普通烟草外向整流Shaker K^(+)通道NtSKOR1的组织表达分析

外向整流Shaker K^(+)通道SKOR(Stelar K^(+)outward rectifier)是一类定位于植物根部中柱细胞质膜的外向整流Shaker K^(+)通道。为探究普通烟草NtSKOR1的启动子活性和不同时期组织表达情况,克隆该基因上游2439 bp的启动子序列,创制启动子驱动β-葡萄糖苷酸酶基因(GUS)的烟草材料并进行组织化学染色,通过RT-qPCR验证该基因的表达。结果表明NtSKOR1启动子含有光响应、逆境胁迫和激素等相关的顺式作用元件;转ProNtSKOR1::GUS烟草的组织化学染色试验表明:萌发期至子叶展平期未检测到GUS活性;小十字期,在真叶叶脉和茎尖分生组织开始检测到GUS活性;生根期,除茎和叶脉的维管组织外,在根部维管组织也开始检测到明显GUS活性,且活性随烟株生长而逐渐增强;盛花期,主要在烟草的根、茎和叶脉维管组织检测到活性,且上部叶叶脉中的GUS活性高于下部叶叶脉。RT-qPCR与GUS活性检测结果基本一致。综上可知,NtSKOR1主要在烟草小十字期及后续发育阶段的根、茎、叶的维管组织中表达,烟草进入盛花期后,该基因在光合作用较强的上部叶中的表达高于下部叶,推测该基因可能参与烟草K^(+)转运和同化产物协同运输。

Na^(+)/K^(+)-ATPase:ion pump,signal transducer,or cytoprotective protein,and novel biological functions

Na^(+)/K^(+)-ATPase is a transmembrane protein that has important roles in the maintenance of electrochemical gradients across cell membranes by transporting three Na^(+)out of and two K^(+)into cells.Additionally,Na^(+)/K^(+)-ATPase participates in Ca^(2+)-signaling transduction and neurotransmitter release by coordinating the ion concentration gradient across the cell membrane.Na^(+)/K^(+)-ATPase works synergistically with multiple ion channels in the cell membrane to form a dynamic network of ion homeostatic regulation and affects cellular communication by regulating chemical signals and the ion balance among different types of cells.Therefo re,it is not surprising that Na^(+)/K^(+)-ATPase dysfunction has emerged as a risk factor for a variety of neurological diseases.However,published studies have so far only elucidated the important roles of Na^(+)/K^(+)-ATPase dysfunction in disease development,and we are lacking detailed mechanisms to clarify how Na^(+)/K^(+)-ATPase affects cell function.Our recent studies revealed that membrane loss of Na^(+)/K^(+)-ATPase is a key mechanism in many neurological disorders,particularly stroke and Parkinson's disease.Stabilization of plasma membrane Na^(+)/K^(+)-ATPase with an antibody is a novel strategy to treat these diseases.For this reason,Na^(+)/K^(+)-ATPase acts not only as a simple ion pump but also as a sensor/regulator or cytoprotective protein,participating in signal transduction such as neuronal autophagy and apoptosis,and glial cell migration.Thus,the present review attempts to summarize the novel biological functions of Na^(+)/K^(+)-ATPase and Na^(+)/K^(+)-ATPase-related pathogenesis.The potential for novel strategies to treat Na^(+)/K^(+)-ATPase-related brain diseases will also be discussed.

New perspectives in prognostication of hepatocellular carcinoma:The role and clinical implications of transient receptor potential family genes

The study titled“Transient receptor potential-related risk model predicts prognosis of hepatocellular carcinoma patients”is a significant contribution to hepatocellular carcinoma(HCC)research,highlighting the role of transient receptor potential(TRP)family genes in the disease’s progression and prognosis.Utilizing data from The Cancer Genome Atlas database,it establishes a new risk assessment model,emphasizing the interaction of TRP genes with tumor proliferation pathways,key metabolic reactions like retinol metabolism,and the tumor immune microenvironment.Notably,the overexpression of the TRPC1 gene in HCC correlates with poorer patient survival outcomes,suggesting its potential as a prognostic biomarker and a target for personalized therapy,particularly in strategies combining immunotherapy and anti-TRP agents.

Expression levels of K_(ATP)channel subunits and morphological changes in the mouse liver after exposure to radiation

BACKGROUND ATP sensitive K+(K_(ATP))channels are ubiquitously distributed in various of cells and tissues,including the liver.They play a role in the pathogenesis of myocardial and liver ischemia.AIM To evaluate the radiation-induced changes in the expression of K_(ATP)channel subunits in the mouse liver to understand the potential role of K_(ATP)channels in radiation injury.METHODS Adult C57BL/6 mice were randomly exposed toγ-rays at 0 Gy(control,n=2),0.2 Gy(n=6),1 Gy(n=6),or 5 Gy(n=6).The livers were removed 3 and 24 h after radiation exposure.Hematoxylin and eosin staining was used for morphological observation;immunohistochemical staining was applied to determine the expression of K_(ATP)channel subunits in the liver tissue.RESULTS Compared with the control group,the livers exposed to 0.2 Gyγ-ray showed an initial increase in the expression of Kir6.1 at 3 h,followed by recovery at 24 h after exposure.Exposure to a high dose of 5.0 Gy resulted in decreased expression of Kir6.1 and increased expression of SUR2B at 24 h.However,the expression of Kir6.2,SUR1,or SUR2A had no remarkable changes at 3 and 24 h after exposure to any of these doses.CONCLUSION The expression levels of Kir6.1 and SUR2B in mouse liver changed differently in response to different radiation doses,suggesting a potential role for them in radiation-induced liver injury.

Roles of TRESK,a novel two-pore domain K+channel,in pain pathway and general anesthesia

TRESK is the most recently reported two-pore domain K^＋ channel, and different from other two-pore domain channels in gene, molecular structure, electrophysiological and pharmacological properties. Although the current knowledge of this potassium channel is inadequate, researches have demonstrated that TRESK is remarkablely linked to acute and chronic pain by activation of calcineurin. The fact that TRESK is sensitive to volatile anesthetics and localization in central nerve system implies that TRESK may play a very important role in the mechanism mediating general anesthesia. The further research of TRESK may contribute to explore the underlying mechanism of some pathological conditions and yield novel treatments for some diseases.

Involvement of leak K^+ channels in neurological disorders

TWIK-related acid-sensitive K+(TASK) channels give rise to leak K+ currents which influence the resting membrane potential and input resistance. The wide expression of TASK1 and TASK3 channels in the central nervous system suggests that these channels are critically involved in neurological disorders. It has become apparent in the past decade that TASK channels play critical roles for the development of various neurological disorders. In this review, I describe evidence for their roles in ischemia, epilepsy, learning/memory/cognition and apoptosis.

An expert system for predicting shear stress distribution in circular open channels using gene expression programming

The shear stress distribution in circular channels was modeled in this study using gene expression programming(GEP). 173 sets of reliable data were collected under four flow conditions for use in the training and testing stages. The effect of input variables on GEP modeling was studied and 15 different GEP models with individual, binary, ternary, and quaternary input combinations were investigated. The sensitivity analysis results demonstrate that dimensionless parameter y/P, where y is the transverse coordinate, and P is the wetted perimeter, is the most influential parameter with regard to the shear stress distribution in circular channels. GEP model 10, with the parameter y/P and Reynolds number(Re) as inputs, outperformed the other GEP models, with a coefficient of determination of 0.7814 for the testing data set. An equation was derived from the best GEP model and its results were compared with an artificial neural network(ANN) model and an equation based on the Shannon entropy proposed by other researchers. The GEP model, with an average RMSE of 0.0301, exhibits superior performance over the Shannon entropy-based equation, with an average RMSE of 0.1049, and the ANN model, with an average RMSE of 0.2815 for all flow depths.

Identification and Functional Analysis on Abiotic Stress Response of Soybean Cl^- Channel Gene GmCLCnt

The Cl^- homeostasis was known as the major mechanism of soybean to achieve NaCl tolerance, but studies on the role of chloride channel under abiotic stress were relatively few. We cloned a putative CLC-type chloride channel gene GmCLCnt from soybean via RACE and it was predicted to encode a protein of 783 amino acids with 9 possible transmembrane domains and 2 tandem CBS domains. Real-time RT-PCR analysis showed that the GmCLCnt gene was expressed in all tissues of soybean but enriched in leaves and its expression was induced by NaCl, polyethylene glycol （PEG）, coldness and ABA treatments. The Arabidopsis seedlings overexpressing GmCLCnt were more tolerant to higher concentration of NaCl than those of wild type. The results suggested that the GmCLCnt may be a CLC-type chloride channel and play an important role in salt tolerance.

Epigenetics of epithelial Na^+ channel-dependent sodium uptake and blood pressure regulation

The epithelial Na^＋ channel （ENaC） consists of α, β, γ subunits. Its expression and function are regulated by aldosterone at multiple levels including transcription. ENaC plays a key role in Na^＋ homeostasis and blood pressure. Mutations in ENaC subunit genes result in hypertension or hypotension, depending on the nature of the mutations. Transcription of αENaC is considered as the rate-limiting step in the formation of functional ENaC. As an aldosterone target gene, αENaC is activated upon aldosterone- mineralocorticoid receptor binding to the cis-elements in the αENaC promoter, which is packed into chromatin. However, how aldosterone alters chromatin structure to induce changes in transcription is poorly understood. Studies by others and us suggest that Dot1a-Af9 complex represses αENaC by directly binding and regulating targeted histone H3 K79 hypermethylation at the specific subregions of αENaC promoter. Aldosterone decreases Dot1a-Af9 formation by impairing expression of Dot1a and Af9 and by inducing Sgk1, which, in turn, phosphorylates Af9 at S435 to weaken Dot1a-Af9 interaction. MR attenuates Dot1a-Af9 effect by competing with Dot1a for binding Af9. Af17 relieves repression by interfering with Dot1a-Af9 interaction and promoting Dot1a nuclear export. Af17^-/- mice exhibit defects in ENaC expression, renal Na^＋ retention, and blood pressure control. This review gives a brief summary of these novel fndings.

Voltage-dependent K^+-channel Responses during Activation and Damage in Alveolar Macrophages Induced by Quartz Particles

The roles of voltage-dependent K^＋ channels during activation and damage in alveolar macrophages （AMs） exposed to different silica particles were examined. Rat AMs were collected by means of bronchoalveolar lavage, and were adjusted to 5× 10^5/mL. After AMs were exposed to different concentrations （0, 25, 50, 100, 200 μg/mL） of quartz particles and 100 μg/mL amorphous silica particles for 24 h, the voltage-depended K^＋ current in AMs was measured by using patch clamp technique. Meanwhile the leakage of lactate dehydrogenase （LDH） and the viability of AMs were detected respectively. Patch clamp studies demonstrated that AMs possessed outward delayed and inward rectifying K^＋ current. Exposure to quartz particles increased the outward delayed K^＋ current but it had no effect on inward rectifier K^＋ current in AMs. Neither of the two K^＋ channels in AMs was affected by amorphous silica particles. Cytotoxicity test showed that both silica particles could damage AM membrane and result in significant leakage of LDH （P〈0.05）. MTT studies, however, showed that only quartz particles reduced viability of AMs （P〈0.05）. It is concluded that quartz parti- cles can activate the outward delayed K^＋ channel in AMs, which may act as an activating signal in AMs to initiate an inflammatory response during damage and necrosis in AMs induced by exposure to quartz particle. K^＋ channels do not contribute to the membrane damage of AMs.

Localization of ATP-sensitive K^+ channel subunits in rat liver

BACKGROUND ATP-sensitive K^+(KATP)channels were originally found in cardiac myocytes by Noma in 1983.KATP channels were formed by potassium ion-passing poreforming subunits(Kir6.1,Kir6.2)and regulatory subunits SUR1,SU2A and SUR2B.A number of cells and tissues have been revealed to contain these channels including hepatocytes,but detailed localization of these subunits in different types of liver cells was still uncertain.AIM To investigate the expression of KATP channel subunits in rat liver and their localization in different cells of the liver.METHODS Rabbit anti-rat SUR1 peptide antibody was raised and purified by antigen immunoaffinity column chromatography.Four of Sprague-Dawley rats were used for liver protein extraction for immunoblot analysis,seven of them were used for immunohistochemistry both for the ABC method and immunofluorescence staining.Four of Wistar rats were used for the isolation of hepatic stellate cells(HSCs)and Kupffer cells for both primary culture and immunocytochemistry.RESULTS Immunoblot analysis showed that the five kinds of KATP channel subunits,i.e.Kir6.1,Kir6.2,SUR1,SUR2A,and SUR2B,were detected in liver.Immunohistochemical staining showed that Kir6.1 and Kir6.2 were weakly to moderately expressed in parenchymal cells and sinusoidal lining cells,while SUR1,SUR2A,and SUR2B were mainly localized to sinusoidal lining cells,such as HSCs,Kupffer cells,and sinusoidal endothelial cells.Immunoreactivity for SUR2A and SUR2B was expressed in the hepatocyte membrane.Double immunofluorescence staining further showed that the pore-forming subunits Kir6.1 and/or Kir6.2 colocalized with GFAP in rat liver sections and primary cultured HSCs.These KATP channel subunits also colocalized with CD68 in liver sections and primary cultured Kupffer cells.The SUR subunits colocalized with GFAP in liver sections and colocalized with CD68 both in liver sections and primary cultured Kupffer cells.In addition,five KATP channel subunits colocalized with SE-1 in sinusoidal endothelial cells.CONCLUSION Observations from the present study indicated that KATP channel subunits expressed in rat liver and the diversity of KATP channel subunit composition might form different types of KATP channels.This is applicable to hepatocytes,HSCs,various types of Kupffer cells and sinusoidal endothelial cells.

A novel mutation in the sodium channel α1 subunit gene in a child with Dravet syndrome in Turkey

Dravet syndrome is a rare epileptic encephalopathy characterized by frequent seizures beginning in the first year of life and behavioral disorders. Mutations in the sodium channel α1 subunit gene are the main cause of this disease. We report two patients with refractory seizures and psychomotor retardation in whom the final diagnosis was Dravet syndrome with confirmed mutations in the sodium channel α1 subunit gene. The mutation identified in the second patient was a novel frame shift mutation, which resulted from the deletion of five nucleotides in exon 24.

A novel frameshift mutation in the beta-subunit of the epithelial sodium channel gene caused Liddle syndrome

Objective To characterize a novel frameshift mutation of the epithelial sodium channel(ENaC)βsubunit in a Chinese family with clinical suspicion of Liddle syndrome.And to emphasize that genetic testing is a confirmatory evidence of the diagnosis of Liddle syndrome.Methods DNA samples from the proband with early-onset,treatment-resistant hypertension and hypokalemia and 31 additional relatives were all sequenced for mutations in exon 13 of theβ-ENaC andγ-ENaC genes,using amplification by polymerase chain reaction and direct DNA sequencing.

Arsenic exposure decreases rhythmic contractions of vascular tone through sodium transporters and K^+ channels

Arsenic-contaminated drinking water is a public health problem in countries such as Taiwan, Bangladesh, United States, Mexico, Argentina, and Chile. The chronic ingestion of arsenic-contaminated drinking water increases the risk for ischemic heart disease, cerebrovascular disease, and prevalence of hypertension. Although toxic arsenic effects are controversial, there is evidence that a high concentration of arsenic may induce hypertension through increase in vascular tone and resistance. Vascular tone is regulated by the rhythmic contractions of the blood vessels, generated by calcium oscillations in the cytosol of vascular smooth muscle cells. To regulate the cytosolic calcium oscillations, the membrane oscillator model involves the participation of Ca2+ channels, calcium-activated K+ channels, Na+/Ca2+exchange, plasma membrane Ca2+-ATPase, and the Na+/K+-ATPase. However, little is known about the role of K+ uptake by sodium transporters [Na+/K+-ATPase or Na+-K+-2Cl-(NKCC1)] on the rhythmic contractions.Vascular rhythmic contractions, or vasomotion are a local mechanism to regulate vascular resistance andblood flow. Since vascular rhythmic contractions of blood vessels are involved in modulating the vascular resistance, the blood flow, and the systemic pressure,we suggest a model explaining the participation of the sodium pump and NKCC1 co-transporter in low dose arsenic exposure effects on vasomotion and vascular dysfunction.

Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons

Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knock- down of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.

The genetic study of ischemia induced arrhythmia and potassium channels

Objectives Ischemia induced arrhythmia(ventricular tachycardia/ventricular fibrillation) is one of the major causes of death.Potassium channels change are likely to be responsible for the ischemia-related arrhythmias.Cardiac potassium current is the major outward current involved in cardiac repolarization.The properties of potassium channels have been intensively studied.Here,we investigated the association between ischemia induced arrhythmia and potassium channels genetic variations.Methods 23 patients with ventricular tachycardia/ventricular fibrillation induced by ischemia were selected as objects.5ML peripheral blood were taken from each person,from which DNA was extracted us- ing a standard enzymatic phenol-chloroform method.Candidate genes(HERG、KCNJ2、KCNQ1、Mink、Mirp1、Kir2.1、KV4.3、Kir3.1、KV1.5、Kir6.1、Kir6.2、Kir2.1) Were screened for potassium channels gene mutations with direct sequencing methods.Results Here 4 potassium channels gene mutations have been discovered.In the gene coding for the ATP-sensitive K^+ channels subunit Kir6.2,there is a change from valine to isoleucine at the position of 326(V326I).At the position 448,arginine substitutes proline(P448R) in the KC-NQ1 gene.In the gene KCNJ2 two mutations have been found(P156L,Q193H).Conclusions This study implicated that there is a high relationship between ischemia induced arrhythmia and the mutation of potassium channels.In order to identify the precisely relationship there is need functional analysis.

Salt Stress Affects the Growth and Yield of Wheat (Triticum aestivum L.) by Altering the Antioxidant Machinery and Expression of Hormones and Stress- Specific Genes

Understanding physiological responses in saline agriculture may facilitate wheat breeding programs.Based on a screening test,the Ningmai-14(NM-14)and Yangmai-23(YM-23)wheat cultivars were selected for further experiments to understand the underlying salinity tolerance mechanism.This study investigated the effects of five salinity levels such as Control(CK)=0(without NaCl stress),S1=0.20%,S2=0.25%,S3=0.30%and S4=0.35%of NaCl concentrations of soil on wheat plants.The results showed that increased salinity concentration reduced the growth and yield of wheat cultivars(NM-14 and YM-23).However,YM-23(12.7%)yielded more than NM-14 at maximum salinity stress.The higher salinity(S4)increased the concentration of Na^(+)(4.3 to 5.8-fold)and P contents(2.5 to 2.2-fold),while reducing the average concentrations of K^(+),Cu,and K^(+)/Na^(+)ratio.The higher salinity(S4)reduced the spikelet length by 21.35%(followed by grain spike−1),and the starch content by 18.81%.In the YM-23 cultivar,higher salinity increased superoxide dismutase(SOD),total antioxidant capacity(TAC),and amylase.Compared to NM-14,induced expression of TaYUC2,6,and TaGA13ox,20ox genes were recorded in YM-23.Similarly,in YM-23 the stress-specific genes such as TaHSP70,90 were enhanced whereas,TaSOS1,2 were suppressed.Overall,our study revealed that salt tolerant cultivars modulate hormonal and antioxidant activities,thus maintaining high growth.

神经元限制性沉默因子REST/NRSF参与调控癫痫作用及机制研究

目的探究神经元限制性沉默因子(REST/NRSF)参与调控癫痫作用及分子机制。方法采用免疫组织化学染色、免疫荧光、Western blot和qPCR检测癫痫患者病灶组织和海人藻酸(kainic acid,KA)癫痫小鼠海马脑区REST/NRSF表达水平的变化;采用病毒注射,脑电图记录和行为学方法检测在海马CA1区分别敲低或过表达REST/NRSF后对小鼠癫痫发作的影响。结果癫痫患者病灶REST/NRSF表达水平相对于脑外伤患者脑组织明显升高;KA模型组小鼠海马CA1区REST/NRSF蛋白和mRNA水平均明显升高,Kv7.2、Kv7.3钾通道mRNA表达水平明显下调;脑内注射NMDA兴奋海马脑区小鼠REST/NRSF表达水平明显上调;海马CA1区敲低REST/NRSF明显升高Kv7.2、Kv7.3钾通道mRNA表达水平,明显降低小鼠脑电图棘波、尖波发放频率以及癫痫发作等级;海马CA1区过表达REST/NRSF明显降低Kv7.2、Kv7.3钾通道mRNA表达水平,明显升高小鼠棘波、尖波发放频率,癫痫症状明显加重。结论小鼠海马脑区REST/NRSF通过转录调控Kv7.2、Kv7.3钾通道参与癫痫疾病发生发展。

筇竹钾离子通道QtSKOR1基因的克隆及表达分析

【目的】外向整流型钾离子通道SKOR(Stelar K+outward rectifier)家族是参与植物钾离子转运和分配的重要通道,且K+在植物生理过程和响应非生物胁迫中有决定性作用。旨在研究外向整流型钾离子通道SKOR在富含钾元素的筇竹(Qiongzhuea tumidinoda)发育及受到盐胁迫过程中的功能。【方法】利用RACE技术从筇竹的幼苗中获得SKOR基因并命名为QtSKOR1(基因号:MT078984),并对其进行生物信息分析及表达特征分析。【结果】QtSKOR1基因开放阅读框(1923 bp)编码了641个氨基酸。QtSKOR1蛋白存在环核苷酸(cNMP)和锚蛋白重复结构域(ANK)属于Shake亚家族,其相对分子质量为157.27 kD,理论等电点为4.94,含有3个跨膜区但不存在信号肽,属于疏水性蛋白。亚细胞定位分析表明QtSKOR1蛋白主要定位于线粒体(30.4%)和细胞质(26.1%)中。同源分析和进化分析显示,QtSKOR1与二穗短柄草(Brachypodium distachyum)和玉米(Zea mays)的同源性较高,分别为89.06%和87.91%。qPCR结果显示,QtSKOR1基因在筇竹所有组织中均有表达,且表达量从高到低依次为叶、根、茎、笋。与对照相比,随着钾胁迫时间的增加,QtSKOR1的表达量在根中显著增加,而在茎叶中显著降低。随着钠胁迫时间的增加,QtSKOR1的表达量在根茎叶中均呈下降趋势。【结论】QtSKOR1基因属于Shake亚家族,均参与了筇竹各个组织特别是叶的钾运输。同时,在钾胁迫下QtSKOR1基因在根中发挥了积极作用。

K^+通道基因MIRK在网纹甜瓜植株中的表达分析

在网纹甜瓜(Cucumis melovar.reticulatus Naud.)叶片中克隆了一个cDNA片段,片段长1 330 bp。这段cDNA序列及由此推测的氨基酸序列分析和多序列比对表明扩增的片段是编码K+通道MIRK(Melon Inward Rectifying K+Channel)基因的5′端。由此cDNA片段推测的氨基酸序列包含了6个跨膜片段S1到S6,其间有一个对离子选择性有重要作用的特征序列结构GYGD(Gly-Tyr-Gly-Asp)。进化树分析显示MIRK属于KAT1亚家族,和在葡萄叶片中克隆的K+通道SIRK最相近。半定量RT-PCR试验表明,MIRK在甜瓜植株中根、茎、叶、花、果实等各器官中均有表达,主要在叶片和初期发育的果实中表达,在雌花和茎中也有表达,在根中的表达量最低。MIRK在甜瓜不同器官中的表达模式显示MIRK可能在甜瓜植株发育过程中起重要作用。