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Objective@#To investigate the toxic effect of HIV-1 Vpr protein on neurons.@*Methods@#HIV-1 vpr gene was amplified by nested PCR in four parts of peripheral spleen (SPL) and central nervous tissue meninges (MG) of HIV-associated dementia (HAD) patients and non-HAD patients. Eukaryotic expression vector pEGFP-N1-vpr was constructed. The gene sequence and key amino acid sites were analyzed by BLAST and MEGA6. The expression of Vpr protein in N2a cells was detected by Western-blotting. The effects of Vpr proteins from different sources on the activity and cell cycle of N2a cells were studied by flow cytometry.@*Results@#HIV-1 vpr gene was successfully amplified by PCR. Sequence analysis showed that the vpr gene sequence belonged to HIV-1B subtype. There were amino acid mutations at C-terminal 84, 86 and 87 sites of central Vpr protein from HAD and non-HAD patients. Vpr protein could inhibit the activity of nerve cells, leading to G2 phase arrest. Different sources of Vpr had different intensity of action. Compared with other groups, Vpr protein from the meninges of HAD patients showed stronger inhibition of cell activity and G2 phase arrest ability.@*Conclusions@#Variations in key amino acid sites of Vpr protein could cause significant changes in its biological functions, and its significance in the pathogenesis of HAD remains to be further studied.
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Objective@#BG-derived HIV-1 Tat protein from an HIV-associated dementia (HAD) patient was expressed in E. coli BL21(DE3) and purified in order to research the effects on human umbilical vein endothelial cells (HUVECs) activity.@*Methods@#The recombinant plasmid pGEX-KG-tat with HIV-1 tat stored in our laboratory was amplified by PCR. The PCR product was cloned into pET-32a-tat. The recombinant plasmid pET-32a-tat was transfected into E. coli, and Tat protein was expressed in BL21(DE3), which was induced by IPTG. Then it was purified by Ni-chelating chromatography column and gel filtration preloaded column, and identified by SDS-PAGE and Western blot(WB). The concentration was determined by BCA Kit. Different concentrations of Tat were added into HUVECs to detect their effects on cell activity by cck-8.@*Results@#The Tat with high purity was efficiently expressed in BL21 (DE3) and obtained by using the Ni-chelating chromatography column and gel filtration preloaded column. The concentration was 0.47 mg/ml by using BCA Kit. As the concentration of Tat increased, HUVECs activity decreased. There was no significant difference in cells viability between negative control with 100 ng/ml and 200 ng/ml group (P>0.05). There was significant difference in cells viability between negative control with 300 ng/ml, 400 ng/ml, 500 ng/ml and 1000 ng/ml group (P<0.05). But the difference between 300 ng/ml, 400 ng/ml, 500 ng/ml and 1000 ng/ml group was not statistically significant (P>0.05).@*Conclusions@#The HIV-1 Tat with biological activity was efficiently expressed in BL21 (DE3), and the activity of HUVECs was significantly decreased when the concentration reached 300 ng/ml.
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Human immunodeficiency virus-1 (HIV-1) infects the human body and acts on the related cells of blood brain barrier, causing structural and functional abnormalities of blood brain barrier, which will be beneficial to the invasion of the virus into central nervous system. There are many types of the molecular mechanism of damage of blood brain barrier. Tat protein, as HIV-1 trans-activator of transcription, can damage the vascular endothelial cells, pericytes and astrocytes, and reduce the tight junction protein expression. Some chemicals such as methamphetamine can cooperate with HIV-1 Tat protein damage blood brain barrier. In this paper, the effects and its mechanism of HIV-1 Tat protein on blood brain barrier are reviewed.
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Objective@#To study the sequence characteristics and variation of HIV-1 Vpr gene in different parts of an AIDS dementia complex (ADC) patient and provide basis for the study of the neurologic pathogenesis of HIV-1-assciatd dementia.@*Methods@#Genomic DNA was extracted from peripheral samples (lymph nodes, spleen, liver) and central nervous system (meninges, frontal lobe, temporal lobe gray matter, frontal white matter, basal ganglia cortex) of an ADC patient, The Vpr gene was amplified with nested polymerase chain reaction (PCR). PCR products were cloned into the pMD19-T vector. After transformation into DH5α competent E. coli, five positive clones were sequenced. The phylogenetic tree was built and genetic distance was calculated through MEGA6, and the values of ds/dn was calculated through SNAP, then the changes of the amino acid sites were analyzed.@*Results@#HIV-1 Vpr genes isolated from different tissues of the ADC patient had variations. Vpr HIV-1 gene sequences from central nervous system and peripheral tissues were intercrossed together in the phylogenetic tree. Central nervous system and peripheral HXB2 Vpr had no significant differences in genetic distance. The ds/dn of all the HIV-1 Vpr gene sequences were 3.3749.@*Conclusions@#The HIV-1 Vpr sequences were different in the ADC patient, and there were different variations in different parts of the peripheral and central regions. Whether these variations are related to the pathogenesis of ADC remains to be further studied.
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[ ABSTRACT] AIM:To investigate the effect of Jia-jian-yi-yin decoction on endothelium-dysfunction in ovariecto-mized rats.METHODS:The ovariectomized rats were treated with Jia-jian-yi-yin decoction or turbid liquid of estradiol va-lerate for 8 weeks.The vascular ring tension was measured.Scanning electron microscopy and Western blotting were ap-plied to assess the function of endothelium-dependent dilation, aortic endothelial morphology and the expression of endothe-lial lipase.The pathologic sections were prepared to observe the effect of Jia-jian-yi-yin decoction on the uterus.RE-SULTS:In ovariectomized rats, the decrease in endothelium-dependent relaxation to acetylcholine ( ACh) was reversed to normal level, the endothelial morphology returned to normal without lipid accumulation and the endothelial lipase expression was decreased by Jia-jian-yi-yin decoction.Furthermore, no obvious change of the wet weight of uterine between the ovari-ectomized rats with or without Jia-jian-yi-yin decoction treatment was observed.CONCLUSION:Jia-jian-yi-yin decoction may have protective effects on endothelium-dependent vasodilation and aortic endothelial morphology in estrogen-deficient animals.
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Objective To study the clinical effects and side effects of recombinant human vascular endostatin combined with pemetrexed and cisplatin in the treatment of advanced lung adenocarcinoma.Methods Forty-seven patients with advanced lung adenocarcinoma were recruited from Jan 2011 to Jan 2013,and they were randomly divided into experimental group and control group.The experimental group (n =24) was added with recombinant human vascular endostatin based on pemetrexed and cisplatin,whereas the control group(n =23) was administered with pemetrexed and cisplatin only.The objective response rate (ORR),disease control rate (DCR),progressive disease (PD) rate,progression-free survival (PFS),overall survival (OS) and the side effects of 2 groups were evaluated.Results In the experimental group,ORR,DCR,PD rate,PFS and OS were 41.7 ％ (10/24),79.2 ％ (19/24),20.8 ％ (5/24),8.0 months and 12.5 months respectively,while those of control group were 34.7 ％ (8/23),47.8 ％ (11/23),52.2 ％ (12/23),6.2 months and 10.0 months.DCR,PD rate and PFS of experimental group had significant differences compared with control group (P ＜ 0.05).OS of experimental group had no significant difference compared with control group (P ＞ 0.05).The side effects of 2 groups were mainly hematologic toxicities,digestive reactions and fatigue,and the incidence rates were not significantly different between 2 groups (P ＞ 0.05).Conclusions Recombinant human vascular endostatin combined with pemetrexed and cisplatin in treatment of patients with advanced lung adenocarcinoma improves the DCR,decreases the PD rate,prolongs the PFS.There is an increasing trend in the OS of experimental group,and with tolerable side effects.
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Objective To evaluate the effects of various head and neck postures and phonation on oropharyngeal view(Mallampati score),and ex-plore the correlation with laryngoscopic view tested by the improved Cormach-Lehane score(MCLS). Methods Following the local ethics commit-tee approval and patients′informed consent to anesthesia,124 patients were enrolled for this study. Prior to anesthesia,these patients were placed in supine position with various head and neck positions for oropharyngeal structures visualization and evaluation,including two head positions(neu-tral and full extension),two tongue positions(in and out),and with and without phonation,according to the modified Mallampati test score(MMT). Following induction,laryngoscopic view scores according to MCLS were recorded,then the sensitivity,specificity,positive and negative predictive value and accuracy of the various MMT scores were calculated. Correlation coefficient(r)of MMT scores with MCLS were also studied. Results Mallampati score was decreased in all the postures of head full extension,tongue outside and phonation,which makes oropharyngeal structures to be better visualized. There is no correlation between MMT scores and MCLS. The sensitivity,specificity,positive and negative predictive value and accu-racy of the various MMT scores is most satisfied in the posture of head full extension,tongue outside and phonation. Conclusion During airway as-sessment in supine position,the best posture is head full extension,tongue outside and phonation.