Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.     

Formation of covalent beta-linked carbohydrate-enzyme intermediates during the reactions catalyzed by alpha-amylases

Porcine pancreatic and Bacillus amyloliquefaciens alpha-amylases were examined for the formation of covalent carbohydrate intermediates during reaction. The enzymes were precipitated and denatured by adding 10 volumes of acetone. When these denatured enzymes were mixed with methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and chromatographed on BioGel P-2, no carbohydrate was found in the protein void volume peak. When the enzymes were added to the methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and allowed to react for 15s at 1 degrees C and then precipitated and denatured with 10 volumes of acetone, (3)H-labeled carbohydrates were found in the BioGel P-2 protein void volume peak, indicating the formation of enzyme-carbohydrate covalent intermediates. (1)H NMR analysis of the denatured enzyme from the reaction with methyl alpha-maltooligosaccharide glycosides confirmed that carbohydrate was attached to the denatured enzyme. (1)H NMR saturation-transfer analysis further showed that the carbohydrate was attached to the denatured enzyme by a beta-configuration. This configuration is what would be expected for an enzyme that catalyzes the hydrolysis of alpha-(1-->4) glycosidic linkages by a two-step, S(N)2 double-displacement reaction to give retention of the alpha-configuration of the substrates at the reducing-end of the products.     

Uneven modulation of the annexin 1 system in osteoblast-like cells by dexamethasone

We tested whether glucocorticoids modulated osteoblast expression of the annexin 1 system, including the ligand and two G-coupled receptors termed formyl-peptide receptor (FPR) and FPR-like-1 (FPRL-1). In Saos-2 cells, rapid up-regulation of FPR mRNA upon cell incubation with dexamethasone (0.01-1 microM) was observed, with significant changes as early as 2h and a more marked response at 24h; annexin 1 and FPRL-1 mRNA changes were more subtle. At the protein level, dexamethasone provoked a rapid externalization of annexin 1 (maximal at 2h) followed by delayed time-dependent changes in the cell cytosol. Saos-2 cell surface expression of FPR or FPRL-1 could not be detected, even when dexamethasone was added with the bone modelling cytokines interleukin-6 or interleukin-1. The uneven modulation of the annexin 1 system (mediator and its putative receptors) in osteoblasts might lead to a better understanding of how these complex biochemical pathways become operative in bone.     

Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.     

我国男子橄榄球运动员有氧能力的评定和比赛时能耗的初探*

目的:探究我国男子橄榄球运动员的有氧能力和比赛时的能量消耗,为精准化训练和比赛时营养策略的制定提供理论依据。方法:以18名男子橄榄球运动员(健将级)为研究对象,分别进行最大摄氧量(VO₂max),乳酸阈(LT)和conconi场地测试来评定其有氧能力,并比较前、后锋之间的供能差异,用wGT3X加速度计结合团队心率探究比赛时的能量消耗,所得数据进行独立样本t检验。结果:本次检测的橄榄球运动员相对最大摄氧量较差为(42.05±3.69) ml/min·kg^-1,前峰队员相对最大摄氧量为(38.83±3.52) ml/min·kg^-1、后锋队员相对最大摄氧量为(47.31±3.17)ml/min·kg^-1,二者之间的差异有统计学意义(P＜0.05);血乳酸阈测定在7 min时出现血乳酸拐点,后锋的血乳酸阈值要高于前锋运动员;conconi场地测试与实验室VO₂max有较高的相关性(r=0.772)。比赛中平均能量消耗上半场约为(276.94±18.08) kcals,下半场约为(225.58±22.86) kcals,下半场的能耗小于上半场(P＜0.05)。结论:英式7人制橄榄球运动员有氧能力较弱,且前、后锋队员存在差异。     

Recent Advances of Natural Polyphenols Activators for Keap1‐Nrf2 Signaling Pathway

The Keap1‐Nrf2/ARE signaling pathway is an important defense system against exogenous and endogenous oxidative stress injury. The dysregulation of the signaling pathway is associated with many diseases, such as cancer, diabetes, and respiratory diseases. Over the years, a wide range of natural products has provided sufficient resources for the discovery of potential therapeutic drugs. Among them, polyphenols possess Nrf2 activation, not only inhibit the production of ROS, inhibit Keap1‐Nrf2 protein–protein interaction, but also degrade Keap1 and regulate the Nrf2 related pathway. In fact, with the continuous improvement of natural polyphenols separation and purification technology and further studies on the Keap1‐Nrf2 molecular mechanism, more and more natural polyphenols monomer components of Nrf2 activators have been gradually discovered. In this view, we summarize the research status of natural polyphenols that have been found with apparent Nrf2 activation and their action modes. On the whole, this review may guide the design of novel Keap1‐Nrf2 activator.     

MicroRNA‐351 eases insulin resistance and liver gluconeogenesis via the PI3K/AKT pathway by inhibiting FLOT2 in mice of gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is known as different degree glucose intolerance that is initially identified during pregnancy. MicroRNAs (miRs) may be a potential candidate for treatment of GDM. Herein, we suggested that miR‐351 could be an inhibitor in the progression of GDM via the phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway. Microarray analysis was used to identify differentially expressed genes and predict miRs regulating flotillin 2 (FLOT2). Target relationship between miR‐351 and FLOT2 was verified. Gestational diabetes mellitus mice were treated with a series of mimic, inhibitor and small interfering RNA to explore the effect of miR‐351 on insulin resistance (IR), cell apoptosis in pancreatic tissues and liver gluconeogenesis through evaluating GDM‐related biochemical indexes, as well as expression of miR‐351, FLOT2, PI3K/AKT pathway‐, IR‐ and liver gluconeogenesis‐related genes. MiR‐351 and FLOT2 were reported to be involved in GDM. FLOT2 was the target gene of miR‐351. Gestational diabetes mellitus mice exhibited IR and liver gluconeogenesis, up‐regulated FLOT2, activated PI3K/AKT pathway and down‐regulated miR‐351 in liver tissues. Additionally, miR‐351 overexpression and FLOT2 silencing decreased the levels of FLOT2, phosphoenolpyruvate carboxykinase, glucose‐6‐phosphatase, fasting blood glucose, fasting insulin, total cholesterol, triglyceride, glyeosylated haemoglobin and homeostasis model of assessment for IR index (HOMA‐IR), extent of PI3K and AKT phosphorylation, yet increased the levels of HOMA for islet β‐cell function, HOMA for insulin sensitivity index and glucose transporter 2 expression, indicating reduced cell apoptosis in pancreatic tissues and alleviated IR and liver gluconeogenesis. Our results reveal that up‐regulation of miR‐351 protects against IR and liver gluconeogenesis by repressing the PI3K/AKT pathway through regulating FLOT2 in GDM mice, which identifies miR‐351 as a potential therapeutic target for the clinical management of GDM.     

Germline variants in oculocutaneous albinism genes and predisposition to familial cutaneous melanoma

Approximately 1%–2% of cutaneous melanoma (CM) is classified as strongly familial. We sought to investigate unexplained CM predisposition in families negative for the known susceptibility genes using next‐generation sequencing of affected individuals. Segregation of germline variants of interest within families was assessed by Sanger sequencing. Several heterozygous variants in oculocutaneous albinism (OCA) genes: TYR, OCA2, TYRP1 and SLC45A2, were present in our CM cohort. OCA is a group of autosomal recessive genetic disorders, resulting in pigmentation defects of the eyes, hair and skin. Missense variants classified as pathogenic for OCA were present in multiple families and some fully segregated with CM. The functionally compromised TYR p.T373K variant was present in three unrelated families. In OCA2, known pathogenic variants: p.V443I and p.N489D, were present in three families and one family, respectively. We identified a likely pathogenic SLC45A2 frameshift variant that fully segregated with CM in a family of four cases. Another four‐case family harboured cosegregating variants (p.A24T and p.R153C) of uncertain functional significance in TYRP1. We conclude that rare, heterozygous variants in OCA genes confer moderate risk for CM.