 days. However, when the dietary TAG oil (30 g/100 g diet) was replaced with the same concentration of DAG oil with the same fatty acid composition, the increase of the TAG concentration and the MTP activity in the liver were significantly less and the mRNA levels remained unchanged. The MTP activity levels correlated significantly with hepatic TAG concentration.These results showed that dietary DAG may suppress high fat diet-induced MTP activity in the liver, and indicated the possibility that hepatic TAG concentration may regulate hepatic MTP activity.     

Thermally reversible hydration of beta-chitin

Thermally induced transition between anhydrous and hydrated forms of highly crystalline beta-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet beta-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 degrees C on heating and one broad exothermic peak at between 60 and 0 degrees C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet beta-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the beta-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process.     

A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana

Met-overaccumulating mutants provide a powerful genetic tool for examining both the regulation of the Met biosynthetic pathway and in vivo developmental responses of gene expression to altered Met levels. We have previously reported the identification of two Arabidopsis thaliana Met over-accumulation (mto) mutants, mto1-1 and mto2-1, that carry mutations in the genes encoding cystathionine gamma-synthase (CGS) and threonine synthase (TS), respectively. A third mutant, mto3-1, has recently been reported to carry a mutation in the gene encoding S-adenosylmethionine synthetase 3 (SAMS3). Here, we report the isolation of a new ethionine-resistant A. thaliana mutant that over-accumulates soluble Met approximately 20-fold in young rosettes. The causal mutation was determined to be a single, recessive mutation that was mapped to chromosome 3. Sequence analysis identified a single nucleotide change in the gene encoding SAMS3 that was distinct from the mto3-1 mutation and altered the amino acid sequence of the enzyme active site. This mutation was therefore referred to as mto3-2. Although Met over-accumulation in the mto3-2 mutant was similar to that in the mto2-1 mutant, CGS mRNA levels did not respond to the mto3-2 mutation and were similar to that in equivalent wild-type plants.     

Enhanced UVfemale1 tumor growth in CBF1 mice infected with Schistosoma mansoni due to modulation of Th1-like responses

Effects of Schistosoma mansoni infection on anti-tumor immunity were examined in CBF1 mice with ultraviolet-induced UVfemale1 fibrosarcoma cells. Although many laboratory established tumor cells had rejection mechanisms independent of CD4(+) T cells, we confirmed that CD4(+) cells had significant roles in rejection of UVfemale1 cells in the syngeneic CBF1 mice. When we prepared two CBF1 mouse groups, S. mansoni-infected and schistosome-free, the former group showed up-regulation of Th2-like response to UVfemale1 cells, whereas the latter group mice showed rather type 1-dominant patterns. Cytotoxic activity against UVfemale1 cells tested in vitro, which was attributed to CD8(+) cells, was significantly weaker in S. mansoni-infected mice compared with infection-free mice. In tumor challenge experiments in vivo, we observed that rapid and complete rejection of UVfemale1 cells required the presence of CD8(+) T cells. Under only CD4-depleted situation, survival of tumor cells in schistosome-free mice was prolonged up to 1 month or more. Under the presence of both CD4(+) and CD8(+) cells, S. mansoni infected mice rejected the challenged UVfemale1 cells as was seen in normal mice. However, when CD8(+) cells were depleted from S. mansoni-infected mice, inoculated UVfemale1 cells grew more rapidly than in infection-free mice. Our results suggest that functionally polarized cytokine patterns in schistosome-infected hosts promote rapid tumor growth.     

4-hydroxy-2-nonenal as a COX-2 inducer

4-hydroxy-2-nonenal (HNE) activates a variety of signaling pathways. We have recently evaluated the effect of oxidized fatty acid metabolites on cyclooxygenase-2 (COX-2) induction in rat liver epithelial RL34 cells and found that, among the compounds tested, HNE most dramatically induced COX-2. A p38 mitogen-activated protein kinase (p38 MAPK) pathway has been shown to play a key role in the mechanism of the HNE-induced COX-2 expression. It appears that the HNE-induced activation of p38 MAPK leads to the stabilization of COX-2 mRNA.     

A gene for a Class II DNA photolyase from<Emphasis Type="Italic"> Oryza sativa</Emphasis>: cloning of the cDNA by dilution-amplification

Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class II) repair CPDs. A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells.