Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

The Amino Acid Content in Gamma-irradiated Rice†

High phosphate accumulating bacteria were isolated by autoradiography. One isoate, Arthrobacter globiformis PAB-6 accumulated phosphate intracellularly at 20% of dry cell mass in a simple synthetic medium. This amount was 3~7 times higher than type cultures examined. Almost no phosphate was released into the medium after cessation of growth. Fifty percent of total intracellular phosphate was fractionated as nucleic acids, while 20% each was recovered from cold PCA soluble fractions and polyphosphate fractions. The large content of nucleic acids in this bacterium appeared due to increased RNA content, specifically 4 S RNA fraction.     

Serotonin transporter gene,childhood emotional abuse and cognitive vulnerability to depression

Meta‐analyses evaluating the association between the serotonin transporter polymorphism (5‐HTTLPR) with neuroticism and depression diagnosis as phenotypes have been inconclusive. We examined a gene–environment interaction on a cognitive vulnerability marker of depression, cognitive reactivity (CR) to sad mood. A total of 250 university students of European ancestry were genotyped for the 5‐HTTLPR, including SNP rs25531, a polymorphism of the long allele. Association analysis was performed for neuroticism, CR and depression diagnosis (using a self‐report measure). As an environmental pathogen, self‐reported history of childhood emotional abuse was measured because of its strong relationship with depression. Participants with the homozygous low expressing genotype had high CR if they had experienced childhood emotional maltreatment but low CR if they did not have such experience. This interaction was strongest on the Rumination subscale of the CR measure. The interaction was not significant with neuroticism or depression diagnosis as outcome measures. Our results show that 5‐HTTLPR is related to cognitive vulnerability to depression. Our findings provide evidence for a differential susceptibility genotype rather than a vulnerability genotype, possibly because of the relatively low levels of abuse in our sample. The selection of phenotype and environmental contributor is pivotal in investigating gene–environment interactions in psychiatric disorders.     

Immunohistochemical localization of Na+-dependent glucose transporter in rat jejunum

Summary Glucose is actively absorbed via a Na⁺-dependent active glucose transporter (Na-GT) in the small intestine. We raised a polyclonal antibody against the peptide corresponding to amino acids 564–575 of rabbit intestinal Na-GT, and localized it immunohistochemically in the rat jejunum. By means of immunofluorescence staining, Na-GT was located at the brush border of the absorptive epithelial cells of the intestinal villi. Electron-microscopic examination showed that Na-GT was localized at the plasma membrane of the apical microvilli of these cells. Little Na-GT was found at the basolateral plasma membrane. Along the crypt-villus axis, all of the absorptive epithelial cells in the villus were positive for Na-GT. In addition to the brush border staining, the supranuclear positive staining, which was shown to be the Golgi apparatus by use of electron microscopy, was seen in cells located between the base to the middle of the villus. Cells in crypts exhibited little or no staining for Na-GT. Goblet cells scattered in the intestinal epithelium were negative for Na-GT staining. These observations show that Na-GT is specific to the apical plasma membrane of the absorptive epithelial cells, and that the onset of Na-GT synthesis may occur near the crypt-villus junction.     

Vanadate induces the recruitment of glut-4 glucose transporter to the plasma membrane of rat adipocytes

Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter.     

去甲肾上腺素在大鼠缰核引起的心血管效应及其机制

在乌拉坦麻醉的大鼠,研究了去甲肾上腺素(NE)在缰核(Hb)引起的心血管效应及其机制。Hb内微量注射NE使平均动脉压和心率呈剂量依赖性增加。用α受体阻断剂酚妥拉明预处理Hb,可明显减弱NE在Hb引起的心血管效应,但β受体阻断剂心得安或生理盐水不引起任何影响。Hb内微量注射海人酸使平均动脉压和心率明显增加,但Hb内微量注射利多卡因则不能引起明显的心血管效应。上述结果表明,Hb内NE在调节心血管活动中起重要作用,而这种效应可能是通过激活α受体使Hb兴奋的结果。     

脑室注射6-羟多巴胺对黄鼠冬眠入眠的影响

采用脑室内注射化学去交感药物6-羟多巴胺(6-OHDA)的方法,观察了人为地降低脑内去甲肾上腺素(NE)系统活动对达乌尔黄鼠入眠的影响。结果表明:脑室注射100-200μg6-OHDA使脑内NE含量减少50％以上,明显促进黄鼠入眠,平均入眠诱导期比自然冬眠动物明显缩短,整个冬眠季内冬眠时间延长,冬眠黄鼠仍具有正常的入眠觉醒周期。这些结果提示脑内NE系统活动水平降低是触发动物入眠的重要因素之一。     

Brain injury: New insights into neurotransmitter and receptor mechanisms

The studies reviewed here represent a continuing search for mechanisms which play a role in neurological disturbances resulting from brain injury. Focal cortical freezing lesions in rats were shown to cause a widespread decrease in local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere and this was interpreted as reflecting a depression of cortical activity. Such an interpretation was supported by the finding that in lesioned brain reduction of cerebral metabolism by pentobarbital and isoflurane was limited by the metabolic depression that has already occurred as a result of injury and by the demonstration that the energy status and substrate (glucose) supply in the cortical areas in the injured brain have not been compromised at the time when LCGU was decreased. Both the serotonergic and the noradrenergic neurotransmitter systems were implicated in functional alterations associated with injury. Cortical serotonin (5-HT) metabolism was increased throughout the lesioned hemisphere and complete inhibition of 5-HT synthesis withp-chlorophenylalanine ameliorated the decrease in cortical LCGU, interpreted as reflecting cortical functional depression. Cortical norepinephrine metabolism was bilaterally increased in focally injured brain, while prazosin, a selective ₁-noradrenergic receptor blocker, normalized cortical LCGU in the lesioned hemisphere. Low-affinity in vivo binding of [¹²⁵I]HEAT, another selective ₁-receptor ligand, was specifically increased in cortical areas of the lesioned hemisphere at the time of the greatest depression in LCGU, suggesting that ₁-adrenoreceptors may be of functional importance in injured brain. The general conclusion from this series of studies on mechanisms underlying functional disturbances in injured brain is that both the serotonergic and the noradrenergic neurotransmitter systems are involved in the widespread cortical depression which develops with time as a consequence of a focal lesion. The data are compatible with the inhibitory effects of NE and 5-HT in the cortex and with the hypothesis that these two transmitter systems affect cortical information processing.     

内皮素对大鼠肠系膜微血管的作用

应用显微电视录象技术可观察到内皮素(ET)对戊巴比妥钠麻醉的 Wistar 大鼠的肠系膜微血管有收缩作用,血液流速减慢,甚至产生微循环障碍,这个作用相当持久,并呈剂量依赖关系。ET 的这个作用远高于去甲肾上腺素。这些结果提示,ET 作为强烈的微血管收缩剂,在微循环紊乱疾病的发病学中可能具有重要意义。     

大鼠离体乳头肌收缩末期张力—长度关系的曲线性

大鼠离体左室乳头肌固定于最适初长位,逐步递减“后荷”获得一系列等张收缩的张力、长度缩短程度和速度。结果发现:(1)收缩末期张力-长度关系(ESTLR)为指数曲线,回归方程 T=ar~(-bL)-K 拟合的优度明显高于线性方程拟合的优度(P<0.001),其中 a,k 分别代表总张力和静息张力,b 为曲线的弯曲度;(2)在高钙(4mmol/L)或去甲肾上腺素(NE10~(-6)mol/L)作用下,ESTLR 右上移位,a,b 和无张力缩短速度 L_O 均增大(P均<0.01),尤以高钙时的变化更明显,(3)NE 使张力-速度曲线的右上移位比高钙显著。这提示大鼠离体心肌的 ESTLR 呈非线性特征,参数 a,b 及长度轴截距 L_O 对收缩强度的变化敏感,但对收缩速度改变的敏感性可能比经典的力学指标低。