Effect of hemX gene deletion on heme synthesis in Bacillus amyloliquefaciens / 生物工程学报

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.

Investigation of canine mesenchymal stem cells differentiation to vascular endothelial cell in vitro / 生物医学工程学杂志

To induce endothelial cell, canine bone marrow-derived mesenchymal stem cells (MSCs) were separated from bone marrow by density gradient centrifugation. The isolated MSCs were induced to form endothelial-like cell in the presence of vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and so on. These results showed that the cells uniformly took on a cobblestone morphology under the light microscope, and cell nucleolus was in the middle of the cells. The cells displayed Weibel-Palade bodies under the transmission electron microscope. vWF, a specific marker of endothelial cell was positive in the cells. The above results demonstrate that MSCs may be differentiated into endothelial cells in vitro.

Effects of progesterone on the proliferation of neural stem cells in rats with brain trauma / 中国组织工程研究

BACKGROUND: Brain trauma can stimulate the proliferation of neural stem cells (NSCs) to some extent, while progesterone can ameliorate the learning and memory function following brain trauma, which can also promote the neurofunctional recovery after brain trauma by stimulating the proliferation of NSCs.OBJECTIVE: To observe the effects of progesterone on the proliferation of NSCs after diffuse brain injury (DBI).DESIGN: Randomized control animal experiment.SETTING: Xinxiang Medical College.MATERIALS: Forty-eight healthy male SD rats at 4-5 months with the body mass of 280-330 g were selected.METHODS: The experiment was conducted in Xinxiang Medical College from September 2004 to January 2005. Forty-eight rat models of Marmarou DBI were selected and randomly divided into 4 groups with 12 rats in each group: ①Sham-operation group: rats were cut open the scalp and then sutured.②Brain trauma group: rats were made into animal models of brain trauma.③Dimethyl sulphoxide (DMSO) group: rats were given intraperitoneal injection of DMSO at the same volume as progesterone group at one hour after brain trauma and then the same administration was performed daily. ④Progesterone group: rats were intraperitoneally injected with 4 mg/kg progesterone at one hour after brain trauma and then the same administration was performed daily. Rats were executed respectively at 3 and 6 days after sham operation or brain trauma operation, and hematoxylin-eosin staining was conducted to observe the morphological changes of cortical neurons in brain. The expressions of nestin in dentate gyrus and hippocampus were detected with immunohistochemical staining.MAIN OUTCOME MEASURES: Observation of histomorphological changes of neurons and detection of the expressions of nestin in hippocampus and dentate gyrus.RESULTS: ①There was no injury in cortical neurons in the sham-operation group, while obvious neuronal injury and loss in cortex of rats were found in the 3-day and 6-day brain trauma groups, and the neuronal injury was significantly severer in brain trauma than in 3-day and 6-day progesterone groups. ②The expressions of nestin in hippocampal CA4 region or dentate gyrus of sham-operation group were in low level or little, and the expression of nestin could be seen occasionally in hippocampal CA4 region. The expressions of nestin in hippocampal CA4 region and dentate gyrus of the brain trauma group significantly increased (P ＜ 0.05), while those in the progesterone group increased more than the brain trauma group remarkably (P ＜ O.05).③There were no differences in neuronal injury and nestin expression between braintrauma group and DMSO group(P ＞ 0.05).CONCLUCION: Progesterone for brain trauma may be related with its promoting effects on the proliferation of NSCs.

IL-12 signaling pathway through Lck/ p38 /c-jun in splenocytes from systemic lupus erythematosus mouse / 细胞与分子免疫学杂志

Aim To investigate whether there is IL-12 signaling pathway through lck/P38/c-jun in splenic cells obtained from lupus mouse and its effect on splenic cells. Methods Mice with graft versus host disease were used as lupus nephritis model. Activity of Lck tyrosine kinase, p38 phosphorylation and mRNA expression of c-jun in splenic cells were determined by autoradiography, Western blot and Northern blot, respectively. Results There were higher levels of Lck activity, p38 phosphorylation and c-jun expression of IL-12-stimulated splenic cells from lupus model when compared with that observed in similarly treated splenic cells from normal control. The Lck activity and p38 phosphorylation were almost inhibited by Lck inhibitor PP1, on the other hand, p35 specific inhibitor SB203580 decreased phosphorylation of p38. In addition, expression of c-Jun was also inhibited by PP1 or SB203580 although splenic cells were stimulated with IL-12. Conclusion Aberrant murine splenic cell, IL-12-me-diated intracelluar signaling pathway through lck/p38/c-Jun were involved in immunologic damage.