Protective immune responses in mice induced by intramuscular and intranasal immunization with a Mycoplasma pneumoniae P1C DNA vaccine

Mycoplasma pneumoniae is an important causative agent of atypical pneumonia. This study was to determine the ability of a DNA expression vector, which encodes the carboxy terminal region of the M. pneumoniae P1 protein (P1C), to induce humoral and cellular immune responses and to protect against M. pneumoniae infection in BALB/c mice. Mice were immunized with pcDNA3.1/P1C by either intramuscular injection (i.m.) or intranasal inoculation (i.n.). Our results showed that p1c DNA immunization generates detectable antibodies specific to M. pneumoniae, and elicits high levels of IgG1, IgG2a, and IgG2b isotypes (P?< 0.01). The levels of IFN-γ and IL-4 in spleen cells of the immunized mice were significantly elevated by immunization via both the i.m. and i.n. methods. Moreover, p1c DNA-immunized mice exhibited detectable protection against M. pneumoniae infection. The lung tissue inflammation was relieved and the histopathologic score (HPS) of pcDNA3.1/P1C-immunized mice was significantly decreased than those in phosphate-buffed saline (PBS) or vaccine-vector-immunized mice (P?< 0.01), whereas there were no significant differences in HPS between i.m. and i.n. vaccination (P?> 0.05). Our results suggest that pcDNA3.1/P1C could be useful for developing a vaccine against M. pneumoniae infection.     

Macrophage-Activating Lipopeptide-2 Requires Mal and PI3K for Efficient Induction of Heme Oxygenase-1

Aims

This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes.

Methods

Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay.

Results

MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions

These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.     

树木胸径大小对树干液流变化格局的偏度和时滞效应

通过分析具不同水力结构的马占相思、荷木和粉单竹液流变化格局的偏度和时滞,探讨液流的空间分布特征及对冠层蒸腾的影响。结果表明:荷木的液流格局偏度和时滞随树木胸径的增加呈减小的趋势,但马占相思由于冠层开阔和林分分化程度高而规律不明显,粉单竹液流偏度随胸径减少,由于冠幅较小,接受的光照较均匀,个体间的时滞差异不明显,但时滞值比胸径近似的荷木小。树干水分传输过程中存在液流再分配的现象,边材的导水效率可能是影响时滞的重要原因。冠层蒸腾的空间异质性与树木储存水有关,大树储存水较多,冠层蒸腾的异质性小;小树储存水较少,液流被优先分配到光照充足的东南方位,导致冠层蒸腾较高的异质性。旱季受土壤水分的限制,大树储存水对蒸腾的贡献大于湿季,而小树蒸腾由于受到储水容量的制约,储存水对蒸腾的贡献小于湿季。冠层接受光照的迟或早以及辐射量的大小是引起蒸腾时间变化格局和树干不同方位液流格局差异的重要原因,但液流的横向交换弱化了这种现象,往往是个体间的差异掩盖了方位的差异。湿季较小胸径的树木比偏值(枝下高与胸高处液流偏度的比值)大于旱季,而较大胸径的树木比偏值恰好相反,总体而言,比偏值随着胸径的增加而逐渐下降。     

Screening and Identification of the Binding Peptides of Mycoplasma genitalium Protein of Adhesion

International Journal of Peptide Research and Therapeutics - Mycoplasma genitalium protein of adhesion (MgPa) is a vital membrane protein, which plays an important role in mediating the adhesion...     

Genetic confirmation of 2 independent genes for resistance to soybean mosaic virus in J05 soybean using SSR markers

J05 soybean was previously identified to carry 2 independent genes, Rsv1 and Rsv3, for "soybean mosaic virus" (SMV) resistance by inheritance and allelism studies. The objective of this research was to confirm the 2 genes in J05 using molecular markers so that a marker-assisted selection can be implemented. The segregation of F(2) plants from J05 x Essex exhibited a good fit to a 3:1 ratio when inoculated with SMV G1. Three simple sequence repeat (SSR) markers near Rsv1, Satt114, Satt510, and Sat_154, amplified polymorphic DNA fragments between J05 and Essex and were closely linked to the gene on soybean molecular linkage group (MLG) F, thus verifying the presence of Rsv1 in J05 for resistance to SMV G1. The presence of Rsv3 in J05 was confirmed by 2 closely linked SSR markers on MLG B2, Satt726 and Sat_424, in F(2:3) lines that were derived from the SMV G1-susceptible F(2) plants and segregated in a 1:2:1 ratio for reaction to SMV G7. Two closely linked markers for Rsv4, Satt296 and Satt542, segregated independently of SMV resistance, indicating the absence of Rsv4 in J05. These SSR markers for Rsv1 and Rsv3 can serve as a useful molecular tool for selection and pyramiding of genes in J05 for SMV resistance.     

Genetic diversity among mulberry genotypes from seven countries

Mulberry (Morus alba) is an economically and ecologically important, widespread woody plant. It has served human beings for over hundreds of years, and it is still widely used in pharmaceuticals, food industry and farming nowadays. Using modern techniques, deeper understanding in classification and conservation resources of mulberry leads to higher-efficiency hybrids among populations. Genetic polymorphisms among 42 mulberry genotypes from seven countries over Asia and South America were detected using 17 inter simple sequence repeat (ISSR) primers. A dendrogram was constructed using the similarity matrix among genotypes and a principal component analysis (PCA) was carried out to further identify and cluster the mulberry genotypes. In the 42 genotypes, 175 distinct bands were displayed, among which 169 were polymorphic bands (96.57%). The polymorphic information content of 17 ISSR primers ranged from 0.2921 to 0.3746 with the mean of 0.3494. And Nei’s index and Shanon’s information index averaged 0.116 and 0.174, respectively, indicating low diversity of mulberry. For further study, cluster analysis and PCA were carried out and the results were similar. 42 genotypes were grouped, showing some hybridized combinations. Additionally, a connection between mulberry diseases and their genotypes was noted, which indicates possible application for ISSR in studying disease resistance of mulberry.     

Protective efficacy of a Mycoplasma pneumoniae P1C DNA vaccine fused with the B subunit of Escherichia coli heat-labile enterotoxin

In the present study, we investigated the immunomodulatory responses of a DNA vaccine constructed by fusing Mycoplasma pneumoniae P1 protein carboxy terminal region (P1C) with the Escherichia coli heat-labile toxin B subunit (LTB). BALB/c mice were immunized by intranasal inoculation with control DNAs, the P1C DNA vaccine or the LTB-P1C fusion DNA vaccine. Levels of the anti-M. pneumoniae antibodies and levels of interferon-γ and IL-4 in mice were increased significantly upon inoculation of the LTB-P1C fusion DNA vaccine when compared with the inoculation with P1C DNA vaccine. The LTB-P1C fusion DNA vaccine efficiently enhanced the M. pneumoniae-specific IgA and IgG levels. The IgG2a/IgG1 ratio was significantly higher in bronchoalveolar lavages fluid and sera from mice fusion with LTB and P1C than mice receiving P1C alone. When the mice were challenged intranasally with 10(7) CFU M. pneumoniae strain (M129), the LTB-P1C fusion DNA vaccine conferred significantly better protection than P1C DNA vaccine (P < 0.05), as suggested by the results, such as less inflammation, lower histopathological score values, lower detectable number of M. pneumoniae strain, and lower mortality of challenging from 5 × 10(8) CFU M. pneumoniae. These results indicated that the LTB-P1C fusion DNA vaccine efficiently improved protective efficacy against M. pneumoniae infection and effectively attenuated development of M. pneumoniae in mice.     

淋球菌porB和大肠杆菌ltB融合基因的构建、表达及其免疫活性

【目的】构建融合基因原核表达载体pET-30a/ltB-porB,并表达重组融合蛋白LTB-PorB,鼻饲途径免疫雌性BALB/c小鼠,分析重组融合蛋白的免疫活性,为研制抗淋病蛋白疫苗提供实验依据。【方法】构建大肠杆菌不耐热肠毒素B亚单位(LTB)与淋球菌外膜孔蛋白B(PorB)融合基因及LTB、PorB单基因pET-30a原核表达载体,在大肠杆菌BL21中表达重组蛋白;鼻饲途径免疫雌性BALB/c小鼠,检测体液免疫和细胞免疫水平。【结果】在大肠杆菌BL21中获得高效表达的重组蛋白;经鼻饲免疫小鼠后,重组融合蛋白LTB-PorB组生殖道黏膜产生的PorB特异性sIgA水平随免疫时间呈上升趋势,第42天A450值达0.66,明显高于对照组(P0.01),效价高达1∶1280;血清中产生的PorB特异性IgG第28天达最高,A450值为0.60,明显高于LTB和蛋白溶解液(Solution Buffer)对照组(P0.01),效价高达1:2560,但与PorB对照组血清IgG水平(A450:0.57)无明显差异(P0.05)。LTB-PorB组脾淋巴细胞刺激指数明显高于LTB和SolutionBuffer对照组(P0.05),但脾淋巴细胞诱生的IFN-γ水平与对照组无明显差异(P0.05)。【结论】重组融合蛋白LTB-PorB通过鼻饲途径免疫雌性BALB/c小鼠后,能诱导产生高水平的体液免疫和一定水平的细胞免疫。首次证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道粘膜免疫。     

Genetic analysis of resistance to soybean mosaic virus in j05 soybean

Soybean cultivar J05 was identified to be resistant to the most virulent strain of soybean mosaic virus (SMV) in northeastern China. However, the reaction of J05 to SMV strains in the United States of America is unknown, and genetic information is needed to utilize this germplasm in a breeding program. The objectives of this study were to determine the reaction of J05 to all US strains of SMV (G1-G7), the inheritance of SMV resistance in J05, and the allelic relationship of resistance genes in J05 with other reported resistance genes. J05 was crossed with susceptible cultivar Essex (rsv) to study the inheritance of SMV resistance. J05 was also crossed with PI 96983 (Rsv1), L29 (Rsv3), and V94-5152 (Rsv4) to test the allelism of resistance genes. F(2) populations and F(2:3) lines from these crosses were inoculated with G1 or G7 in the greenhouse. Inheritance and allelism studies indicate that J05 possesses 2 independent dominant genes for SMV resistance, one at the Rsv1 locus conferring resistance to G1 and necrosis to G7 and the other at the Rsv3 locus conditioning resistance to G7 but susceptibility to G1. The presence of both genes in J05 provides resistance to G1 and G7. J05 is unique from the previous sources that carry 2 genes of Rsv1Rsv3 and will be useful in breeding for SMV resistance.