闽东地区畲族人群CYP2C19基因多态性分析

目的：调查闽东地区汉族与畲族人群CYP2C19基因多态性分布情况。方法：采用限制性片段长度多态性聚合酶链反应（PCR-RFLP）,对汉族（n=140）与畲族（n=180）人群CYP2C19两个多态性位点CYP2C19·2和 CYP2C19·3的基因多态性进行分析,用SPSS16.0软件进行统计分析,Hardy-Weinberg遗传平衡定律验证基因型分布情况。结果：汉族与畲族人群各种基因型出现频率分别为：·1/·1 14.3%,28.9%;·1/·2 28.6%,30.0%;·1/·3 11.4%,16.7%;·2/·2 21.4%,9.4%;·2/·3 21.4%,12.8%;·3/·3 2.9%,2.2%。汉族与畲族CYP2C19不同等位基因出现频率：·1为34.3%,52.2%;·2为46.4%,30.8%;·3为19.3%,16.9%。结论：闽东地区畲族与汉族CYP2C19·2与·3等位基因突变率存在差异,畲族人群基因突变率低于汉族（P＜0.05）。     

Inhalation delivery dramatically improves the efficacy of topotecan for the treatment of local and distant lung cancer

Topotecan is potent anti-cancer drug approved for various malignancies but hematopoietic toxicities undermine its wider application and use of its most effective dose. This study aims to improve these limitations through inhalation-delivery. The pharmacokinetics, efficacy, and toxicity of 2–5 times lower inhalation doses of topotecan dry-powder were compared with the standard intravenous (IV) delivery once/twice-a-week. Human-derived EGFR-mutant (H1975), KRAS-mutant (A549), and EGFR/KRAS wild-type (H358) orthotopic and distant lung tumors were evaluated in murine models. Inhalation of 1 mg/kg topotecan significantly improved the half-life and drug exposure (area under the curve, AUC) compared to 5 mg/kg via IV-delivery. AUCs (h*ng/mL) for inhaled/IV topotecan in plasma, lung, liver, and brain were, 831/888, 60,000/1080, 8380/4000, and 297/15, respectively; while the half-life was also greatly increased in these tissues. The average lung tumor burden of H358-derived tumors was reduced from 15.0 g to 8.4 g (44%) in rats treated once-a-week with 2 mg/kg IV and 1.8 g (88%) with 1 mg/kg inhaled topotecan, corroborating previous findings using A549- and H1975-derived orthotopic lung tumors. Importantly, inhaled topotecan showed superior efficacy in suppressing lung tumors at distant sites. The growth of H1975- and H358-derived subcutaneous xenografts were completely arrested and A549-derived tumors were significantly reduced in mice treated twice-a-week with 1 mg/kg inhaled topotecan compared to a minor (H1975 and H358) or no reduction (A549) with twice-a-week 5 mg/kg IV topotecan.     

mCPP-induced hyperactivity in 5-HT2C receptor mutant mice is mediated by activation of multiple 5-HT receptor subtypes

The serotonin receptor agonist mCPP induces hyperlocomotion in 5-HT2C receptor knockout (KO) mice or in the presence of a 5-HT2C receptor antagonist. In the present group of experiments, we evaluate the role of 5-HT1A, 5-HT1B and 5-HT2A receptors in mCPP-induced hyperactivity in 5-HT2C KO mice. We also assess the ability of agonists at these receptors to induce hyperactivity in wildtype (WT) mice pre-treated with a selective 5-HT2C receptor antagonist. As previously reported, mCPP (3 mg/kg) induced hyperactivity in 5-HT2C KO mice. A combination of the 5-HT1B receptor agonist CP-94,253 (20 mg/kg) and the 5-HT1A receptor agonist 8-OH-DPAT (0.5 mg/kg) induced marked hyperactivity in WT but not in 5-HT2C KO mice, nor in mice treated with the selective 5-HT2C receptor antagonist, SB 242084 (1.5 mg/kg). Neither CP-94,253 nor 8-OH-DPAT had any intrinsic effect on locomotion in WTs. mCPP-induced hyperactivity was attenuated in 5-HT2C KO mice by the 5-HT1B receptor antagonist SB 224289 (2.5 mg/kg), and the 5-HT2A receptor antagonists ketanserin (0.3 mg/kg) and M100907 (0.01 mg/kg) but not by the 5-HT1A receptor antagonist WAY 100635 (1 mg/kg). The 5-HT(2A/2B/2C) receptor agonist, Ro 60-0175 (3 mg/kg), induced a modest increase in locomotor activity in WT mice pre-treated with SB 242084. However, the combination of Ro 60-0175 with CP-94,253 induced a substantial increase in activity in 5-HT2C KO mice, an effect comparable to mCPP-induced hyperactivity. Thus, joint activation of 5-HT1A and 5-HT1B receptors stimulates locomotion in WT mice but this response is dependent on a functional 5-HT2C receptor population and hence is absent in 5-HT2C KO mice. By contrast, mCPP-induced hyperactivity depends on the inactivation of a separate 5-HT2C receptor population and is mediated by 5-HT2A and 5-HT1B receptor activation.     

Identification of a zebrafish model of muscular dystrophy

1. Large-scale mutagenic screens of the zebrafish genome have identified a number of different classes of mutations that disrupt skeletal muscle formation. Of particular interest and relevance to human health is a class of recessive lethal mutations in which muscle differentiation occurs normally, but is followed by tissue-specific degeneration reminiscent of human muscular dystrophies. 2. We have shown that one member of this class of mutations, sapje (sap), results from mutations within the zebrafish orthologue of the human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells. 3. We have found that the progressive muscle degeneration phenotype of sapje-mutant zebrafish embryos is caused by the failure of somitic muscle attachments at the embryonic myotendinous junction (MTJ). 4. Although a role for dystrophin at the MTJ has been postulated previously and MTJ structural abnormalities have been identified in the dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure is thus far lacking. Therefore, the sapjre mutation may provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases. In the present review, we discuss this finding in light of previously postulated models of dystrophin function.     

复合诱变法选育普那霉素高产菌株

以始旋链霉菌(Streptomyces pristinaespiralis)ZP2为出发菌株,经紫外线(UV)、亚硝基胍(NTG)复合诱变,并结合普那霉素抗性突变株的理化筛选,选育到一株高产突变菌株UN2056,其普那霉素产量达到1490 mg/L,比出发菌株提高了45.7%.传代试验表明该高产突变菌株的高产性能遗传特性稳定.高产突变株UN2056在5 L发酵罐中进行发酵试验,其平均发酵产量达到1645 mg/L,比出发菌株提高了60.8%.     

突变SOD1cDNA亚克隆入酵母穿梭表达质粒pGBKT7中

目的将突变SOD1cDNA亚克隆入酵母穿梭表达质粒pGBKT7中。方法利用PCR方法从重组子pEGFP—N2-mSOD1中扩增突变的SOD1cDNA片断。以EcoRⅠ／salⅠ双酶切突变SOD1cDNA片断和酵母穿梭表达质粒pGBKT7，在T4DNA连接酶的作用下，连接突变SOD1cDNA片断和酵母穿梭表达质粒pGBKT7。转化感受态DH5α，筛选重组子，进行双酶切和测序鉴定。结果经酶切和测序鉴定，突变SOD1cDNA成功地亚克隆入酵母穿梭表达质粒pGBKT7中。结论亚克隆成功，可以对目的基因进行下一步研究。     

β-地中海贫血CD41-42型突变基因重组载体的构建

目的:构建β-地中海贫血CD41-42突变基因和基因调控区HS2、HS3全基因片段的真核表达重组载体,为β-地中海贫血CD41-42突变基因治疗的细胞模型奠定基础.方法:设计合成β-地中海贫血CD41-42突变纯合子患者的DNA特异性引物,扩增基因座控制区(LCR)和CD41-42突变基因,插入载体peDNA3.1-中,筛选阳性重组体,通过酶切分析及PCR进行鉴定.结果:扩增出2.1 kb CD41-42突变基因及5.5 kb LCR片段,酶切及测序结果证明重组载体构建成功,测序结果和引入方向正确.结论:成功构建了含人β-地中海贫血CD41-42基因的重组载体.     

Melatonin Attenuates Memory Impairment Induced by Klotho Gene Deficiency Via Interactive Signaling Between MT2 Receptor,ERK, and Nrf2-Related Antioxidant Potential

Background:

We demonstrated that oxidative stress plays a crucial role in cognitive impairment in klotho mutant mice, a genetic model of aging. Since down-regulation of melatonin due to aging is well documented, we used this genetic model to determine whether the antioxidant property of melatonin affects memory impairment.

Methods:

First, we examined the effects of melatonin on hippocampal oxidative parameters and the glutathione/oxidized glutathione (GSH/GSSG) ratio and memory dysfunction of klotho mutant mice. Second, we investigated whether a specific melatonin receptor is involved in the melatonin-mediated pharmacological response by application with melatonin receptor antagonists. Third, we examined phospho-extracellular-signal-regulated kinase (ERK) expression, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, Nrf2 DNA binding activity, and glutamate-cysteine ligase (GCL) mRNA expression. Finally, we examined effects of the ERK inhibitor SL327 in response to antioxidant efficacy and memory enhancement mediated by melatonin.

Results:

Treatment with melatonin resulted in significant attenuations of oxidative damage, a decrease in the GSH/GSSG ratio, and a significant amelioration of memory impairment in this aging model. These effects of melatonin were significantly counteracted by the selective MT2 receptor antagonist 4-P-PDOT. Importantly, 4-P-PDOT or SL327 also counteracted melatonin-mediated attenuation in response to the decreases in phospho-ERK expression, Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCL mRNA expression in the hippocampi of klotho mutant mice. SL327 also counteracted the up-regulation of the GSH/GSSG ratio and the memory enhancement mediated by melatonin in klotho mutant mice.

Conclusions:

Melatonin attenuates oxidative stress and the associated memory impairment induced by klotho deficiency via signaling interaction between the MT2 receptor and ERK- and Nrf2-related antioxidant potential.     

Simulated digestion of dried leaves of Artemisia annua consumed as a treatment (pACT) for malaria

Ethnopharmacological relevance

Artemisinin (AN) is produced by Artemisia annua, a medicinal herb long used as a tea infusion in traditional Chinese medicine to treat fever; it is also the key ingredient in current artemisinin-based combination therapies (ACTs) effective in treating malaria. Recently we showed that dried leaves from the whole plant Artemisia annua that produces artemisinin and contains artemisinin-synergistic flavonoids seem to be more effective and less costly than ACT oral malaria therapy; however little is known about how digestion affects release of artemisinin and flavonoids from dried leaves.

Material and methods

In the current study we used a simulated digestion system to determine how artemisinin and flavonoids are released prior to absorption into the bloodstream. Various delivery methods and staple foods were combined with dried leaves for digestion in order to investigate their impact on the bioavailability of artemisinin and flavonoids. Digestate was recovered at the end of the oral, gastric, and intestinal stages, separated into solid and liquid fractions, and extracted for measurement of artemisinin and total flavonoids.

Results

Compared to unencapsulated digested dried leaves, addition of sucrose, various cooking oils, and rice did not reduce the amount of artemisinin released in the intestinal liquid fraction, but the amount of released flavonoids nearly doubled. When dried leaves were encapsulated into either hydroxymethylcellulose or gelatin capsules, there was >50% decrease in released artemisinin but no change in released flavonoids. In the presence of millet or corn meal, the amount of released artemisinin declined, but there was no change in released flavonoids. Use of a mutant Artemisia annua lacking artemisinin showed that the plant matrix is critical in determining how artemisinin is affected during the digestion process.

Conclusions

This study provides evidence showing how both artemisinin and flavonoids are affected by digestion and dietary components for an orally consumed plant delivered therapeutic and that artemisinin delivered via dried leaves would likely be more bioavailable if provided as a tablet instead of a capsule.     

Booster vaccination with a fractional dose of an oral cholera vaccine induces comparable vaccine-specific antibody avidity as a full dose: A randomised clinical trial

Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1–2 years later occurred when boosting after a shorter interval.To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS).We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower.Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations.ISRCTN registry identifier 11806026.