Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system

Abstract: The cannabinergic system is present in a variety of organs and tissues that perform a wide range of essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane‐bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)‐terminal His‐tagged CB2 construct displayed a B_max value of 9.3 pmol/mg with a K_D of 7.30 nm using [3_H]CP–55⁹⁴⁰, a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high‐level expression systems when developing membrane‐bound protein purification strategies. This work will aid in the identification of receptor–ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.     

Mechanism of action of lonidamine in the 9L brain tumor model involves inhibition of lactate efflux and intracellular acidification

Malignant gliomas have been associated with a high rate of glycolytic activity which is believed necessary to sustain cellular function and integrity. Since lonidamine (LND) is believed to reduce tumor glucose utilization by inhibition of the mitochondrially-bound glycolytic enzyme hexokinase (HK), ³¹P magnetic resonance spectroscopy (MRS) was used to noninvasively follow the effects of LND on both tumor pH and the high-energy phosphate metabolites; ATP, phosphocreatine (PCr) and inorganic phosphate (P_i) in subcutaneous rat 9L gliosarcomas. ³¹P tumor spectra acquired in 5 min intervals pre- and post LND administration of 50 and 100 mg/kg, i.p. revealed an acidotic pH shift of – 0.25 and – 0.45 pH units, respectively within 30 min post administration. The ATP/P_i ratio of 9L tumors decreased to 40% of control and P_i levels increased to 280% of control over a 3 hr period. LND exerted no effect on tumor blood flow and mean arterial blood pressure. Brain and muscle metabolite levels and pH were also unaffected by LND. In vitro measurements of cultured 9L tumor cell intra- and extracellular lactate, pentose phosphate pathway (PPP) and hexokinase (HK) activities suggest that the mode of action of LND involves inhibition of lactate efflux and intracellular acidification. The selective reduction of tumor energy metabolites and pH by LND may be exploitable for sensitizing gliomas to radiation, chemotherapy or hyperthermia.     

Chemical shift imaging spectroscopy and memory function in temporal lobe epilepsy

PURPOSE: Hippocampal neuron loss and associated memory deficits are characteristic of intractable temporal lobe epilepsy (TLE). Proton chemical shift imaging (CSI) spectroscopy is a sensitive tool for detecting neuronal loss. The aim of this study was to investigate the correlation between memory functions and results provided by CSI spectroscopy of the hippocampal structures. METHODS: Ten patients with cryptogenic TLE participated. The study protocol involved both the acquisition of high-spatial-resolution CSI spectroscopy and neuropsychological evaluation, including memory testing and intracarotid sodium amytal test (IAT). The analysis of the CSI data was based on normative data obtained in 30 healthy volunteers. Memory functions were represented by verbal, visual, and general memory indices. RESULTS: A significant correlation was found between CSI spectroscopy of the hippocampal formation and the verbal memory indices for the dominant hemisphere. In addition, there was a significant correspondence of the qualitative judgment "hippocampal pathology indicated by CSI spectroscopy" and both "material specific memory deficit" and "memory deficit in the IAT." CONCLUSIONS: Our results demonstrate that CSI spectroscopy of the hippocampal structures is strongly related to lateralized memory deficits in patients with TLE. This suggests that CSI spectroscopy may be useful in the prediction of postoperative outcome in respect of seizure control and memory.     

运用共振拉曼散射光谱法测定皮肤类胡萝卜素

[目的]用共振拉曼散射光谱法测试皮肤类胡萝卜素水平,观察机体类胡萝卜素变化情况。[方法]120名成年受试者按照其年龄、性别和试验前皮肤类胡萝卜素指数配对后,随机分为试验组和对照组。试验组人群每天补充β-胡萝卜素12.6mg,共8周。两组人群在试验前、试验第4周末和第8周末,分别运用共振拉曼散射光谱测试受试者皮肤类胡萝卜素,观察皮肤中类胡萝卜素水平变化情况。同时进行膳食调查,了解膳食摄入情况。[结果]试食组人群补充β-胡萝卜素第4周时皮肤类胡萝卜素指数比试验前的基础值上升了24.3%、第8周时比试验前的基础值上升了44.0%,且与对照人群相比差异有统计学意义(P<0.001)。[结论]由于两组人群在试验期间的膳食摄入情况没有差异,实验中试食组人群皮肤类胡萝卜素指数的增高可认为与补充β-胡萝卜素有关。共振拉曼散射光谱作为一种非创伤性的检测方法,可用于反映皮肤类胡萝卜素水平,进而提示机体类胡萝卜素的营养状况。     

油炸及烧烤食品中丙烯酰胺的HPLC测定

目的了解市售及家庭自制油炸烧烤食品中丙烯酰胺的含量,为食品安全研究提供实验数据。方法采用紫外吸收光谱确定检测波长,运用高效液相色谱(high performance liquid chromatography,HPLC)技术,结合溶剂提取的摇床振摇、萃取脱脂、低温浓缩等前处理方法。结果22种油炸烧烤食品中均检测到丙烯酰胺,含量范围为2.09×10-1~1.86×10μg/g。结论采用中国人习惯的油炸,烧烤的方式加工富含淀粉、蛋白质的食品同样易产生人类可能致癌物丙烯酰胺,长期食用此含量的食品将对人体产生怎样的危害值得进一步研究。     

水杨酸、对甲氧基苯甲酸和甲硝唑的拉曼光谱

目的研究水杨酸、对甲氧基苯甲酸和甲硝唑等化合物拉曼光谱的波长选择性。方法分别采用近红外、可见和紫外区3种波长的激光激发获得不同的拉曼光谱,受照样品包括水杨酸、对甲氧基苯甲酸以及临床常用药物甲硝唑。结果水杨酸、对甲氧基苯甲酸的水溶液在可见和近红外激发下无法获得任何拉曼信息,但在紫外激发下可获得信息丰富的拉曼光谱,且与其固体拉曼谱对应良好。而甲硝唑在紫外激发下没有明显的拉曼峰但在近红外和可见光激发下能获得一致的拉曼信息。结论水杨酸、对甲氧基苯甲酸水溶液的拉曼光谱证明了紫外激发的优越性,甲硝唑的拉曼光谱提示样品对激发波长的选择性。     

金钱草的化学成分研究

目的研究金钱草的化学成分.方法利用色谱手段分离化学成分,用理化常数和波谱技术鉴定结构.结果分离鉴定了7个化合物:山萘酚(1)、山萘酚3-O-β-D-吡喃葡萄糖苷(2)、山萘酚3-O-α-L-鼠李糖-(1→6)-β-D-吡喃葡萄糖苷(3)、山萘酚3-O-α-L-鼠李糖(1→2)-β-D-吡喃葡萄糖苷(4)、槲皮素(5)、β-谷甾醇(6)、胡萝卜苷(7).结论化合物4为首次从该属植物中分离得到,化合物7为首次从该种植物中分离得到.     

琥珀酸胆固醇酯作为脂质体膜稳定剂的研究及其在制备柴胡皂苷-D脂质体中的应用

目的研究琥珀酸胆固醇酯(CHEMS)对二棕榈酰磷脂酰胆碱(DPPC)脂质体的膜稳定作用以及作用机制；以CHEMS和DPPC为膜材制备柴胡皂苷-D(SSD)脂质体，考察其包封率和溶血性。方法差示扫描量热法(DSC)和荧光释放实验考察CHEMS的膜稳定作用，傅立叶红外(FT-IR)研究CHEMS与DPPC膜的作用机制，沉降实验研究CHEMS与SSD相互作用，溶血实验考察了以CHEMS为膜材包裹SSD脂质体的溶血性。结果CHEMS在膜稳定性上优于胆固醇(CHOL)，CHEMS与DPPC的极性端头同时有氢键和静电作用；CHEMS与SSD不会形成不溶性复合物(INCOM)，用DPPC和CHEMS为膜材制备了稳定的SSD包裹的脂质体，其溶血性大大降低。结论CHEMS对DPPC膜的稳定作用大于CHOL，并可取代CHOL作为制备胆固醇依赖性的溶血性皂苷脂质体的膜稳定剂。     

A Raman spectroscopic study of the diclofenac sodium–β-cyclodextrin interaction

The inclusion complex of diclofenac sodium (DCFNa) and β-cyclodextrin (βCD) was prepared and analyzed by using Raman and SERS spectroscopy. The interaction between DCFNa and βCD molecules was evidenced by monitoring the change in the peak positions and the widths of some guest molecule bands relative to those observed in the spectra of the free compound and 1:1 DCFNa–βCD physical mixture. Raman data have shown that the interaction between the guest and host molecules in the 1:1 DCFNa–βCD complex is maintained both through the dichlorophenyl ring and the phenylacetate group. SER spectra revealed that depending on the pH value of the solution different isomeric forms of the 1:1 guest–host complex are preferentially adsorbed on the silver surface via the nonbonding electrons of the oxygen atom. The orientation of the adsorbed species with respect to the metal surface was also elucidated.