Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene

Increasing soil salinization of arable land has a major impact on the global ecosystem. One approach to increase the usable global forest area is to develop transgenic trees with higher tolerance to conditions of salt stress. An allene oxide cyclase homolog, mangrin, contains a core protein domain that enhances the salt tolerance of its host. We utilized this feature to develop improved salt-tolerant eucalyptus trees, by using transgenic Eucalyptus camaldulensis carrying the mangrin gene as a model. Since the Japanese government requires an environmental biosafety assessment for the surrounding biosphere, we performed experiments on trees grown in a special netted-house. This study examined the transgenic E. camaldulensis carrying the mangrin gene to assess the feasibility of using these transformants, and assessed their salt tolerance and environmental biosafety. We found that seven of 36 transgenic genotypes had significantly higher salt tolerance than non-transformants, and more importantly, that these plants had no significant impact on environmental biosafety. These results suggest that introduction of the mangrin gene may be one approach to safely enhance salt tolerance in genetically modified Eucalyptus species, and that the transformants have no apparent risks in terms of environmental biosafety. Thus, this study provides valuable information regarding the use of transgenic trees in situ.     

Effective selection system for generating marker-free transgenic plants independent of sexual crossing

 In a previous report, a novel selection protocol termed "the MAT-vector system" for generating marker-free transgenic plants (MFTPs) was presented. The first stage of the system is visual selection of morphologically abnormal transgenic shoots, ipt-shooty, that have lost apical dominance and rooting ability. The second stage involves elimination of the ipt gene and the appearance of MFTPs free of ipt gene influence. The present report describes a practical MAT-vector in which removal of the ipt gene is efficiently mediated by the site-specific recombination system R/RS from Zygosaccharomyces rouxii, in place of the maize transposable element Ac, used previously. This improved MAT-vector produced MFTPs from 39% of moderate ipt-shooty and 70% of extreme ipt-shooty lines. These results are superior to the previous MAT-vector which produced MFTPs from only 5% of ipt-shooty lines. The present novel system also induced direct development of MFTPs from adventitious buds without production of ipt-shooty intermediates. The presence of β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) genes of interest, and the absence of the ipt gene were verified by a GUS histochemical assay, NPTII assay, and molecular analysis. Received: 19 June 1998 / Revision received: 4 December 1998 / Accepted: 18 December 1998     

A new dipeptide,O-phosphoserylethanolamine isolated from Agkistroden blomhoffi (mamushi)

A new dipeptide was isolated from several tissues of Agkistroden blomhoffi (mamushi: a venomous snake in Japan), using ion-exchange resins and thin-layer chromatography. It was identified as O-phosphoserylethanolamine by mass spectrometry and comparison with synthetic compounds using several methods. This compound was contained in several mamushi tissues including the liver, heart, brain, bile, and muscle. The concentrations of O-phosphoserylethanolamine in the liver, brain, muscle, skin, heart, and bile were 7.17+/-3.11,16.98+/-4.25,37.37+/-7.88,37.56+/-8.97,23.93+/-6.11, and 22.21+/-5.76 micromol/g, respectively.     

Contents of sulfur amino acids, and cystathionine beta-synthase and gamma-lyase activities in various tissues from agkistroden blomhoffi (mamushi)

The concentrations of sulfur-containing amino acids, taurine, cystathionine, methionine and cystine, as well as cystathionine beta-synthase and gamma-lyase activities in various tissues of Agkistrodon blomhoffi (mamushi) were measured. The concentration of taurine in examined tissues was greater than the concentration of other sulfur-containing amino acids. The concentration of cystathionine in various tissues was also much higher than those of methionine and cystine, but the concentration of cystathionine in the brain was lower than that of methionine. In all tissues examined in this study, cystathionine beta-synthase activity was much higher than that of cystathionine gamma-lyase. The ratios of cystathionine beta-synthase to gamma-lyase activities in various tissues were 5.6 to approximately 85.6. The concentration of sulfur-containing amino acids in muscle and skin divided into eight portions of the body were also determined. The concentrations of methionine and cystine in each portion of muscle and skin were almost the same, but the concentrations of taurine and cystathionine in each portion of the body were varied.     

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two‐phase polymer systems

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two‐phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG‐rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (K_C) was obtained in the presence of NaCl 0.1 M (K_C = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na₂SO₄ 0.5 M showed the highest K_C (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%η_TOP) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0–3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1295–1304, 2015     

Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/ Ribavirin Therapy in Chronic Hepatitis C

Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3− NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed.　Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.     

A new GST-MAT vector containing both ipt and iaaM/H genes can produce marker-free transgenic tobacco plants with high frequency

. Agrobacterium-mediated transformation is highly dependent upon competency of the target plant tissues. It is important to develop the capacity of transformed cells to include cell proliferation and differentiation. A system which results in cell proliferation and differentiation only of transformed cells is highly desirable for plant transformation. We report here a new GST-MAT vector system (MATIMH), in which the ipt gene combined with iaaM/H genes was used as the selectable marker gene and the GST-II promoter was used as the promoter of the R gene in a site-specific recombination system. In tobacco transformation, the combination of the ipt gene and the iaaM/H genes can result in the production of both auxin and cytokinin in transformed tissues and induce regeneration of transgenic shoots exhibiting an ipt-shooty phenotype more efficiently than the ipt gene alone. When we transformed 20 tobacco leaf discs with the MATIMH vector, marker-free transgenic plants were produced from five (41.6%) out of 12 ipt-shooty lines. These results indicated that the combination of the iaaM/H genes and the ipt gene can more efficiently produce both transgenic plants and marker-free transgenic plants.     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

Production of trans-10, cis-12 Conjugated Linoleic Acid in Rice

Conjugated linoleic acid (CLA) has anti-carcinogenic and anti-atherosclerosis activity, and modulatory effects on the immune system and lipid metabolism. To produce a transgenic rice plant that can accumulate CLA, a linoleate isomerase gene that can convert linoleic acid to trans-10, cis-12 CLA was introduced and expressed under the control of seed-specific promoters from the oleosin and globulin genes. The fatty acid composition of the transgenic rice grain was analyzed by gas chromatography. Although there was no clear difference in the fatty acid composition between seeds from transformed versus untransformed plants, a peak of trans-10, cis-12 CLA methyl ester, which was not present in seeds from untransformed plants, was found in transformed plants. The trans-10, cis-12 CLA comprised an average of 1.3% (w/w) of the total fatty acids in seeds carrying the oleosin promoter in comparison to 0.01% (w/w) in seeds carrying the globulin promoter. In addition, approximately 70 and 28% of the total amount of the CLA isomer were present in the triacylglycerol and free fatty acid fractions, respectively. These results demonstrate the ability to produce fatty acid components of vegetable oils with novel physiological activities in crops.     

Transgene stacking in plants in the absence of sexual crossing

A transgene stacking system is a prerequisite for the introduction of multiple genes and for the modification of complex metabolic pathways in plants. We demonstrate here that the MAT-vector system previously used for generating marker-free transgenic plants is also an efficient and reliable transformation system for the repeated introduction of multiple transgenes independent of sexual crossing. We previously reported that the GST-MAT vector system, in which excision of the yeast site-specific recombination R/RS system is regulated by the maize GST-II-27 promoter, could generate marker-free transgenic plants containing a single transgene with high frequency. Here we show that the GST-MAT vector can be used successfully to introduce a second transgene (GFP) into a marker-free transgenic tobacco line containing single copies of the first transgenes (nptII and uidA genes). The transgene-stacked marker-free transgenic tobacco plants were generated from ca. 20% of excision-positive ipt-shooty explants within 5 months of Agrobacterium infection. The presence of uidA, nptII, GFP genes and the absence of the ipt gene were verified by PCR analyses. Furthermore, Southern blot analysis showed that no chromosomal rearrangements were introduced between the first and second transformations.