Effect of pre-electroacupuncture at "Zusanli" (ST 36) on DA and 5-HT contents and their ratio in hypothalamus and striatum in exercise rats / 中国针灸

<p><b>OBJECTIVE</b>To observe anti-fatigue effect and mechanisms of pre-electroacupuncture (EA) at "Zusanli" (ST 36) in rats undergoing acute treadmill running.</p><p><b>METHODS</b>Fifty male SD rats were randomly divided into three groups: a quiet group (group Q, n = 10), a model group (group M, n = 20) and an EA preconditioning group (group EAP, n = 20). After adaptation for undergoing treadmill running, all the rats in group M and group EAP were trained on acute treadmill running. Besides, EA with continuous waves, 2 Hz in frequency and 2 mA in intensity was applied at bilateral "Zusanli" (ST 36) for 30 min, which was applied once daily for continuous 6 days before treadmill running for the rats in Group EAP. Plasma lactate contents were measured immediately and 3 hours after treadmill running, respectively. Changes of dopamine (DA) and serotonin (5-HT) contents obtained immediately and 3 hours after treadmill running, respectively, in hypothalamus and striatum, were detected and compared, and DA/5-HT ratios were calculated.</p><p><b>RESULTS</b>Compared with group Q, the levels of blood lactate and hypothalamic 5-HT tented to increase in rats of group M, and the contents of hypothalamic DA increased significantly (P < 0.01), while the contents of striatal DA and 5-HT in group M decreased significantly (both P < 0.01) at 3 h after treadmill running. Immediately after treadmill running, the contents of DA and 5-HT increased significantly in hypothalamus (both P < 0.01), but decreased significantly in striatum (both P < 0.01) in group EAP, compared with those in group M. Moreover, EA pretreatment markedly decreased the levels of blood lactate (P < 0.05) and hypothalamic 5-HT (P < 0.01), and obviously elevated the ratio of DA/5-HT in the hypothalamus (P < 0.01) at 3 h after treadmill running.</p><p><b>CONCLUSION</b>Preventive EA at "Zusanli" (ST 36) can accelerate recovery from fatigue, which may be related to its reducing accumulation of blood lactate, elevating DA/ 5-HT ratio in the hypothalamus of the rats undergoing treadmill running.</p>

Prokaryotic Expression and Functional Study of HIV-1 Envelope Glycoprotein gp41 Helical Bundle / 中国生物工程杂志

HIV-1 envelope glycoprotein gp41,which is a hopeful target for HIV-1 fusion inhibitors,plays a critical role in the fusion of viral and cellular membranes.In order to build up the screening assay of HIV-1 fusion inhibitors targeting gp41,HIV-1 gp41 5-helix and 6-helix were expressed in prokaryotic cells.Gp41 5-helix and 6-helix recombined plasmids were constructed by using PCR,enzyme digestion and ligation taking the clade B HIV-1 genome as a template.The plasmid was transferred into E.coli BL21(DE3)and then induced by IPTG.The expressed protein was purified by affinity chromatography after denaturation and renaturation.The SDS-PAGE analysis was used during expression and purification.Native-PAGE was used to identify the interaction between gp41 5-helix and T-20.The result will be helpful to build up the screening assay of HIV-1 fusion inhibitors targeting gp41.

Two-Step MS-PCR Combined With ELISA Method for the Detection of Drug Resistance Mutations in HIV-1 RT Gene / 中国生物工程杂志

Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years,the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. A novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR),and point mutations including M41L,K70R,K103N,Y181C,T215F were detected. A longer mutant type primer was designed,using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity,low cost,relatively less time consumption and high-throughput screening. It will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.

Prokaryotic Soluble Expression and Functional Study of HIV-1 Integrase Protein / 中国生物工程杂志

The pol gene of HIV-1 encodes mainly three enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no anti-IN drug was approved. HIV-1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multi-drug therapy of AIDS. The 288 amino acids of IN (32kDa) recognizes specific sequences in the long terminal repeats (LTRs) of the retroviral DNA. The IN protein catalyzes the 3′-processing step and the 5′-strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV-1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wild-type enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV-1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV-1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and NdeI/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET-28a(+) to form a recombined plasmid, transferred into the host cell E.coli(BL21 DE3). The recombined clones were identified by PCR and Nde I/Xho I digestion .The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was puriied sy the Co+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and expressed highly and stably in E.coli. The molecular weight of the expression product was identical to the expectation. The IN protein was proved to be functional in 3′ processing and 5′strand transfer by ELISA. It will be helpful to build the platform of screening inhibitors against HIV-1 integrase.