SummaryWe isolated 76 high-light and heat-shock (HL þ HS) stress-inducible genes, including a putative heat-shock transcription factor (HsfA2), by suppression-subtractive hybridization from Arabidopsis. The transcript level of HsfA2 was significantly increased under the several stress conditions or by the H 2 O 2 treatment. Furthermore, the induction of HsfA2 expression was highest among those of other class A HSFs in response to HL þ HS stress conditions. The promoter assay revealed that HsfA2 is induced mainly in rosette leaves under HL þ HS stress conditions. In the HsfA2-overexpressing Arabidopsis (Pro 35S :HsfA2) plants, 46 genes, including a large number of heat-shock proteins, ascorbate peroxidase 2 and galactinol synthase 1 and 2, were highly expressed compared with those in the wild-type plants. The transcript levels of the HsfA2 target genes are highly correlated with those of HsfA2 in the Pro 35S :HsfA2 plants. The transcript levels of the HsfA2 target genes, as well as HsfA2 transcripts, were induced by treating with exogenous H 2 O 2 . In the knockout HsfA2 Arabidopsis plants, the induction of 26 HsfA2 target genes was strongly reduced for up to 2 h under HL þ HS stress conditions. Furthermore, the Pro 35S :HsfA2 plants showed increased tolerance to combined environmental stresses. Our present results indicate that HsfA2 is a key regulator in the induction of the defence system under several types of environmental stress.
ABSTRACT. Paramylon is a -1,3-D-glucan isolated from Euglena gracilis Z. This study was designed to evaluate the suppressive effects of the oral administration of paramylon on the development of atopic dermatitis (AD)-like skin lesions induced by repeated application of 2,4,6-trinitrochlorobenzene (TNCB) in sensitized NC/Nga mice. The effects of paramylon were assessed by measuring macroscopical and histopathological findings of skin, ear swelling, serum levels of total IgE, interleukin-4 (IL-4) and interferon- (IFN-) and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon inhibited the development of AD-like skin lesions as exemplified by a significant decrease in dermatitis scores for the back, ear swelling and hypertrophy of the skin, infiltration of inflammatory cells in the skin, and serum IgE levels. Oral administration of paramylon reduced serum levels of both IL-4 and IFN- and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon did not cause weight loss, as was observed with prednisolone. These results suggest that paramylon inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing both the T-helper (Th) 1 and Th 2 cell responses. Our results indicate that paramylon treatment could provide an effective alternative therapy for the management of AD.KEY WORDS: atopic dermatitis, -1,3-D-glucan, interferon-, interleukin-4, paramylon.
BackgroundMicroalgae have recently been attracting attention as a potential platform for the production of biofuels. Euglena gracilis, a unicellular phytoflagellate, has been proposed as an attractive feedstock to produce biodiesel because it can produce large amounts of wax esters, consisting of medium-chain fatty acids and alcohols with 14:0 carbon chains. E. gracilis cells highly accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. When grown aerobically and then transferred into anaerobic conditions, E. gracilis cells degrade paramylon to actively synthesize and accumulate wax esters. Thus, the enhanced accumulation of paramylon through the genetic engineering of photosynthesis should increase the capacity for wax ester production.ResultsWe herein generated transgenic Euglena (EpFS) cells expressing the cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), which is involved in the Calvin cycle, to enhance its photosynthetic activity. FBP/SBPase was successfully expressed within Euglena chloroplasts. The cell volume of the EpFS4 cell line was significantly larger than that of wild-type cells under normal growth conditions. The photosynthetic activity of EpFS4 cells was significantly higher than that of wild type under high light and high CO2, resulting in enhanced biomass production, and the accumulation of paramylon was increased in transgenic cell lines than in wild-type cells. Furthermore, when EpFS cell lines grown under high light and high CO2 were placed on anaerobiosis, the productivity of wax esters was approximately 13- to 100-fold higher in EpFS cell lines than in wild-type cells.ConclusionOur results obtained here indicate that the efficiency of biomass production in E. gracilis can be improved by genetically modulating photosynthetic capacity, resulting in the enhanced production of wax esters. This is the first step toward the utilization of E. gracilis as a sustainable source for biofuel production under photoautotrophic cultivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0264-5) contains supplementary material, which is available to authorized users.
Heat shock transcription factor A2 (HsfA2) is induced under environmental stress and regulates transcription of various defense-related genes. Thus HsfA2 plays an important role in induction of defenses against different types of environmental stress, but its mode of regulation remains unknown. To clarify the signal transduction pathway involved in the regulation of HsfA2 expression, we investigated the effect of MG132, a 26S proteasome inhibitor, or geldanamycin (GDA), a heat shock protein 90 (Hsp90) inhibitor, on the transcription of HsfA2 and its targets, Hsp18.1-CI and ascorbate peroxidase 2 (Apx2), in Arabidopsis T87 cells. The levels of transcripts were significantly increased by treatment with MG132 or GDA. Overexpression of a dexamethazone-inducible dominant-negative form of Hsp90.2 in Arabidopsis plants caused significant expression of HsfA2 and its target gene on treatment with the compound. Treatment with MG132 or GDA had no effect on intracellular levels of reactive oxygen species (ROS). Interestingly, the levels of polyubiquitinated proteins as well as the levels of HsfA2 transcript were rapidly increased under oxidative stress derived from treatment with H2O2 or methylviologen, while they were completely suppressed by pre-treatment with ascorbate, a scavenger of ROS, under oxidative stress. The present findings suggest that the inhibition of 26S proteasome function and/or Hsp90 activity is involved in the induction of HsfA2 expression in response to oxidative stress.
In the present study, the effects of β-glucans isolated from Euglena on the formation of preneoplastic aberrant crypt foci (ACF) in the colon were examined in mice. Mice were fed a semi-purified AIN-93M diet containing cellulose or the same diet but with the cellulose replaced with β-glucans in the form of Euglena, paramylon, or amorphous paramylon, for 11 weeks. After consuming these dietary supplements for 8 days, half of the mice were intraperitoneally administered 1,2-dimethylhydrazine (DMH) at a dose of 20 mg kg(-1) body weight every week for 6 weeks. Among the DMH-treated groups, the paramylon- and amorphous paramylon-fed mice displayed a significantly lower number of ACF than the control group. Also, the liver weight of the paramylon group was markedly decreased compared with those of the control and Euglena groups, whereas the cecal content weight and fecal volume of the paramylon group were significantly increased. As for the levels of organic acids in the cecal contents, the paramylon group displayed significantly increased lactic acid levels compared with the control and Euglena groups. From these findings, although the mechanism of the ACF-inhibiting effects of paramylon remains unclear, it is considered that β-glucans, such as paramylon and its isomer amorphous paramylon, have preventive effects against colon cancer and are more effective against the condition than Euglena.
ABSTRACT. Paramylon is a -(1-3)-D-glucan isolated from Euglena gracilis Z. This study was designed to evaluate the protective effects of paramylon on liver injury induced by carbon tetrachloride (CCl 4 ) in rats. Wistar stain male rats were orally administered paramylon (500, 1,000 and 2,000 mg/kg body weight) before treatment with a single intraperitoneal dose of 50% CCl 4 (2 ml/kg body weight). The rats were sacrificed 24 hr later, and blood samples were collected for assay of serum biochemical parameters. The livers were excised to evaluate the activity of antioxidant enzymes. Histopathological examination of the livers was also performed. The results showed that the treatment of paramylon prevented elevation of the serum levels of hepatic enzyme markers and inhibited fatty degeneration and hepatic necrosis induced by CCl 4 . Pre-administration of paramylon reduced the liver apoptotic index. The treatment of paramylon recovered reductions of activity of hepatic superoxide dismutase, catalase and glutathione peroxidase induced by CCl 4 . These results demonstrate that paramylon exhibits protective action on acute hepatic injury induced by CCl 4 via an antioxidative mechanism. To the best of our knowledge, this is the first report of a hepatoprotective effect based on the antioxidative action of paramylon.
Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1–5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.
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