An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.

INTRODUCTION: High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim. OBJECTIVE: To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol. METHODOLOGY: Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added ß-mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at -20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH3COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high-purity RNA. RESULTS: The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8-2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size. CONCLUSION: The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. High-quality RNA has more advantages in molecular biology study of E. senticosus Maxim.

Effects of Different Doses of sUV-B Exposure on Taxane Compounds' Metabolism in Taxus wallichiana var. Mairei.

UV-B is an important environmental factor that differentially affects plant growth and secondary metabolites. The effects of supplemental ultraviolet-B (sUV-B) exposure (T1, 1.40 kJ·m-2·day-1; T2, 2.81 kJ·m-2·day-1; and T3, 5.62 kJ·m-2·day-1) on the growth biomass, physiological characteristics, and secondary metabolites were studied. Our results indicated that leaf thickness was significantly (p < 0.05) reduced under T3 relative to the control (natural light exposure, CK); The contents of 6-BA and IAA were significantly reduced (p < 0.05); and the contents of ABA, 10-deacetylbaccatin III, and baccatin III were significantly (p < 0.05) increased under T1 and T2. The paclitaxel content was the highest (0.036 ± 0.0018 mg·g-1) under T3. The cephalomannine content was significantly increased under T1. Hmgr gene expression was upregulated under T1 and T3. The gene expressions of Bapt and Dbtnbt were significantly (p < 0.05) upregulated under sUV-B exposure, and the gene expressions of CoA, Ts, and Dbat were significantly (p < 0.05) downregulated. A correlation analysis showed that the 6-BA content had a significantly (p < 0.05) positive correlation with Dbat gene expression. The IAA content had a significantly (p < 0.05) positive correlation with the gene expression of Hmgr, CoA, Ts, and Dbtnbt. The ABA content had a significantly (p < 0.05) positive correlation with Bapt gene expression. Dbat gene expression had a significantly (p < 0.05) positive correlation with the 10-deacetylbaccatin content. Hmgr gene expression was positively correlated with the contents of baccatin III and cephalomannine. Bapt gene expression had a significantly (p < 0.01) positive correlation with the paclitaxel content. A factor analysis showed that the accumulation of paclitaxel content was promoted under T2, which was helpful in clarifying the accumulation of taxane compounds after sUV-B exposure.

CD83-stimulated monocytes suppress T-cell immune responses through production of prostaglandin E2.

CD83 is commonly known as a specific marker for mature dendritic cells. It has been shown to be important for CD4(+) T-cell development in the thymus. However, its function in the peripheral immune system remains enigmatic. Here, we show that CD83 inhibits proliferation and production of IL-2 and IFN-γ by T cells, and the inhibitory effect of CD83 is mediated by monocytes. Prostaglandin E2 (PGE(2)), but not IL-10 or TGF-ß, was up-regulated specifically by CD83 in monocytes. Consistent with high levels of PGE(2), expression of COX-2 also was increased upon CD83 treatment. NF-κB activation also is required for induction of PGE(2) by CD83. Finally, application of the COX-2-selective inhibitor NS-398 fully prevented CD83-triggered inhibition of T-cell responses. Our study establishes an immune-regulatory mechanism by CD83 via stimulation of PGE(2) production in monocytes.

Two novel monoclonal antibodies produced against human CD83 molecule.

CD83, a maturation marker for human and mouse dendritic cells (DCs), plays a critical role in CD4(+) T cell development as well as peripheral immune regulation. Here, two novel mouse anti-human CD83 monoclonal antibodies (MAbs) were prepared and their immunological characteristics were determined. Among the two MAbs, 8B4 binds to a linear epitope whereas 1E11 recognizes a conformational epitope. Cross-linking of 8B4 but not 1E11 with CD83-Ig augments the fusion protein mediated inhibition of peripheral blood mononuclear cells (PBMCs). Thus the two MAbs may be good candidates for immunoassaying and functional exploration of CD83 molecule.

[Preparation and characterization of a novel functional anti-human CD83 monoclonal antibody].

AIM: To prepare and characterize a novel anti-human CD83 monoclonal mAb. METHODS: Female BALB/c mice of 6-8 weeks old were immunized with CD83 transfectant (L929/CD83) as immunogen. The spleen B cells of the mice were fused with Sp2/0 myeloma cells.The hybridoma cells were screened with CD83 transfectant (L929/CD83 and 293/CD83) by FCM. The biological characteristics of antibody were investigated by rapid isotyping analysis, karyotype analysis, competitive inhibition test and Western blot. RESULTS: One hybridoma cell line named 9D8 was obtained, which had the property of secreting anti-human CD83 monoclonal antibody steadily, This mAb specifically recognized CD83 molecule expressed on human mature DC, activated T cells, and tumor cell line Daudi, myeloma cell line 8226.This mAb can recognize a distinct epitope from commercial mAb (HB15e). CONCLUSION: One hybridoma cell line has been developed successfully, which may lay a foundation for further study on the function of this molecule.

Transcriptional profiles during the differentiation and maturation of monocyte-derived dendritic cells, analyzed using focused microarrays.

Dendritic cells (DC) are professional antigen-presenting cells capable of initiating primary immune responses. They have been intensively studied and are used in both basic immunology research and clinical immunotherapy. However, the genetic pathways leading to DC differentiation and maturation remain poorly understood. Using focused microarrays with oligonucletotide probes for 120 genes encoding co-stimulatory molecules, chemokines, chemokine receptors, cytokines, cytokine receptors, TLRs, and several other related molecules, we analyzed the kinetics of gene expression for the overall differentiation process of monocytes into mature DC. In parallel, we compared the transcriptional profiles in DC maturation in the presence of LPS, TNF-alpha or trimeric CD40L. We found similar transcriptional profiles for early immature DC and immature DC, respectively generated by culturing monocytes with GM-CSF and IL-4 for three or six days. We identified sets of common and stimuli-specific genes, the expression of which changed following stimulation with LPS, TNF-alpha or CD40L. A dynamic analysis of the entire DC differentiation and maturation process showed that some important inflammatory and constitutive chemokines are transcribed in both immature and mature DC. The correlative expression kinetics of the gene pairs IL1R1/IL1R2, IL15/IL15RA, DC-SIGN/ICAM-2 and DC-SIGN/ICAM-3 imply that they all play crucial roles in mediating DC functions. Thus, our analysis with focused microarrays shed light on the transcriptional kinetics of DC differentiation and maturation, and this method may also prove useful for identifying novel marker genes involved in DC functions.

Association of Graves' disease and prevalence of circulating IFN-gamma-producing CD28(-) T cells.

BACKGROUND: Peripheral blood CD4(+) and CD8(+) T-cell subsets lacking surface CD28 have been suggested to predispose patients to immune-mediated disorders. MATERIALS AND METHODS: To determine the role of CD28(-) T-cell subset in Graves' disease (GD), we characterized peripheral blood CD4(+)CD28(-) and CD8(+)CD28(-) T cell from early onset GD patients. RESULTS AND DISCUSSION: GD patients had significantly higher percentages of CD4(+)CD28(-) and CD8(+)CD28(-) T cells than did healthy donors. Both CD28(-) T cells expressed mostly CD45RO, suggesting that they are activated and/or are memory T cells. GD patient-derived CD4(+)CD28(-) and CD8(+)CD28(-) T cells produced more intracellular IFN-gamma than their counterparts from healthy donors. Furthermore, CD4(+)CD28(-) and CD8(+)CD28(-) T cells from GD patients with Graves' ophthalmopathy (GO) secreted higher level of intracellular IFN-gamma than those CD28(-) T cells from GD patients without GO. Retrospective analysis showed that the increased levels of CD4(+)CD28(-) T cells and their IFN-gamma-producing subgroups were positively correlated to the serum anti-thyrotropin receptor (TSHR) autoantibodies (TRAb). Our observations suggest that increased IFN-gamma-producing CD28(-) T cells in GD patients may play an important role in the pathogenesis of GD.