AIM: The main purpose of this study is to investigate the regulatory role of C/EBPα in mouse vesicular glutamate transporter 2(mVGLUT2) gene expression. METHODS: The promoter region of mVGLUT2 was cloned to PGL3-basic vector. Site-direction mutation was used to identify the CCAAT-enhancer-binding protein α(C/EBPα) binding site. The promoter activity was observed by luciferase system. The binding between C/EBPα protein and mVGLTU2 promoter region was determined by EMSA. The C/EBPα gene expression was inhibited by its specific siRNA. RESULTS: mVGLUT2 promoter activity decreased about 50% after mutation of C/EBPα binding site. EMSA showed that C/EBPα protein bound onto mVGLUT2 promoter region. Meanwhile, when C/EBPα gene expression was inhibited by its specific siRNA, mVGLUT2 promoter activity, mRNA level and protein level were decreased about 60%, 40% and 45%, respectively. CONCLUSION: C/EBPα is involved in the regulation of mVGLUT2 gene expression.

Marburg virus (MARV) is a member of the Filoviridae family and belongs to a non-segmented, single-strand and negative-sense RNA virus.Since the first discovery of virus in 1967, infections have broken out 14 times, causing the infection of 588 people and 482 deaths.The mortality is up to 82%.Marburg virus results in multiple organ infections , severe hemorrhagic fever and death .Currently, there are no available licensed vaccines or post-exposure treatment , but the vaccines have proved effective in experimental animals .This review briefly summarizes the structure , infection mechanism and the progress in vaccines of this virus .

AIM:The main purpose of this study is to investigate the regulatory role of C/EBP? in mouse vesicular glutamate transporter 2(mVGLUT2) gene expression. METHODS:The promoter region of mVGLUT2 was cloned to PGL3-basic vector. Site-direction mutation was used to identify the CCAAT-enhancer-binding protein ?(C/EBP?) binding site. The promoter activity was observed by luciferase system. The binding between C/EBP? protein and mVGLTU2 promoter region was determined by EMSA. The C/EBP? gene expression was inhibited by its specific siRNA. RESULTS:mVGLUT2 promoter activity decreased about 50% after mutation of C/EBP? binding site. EMSA showed that C/EBP? protein bound onto mVGLUT2 promoter region. Meanwhile,when C/EBP? gene expression was inhibited by its specific siRNA,mVGLUT2 promoter activity,mRNA level and protein level were decreased about 60%,40% and 45%,respectively. CONCLUSION:C/EBP? is involved in the regulation of mVGLUT2 gene expression.

Objective To clone a new conotoxin Bt14.10 from Conus betulinus derived from the South China Sea, synthesize the peptide , and to determine linkage of its disulfide bridges .Methods The genomic DNA was extracted from C.betulinus venom duct while the Bt14.10 sequence was cloned using primers designed based on the untranslated region and intron.The peptide was then synthesized using solid-phase method and folded into the target product whose disulfide bridge connection was further determined by two-step oxidative folding .Results A novel conotoxin designated as Bt 14.10 (CAHSVPGMHPCKCNNTC-NH2) was obtained,the disulfide connectivity of which was C1-C3,C2-C4.Conclusion Bt14.10 is a new A-superfamily conotoxin and has a distinct loop spacing pattern between cysteines in A-superfamily conotoxins.

Objective To screen small fragments with high antigenicity in order to overcome the defects of the full-length Marburg virus GP as a vaccine antigen.Methods Based on the structure and function of GP sequence [amino acids(aa) 1-681], three small fragments,including GP1△(aa 25-239), GPM(aa 250-520) and GP2△(aa 436-648), were expressed by prokaryotic cells and immunized into mice, and the serum specific antibodies were detected after different immunization time.The proliferation of spleen lymphocytes and the concentration of cytokines of immunized mice were also measured.Results ELISA test results showed that high humoral immunity of GP2△ was produced as the full-length GP group did, and was higher than that of GP1△ and GPM (P<0.05).GP2△ immunization groups exhibited a higher SI value in mouse splenic lymphocytes stimulated by ConA than the mixed GP immunization groups did(P<0.05), but the effect was the opposite when mouse splenic lymphocytes were stimulated by the mixed GP.In addition, the amount of cytokines IL-2 and IFN-γ of mouse splenic lymphocytes in the GP2△ group was larger than that of the saline group (P<0.05), but smaller than that of the mixed GP group.Conclusion The fragment GP2△ can induce not only the high humoral immunity as GP does,but also moderate cellular immunity, which can be used for vaccine design.

Objective To discover novel conopeptides which are the antagonists of neuronal nicotinic acetylcholine receptors (nAChRs) in order to contribute to the development of novel analgesic drugs and neuropharmacological probes.Methods Based on the conserved untranslated region and intron of A-superfamily conotoxins,a novel α-conotoxin Lt1.1 was cloned from Conus litteratus.The peptide-resin was synthesized using the solid-phased method and was cleaved.The resulting linear peptide was oxidized by air to give the product containing disulfide bridges.The folding product was finally purified by HPLC.The disulfide bond connectivity was determined using the two-step oxidative folding methods.The cRNA of rat nAChRs was expressed on the membrane of Xenopus oocyte.Membrane currents were recorded using the two electrode voltage-clamp technique.Results A novel α-conotoxin designated as Lt1.1(GCCSHPACNVNNPDIC-NH2) was cloned and its disulfide connectivity was C1-C3,C2-C4.Lt1.1 selectively inhibited the α3β2 and α3β4 nAChRs with an IC50 of 166.76 and 190.00 nmol/L,respectively.Conclusion Lt1.1 is a novel 4/7 α-conotoxin that selectively targets α3β2 and α3β4 nAChRs.

Objective:Our aim was to study the levels of atrial natriuretic peptide (ANP) and pulmonary perfusion imaging in determining of pulmonary artery hypertension (PAH). Methods: We measured ANP levels by using radioimmunoassay after making various types of PAH in rabbits. Catheterization and pulmonary perfusion tomographic imaging were used in the control group. Results: When the pulmonary artery pressure increased slightly, the ANP levels were lower in PAH group than that of control group, but the difference was not significant (t=1, P>0.05). At the same time, pressure measured by catheterization did not change as the control group, but the ratio of back/abdomen radiopharmaceuticals distribution which was measured with pulmonary perfusion tomographic imaging was significantly higher in PAH group than that of the control (t=2.5, P<0.05). The difference between the ANP levels when the pulmonary artery pressure increased medially and seriously was significant (t＝4.0 and 6.5, P＜0.05). The other two methods got the same result (P＜0.05). Results of three examining methods were positively related. Conclusion:The ANP levels can determine the degree of PAH, and it was simple. But it was not as sensitive as that of pulmonary perfusion tomographic imaging.

To generate a mWAP-hLF hybrid locus that the transcription of human lactoferrin (hLF) genomic sequence is directed by the up & down stream regulatory sequence of murine whey acidic protein (mWAP) gene locus, we describe here a successive three-step 'Gap-repair' method. First, a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed. Then using 'Gap-repair 'method mediated by Red recombination system of lambda-prophage in Escherichia coli, in the first step, the 8 kb 3' flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus(mWAP BAC) into the gap-repair vector; in the second step, the 29 kb hLF genomic sequence from the ATG code to the TAA code was subcloned from the hLF BAC; in the third step, the 12 kb 5' flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a 49 kb mWAP-hLF hybrid locus that the hLF genomic sequence was flanked by the 5' & 3' flanking region of mWAP gene locus was constructed. The result was confirmed by PCR, restriction enzyme digestion and sequencing. Our method provide a new way for the construction of large mammary-gland expression vector.
Animals ; Bioreactors ; DNA Repair ; genetics ; Genetic Engineering ; methods ; Humans ; Hybridization, Genetic ; Lactoferrin ; genetics ; Mammary Glands, Animal ; metabolism ; Mice ; Mice, Transgenic ; Milk Proteins ; genetics

Objective To detect the expression of Beclin1 and Bcl2 in invasive pituitary adenomas and to explore the relationship of Beclin1 and Bci2 in invasive pituitary adenomas and the relativity between the 2 genes.Methods 61 specimens were classified into invasive group (32 cases) and non-invasive group (29 cases) according to the comprehensive evaluation of invasive pituitary adenomas.lmmunofluorescence analysis and RT-PCR were adopted respectively to detect the protein and mRNA expressions of Beclinl and Bcl2.The difference and relativity of Beclin1 and Bcl2 expression in invasive group and non-invasive group were analyzed.Results 32 specimens of pituitary adenoma were invasive and 29 were non-invasive.Beclin1 protein and mRNA expressions were lower in the invasive group than in the non-invasive group (P ＜0.01 ).Bcl2 protein and mRNA expressions were higher in the invasive group than in the non-invasive group (P ＜0.01 ).Pearson related analysis showed that Beclin1 mRNA expression was negtively correlated with Bcl2 mRNA expression in the invasive group ( r =-0.42,P =0.028 ).Conclusions Beclinl expression is decreased in invasive pituitary adenomas.The invasiveness of pituitary adenoma is closely related to the high expression of Bcl2 protein and mRNA,and the low expression of Beclin1 protein and mRNA.The inhibition of the autophagy may lead to the enhancement of the invasiveness of pituitary adenomas and that inhibition may come from the interaction of Beclin1 and Bcl2.