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ABSTRACT: To construct the recombinant prokaryotic expression plasmid pET/c-ABCSP-Aβ(15-c);, and evaluate the immunogenicity of the fusion protein expressed in E.coli.
The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The APP beta cleavage site peptide(ABCSP) and Aβ(1-15); gene(ABCSP-Aβ(15);) was amplified by PCR and inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-17-ABCSP-Aβ(15); were connected with HBc88-144, yielding the recombinant gene c-ABCSP-Aβ(15-c);. c-ABCSP-Aβ(15-c); gene was subcloned into pET-28a(+).The fusion protein expressed in transformed E.coli BL21 was induced with IPTG and analyzed by SDS-PAGE. The virus-like particles (VLP) formed by fusion protein was observed with Transmission Electron Microscope (TEM). 4 Kunming (KM) mice received intraperitoneal injection (i.p) of fusion protein VLP. The antibody was detected by indirect ELISA.
The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, fusion protein was expressed and mainly existed in the sediment of the bacterial lysate. The expression level was 40% of all the proteins in the sediment. The fusion protein could form VLP. After 5 times of immunization, the titer of anti-ABCSP and anti-Aβantibody in sera of KM mice reached up to 1:5 000 and 1:10 000 respectively, while the anti-HBc antibody was undetectable.
Recombinant c-ABCSP-Aβ(15-c); gene can be expressed in E.coli. The expressed protein could form VLP and has a strong immunogenicity. This study lays the foundation for the study of AD genetic engineering vaccine.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 11/2011; 27(11):1169-72.
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ABSTRACT: To study the effects of immunization with the fusion protein CAC (a product of prokaryotic expression of recombinant HBcAg and β-amyloid peptide fusion gene) against the toxicity induced by intrahippocampal injection of aggregated β-amyloid peptide (Aβ) in rats.
SD rats were immunized intraperitoneally with the fusion protein CAC, and the titer of anti-Aβ antibody was evaluated by ELISA. When the titers of the anti-Aβ antibody reached 1:3 000, aggregated Aβ was injected into the CA1 region of the rat hippocampus. Two weeks after Aβ injection, the rats underwent morris water maze test before sacrificed to prepare the brain slices with Congo red and haematoxylin staining.
The titer of anti-Aβ antibody reached 1:3 000 after 5 immunizations with the fusion protein. After Aβ injection, the saline-immunized rats showed a reduced cognitive behavior in the Morris water maze test compared to the CAC-immunized rats. In the saline-immunized rats, the neurons around the site of Aβ injection exhibited obvious cell damages with Aβ deposits and glial infiltration, whereas in CAC-immunized rats, Aβ deposits were significantly reduced or even absent.
Immunization with the fusion protein CAC can inhibit the toxicity induced by intrahippocampal aggregated Aβ injection.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 07/2011; 31(7):1236-9.
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ABSTRACT: To investigate the effect of [Gly14]-Humanin overexpression on Abeta(25-35);-induced PC12 cell apoptosis.
Recombinant plasmid pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells by liposome method. The subclone cell lines were obtained by persistent G418 selection. The HNG gene expression of PC12 cells was detected by immunocytochemistry. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability was determined by MTT assay, and apoptosis rate was detected by using flow cytometric analysis. Hochest33258 staining was used to observe the morphological changes of cellular nuclei.
PC12 cell lines stably expressing HNG gene was successfully selected. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability of PC12 cells overexpression HNG was significant elevated compared with empty plasmid transfected cells (P<0.05), and the apoptosis rate was lower significantly (P<0.05). By Hoechst 33258 staining, the nuclei of empty plasmid transfected PC12 cells exhibited highly condensed and fragmented nuclei morphology which was the typical characteristics of apoptosis, and the nuclei of PC12 cells overexpression HNG were round and homogeneously stained.
Overexpression of HNG prevented the cell apoptosis induced by Abeta(25-35); in PC12 cells.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2010; 26(5):427-30.
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ABSTRACT: Mitochondrial dysfunction is a hallmark of beta-amyloid (Aβ)-induced neuronal toxicity in Alzheimer's disease (AD), and is considered as an early event in AD pathology. Humanin (HN) and its derivative, [Gly14]-Humanin (HNG), are known for their ability to suppress neuronal death induced by AD-related insults in vitro and in vivo. In the present study, we investigated the neuroprotective effects of HNG on Aβ25–35-induced toxicity and its potential mechanisms in PC12 cells. Exposure of PC12 cells to 25μM Aβ25–35 caused significant viability loss and cell apoptosis. In addition, decreased mitochondrial membrane potential and increased cytochrome c releases from mitochondria were also observed after Aβ25–35 exposure. All these effects induced by Aβ25–35 were markedly reversed by HNG. Pretreatment with 100nM HNG 6h prior to Aβ25–35 exposure significantly elevated cell viability, reduced Aβ25–35-induced cell apoptosis, stabilized mitochondrial membrane potential, and blocked cytochrome c release from mitochondria. Furthermore, HNG also ameliorated the Aβ25–35-induced Bcl-2/Bax ratio reduction and decreased caspase-3 activity in PC12 cells. These results demonstrate that HNG could attenuate Aβ25–35-induced PC12 cell injury and apoptosis by preventing mitochondrial dysfunction. Furthermore, these data suggest that mitochondria are involved in the protective effect of HNG against Aβ25–35.
Neurochemistry International - NEUROCHEM INT. 01/2010; 56(3):417-423.
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ABSTRACT: Mitochondrial dysfunction is a hallmark of beta-amyloid (Abeta)-induced neuronal toxicity in Alzheimer's disease (AD), and is considered as an early event in AD pathology. Humanin (HN) and its derivative, [Gly14]-Humanin (HNG), are known for their ability to suppress neuronal death induced by AD-related insults in vitro and in vivo. In the present study, we investigated the neuroprotective effects of HNG on Abeta(25-35)-induced toxicity and its potential mechanisms in PC12 cells. Exposure of PC12 cells to 25 microM Abeta(25-35) caused significant viability loss and cell apoptosis. In addition, decreased mitochondrial membrane potential and increased cytochrome c releases from mitochondria were also observed after Abeta(25-35) exposure. All these effects induced by Abeta(25-35) were markedly reversed by HNG. Pretreatment with 100 nM HNG 6h prior to Abeta(25-35) exposure significantly elevated cell viability, reduced Abeta(25-35)-induced cell apoptosis, stabilized mitochondrial membrane potential, and blocked cytochrome c release from mitochondria. Furthermore, HNG also ameliorated the Abeta(25-35)-induced Bcl-2/Bax ratio reduction and decreased caspase-3 activity in PC12 cells. These results demonstrate that HNG could attenuate Abeta(25-35)-induced PC12 cell injury and apoptosis by preventing mitochondrial dysfunction. Furthermore, these data suggest that mitochondria are involved in the protective effect of HNG against Abeta(25-35).
Neurochemistry International 11/2009; 56(3):417-23. · 2.86 Impact Factor
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ABSTRACT: The gentamicin-induced pathological alteration in the cochlear nucleus is not exclusively a secondary consequence of the damage in the cochlea. Instead, the toxic effect of gentamicin on the cochlear nucleus may occur simultaneously or even earlier than that on the cochlea.
To investigate the pathological alteration of cochlear nucleus neurons in guinea pigs following systemic application of gentamicin.
Guinea pigs were injected with gentamicin for 1 day, 3 days, 1 week, 2 weeks, and 3 weeks, respectively. In gentamicin-treated animals, the hearing function was evaluated by measuring the auditory brainstem response (ABR). The number and cross-sectional area of substance P-positive neurons in the cochlear nucleus were also measured.
The threshold of ABR and the number of substance P-positive neurons in the cochlear nucleus were significantly increased after 1 week and 3 days of injection of gentamicin, respectively. The cross-sectional area of substance P-positive neurons in the cochlear nucleus was significantly reduced after 1-day injection of gentamicin.
Acta oto-laryngologica 11/2008; 129(7):745-8. · 0.98 Impact Factor
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ABSTRACT: To explore the neuroprotective action of vasoactive intestinal peptide (VIP) on ischemia and reperfusion in the rat.
VIP was given via intracerebroventriclar injection after a 2 hour transient middle cerebral artery occlusion using filament model. The infarct volume was investigated with TTC stain. Apoptosis in the ischemic boundary zone were evaluated with TUNEL stain. Western blotting were used to analyze the iNOS protein expression as well.
After VIP injection, the relative infarct volume of rats was significantly reduced by approximately 28% campared to that of the control groups at 1 day (P < 0.05). The number of TUNEL positive cells was significantly decreased in the ischemic boundary zone, and then the expression of iNOS was remarkablely decreased as well (P < 0.05).
VIP has a neuroprotective effect on cerebral ischemia and reperfusion. The mechanism seems to involve decreasing the apoptosis and down-regulating the iNOS expression.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 08/2008; 39(4):563-6.
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ABSTRACT: The present study aimed at investigating the effects of chronic multiple stress on learning and memory functions of rats. Adult male Wistar rats were randomly divided into stressed and control groups. Rats in the stressed group were irregularly and alternately exposed to the situation of vertical revolution, sleep deprivation, noise stimulation, and night illumination 6 h per day for 6 weeks to prepare a chronic multiple stressed model. Learning and memory performance of rats was measured by using Morris water maze first and Y-maze afterwards. Neurons in the dentate gyrus(DG), CA3 and CA1 regions of the hippocampus were stained by using Cresyl violet method and counted. The results showed that: (1) After chronic multiple stress, compared with the control rats, the escape latency to the hidden platform in Morris water maze was significantly shortened in stressed rats. In stressed and control groups, the escape latency periods were (15.89+/-9.15) s and (27.30+/-12.51) s, respectively, indicating that spatial memory of the stressed rats was stronger than that of the control ones. In brightness-darkness discrimination learning in the Y- maze, the correct trials and correct percentage of entering safe arm was remarkably increased in the stressed rats, the correct rates of stressed and control groups were (79.01+/-1.23)% and (66.12+/-1.61)%, respectively, indicating that brightness-darkness discrimination learning ability of the stressed rats was better than that of the control ones. (2) After chronic multiple stress, nerve cell density in DG, CA1 and CA3 of the hippocampus in stressed rats was higher than that of the control group, the cell densities in DG, CA1 and CA3 of the stressed and the control group were (223.78+/-26.52), (112.07+/-14.23) and (105.55+/-18.12) as well as (199.13+/-15.36), (92.89+/-13.69), and (89.02+/-15.77) respectively. These results suggest that the chronic multiple stress may enhance the capability of spatial memory and brightness-darkness discrimination learning of rats. Possible reasons for the chronic multiple stress-induced learning and memory enhancement of rats were also discussed.
Sheng li xue bao: [Acta physiologica Sinica] 11/2004; 56(5):615-9.
