Chloramphenicol improved expression of recombinant cholera toxin B subunit in Escherichia coli and its adjuvanticity.

BACKGROUND: Cholera toxin B subunit (CTB) was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine. METHODS: Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl ß-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ (IFN-γ) enzyme-linked immunospot (Elispot) assay was used to read out the specific T-cell immunity. RESULTS: Chloramphenicol was essential for the efficient expression of recombinant CTB (rCTB) in pET-30a/BL21 (DE3) system and could be optimized at the concentration of 0.625 µg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group ((734 ± 240) spot forming cells/10(6) splenocytes) was higher than that induced by non-adjuvanted ((520 ± 150) spot forming cells/10(6) splenocytes), all responses against different antigens were enhanced in parallel. CONCLUSION: CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancing the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.

[In vitro co-cultivation of Toxoplasma gondii tachyzoites with rat brain astrocytes].

Purified astrocytes were cultured in plates. When astrocytes grew over 80% of the plate, tachyzoites of Toxoplasma gondii RH strain were added for co-culture. In the period of 0-72 h, change of the astrocytes and tachyzoites was observed after Giemsa staining. In 0-48 h, monodansylcadaverine (MDC) was used to study the action of autophagy in the process of tachyzoites invading astrocytes. At 1 h co-culture, tachyzoites had entered in astrocytes and the autophagosomes appeared. At 4 h, the autophagosomes increased pronouncedly. However, after 12 h, number of autophagosomes considerably decreased and damage of the cells occurred. 48 h later, autophagosomes disappeared and more astrocytes were destroyed. At 72 h most cells destroyed and tachyzoites were released. The result showed that autophagy is inhibited when the astrocytes were in vitro infected by tachyzoites.

[Tropism of bone marrow mesenchymal stem cells in neurogenic differentiation for C6 conditioned medium and SDF-1alpha].

AIM: To investigate the tropism of bone marrow mesenchymal stem cells (BMSCs) in neurogenic differentiation for glioma and SDF-1alpha. METHODS: BHA, bFGF, and DMSO were used to induce the neural differentiation of BMSCs, and the expression of neural markers, Nestin, beta-III-Tubulin and NSE were analyzed by immunocytochemistry. We investigated, by using the Dunn chamber, the migration of BMSCs in neurogenic differentiation towards glioma and SDF-1alpha. RESULTS: BMSCs could be induced to differentiate into neuron-like cells, while the control remained the morphology of BMSCs. Dunn chamber analysis revealed that both the migration speed and efficiency of cells induced by C6 conditioned medium and SDF-1alpha were higher than control, demonstrating the chemotactic effect of both C6 conditioned medium and SDF-1alpha on BMSCs. These results were confimed by examination of migration tracks of individual cells. Moreover, BMSCs at different states of differentiation showed different degree of tropism for C6 conditioned medium and SDF-1alpha. CONCLUSION: The directed migration of BMSCs is closely related to the differentiation states of these cells.

Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle.

In this study, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are used as models of agamogony and syngamy, respectively. In order to elucidate the reasons of low efficiency of somatic cell cloning, cytoskeletal and nuclear organization in cloned mouse embryos was monitored before and during the first cell cycle, and compared with the pattern of ICSI zygote. A metaphase-like spindle with alignment of condensed donor chromosomes was assembled within 3 hr after NT, followed by formation of pronuclear-like structures at 3-6 hr after activation, indicating that somatic nuclear remodeling depends on microtubular network organization. The percentage of two (pseudo-) pronuclei in cloned embryos derived from delayed activation was greater than that in immediate activation group (68.5% vs. 30.8%, P<0.01), but similar to that of ICSI group (68.5% vs. 65.5%, P>0.05). The 2-cell rate in NT embryos was significantly lower than that in zygotes produced by ICSI (64.8% vs. 82.5%, P<0.01). Further studies testified that the cloned embryos reached the metaphase of the first mitosis 10 hr after activation, whereas this occurred at 18 hr in the ICSI zygotes. Comparision of the pattern of microfilament assembly in early NT embryos with that in syngamic zygotes suggested that abnormal microfilamental pattern in cloned embryos may threaten subsequent embryonic development. In conclusion, agamogony, in contrast to syngamy, displays some unique features in respect of cytoskeletal organization, the most remarkable of which is that the first cell cycle is initiated ahead distinctly, which probably leads to incomplete organization of the first mitotic spindle, and contributes to low efficiency of cloning.

Attachment and growth of human bone marrow derived mesenchymal stem cells on regenerated antheraea pernyi silk fibroin films.

Silk fibroin of the silkworm Bombyx mori has been studied extensively, while the research on Antheraea pernyi silk fibroin (A. pernyi SF) in biomaterials is only at an early stage. In this study, the attachment, morphology, growth and phenotype of human bone marrow derived mesenchymal stem cells (hBMSCs) cultured on the regenerated A. pernyi SF films were studied in vitro. The results indicated that the attachment of hBMSCs on the regenerated A. pernyi SF films was almost the same as that on the collagen films. MTT and cell counting analyses demonstrated that the growth of hBMSCs on the regenerated A. pernyi SF films was better than that on controls. Moreover, electron scanning microscopy and fluorescence-activated cell sorting assays showed that the regenerated A. pernyi SF supported hBMSCs growth and functional maintenance compared with the controls. These data suggest that the regenerated A. pernyi SF, like Bombyx mori silk fibroin (B. mori SF) and collagen, can support hBMSCs attachment, growth and phenotypic maintenance, and has better biocompatibilities for hBMSCs in vitro culture.

Effects of anti-leukemia drug Harringtonine on the levels of centromere proteins and gene expression of CenpB in L1210 cells.

In this study, we investigated the effects of the anti-leukemia drug Harringtonine (HT) on the levels of centromere proteins and gene expression of centromere protein CenpB in L1210 cells. The intensity of centromere fluorescence, shown by indirect immunofluorescence staining, decreased with HT treatment. Western blot showed that the anti-centromere antibody (ACA) serum used in this study recognized 8 proteins with different molecular weights: 140, 80, 70, 56, 37, 34, 32 and 17 kD. The amounts of some of these proteins were reduced to different extents by HT treatment. The ACA antibodies that recognized the 17, 80 and 140 kD proteins in L1210 cells also cross-reacted with 3 proteins with similar molecular weights which are known to be CenpA, CenpB and CenpC, respectively. Northern and Dot blot analyses revealed that the level of CenpB mRNA in HT-treated cells was markedly lower than that in the untreated cells. These results suggest that HT may cause a decrease in the intracellular level of some centromere proteins, probably by inhibiting mRNA expression of corresponding genes. Moreover, it is possible that the cell-killing and appotosis-inducing effects of HT may be mediated by the inhibition of the expression of CenpB and other centromere protein genes.