东亚钳蝎氯离子通道毒素BmK CTa在大肠杆菌中的异源表达及其对宿主菌增殖的影响

目的在大肠杆菌中异源表达东亚钳蝎氯离子通道毒素BmK CTa,并观察其对宿主菌增殖的影响。方法构建BmK CT基因原核表达质粒pExSecI-rBmK CTa,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot进行鉴定。光密度法检测含不同质粒的大肠杆菌BL21(DE3)及空菌在37℃,LB液体培养基中的生长速率。结果重组原核表达质粒pExSecI-rBmK CTa经PCR、双酶切和测序证明构建正确。目的蛋白的表达量占全菌总蛋白的19.94%,为可溶性表达,且具有良好的反应原性。rBmK CTa的异源表达显著抑制了大肠杆菌在对数生长期的增殖。结论rBmK CTa在大肠杆菌BL21(DE3)中获得了可溶性表达,且对大肠杆菌的生长具有显著的抑制作用。提示BmK CT可能特异性地作用于宿主菌的氯离子通道,对原核生物的氯离子通道有抑制作用。     

综采工作面回撤通道锚网索联合支护技术

针对盛鑫煤业有限责任公司51101综采工作面回撤通道支护难题,分析工作面过回撤通道时围岩变形与破坏特征,阐述其支护要点,并结合现场实际地质生产条件,提出回撤通道锚网索联合支护的方案。现场矿压观测表明,主辅回撤通道顶板最大下沉量均在390 mm左右,最大下沉量出现在回撤通道的中部,为517 mm,有效控制了回撤通道围岩变形破坏。     

高分子量聚丙二醇在微通道反应器中的制备

高分子量聚醚多元醇应用广泛,但用传统的半连续釜式法制备不仅反应热风险大,而且反应时间漫长。采用微通道反应器(MCR)具有传热效率高、过程安全的特点,但反应物料黏稠、放热量大也使聚醚多元醇的分子量难以提高。本研究以双金属氰化络合物(DMC)为催化剂、正己烷为溶剂,在MCR中进行了环氧丙烷的开环聚合,制备出了分子量在2000～8000之间的聚丙二醇(PPG)。通过对流速、通道长度、温度和进料方式影响的考察,发现在停留时间足够长时,产品分子量基本等于理论分子量;当起始剂流速固定,单体流速增加时,分子量分布(MWD)先变宽再变窄;当管长较长时,分子量较高且MWD较宽;温度升高会使聚合反应的诱导期缩短;分段进料比一段进料更易制备高分子量PPG,但MWD变宽。这些均可用DMC催化的环氧丙烷的开环聚合机理和物料在MCR中微观混合强度的变化来解释。微观混合强度越低,聚合反应中链转移与链增长的速率比越小,聚合物的分子量分布也越宽。     

微通道反应器在有机合成中的应用研究

微通道反应器是一类具有极高的传质和传热效率的新型反应器,具有许多常规反应器无法比拟的优势,近年来已被广泛应用于科学研究和化工生产等领域。综述了微通道反应器的优点及其在均相反应、非均相反应、生物有机反应、气-液两相反应、液-液两相反应、气-液-固三相反应等方面的应用研究。微通道反应器在化学与化工行业具有巨大的开发潜力和广泛的应用前景。     

微通道技术在氯化反应工艺中的应用

本文介绍了氯化反应机理,从直接利用氯气源和原位生成氯气开展微通道氯化反应研究两方面,综述了近几年微通道技术在氯化反应中的应用,详述了不同类型氯化反应在微反应器的选择、工艺流程设计和工艺改良方面的内容.并总结了微通道技术在氯化反应中的应用,指出了存在的不足及对未来的展望.     

微通道反应器在精细化工产品合成中的应用研究进展

综述了近2年微通道反应器在精细化工产品合成中的研究进展,包括硝化反应、酯化反应、氯磺化反应、氧化反应、加成反应、分解反应和氧化还原反应等.在微通道反应器中,以烧碱和液氯为原料生产次氯酸钠的氧化还原反应已实现工业化应用.     

微通道反应器在聚合领域中的应用研究进展

微反应器也被称为微通道反应器,因其具有较传统釜式反应器更优异的传热、传质性能,可实现化工连续化生产,在化工行业的应用引起了广泛关注.微通道反应器可实现对反应条件的精确控制,从而实现对聚合反应的精确调控,提高聚合反应速率和效率,优化产物分子结构,得到的聚合物分子量具有分布系数较窄的特性.本文综述了微通道反应器在聚合反应中...     

钯在硝酸高铈铵溶液中的溶解工艺及宏观动力学研究

钯(Pd)是重要的战略金属,具有优异的理化性质,广泛应用于石油化工、汽车制造、航空航天和电子信息等领域。因其自然储量低,难以满足社会发展的需求,研究含钯二次资源回收技术,实现金属钯的循环利用具有重要意义。本工作针对传统湿法回收钯技术存在的酸耗高、NO_x排放量大等问题,提出了环境友好的硝酸高铈铵浸出新体系,考察了浸出剂浓度、酸度、添加剂浓度、反应温度、搅拌速度等因素对钯粉在硝酸高铈铵溶液中的溶解率的影响。结果表明,金属钯在混合体系中的溶解受到Pd表面吸附的显著影响。在没有Cl^-的情况下,金属钯表面吸附氧或者吸附NO₃^-形成了表面钝化,阻碍了Ce⁴⁺与Pd原子的反应,5 h溶出率仅为0.2%。添加Cl^-后,通过竞争吸附能够有效破坏表面钝化层,构成突破点,从而加速钯的溶解反应。NO₃^-与Cl^-构成竞争吸附,更高浓度的硝酸根需要更高浓度的Cl^-启动溶解反应。优选浸出反应条件如下...     

国产约翰逊网在固定床反应器中的应用

通过对固定床反应器中所用的几种筛网形式进行比较,说明了约翰逊网的特点,简要介绍了约翰逊网国产化的过程、遇到的问题和所作的改进,以及在固定床反应器中的应用情况。     

微通道反应器在芳香化合物硝化反应中的应用进展

芳香化合物的硝化是常用的生产单元,采用传统釜式反应器进行硝化反应存在放热量大、选择性低、浪费资源、污染环境、存在安全隐患等问题.微通道反应器具有优良的传热、传质性能,可有效解决传统釜式反应器存在的问题,是一种安全、环保、高效的新型反应设备.综述了微通道反应器在芳香化合物硝化反应中的应用进展,包括以苯型芳香烃为底物的硝化...     

Chloride Channelopathies of ClC-2

Chloride channels (ClCs) have gained worldwide interest because of their molecular diversity, widespread distribution in mammalian tissues and organs, and their link to various human diseases. Nine different ClCs have been molecularly identified and functionally characterized in mammals. ClC-2 is one of nine mammalian members of the ClC family. It possesses unique biophysical characteristics, pharmacological properties, and molecular features that distinguish it from other ClC family members. ClC-2 has wide organ/tissue distribution and is ubiquitously expressed. Published studies consistently point to a high degree of conservation of ClC-2 function and regulation across various species from nematodes to humans over vast evolutionary time spans. ClC-2 has been intensively and extensively studied over the past two decades, leading to the accumulation of a plethora of information to advance our understanding of its pathophysiological functions; however, many controversies still exist. It is necessary to analyze the research findings, and integrate different views to have a better understanding of ClC-2. This review focuses on ClC-2 only, providing an analytical overview of the available literature. Nearly every aspect of ClC-2 is discussed in the review: molecular features, biophysical characteristics, pharmacological properties, cellular function, regulation of expression and function, and channelopathies.     

A20 Attenuates the Fibrotic Response in the Trabecular Meshwork

Although the extracellular matrix (ECM) in trabecular meshwork (TM) cells is known to be important in intraocular pressure (IOP) regulation, the molecular mechanisms involved in generating a glaucomatous environment in the TM are not completely understood. Recently we identified a molecular pathway, transforming growth factor beta 2 (TGFβ2)–toll-like receptor 4 (TLR4) signaling crosstalk, as an important regulator of glaucomatous damage in the TM, which contributes to fibrosis. Here we evaluate a novel molecular target, A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), which may help to block pathological TGFβ2–TLR4 signaling. Primary human TM cells were analyzed for A20 message and for A20 and fibronectin protein expression after treatment with TGFβ2. A20 message increased when the TLR4 pathway was inhibited in TM cells. In addition, TGFβ2, a known inducer of fibrosis, increased fibronectin expression, while at the same time decreasing the expression of A20. We then overexpressed A20 in TM cells in order to test the effect on treatment with TGFβ2, lipopolysaccharide (LPS), or cellular fibronectin extra domain A (cFN-EDA). Importantly, overexpression of A20 rescued the fibrotic response when TM cells were treated with TGFβ2, LPS, or cFN-EDA. In situ hybridization was used to probe for A20 RNA expression in age-matched control (C57BL/6J) mice and mice that constitutively express the EDA isoform of fibronectin (B6.EDA^+/+). In this novel mouse model of glaucoma, A20 RNA was increased versus age-matched control mice in a cyclic manner at 6 weeks and 1 year of age, but not at 8 months. Overall, these data suggest that A20 may work through a negative feedback mechanism attenuating the ability of TGFβ2–TLR4 signaling to induce fibrosis.     

Relationships between Intraocular Pressure,Effective Filtration Area,and Morphological Changes in the Trabecular Meshwork of Steroid-Induced Ocular Hypertensive Mouse Eyes

We investigated whether an inverse relationship exists between intraocular pressure (IOP) and effective filtration area (EFA) in the trabecular meshwork (TM) in a steroid-induced ocular hypertensive (SIOH) mouse model and the morphological changes associated with the reduction of EFA. C57BL/6 mice (n = 15 per group) received either 0.1% dexamethasone (DEX) or saline eye drops twice daily for five weeks. IOP was measured weekly. Fluorescent tracers were injected into the anterior chamber to label EFA at the endpoint. Injected eyes were fixed and processed for confocal microscopy. EFA in the TM was analyzed. Light and electron microscopy were performed in high- and low-tracer regions of six eyes per group. The mean IOP was ~4 mm Hg higher in DEX-treated than saline-treated control eyes (p < 0.001) at the endpoint. EFA was reduced in DEX-treated eyes compared to controls (p < 0.01) and negatively correlated with IOP (R² = 0.38, p = 0.002). Reduced thickness of juxtacanalicular tissue (JCT) and increased abnormal extracellular matrix in the JCT were found to be associated with reduced EFA. Our data confirm the inverse relationship between EFA and IOP, suggesting that morphological changes in the JCT contribute to the reduction of EFA, thus elevating IOP in SIOH mouse eyes.     

高含量氯离子水样CODcr测定方法研究

有些工厂的废水中Cl~-含量极高,如按常规方法分析,CODcr值大幅度增高,造成很大正误差。另外,有些废水成份很复杂,即有易被氧化的物质,又有难以氧化的物质,在用CODcr法测量水质指标时:温度变化对测定结果产生什么影响,本文对上述问题通过实验进行了研究,做出了可信服的回答。     

All-trans Retinoic Acids Synergistically and Beneficially Affect In Vitro Glaucomatous Trabecular Meshwork (TM) Models Using 2D and 3D Cell Cultures of Human TM Cells

We report herein on the effects of all-trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor β2 (TGF-β2). In the presence of 5 ng/mL TGF-β2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans-endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-β2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-β2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-β2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-β2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-β2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.     

A Novel Mouse Model of TGFβ2-Induced Ocular Hypertension Using Lentiviral Gene Delivery

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2^C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2^C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2^C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2^C226,228S that induces TM dysfunction and outflow resistance.     

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.     

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca²⁺]_i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K⁺ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline—a specific inhibitor of large-conductance Ca²⁺-activated BK channels. The TEA-insensitive component was inhibited by senicapoc—a specific inhibitor of the Ca²⁺-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca²⁺]_i induced by Chl-T. Both KROS and [Ca²⁺]_i increase were inhibited by ACA and clotrimazole—two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca²⁺]_i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.     

Receptor-Dependent and Independent Regulation of Voltage-Gated Ca2+ Channels and Ca2+-Permeable Channels by Endocannabinoids in the Brain

The activity of specific populations of neurons in different brain areas makes decisions regarding proper synaptic transmission, the ability to make adaptations in response to different external signals, as well as the triggering of specific regulatory pathways to sustain neural function. The endocannabinoid system (ECS) appears to be a very important, highly expressed, and active system of control in the central nervous system (CNS). Functionally, it allows the cells to respond quickly to processes that occur during synaptic transmission, but can also induce long-term changes. The endocannabinoids (eCBs) belong to a large family of bioactive lipid mediators that includes amides, esters, and ethers of long-chain polyunsaturated fatty acids. They are produced “on demand” from the precursors located in the membranes, exhibit a short half-life, and play a key role as retrograde messengers. eCBs act mainly through two receptors, CB1R and CB2R, which belong to the G-protein coupled receptor superfamily (GPCRs), but can also exert their action via multiple non-receptor pathways. The action of eCBs depends on Ca²⁺, but eCBs can also regulate downstream Ca²⁺ signaling. In this short review, we focus on the regulation of neuronal calcium channels by the most effective members of eCBs-2-arachidonoylglycerol (2-AG), anandamide (AEA) and originating from AEA-N-arachidonoylglycine (NAGly), to better understand the contribution of ECS to brain function under physiological conditions.