含par位点的重组质粒Psjm3的构建及其稳定性研究

利用自然质粒pSC101par位点的分离稳定性功能,构建了含par位点的质粒pSJM4和pSJM3,通过在同样宿主E.coli HB101中的稳定性比较研究表明,不含par位点的重组质粒pSJ3很不稳定,E.coli G3(pSJ3)在培养到第10代时已开始出现pSJ3的丢失,到培养至50代时则已全部丢失;而含par位点的重组质粒pSJM3则表现得十分稳定,E.coli G3-1(pSJM3)经70代培养,仍无明显的质粒丢失现象,其稳定率保持97%以上。通过对不含par和含par的非重组质粒pUC18和pSJM4的稳定性比较也获得同样的结果。通过对E.coliG3(pSJ3)和E.coli G3-1(pSJM3)的产酶活性比较研究表明,G3-1菌株明显高于G3菌株,说明我们构建的重组质粒pSJM3上的par位点功能不仅没有因外源基因的表达而受影响,而且有利于外源基因的表达。     

红壤茶树根层土壤基础呼吸作用和酶活性

对不同树龄茶树根层土壤的呼吸作用(包括代谢熵qCO2)和土壤酶(脲酶、转化酶和酸性磷酸单酯酶)活性进行了研究、不同树龄茶树根层土壤日基础呼吸作用强度(36．23—58．52mg·kg^-1·d^-1)和日代谢墒(0．30一0．68)都以40和90年茶树较为接近,分别显著大于和小于10年树龄茶树根层土壤;服酶活性(41．48—47、72mg·kg^-1·d^-1)则三者间差异不大,虽然随树龄增长而下降;转化酶活性(189．29—363．40mg·kg^-1·d^-1)也随树龄增长而下降,并且10年茶树根层土壤显著大于40和90年树龄茶树;而酸性磷酸单酯酶活性(444．22—828．32mg·kg^-1·d^-1)相反,随树龄增长而增强．结果表明,土壤基础呼吸作用、代谢熵和3种土壤酶活性都与茶树树龄、土壤pH、土壤有机碳、土壤全氮、土壤可活性酚总量、及土壤微生物生物量密切相关．     

Tissue microarray study for classification of breast tumors

Clinical and pathological heterogeneity of breast cancer hinders selection of appropriate treatment for individual cases. Molecular profiling at gene or protein levels may elucidate the biological variance of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters. We studied the immunohistochemical profile of a panel of seven important biomarkers using tumor tissue arrays. The tumor samples were then classified with a monothetic (binary variables) clustering algorithm. Two distinct groups of tumors are characterized by the estrogen receptor (ER) status and tumor grade (p = 0.0026). Four biomarkers, c-erbB2, Cox-2, p53 and VEGF, were significantly overexpressed in tumors with the ER-negative (ER-) phenotype. Eight subsets of tumors were further identified according to the expression status of VEGF, c-erbB2 and p53. The malignant potential of the ER-/VEGF+ subgroup was associated with the strong correlations of Cox-2 and c-erbB2 with VEGF. Our results indicate that this molecular classification system, based on the statistical analysis of immunohistochemical profiling, is a useful approach for tumor grouping. Some of these subgroups have a relative genetic homogeneity that may allow further study of specific genetically-controlled metabolic pathways. This approach may hold great promise in rationalizing the application of different therapeutic strategies for different subgroups of breast tumors.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Enhancing Specific-Antibody Production to the ragB Vaccine with GITRL That Expand Tfh,IFN-γ+ T Cells and Attenuates Porphyromonas gingivalis Infection in Mice

The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ⁺ T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ⁺ T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ⁺ T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ⁺ T cells and antibody production to P. gingivalis.     

The Molecular Events Involved in Oligodendrocyte Precursor Cell Proliferation Induced by the Conditioned Medium from B104 Neuroblastoma Cells

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of oligodendrocyte progenitor cells (OPCs) in vitro. However, the molecular events that occur during B104CM-induced proliferation of OPCs has not been well clarified. In the present study, using OPCs immunopanned from embryonic day 14 Sprague–Dawley rat spinal cords, we explored the activation of several signaling pathways and the expression of several important immediate early genes (IEGs) and cyclins in OPCs in response to B104CM. We found that B104CM can induce OPC proliferation through the activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2), but not PI3K or p38 MAPK signaling pathways in vitro. The IEGs involved in B104CM-induced OPC proliferation include c-fos, c-jun and Id2, but not c-myc, fyn, or p21. The cyclins D1, D2 and E are also involved in B104CM-stimulated proliferation of OPCs. The activation of Erk results in subsequent expression of IEGs (such as c-fos, c-jun and Id-2) and cyclins (including cyclin D1, D2 and E), which play key roles in cell cycle initiation and OPC proliferation. Collectively, these results suggest that the phosphorylation of Erk1/2 is an important molecular event during OPC proliferation induced by B104CM.     

Neuroprotection of Bone Marrow-Derived Mesenchymal Stem Cell-Derived Extracellular Vesicle-Enclosed miR-410 Correlates with HDAC4 Knockdown in Hypoxic-Ischemic Brain Damage

Neurochemical Research - Evidence exists reporting that miR-410 may rescue neurological deficits, neuronal injury, and neuronal apoptosis after experimental hypoxic ischemia. This study aimed to...     

Interleukin-22 Mediates Early Host Defense against Rhizomucor pusilluscan Pathogens

Background

In recent years, the fungal infectious disease zygomycosis has increased in incidence worldwide, especially among the immunodeficient population. Despite the rates of zygomycosis-related death and deformation being very high, the mechanism(s) by which the fungal pathogens cause these severe manifestations remain unknown.

Methods

Using the associated Rhizomucor variabilis species, which can selectively induce cutaneous zygomycosis in otherwise healthy individuals, we investigated the host mechanisms of infection-related responses, including cytokine and chemokine expression as well as contributions of particular T cell subsets. siRNA specifically targeting IL-22,IL-17 and IFN-γ were used to down-regulate expression of those molecules.

Results

In mouse models of infection, IL-22 was implicated in development of Rhizomucor spp.-induced skin lesions. In cultured human peripheral blood monocytes, R. pusilluscan, which is often found in immunodeficient patients, induced the production of IL-22, while R. variabilis did not. Moreover, Rhizomucor spp.-induced secretion of Il-22 from CCR6⁺CCR4⁺CCR10⁺ cells was down-regulated by knockdown of IL-22 related signaling receptors, RORC and ARH.

Conclusion

Our data strongly suggest that avoidance of IL-22 may be one mechanism by which mucor species produce morbidity and mortality in infected individuals.