[+]-Huperzine A treatment protects against N-methyl-d-aspartate-induced seizure/status epilepticus in rats

The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood–brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [−]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [−]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20 μg/kg NMDA (intracerebroventricular) 20–30 min later. For post-exposure, rats were treated with [+]-Hup A (3 mg/kg, intramuscularly) 1 min after NMDA (20 μg/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3 mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [−]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.     

The effects of biomass removal and N additions on microbial N transformations and biomass at different vegetation types in an old-field ecosystem in northern China

There is an increasing demand for the sustainable management of old-field communities in northern China, which have developed on abandoned cropland on formerly converted natural steppe sites, to regain forage yield, biodiversity, and soil fertility. In thus study we examined how two management options—clipping and nitrogen (N) addition—may affect net >microbial N mineralization (ammonification?+?nitrification), microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and microbial respirations (MR) in grass dominated, herb dominated, and grass-herb mixed patches in an old-field community in northern China.Topsoil (0–10 cm) net N mineralization rate was 177% and 69% higher in mixed grass and herb patches (patch B) as compared to unmixed grass (patch A) or herb (patch C) patches, respectively. Topsoil MBN was significantly different among the three patches with the highest value for soils taken from umixed grass patches. However, patches with mixed grass and herb or herb dominated patches had 12% higher microbial respiration (MR) than unmixed grass patch. Clipping and N addition had no effects on net N mineralization or MBC, but both treatments decreased MBN and MR and increased the ratio between microbial biomass C and microbial biomass N (MBC/MBN) in the growing season. Incubation of soil cores under optimal water and temperature conditions in the laboratory showed that the response of microbial N transformations in soils under different vegetation patches to experimental N addition and clipping was limited by soil water availability. Our results strongly highlight the need to further study the importance of belowground C supply as a control of microbial N cycling processes. It also suggests that during the restoration process of degenerated croplands N cycling rates are stimulated, but that the magnitude of this stimulation is modulated by plant community composition of the old-fields.     

腺病毒载体介导的重组蛋白sTNFRII-gAD快速表达和制备

利用腺病毒载体高效感染哺乳动物细胞及表达外源基因的特性,建立一种快速高效表达及制备重组蛋白的方法,并用该方法获得sTNFRII-gAD (可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白) 蛋白纯品。首先用携带EGFP基因的腺病毒载体rAd5-EGFP以不同的腺病毒用量 (MOI) (0~1 000) 感染BHK21c022细胞,观察比较其转导效率和细胞毒性。用AdMax腺病毒载体系统制备携带融合基因sTNFRII-gAD的重组复制缺陷型5型腺病毒rAd5-sTNFRII-gAD。用rAd5-sTNFRII-gAD以不同的MOI (0~1 000) 感染BHK21c022细胞,收取上清进行Western blotting分析,比较上清中sTNFRII-gAD蛋白的表达量。在此基础上,用rAd5-sTNFRII-gAD以MOI 100感染大量培养的BHK21c022细胞,在无血清培养条件下反复多次收取培养上清,经过硫酸铵浓缩、分子筛柱层析、透析等步骤浓缩和纯化sTNFRII-gAD融合蛋白,并体外测定该融合蛋白拮抗TNFa的活性。结果获得了携带sTNFRII-gAD融合基因的重组腺病毒rAd5-sTNFRII-gAD;用rAd5-EGFP感染BHK21c022细胞结果表明,随着腺病毒用量的增高,表达EGFP蛋白的BHK21c022细胞数量和亮度明显增加;MOI在0~100之间被感染的BHK21c022细胞未表现出明显的细胞毒性,MOI为1 000时可观察到细胞变圆和少量死亡现象。Western blotting分析结果表明,随着腺病毒用量的增高,培养上清中sTNFRII-gAD融合蛋白表达量明显增加,以MOI为1 000时最高。在此基础上,我们用MOI为100的rAd5-sTNFRII-gAD感染5个转瓶培养的BHK21c022细胞以制备sTNFRII-gAD融合蛋白。每个转瓶加无血清培养液100 mL,每48 h收获上清1次并换液,反复6次收取培养上清共约3 L,经过纯化获得了约11 mg的sTNFRII-gAD融合蛋白。体外活性测定实验表明,获得的sTNFRII-gAD融合蛋白能有效拮抗TNFα对L929细胞的杀伤作用。腺病毒载体/BHK21细胞表达系统是一个简便、高效、通用的表达系统,利用该系统成功制备了有生物学活性的sTNFRII-gAD融合蛋白。该表达系统的特点是生产规模易于放大,适应无血清培养,可以反复多次收获目标蛋白。     

Sequence Analysis of cytb Gene in Echinococcus granulosus from Western China

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.     

Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation

The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.     

硝酸咪康唑联合妇炎消生物敷料栓对妊娠期阴道假丝酵母菌病患者的疗效及对阴道微生态的影响

目的探讨硝酸咪康唑联合妇炎消生物敷料栓对妊娠期阴道假丝酵母菌病患者的疗效及对阴道微生态的影响。方法选取2017年3月至2019年3月在西安医学院第二附属医院产科就诊的96例妊娠期阴道假丝酵母菌病患者为研究对象,采用随机数字表法分为观察组和对照组,每组各48例。对照组患者给予妇炎消生物敷料栓,阴道给药,1枚/d。观察组患者在对照组基础上加用硝酸咪康唑栓,阴道给药,1枚/d,两组均为7 d一个疗程。比较两组患者的临床疗效,阴道微生态情况和母婴结局。结果治疗后观察组患者总有效率(91.67%)明显高于对照组(70.83%),差异有统计学意义(χ~2=6.838,P=0.009)。治疗后,观察组患者阴道pH、菌群多样性Ⅱ～Ⅲ级比例高于对照组,差异有统计学意义(χ~2=4.042、4.002,P=0.044、0.045);而两组患者阴道菌群密集度Ⅱ～Ⅲ级比例、乳杆菌占优势比例差异无统计学意义(χ~2=0.889、0.211,P=0.346、0.646)。观察组患者产褥感染、胎膜早破、早产发生率均低于对照组。两组患者胎儿窘迫和新生儿黄疸发生率比较差异无统计学意义(χ~2=0.154、3.010,P=0.695、0.083)。结论硝酸咪康唑联合妇炎消生物敷料栓对妊娠期阴道假丝酵母菌病患者的临床疗效显著,可促进患者阴道微生态的恢复,改善母婴结局。     

TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY‐5Y cells

The light chain (LC) of botulinum neurotoxin B (BoNT/B) is unable to enter target neuronal cells by itself. It is brought into the cell in association with the BoNT/B heavy chain (HC) through endocytosis. The BoNT HC‐LC subunits are held together by a single disulfide bond. Intracellular reduction of this bond and separation of the two subunits activates the endopeptidase activity of the LC. This requirement suggests a strategy to prevent uptake by prophylactic reduction to disrupt the disulfide bond prior to endocytosis of the complex. We examined the utility of tris‐(2‐carboxyethyl)‐phosphine hydrochloride (TCEP), a relatively non‐toxic, non‐sulfur containing disulfide bond reducing agent that lacks the undesirable properties of mercapto‐containing reducing agents. We found that TCEP was as effective as DTT with maximal LC endopeptidase activation occurring at 1 mM, a concentration not toxic to the human neuronal cell line, SHSY‐5Y. In these cells, 1 mM TCEP maximally protected against BoNT/B inhibition of [³H]‐NA release, achieving 72% of the release from un‐intoxicated controls. This effect appears to be due to the sparing of SNARE proteins as the levels of VAMP‐2, the specific target of BoNT/B, were protected. These results show that TCEP disrupts the structure of BoNT/B by reduction of the LC and HC bridging disulfide bond and prevents neuronal intoxication. Since disulfide bond coupling between toxin subunits is a general motif for many toxins, e.g., ricin, snake venom, and all BoNT serotypes, this suggests that TCEP is a promising means to protect against these toxins by preventing cell penetration. J. Cell. Biochem. 107: 1021–1030, 2009. Published 2009 Wiley‐Liss, Inc.