The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels

BACKGROUND

Depletion of the Ca²⁺ store by ryanodine receptor (RyR) agonists induces store-operated Ca²⁺ entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE.

EXPERIMENTAL APPROACH

SOCE and ORAI1-3 currents were recorded by Ca²⁺ imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3.

KEY RESULTS

4-CEP induced a significant release of Ca²⁺ in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca²⁺ release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca²⁺ store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3.

CONCLUSION AND IMPLICATIONS

4-CEP not only acts as a RyR agonist to deplete the Ca²⁺ store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists.     

Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC‐MS/MS bioanalysis of human IgG and Fc‐fusion protein drug candidates

The synthesis of a 16‐residue, stable isotopically labeled peptide is described for use as a LC‐MS/MS (Liquid chromatography‐mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc‐fusion protein drug candidates in animal species used in pre‐clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave‐assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative. The corresponding conventional room temperature SPPS was unsuccessful and gave only mixtures of truncated products. Stable‐labeled leucine was incorporated as a single residue via manual coupling of commercially available Fmoc‐[¹³C₆, ¹⁵N]‐l ‐leucine onto an 11‐unit segment followed by automated microwave‐assisted elaboration of the final four residues. Using this approach, the desired labeled peptide was prepared in high purity and in sufficient quantities for long‐term supplies as a bioanalytical internal standard. The results strongly demonstrate the importance of utilizing both microwave‐assisted peptide synthesis and pseudoproline dipeptide techniques to allow the preparation of labeled peptides with highly lipophilic and sterically hindered side‐chains.     

A novel gastroretentive porous microparticle for anti-Helicobacter pylori therapy: Preparation, in vitro and in vivo evaluation

Gastroretentive drug delivery system is a promising option for the treatment of Helicobacter pylori infection, which can prolong gastric residence time and supply high drug concentration in the stomach. In the present study, a low density system of metronidazole-loaded porous Eudragit^® RS microparticle with high drug loading capacity (>25%) was fabricated via electrospray method. The porous structure and size distribution of microparticles were affected by polymer concentration and flow rate of solution. FTIR and XRD analyses indicated that drug has been entrapped into the porous microparticles. In addition, sustained release profiles and slight cytotoxicity in vitro were detected. Gamma scintigraphy study in vivo demonstrated that ¹³¹I-labeled microparticles retained in stomach for over 8 h, and about 65.50% radioactive counts were finally detected in the region of interest. The biodistribution study confirmed that hotspot of radioactivity was remaining in the stomach. Furthermore, metronidazole-loaded porous microparticles can eradicate H. pylori completely with lower dose and administration frequency of antibiotic compared with pure drug, which were also more helpful for the healing of mucosal damages. These results suggest that prepared porous microparticle has the potential to provide better treatment for H. pylori infection.     

Perfluorooctanoic acid stimulates breast cancer cells invasion and up-regulates matrix metalloproteinase-2/-9 expression mediated by activating NF-κB

Perfluorooctanoic acid (PFOA) is widely used because of its stain-resistant and water-repellant properties. This study aimed to explore the molecular mechanisms undergoing the stimulation effects of PFOA on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Trans-well filter assay showed that PFOA exposure (≥5 nM) evidently enhanced the invasion ability of the breast cancer cells MDA-MB-231. Luciferase reporter assay, quantitative real-time PCR, western blotting and gelatin zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the cells after PFOA treatment (P < 0.05 each). Western blotting revealed that PFOA could activate nuclear factor kappaB (NF-κB) by accelerating NF-κB translocation into the nucleus. Furthermore, addition of NF-κB inhibitor in culture medium could suppress the breast cancer cells invasiveness enhancement and MMP-2/-9 overexpression. This study indicates that PFOA can stimulate breast cancer cells invasion and up-regulate matrix metalloproteinase-2/-9 expression mediated by activating NF-κB, which deserves more environmental health concerns.     

全肠外营养在术后恶性肿瘤患者治疗中的应用与进展

肿瘤患者大多术前已存在不同程度的营养不良及免疫功能障碍,而手术的创伤和术后应激反应引起的分解代谢加强更加重了营养不良和免疫抑制,导致机体抵抗力降低,并发症增多,死亡率增高和住院时间延长。全肠外营养（ TPN）支持可增加肿瘤患者特别是术后恶性肿瘤患者肌肉组织和肝脏内糖原的贮存,使不能正常进食或超高代谢肿瘤患者维持较好的营养状态,增强自身免疫能力,提高其生存质量。此外,很多术后恶性肿瘤患者在术后营养不良的情况下,还在多次进行放、化疗,不但机体耐受能力下降,明显影响化疗效果,还严重影响患者生活及生存质量。因此,关注术后恶性肿瘤患者营养支持需求,特别是营养的平衡供给尤为重要。本文就TPN在术后恶性肿瘤的应用状况与研究进展做一综述。     

低温联合腺苷预处理对脊髓缺血-再灌注的保护作用

目的：观察腺苷联合深低温对大鼠脊髓缺血-再灌注的保护作用及对 Caspase -12/ GRP -78表达的影响,初步探讨其可能的作用机制。方法将27只 SD 大鼠随机均分为3组,即常温37℃手术对照组（A 组）,低温18℃处理组（B 组）,腺苷﹢低温18℃处理组（C组）。通过夹闭前10 min 尾静脉注射腺苷,用量为0.06 mL /100 g,质量浓度为5 g / L。于再灌注6,12,24 h 进行后肢运动功能评分,取再灌注24 h 大鼠的 L2~ L5节段脊髓组织作 HE 染色及 TUNEL 染色,免疫组化法检测 GRP -78和 Caspase -12蛋白的表达。结果 HE染色结果显示,C 组细胞水肿、尼氏体溶解等组织形态学变化较 B 组轻;TUNEL 染色结果显示,与 B 组、C 组比较,A 组的凋亡细胞数量显著增加;与 B 组相比,C 组凋亡细胞数量较少。行为学评分各组早期均有下降,随时间推移逐渐增加,A 组下降程度高于 B 组、C 组,且 C 组评分高于 B 组（ P ﹤0.01）。免疫组化法检测结果显示,相对于 A 组,B 组、C 组 GRP -78蛋白表达增多,且 C 组显著高于 B 组（ P ﹤0.05）;Caspase -12蛋白表达 A 组高于 B 组、C 组,且 B 组高于 C 组（ P ﹤0.05）。结论腺苷联合深低温对大鼠脊髓缺血再灌注有保护作用,低温可能加强了腺苷的保护作用,并可能通过抑制内质网应激引起的细胞凋亡发挥其保护作用。     

广西贵港地区乡镇卫生院医生、药师三阶梯止痛治疗认知度调查

目的：了解广西贵港地区乡镇卫生院医生、药师对三阶梯止痛治疗的认知度。方法：采用自行设计的“镇痛药物三阶梯止痛治疗认知调查表”,对331名乡镇卫生院的医生、药师进行调查分析。结果：82.5%的医生、药师未参加过癌症疼痛治疗的培训,对三阶梯止痛治疗的基本知识认知度不高。医生、药师对麻醉镇痛药的品种选择、首选给药方式、最佳给药时机和理想的镇痛效果等知识的认识存在明显误区,回答正确率分别只有67.4%,56.8%,36.3%和44.4%,此外还普遍表现出对药物依赖的担忧和恐惧。医生与药师相比较,总体上差异无统计学意义（P〉0.05）。结论：贵港地区乡镇卫生院的医生、药师对三阶梯止痛治疗的认识尚有不足,需要加强培训和继续教育,提高疼痛治疗的专业能力。     

免疫增强药用于恶性肿瘤患者辅助治疗现状调查

目的：调查对免疫增强药临床应用现状及合理性,为该类药物的合理使用提供参考。方法：利用医院HIS系统,随机抽取2012年在我院治疗的240例确诊为恶性肿瘤住院患者病历,调查免疫增强药的应用情况,评价临床应用免疫增强药的合理性。结果：不同年龄组及KPS评分组段,免疫增强药使用率的差异均无统计学意义（P〉0．05）。214例患者使用了免疫增强药,使用率为89．2％,其中联合用药病历114份（47．5％）;中药类免疫增强药用药金额占总金额的74．4％;不合理用药医嘱62份,占总病历数的25．8％,主要为溶媒使用不当。结论：我院免疫增强药使用情况总体良好,但也存在部分不合理现象,卫生行政部门及院相关管理部门应加强管理,促进该类药物的合理应用。