短暂脑缺血大鼠海马CA1区锥体细胞大电导Ca2+依赖K+通道活动降低

短暂脑缺血可对随后的损伤性脑缺血表现出明显的耐受.有研究表明大电导Ca2+依赖K+(BKCa)通道活动增强参与了缺血性脑损伤.采用膜片钳的内面向外式,观察了3 min短暂脑缺血后6 h、24 h以及48 h大鼠海马CA1区锥体细胞上BKCa通道活动的动态变化.短暂脑缺血后BKCa通道的单通道电导和翻转电位均未见明显变化,但通道的开放概率则在缺血预处理后的前24 h内显著降低.通道动力学分析显示通道关闭时间变长是短暂脑缺血后通道活动降低的主要原因,因为通道的开放时间未发生明显变化.结果提示短暂脑缺血所致的BKCa通道活动降低可能与缺血耐受的产生有关.     

低剂量伽玛刀照射癫痫大鼠颞叶神经元超微结构的改变及线粒体形态计量分析

目的观察低剂量伽玛刀照射癫痫大鼠颞叶神经元超微结构的变化,探讨线粒体形态改变程度及性质。方法建立大鼠青霉素局灶性癫痫动物模型,将48只SD大鼠分为对照组（A组）、实验组（癫痫模型组,简称B组）和癫痫后伽玛刀照射组（C组）。照射周边剂量12Gy,等剂量曲线为50％。分别于0．5h～60d后取靶区颞叶皮质区制备电镜样本,透射电镜观察,通过计算机图像分析系统对线粒体形态计量分析。结果A组细胞结构基本正常;B组可见神经元细胞质细胞器明显减少空化,线粒体体密度、数密度、比表面和嵴膜密度较对照组明显减少（P〈0．05）,线粒体平均体积和平均截面积较对照组明显增大（P〈0．05）。C组早期与B组基本一致,细胞质内有少量脂褐素,中期和晚期线粒体的各项参数与A组相差不显著,低剂量伽玛刀照射后早期线粒体的平均体积、平均截面积数密度、比表面与A组相差显著（P〈0．05）,圆球度各组间无明显差异。结论大鼠癫痫发作后其线粒体的形态结构发生明显变化,低剂量伽玛刀照射对神经元的修复有重要作用,本研究认为线粒体参与了癫痫的病理过程。     

20-Hydroxyeicosatetraenoic Acid Contributes to the Inhibition of K+ Channel Activity and Vasoconstrictor Response to Angiotensin II in Rat Renal Microvessels

The present study examined whether 20-hydroxyeicosatetraenoic acid (HETE) contributes to the vasoconstrictor effect of angiotensin II (ANG II) in renal microvessels by preventing activation of the large conductance Ca²⁺-activated K⁺ channel (K_Ca) in vascular smooth muscle (VSM) cells. ANG II increased the production of 20-HETE in rat renal microvessels. This response was attenuated by the 20-HETE synthesis inhibitors, 17-ODYA and HET0016, a phospholipase A₂ inhibitor AACOF₃, and the AT₁ receptor blocker, Losartan, but not by the AT₂ receptor blocker, PD123319. ANG II (10^-11 to 10^-6 M) dose-dependently decreased the diameter of renal microvessels by 41 ± 5%. This effect was blocked by 17-ODYA. ANG II (10^-7 M) did not alter K_Ca channel activity recorded from cell-attached patches on renal VSM cells under control conditions. However, it did reduce the NPo of the K_Ca channel by 93.4 ± 3.1% after the channels were activated by increasing intracellular calcium levels with ionomycin. The inhibitory effect of ANG II on K_Ca channel activity in the presence of ionomycin was attenuated by 17-ODYA, AACOF₃, and the phospholipase C (PLC) inhibitor U-73122. ANG II induced a peak followed by a steady-state increase in intracellular calcium concentration in renal VSM cells. 17-ODYA (10^-5 M) had no effect on the peak response, but it blocked the steady-state increase. These results indicate that ANG II stimulates the formation of 20-HETE in rat renal microvessels via the AT₁ receptor activation and that 20-HETE contributes to the vasoconstrictor response to ANG II by blocking activation of K_Ca channel and facilitating calcium entry.     

Association of 14-3-3 proteins to beta1-adrenergic receptors modulates Kv11.1 K+ channel activity in recombinant systems

We identify a new mechanism for the beta(1)-adrenergic receptor (beta(1)AR)-mediated regulation of human ether-a-go-go-related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3epsilon proteins is competed by wild type beta(1)AR by means of a novel interaction between this receptor and 14-3-3epsilon. The association between beta(1)AR and 14-3-3epsilon is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The beta(1)AR/14-3-3epsilon association is direct, since it can be recapitulated using purified 14-3-3epsilon and beta(1)AR fusion proteins and is abolished in cells expressing beta(1)AR phosphorylation-deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that beta(1)AR phosphorylation-deficient mutants do not recruit 14-3-3epsilon away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type beta(1)AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both beta(1)AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness.     

Knockdown of ZFAS1 Inhibits Hippocampal Neurons Apoptosis and Autophagy by Activating the PI3K/AKT Pathway via Up-regulating miR-421 in Epilepsy

Neurochemical Research - Epilepsy is a common neurological disease. The dysregulated long noncoding RNAs (lncRNAs) are implicated in epileptogenesis. The aim of this research is to explore the role...     

Lyt phenotype of cytotoxic T cells: shift from Lyt-1+2+3+ to a mixed population of Lyt-1+2+3+ and Lyt-1-2+3+ during in vitro culture

The Lyt phenotype of cytotoxic T cells generated in the primary H-2 response was investigated kinetically. The cytotoxicity generated in the early stage of culture was abolished by treatment with alpha Lyt-1,2,3, and complement (C), whereas that generated in the late stage was only partially eliminated by alpha Lyt-1, but was abolished by alpha Lyt-2, 3, and C. This suggested late expansion of the Lyt-1-2+3+ population. Lack of Lyt-1 antigen was confirmed with cells that were depleted of Lyt-1+ from primary culture and then stimulated in the secondary response by elimination of cytotoxicity and by direct Lyt typing. Results indicated that the response of proliferative and cytotoxic T cells of the Lyt-1+2+3+ phenotype in the early stage of culture was followed by activation of Lyt-1-2+3+ T cells. Cytotoxic T cells in the late stage were shown to be a mixture of Lyt-1+2+3+ and Lyt-1-2+3+ cells. This was confirmed with cytotoxic T cells from secondary culture and uncloned long-term T cell lines.     

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during proliferation of Chinese hamster V-79 and human HeLaS-3 cells

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during cell proliferation were examined in Chinese hamster V-79 (V-79) cells (normal cells) and human HeLaS-3 (HeLaS-3) cells (malignant cells). For V-79 cells, the Mg2+-ATPase activity per cell (pmol Pi/h/cell) in the confluent phase was higher than that in the logarithmically growing (log) phase. The amount of microsomal protein per cell was also high in the confluent phase. Specific activities (mumol Pi/h/mg protein) of Na+, K+-, Mg2+- and Ca2+-ATPase were significantly lower in the confluent phase than in the log phase. For HeLaS-3 cells, an increase in Ca2+-ATPase activity per cell was observed. The amount of microsomal protein per cell did not change between the log and confluent phase. The specific activity of Ca2+-ATPase in the confluent phase was also markedly higher than in the log phase. The relation between changes in ATPase activities and cell proliferation is discussed.     

Opposite Changes in GABAA Receptor Function in the CA1-3 Area and Fascia Dentata of Kindled Rat Hippocampus

Abstract: Muscimol-stimulated radiotracer ³⁶Cl⁻ uptake in synaptoneurosomes was used to investigate the function of the GABA_A receptor complex in the CA1-3 area and fascia dentata (granular and molecular layers and hilus) of rats kindled by stimulation, twice a day, of the Schaffer collateral fibers. Two kindled groups were studied: (a) 24 h after the last generalized tonic-clonic seizure [fully kindled (FK) stage] and (b) 28 days after the last generalized seizure (long-term stage). Synaptoneurosomes were prepared in parallel from subslices of the CA1-3 area and fascia dentata. In FK animals, the muscimol-stimulated ³⁶Cl⁻ uptake was significantly reduced by 21% in the CA1-3 area in comparison with nonstimulated controls, whereas a significant increase of 29% was found in the fascia dentata. Significant changes were no longer present at 4 weeks after the last generalized seizure. The observed changes in muscimol-stimulated ³⁶Cl⁻ uptake at the FK stage closely parallel the recently observed changes in [³H]muscimol binding in the CA1 area and fascia dentata. These results indicate that kindling causes a transiently decreased GABA_A receptor-mediated function in the CA1-3, in contrast to an increased GABA_A receptor-mediated function in the fascia dentata.     

E3-14.7K is recruited to TNF-receptor 1 and blocks TNF cytolysis independent from interaction with optineurin

Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor κB (NF-κB) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.     

Potential difference responses due to K+, Na+ and Cl- changes in bullfrog antrum with and without HCO3-

The effect of changing [K+], [Na+] and [Cl-] in nutrient solution was studied in bullfrog antrum with and without HCO3- in nutrient. In 25 mM HCO3- (95% O2/5% CO2) and in zero HCO3- (100% O2), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K+ or from 81 to 8.1 mM Cl- gave a decrease 10 min later in transmucosal PD (nutrient became more negative)--a normal response. These responses were less in zero than in 25 mM HCO3-. A decrease from 102 to 8 mM Na+ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO3-. In contrast, change from 4 to 40 mM K+ gave initial anomalous PD response and change from 102 to 8 mM Na+, initial normal PD response with either zero or 25 mM HCO3-. Both responses were associated with (Na+ + K+)-ATPase pump and were greater in zero than in 25 mM HCO3-. Initial PD increases in zero HCO3- are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO3- modifies conductance pathways of nutrient membrane.     

Changes in PTGS1 and ALOX12 Gene Expression in Peripheral Blood Mononuclear Cells Are Associated with Changes in Arachidonic Acid,Oxylipins, and Oxylipin/Fatty Acid Ratios in Response to Omega-3 Fatty Acid Supplementation

Introduction

There is a high degree of inter-individual variability among people in response to intervention with omega-3 fatty acids (FA), which may partly explain conflicting results on the effectiveness of omega-3 FA for the treatment and prevention of chronic inflammatory diseases. In this study we sought to evaluate whether part of this inter-individual variability in response is related to the regulation of key oxylipin metabolic genes in circulating peripheral blood mononuclear cells (PBMCs).

Methods

Plasma FA and oxylipin profiles from 12 healthy individuals were compared to PBMC gene expression profiles following six weeks of supplementation with fish oil, which delivered 1.9 g/d eicosapentaenoic acid (EPA) and 1.5 g/d docosahexaenoic acid (DHA). Fold changes in gene expression were measured by a quantitative polymerase chain reaction (qPCR).

Results

Healthy individuals supplemented with omega-3 FA had differential responses in prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), and interleukin 8 (IL-8) gene expression in isolated PBMCs. In those individuals for whom plasma arachidonic acid (ARA) in the phosphatidylethanolamine (PE) lipid class decreased in response to omega-3 intervention, there was a corresponding decrease in gene expression for PTGS1 and ALOX12. Several oxylipin product/FA precursor ratios (e.g. prostaglandin E₂ (PGE₂)/ARA for PTGS1 and 12-hydroxyeicosatetraenoic acid (12-HETE)/ARA for ALOX12) were also associated with fold change in gene expression, suggesting an association between enzyme activity and gene expression. The fold-change in PTGS1 gene expression was highly positively correlated with ALOX12 gene expression but not with PTGS2, whereas IL-8 and PTGS2 were positively correlated.

Conclusions

The regulation of important oxylipin metabolic genes in PBMCs varied with the extent of change in ARA concentrations in the case of PTGS1 and ALOX12 regulation. PBMC gene expression changes in response to omega-3 supplementation varied among healthy individuals, and were associated with changes in plasma FA and oxylipin composition to different degrees in different individuals.

Trial Registration

clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01838239","term_id":"NCT01838239"}}NCT01838239     

Potential difference responses to nutrient K+, Na+ and Cl- changes with varying nutrient HCO3- in resting frog stomach

The effects of changes in nutrient concentrations of K+, Na+ and Cl- on the transmucosal potential difference (PD) and resistance were compared for 25 and 5 mM nutrient HCO3- in resting fundus. With 25 mM HCO3-, increase of K+ from 4 to 40 mM, decrease of Na+ from 100 to 10 mM and decrease of Cl- from 81 to 8.1 mM gave, 10 min after the change, delta PD values of -23.2, -15.1 and -21.3 mV, respectively. With 5 mM HCO3-, the same changes in nutrient ion concentration gave delta PD values of -11.9, -9.4 and -10.0 mV, respectively. From these results, in going from 25 to 5 mM HCO3-, it follows that the resistances of the ionic pathways for K+, Na+ and Cl- increased. The anomalous PD response following the increase in nutrient K+ from 4 to 40 mM with 5 mM nutrient HCO3- gave further evidence that the resistance of the simple K+ conductance pathway increased prior to the increase to 40 mM K+. The fact that 2 mM Ba2+ in the 25 mM HCO3- nutrient gave a smaller increase in resistance, compared to the decrease in nutrient HCO3- from 25 to 5 mM, supported the inference that resistances of ion pathways other than that of the K+ pathway increased.     

An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.     

Potential difference responses to secretory K+, Na+ and HCO3- changes in secreting and resting states of frog stomach in Cl(-)-free media

The effects of changes in secretory concentrations of K+, Na+ and HCO3- on transmucosal potential difference (PD) and resistance in Cl(-)-free (SO4(2-)) solutions were compared for secreting fundus and resting fundus of Rana pipiens. In the resting fundus experiments, histamine was not present in the nutrient solution and cimetidine was primarily used to obtain acid inhibition. Increase of K+ from 4 to 80 mM, decrease of Na+ from 156 to 15.6 mM and decrease of HCO3- from 25 to 5 mM gave, 10 min after the change, in the secreting fundus delta PD values of 39.7, -11.9 and 3.2 mV, respectively. In the resting fundus, 1.5 to 2 h after the addition of cimetidine, the same changes in secretory ion concentration gave delta PD values of 12.2, -5.6 and 1.5 mV, respectively. Replacement of cimetidine with SCN and without histamine yielded a delta PD somewhat lower than that in cimetidine, namely 9 mV for a K+ change from 4 to 80 mM. Subsequent addition of histamine with SCN present gave a delta PD of about 21 mV. The change in PD was attributed to histamine increasing the secretory membrane area, leading to an increase in K+ conductance. Another possibility is that histamine increases the K+ conductance per se.     

Susceptibility of the guard-cell K+-uptake channel KST1 to Zn2+ requires histidine residues in the S3-S4 linker and in the channel pore

 Potassium channels are inhibited by several mono- and divalent cations. To identify sites involved in the interaction between K⁺ channels and cationic effectors, we expressed the potato (Solanum tuberosum L.) guard-cell K⁺-uptake channel KST1 in Xenopus oocytes. This channel was reversibly blocked by extracellular Zn²⁺ in the micromolar range. In the presence of this heavy metal, steady-state currents were reduced in a pH-dependent but voltage-independent manner. Since Zn²⁺-inhibition was less effective at elevated external proton concentrations, we generated alanine mutants with respect to both extracellular histidines in KST1. Whereas substitution of the pore histidine H271 resulted in a reduced blockade by Zn²⁺, the channel mutant KST1-H160A in the S3-S4 linker lost most of its Zn²⁺ sensitivity. Since both histidines alter the susceptibility of KST1 to Zn²⁺, the block may predominantly result from these two sites. We thus conclude that the S3-S4 linker is involved in the formation of the outer pore. Received: 3 May 1999 / Accepted: 8 July 1999