G-protein-mediated inhibition of the Trp channel TRPM1 requires the Gβγ dimer

ON bipolar cells are critical for the function of the ON pathway in the visual system. They express a metabotropic glutamate receptor (mGluR6) that, when activated, couples to the G(o) class of G protein. The channel that is primarily responsible for the synaptic response has been recently identified as the transient receptor potential cation channel subfamily M member 1 (TRPM1); TRPM1 is negatively coupled to the mGluR6/Go cascade such that activation of the cascade results in closure of the channel. Light indirectly opens TRPM1 by reducing transmitter release from presynaptic photoreceptors, resulting in a decrease in mGluR6 activation. Conversely, in the dark, binding of synaptic glutamate to mGluR6 inhibits TRPM1 current. Closure of TRPM1 by G-protein activation in the dark is a critical step in the process of ON bipolar cell signal transduction, but the precise pathway linking these two events is not understood. To address this question, we measured TRPM1 activity in retinal bipolar cells, in human ependymal melanocytes (HEMs) that endogenously express TRPM1, and in HEK293 cells transfected with TRPM1. Dialysis of the Gβγ subunit dimer, but not Gα(o), closed TRPM1 channels in every cell type that we tested. In addition, activation of an endogenous G-protein-coupled receptor pathway in HEK293 cells that releases Gβγ without activating Go protein also closed TRPM1 channels. These results suggest a model in which the Gβγ dimer that is released as a result of the dissociation from Gα(o) upon activation of mGluR6 closes the TRPM1 channel, perhaps via a direct interaction.     

负载紫红质通道蛋白2重组腺病毒的包装及功能鉴定

背景 紫红质通道蛋白2(ChR2)是从单细胞绿藻莱茵衣藻上分离的一种光敏感通道蛋白,可作为光刺激神经细胞的手段,与电、磁和超声波相比,光在时间和空间上对神经细胞的刺激将更精确. 目的 构建负载ChR2基因的重组腺病毒,并鉴定其功能.方法 将人胚肾293( HEK293)细胞在含质量分数10％胎牛血清的DF12培养基中进行培养及传代.腺病毒穿梭质粒pSB291-hCHR2-GFP与腺病毒包装质粒pBHGlox △E1,3 Cre共转染HEK293,包装得到少量负载ChR2基因的重组腺病毒(Ad-ChR2),经HEK293细胞扩增、CsCl梯度离心,Tris-HCl透析后得到纯化Ad-ChR2.获取4只出生1d的清洁级Long Evans大鼠视皮层组织,用组织块培养法利用无血清培养基原代培养视皮层细胞,用纯化的Ad-ChR2转染培养的视皮层细胞,当转染成功的视皮层细胞表达绿色荧光时给予460 nm蓝光刺激,应用膜片钳技术记录视皮层细胞的动作电位.结果 纯化浓缩后的Ad-ChR2滴度可达7.9×1010PFU/ml,HEK293细胞转染Ad-ChR2 24 h后倒置荧光显微镜下即可见细胞膜上有绿色荧光表达,转染13d可见细胞由扁平变为圆形.无血清培养的视皮层细胞转染纯化的Ad-ChR2后细胞膜上可见绿色荧光蛋白(GFP)的表达,460 nm蓝光刺激后用膜片钳技术可记录到蓝光诱发的视皮质细胞的动作电位.结论 本研究成功构建了负载ChR2基因的重组腺病毒表达载体,并证实ChR2能够感染有功能的视皮质细胞,这对视觉可塑性方面的研究非常重要.     

A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis

Inorganic phosphate (Pi) is stored in the vacuole, allowing plants to adapt to variable Pi availability in the soil. The transporters that mediate Pi sequestration into vacuole remain unknown, however. Here we report the functional characterization of Vacuolar Phosphate Transporter 1 (VPT1), an SPX domain protein that transports Pi into the vacuole in Arabidopsis. The vpt1 mutant plants were stunted and consistently retained less Pi than wild type plants, especially when grown in medium containing high levels of Pi. In seedlings, VPT1 was expressed primarily in younger tissues under normal conditions, but was strongly induced by high-Pi conditions in older tissues, suggesting that VPT1 functions in Pi storage in young tissues and in detoxification of high Pi in older tissues. As a result, disruption of VPT1 rendered plants hypersensitive to both low-Pi and high-Pi conditions, reducing the adaptability of plants to changing Pi availability. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vpt1 compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO₄²⁻, NO₃⁻, Cl⁻, and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.Phosphorus (P) is a critical component of many metabolites and macromolecules, including ATP, phospholipid, and nucleic acid, and participates in numerous biochemical pathways, including gene expression and signal transduction, among many others. A major source of P element in the biological systems comes from the soil from which plants absorb soluble forms of P-containing molecules and metabolize them into organic forms. As the major form of soluble P, inorganic phosphate (Pi) is present in the soil at low levels and is often a limiting factor for plant growth and productivity. The acquisition and translocation of Pi by plant roots have been the focus of numerous studies identifying transport proteins involved in the uptake and distribution of Pi in plants (1–4).Although the Pi concentration in soil is usually low, it can be increased by microbial activity and by the application of fertilizers for agricultural crop production (2). Thus, Pi availability in soil often changes, requiring plants to develop mechanisms to adapt to such changes and maintain Pi homeostasis in the cell. When soil Pi levels are high, excess Pi is sequestered in the vacuole to prevent toxicity to the cytoplasm and serve as a Pi store (5). When soil Pi levels are low, the vacuolar Pi pool will supply Pi to the cytoplasm in support of biochemical pathways (6, 7). According to ³¹P-NMR analyses, Pi concentration is much higher in the vacuole than in the cytosol (7, 8), indicating that the vacuole is the major Pi store. Although a strong mechanism is essential for vacuolar Pi sequestration in higher plants, there is little knowledge regarding molecular identity of transport proteins in the vacuolar membrane (tonoplast).So far, vacuolar phosphate transporters have been studied mainly in the yeast model, Saccharomyces cerevisiae. The yeast vacuolar protein complex responsible for vacuolar polyphosphate accumulation consists of four transmembrane proteins: ScVTC1 (vacuolar transporter chaperone 1), ScVTC2, ScVTC3, and ScVTC4 (9). Another tonoplast Pi transporter, ScPHO91, is mainly responsible for vacuolar Pi efflux to the cytoplasm under low-Pi conditions (10). Interestingly, ScVTC2/3/4 and ScPHO91 share a common feature, the presence of the SPX domain [named after yeast Syg1 and Pho81 and human xenotropic and polytropic retrovirus receptor 1 (XPR1)]. The SPX proteins also exist in plants, and some of them have been shown to play a role in Pi nutrition (11).In Arabidopsis, there are four protein families containing SPX domains, including SPX, SPX-RING, SPX-EXS, and SPX-MFS. The members of the first two families contain no transmembrane domains. The nucleus-localized SPX1 is a negative regulator of Pi starvation responses through inhibiting the activity of PHOSPHATE STARVATION RESPONSE REGULATOR 1 (PHR1) (12). As a member of the SPX-RING family with the RING-type ubiquitin ligase activity, NITROGEN LIMITATION ADAPTATION (AtNLA) may regulate protein degradation of some Pi transporters to maintain Pi homeostasis in a nitrate-dependent manner (13, 14). The SPX-EXS family consists of 11 members that contain multiple transmembrane domains, characteristic of transporters. Indeed, two members of this family, the Golgi-located PHO1 (PHOSPHATE 1) and PHO1;H1, are shown to function in Pi translocation (15, 16). The SPX-MFS family, like SPX-EXS, also contains a group of proteins containing multitransmembrane domains. There are three members in the Arabidopsis SPX-MFS family, but the function of none of them has been characterized to date. Using a combination of genetic and electrophysiological approaches, we show here that one of these, Vacuolar Phosphate Transporter 1 (VPT1), functions as a phosphate transporter essential for vacuolar Pi sequestration, bridging a gap in our understanding of Pi homeostasis in plant cells.     

银杏内酯B诱导骨髓间充质干细胞分化的神经样细胞的电生理及免疫学特征

目的 利用银杏内酯B(GB)诱导大鼠骨髓间充质干细胞(BMSC)分化成熟,研究其分化的神经样细胞的电生理特征及免疫学特征.为临床治疗神经系统免疫性疾病提供最合适种子细胞.方法 应用倒置相差显微镜对GB诱导的细胞进行多个时间点观察其细胞形态学改变,并对诱导后细胞进行免疫荧光染色;应用膜片钳技术,采取全细胞记录方式,对由GB诱导的大鼠BMSC进行诱导前及诱导后多个时间点的电生理测定.结果 免疫荧光染色阳性细胞:CHAT:(34.8％,n=12);GFAP:(31.6％,n=12);NSE:(21.3％,n=12);膜电位随着时间的变化:未分化细胞的峰值电流(1.45 ±0.15PA/PF,n=10),诱导后的神经元样细胞(2.18 ±0.14 PA/PF,n=12)变化显著(P＜0.05).结论 大鼠BMSC经过GB诱导后免疫特性及膜电位均发生改变,显示诱导后细胞有神经干细胞的免疫学特点,可以作为一种调节神经免疫力及修复力的种子细胞.     

Calculating the insulin to carbohydrate ratio using the hyperinsulinaemic-euglycaemic clamp-a novel use for a proven technique

BACKGROUND: In patients with type 1 diabetes, three main variables need to be assessed to optimize meal-related insulin boluses: pre-meal blood glucose (BG), insulin to carbohydrate ratio (I : C), and basal insulin. We are presenting data for a novel use of the hyperinsulinaemic-euglycaemic clamp (HEC) in patients with type 1 diabetes that minimizes the impact of these variables and can be used to determine the I : C. METHODS: Ten subjects (six men and four women) using continuous subcutaneous insulin infusion (CSII) pumps were recruited for this study [24-65 years; BMI 27.1 +/- 4.9 kg/m(2); A1C 7.2 +/- 1.4% (mean +/- SD)]. The HEC used a primed continuous intravenous insulin infusion of 40 mU/m(2)/min and a variable infusion of 20% glucose to maintain BG at 90 mg/dL. After subjects were in steady state (SS) for 50 min, a standardized meal (40% of total calories/day - 30% carbohydrate, 30% protein, 40% fat) was consumed. Subjects gave the insulin bolus with their CSII pump. No changes were made in the glucose infusion rate. RESULTS: Mean BG at SS was 85.7 +/- 10.4 mg/dL. Peak BG was 115.0 +/- 12.7 mg/dL at 68.5 +/- 8.8 min after the meal. Mean I : C was 1 : 9.3 +/- 1.7 (range 1 : 7-1 : 12). Insulin sensitivity varied from 1.9 to 9.1 mg/kg/min. CONCLUSIONS: The HEC can be used to reduce confounding factors and to determine the I : C. As a first estimate of the I : C in patients with type 1 diabetes, it is recommended to start with a ratio of 1 : 9.3 and to measure post-prandial BG at 70 min.     

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Clamp-loader complexes are heteropentameric AAA+ ATPases that load sliding clamps onto DNA. The structure of the nucleotide-free Escherichia coli clamp loader had been determined previously and led to the proposal that the clamp-loader cycles between an inactive state, in which the ATPase domains form a closed ring, and an active state that opens up to form a "C" shape. The crystal structure was interpreted as being closer to the active state than the inactive state. The crystal structure of a nucleotide-bound eukaryotic clamp loader [replication factor C (RFC)] revealed a different and more tightly packed spiral organization of the ATPase domains, raising questions about the significance of the conformation seen earlier for the bacterial clamp loader. We describe crystal structures of the E. coli clamp-loader complex bound to the ATP analog ATPgammaS (at a resolution of 3.5 A) and ADP (at a resolution of 4.1 A). These structures are similar to that of the nucleotide-free clamp-loader complex. Only two of the three functional ATP-binding sites are occupied by ATPgammaS or ADP in these structures, and the bound nucleotides make no interfacial contacts in the complex. These results, along with data from isothermal titration calorimetry, molecular dynamics simulations, and comparison with the RFC structure, suggest that the more open form of the E. coli clamp loader described earlier and in the present work corresponds to a stable inactive state of the clamp loader in which the ATPase domains are prevented from engaging the clamp in the highly cooperative manner seen in the fully ATP-loaded RFC-clamp structure.     

肥胖者与糖尿病患者胰岛素敏感性的变化

按口服葡萄糖耐量试验将研究对象分成正常年轻组、正常成年组、单纯肥胖组与糖尿病组 ,通过高胰岛素正葡萄糖钳夹试验 ,计算胰岛素敏感性指数并进行比较。结果显示肥胖组及糖尿病组患者的胰岛素敏感性下降     

Lack of acute effect of amylin (islet associated polypeptide) on insulin sensitivity during hyperinsulinaemic euglycaemic clamp in humans

Summary It is suggested that amylin (islet associated polypeptide), co-secreted with insulin from the pancreatic beta cells acts as a circulating hormone which opposes the action of insulin on muscle and increases hepatic glucose production. We have tested the effect of amylin in human subjects on postabsorptive glucose homeostasis and on insulin sensitivity using the euglycaemic hyperinsulinaemic clamp. The amylin used opposed insulin-mediated glucose disposal in rat soleus muscle at concentrations of 10 nmol/l. Seven subjects were studied on two occasions and infused with either amylin or placebo for 6 h, initially when postabsorptive and then during a euglycaemic hyperinsulinaemic clamp. Mean plasma amylin concentrations during the first 3 h were 2006±327 pmol/l during amylin infusion and 20±9 pmol/l during the control infusion. Amylin infusion had no effect on postabsorptive plasma concentrations of insulin (control: 32±16 vs amylin: 25±8 pmol/l) or glucose (5.1±0.1 vs 5.3±0.1 mmol/l). During the clamp, amylin concentrations were 1636 ±422 pmol/l when it was infused and 24±6 during control infusions. Plasma glucose and insulin concentrations were well matched during the control and amylin infusions (glucose: 4.7±0.1 vs 4.8±0.1 mmol/l; insulin: 198±37 vs 195±22 pmol/l). Exogenous glucose infusion rates were a mean of 13 % lower than control values during the amylin infusion but were not statistically different (p =0.17). Therefore, an approximately 100-fold elevation of plasma amylin concentration failed to consistently alter glucose metabolism. Our data suggest that amylin does not act as a circulating hormone to influence glucose metabolism in humans. [Diabetologia (1994) 37: 166–169] Received: 1 June 1993 and in revised form: 16 August 1993     

Effects of angiotensin II on membrane current in cardiac Purkinje fibers

We studied the actions of the octapeptide hormone angiotensin II (AII) on isolated cardiac Purkinje fibers. AII (1 to 75 nm) increased the height and duration of the plateau phase of the action potential and increased the strength of contraction in these preparations. These effects were not blocked by propranolol (10^?6m). A two-microelectrode voltage clamp technique combined with simultaneous tension measurements was used to study the AII-induced changes in membrane current and contractile activation. AII enhanced peak tension and promoted an inward shift in the net membrane current-voltage relation at test voltages between ?40 and 0 mV. The inward shift in current was maintained for the duration of the 500 ms test voltage step. The AII-induced current shift was reduced or abolished when external calcium concentration was decreased from 5.4 mm to 1.8 mm, and was inhibited by the calcium antagonist D600.     

Early detection of euglycemic ketoacidosis during thoracic surgery associated with empagliflozin in a patient with type 2 diabetes: A case report

We report the first case of intraoperatively detected euglycemic diabetic ketoacidosis (DKA) associated with sodium–glucose cotransporter 2 inhibitors during thoracic surgery. A 59-year-old man had a 12-year history of type 2 diabetes mellitus treated with insulin and empagliflozin. The patient developed bacterial empyema and was initiated with antibiotics at a local hospital. Owing to the persistence of his symptoms, he was transferred to our hospital after the medication of empagliflozin the day before surgery. After overnight fasting, the patient underwent thoracoscopic debridement and intrathoracic lavage surgery. During this surgery, he was noted to have euglycemic ketosis and acidosis, and diagnosed as euglycemic DKA. Immediately after the consultation in our department, the patient underwent treatment for DKA. He awoke from anesthesia normally and showed no symptoms of DKA. DKA gradually resolved over the next 24 h. Early identification and management are critical for rapid recovery from perioperative euglycemic DKA associated with sodium–glucose cotransporter 2 inhibitors, especially during thoracic surgery.