The effects of protein kinase A catalytic subunit on sperm motility regulation in Pacific abalone Haliotis discus hannai

Protein kinase A plays a central role in the regulation of sperm motility from echinoderms to mammals, but the information about its regulatory role in molluscs is very limited. In this study, a protein kinase A catalytic subunit (designated as HdPKA‐C) was identified from Pacific abalone Haliotis discus hannai. The open reading frame of HdPKA‐C was of 1,077 bp, encoding a peptide of 358 amino acids with a typical protein kinase domain. HdPKA‐C shared 82%–87% sequence similarities with other PKA‐Cs, and it was clustered first with gastropod PKA‐Cs in the phylogenetic tree. The mRNA of HdPKA‐C was constitutively expressed in examined tissues, with the highest level detected in hepatopancreas. The phosphorylated form of HdPKA‐C (p‐HdPKA‐C) was localized at the acrosome, connecting piece and flagellum of spermatozoa with variable intensity. Its phosphorylated substrates were also detected in these regions with much lower intensity at the connecting piece. The inhibition of HdPKA‐C activity with H‐89 led to a significant reduction in the percentage of motile sperm and sperm velocities. p‐HdPKA‐C was detected by Western blot in strip‐spawned sperm, naturally spawned sperm and H‐89‐treated sperm with almost the same intensity. The intensity of p‐HdPKA‐C substrates in naturally spawned sperm was higher than that in strip‐spawned sperm, and it was roughly the same as that in H‐89‐treated sperm except for two bands at 50 and 60 kDa. These results collectively indicated that HdPKA‐C played an important role in the regulation of abalone sperm motility by altering its substrates phosphorylation.     

The effects of naringenin and naringin on the glucose uptake and AMPK phosphorylation in high glucose treated HepG2 cells

BackgroundNaringin and its aglycone naringenin are citrus-derived flavonoids with several pharmacological effects. On the other hand, the mechanism for the anti-diabetic effects of naringenin and naringin are controversial and remain to be clarified further.ObjectiveThis study examined the relationship between glucose uptake and AMP-activated protein kinase (AMPK) phosphorylation by naringenin and naringin in high glucose-treated HepG2 cells.MethodsGlucose uptake was measured using the 2-NBDG fluorescent D-glucose analog. The phosphorylation levels of AMPK and GSK3β (Glycogen synthase kinase 3 beta) were observed by Western blotting. Molecular docking analysis was performed to evaluate the binding affinity of naringenin and naringin to the γ-subunit of AMPK.ResultsThe treatment with naringenin and naringin stimulated glucose uptake regardless of insulin stimulation in high glucose-treated HepG2 cells. Both flavonoids increased glucose uptake by promoting the phosphorylation of AMPK at Thr172 and increased the phosphorylation of GSK3β. Molecular docking analysis showed that both naringenin and naringin bind to the γ-subunit of AMPK with high binding affinities. In particular, naringin showed higher binding affinity than the true modulator, AMP with all three CBS domains (CBS1, 3, and 4) in the γ-subunit of AMPK. Therefore, both naringenin and naringin could be positive modulators of AMPK activation, which enhance glucose uptake regardless of insulin stimulation in high glucose-treated HepG2 cells.ConclusionsThe increased phosphorylation of AMPK at Thr172 by naringenin and naringin might enhance glucose uptake regardless of insulin stimulation in high glucose treated HepG2 cells.     

Effects of lairage after transport on post mortem muscle glycolysis,protein phosphorylation and lamb meat quality

The objective of this study was to investigate the effect of lairage after transport on post mortem muscle glycolysis, protein phosphorylation and lamb meat quality. Two preslaughter animal treatments, transport for 3 h and lairage for 0 h (T3L0) and transport for 3 h and then lairage for 12 h (T3L12), were compared with a control treatment of 0 h transport and 0 h lairage. Data obtained showed that preslaughter transport had a significant effect on lamb meat quality. Loins from lambs of the T3L0 treatment showed higher (P=0.026) pH_{24 h} and higher (P=0.021) pH_{48 h} values, but lower (P<0.001) drip loss and lower (P<0.05) glycolytic potential at 0 h post mortem than those of the T3L12 and control groups. Muscle samples of the T3L0 group showed higher (P=0.046) shear force and lower (P=0.005) b* value than those of the T3L12 group. Muscle glycogen concentration at 0, 2, 4 h post mortem were lower (P<0.05) in the T3L0 group than in control. No significant difference (P>0.05) in most meat quality parameters was determined between the T3L12 group and control, showing lairage for 12 h allowed lambs to recover from the effects of transport for 3 h and resulted in similar meat quality characteristics compared to no transport. Lairage after transport did not affect most meat quality indices in comparison with control, but increased the meat drip loss and b* value of lambs possibly through decreasing glycogen concentration and glycolytic potential.     

不同海拔高度牛宰后牛肉AMPK活性及能量代谢研究

为了研究不同海拔高度生长的牛宰后牛肉一磷酸腺苷活化蛋白激酶(AMPK)活性与糖酵解指标,以探讨宰后肌肉生理机理,测定了玉树牦牛(海拔4 500 m)、甘南牦牛(海拔3 000 m)和西门塔尔杂交肉牛(海拔1 500 m)宰后成熟过程中牛肉AMPK活性、AMPK基因(PRKAA1、PRKAA2)mRNA表达量、糖酵解和能量代谢的变化。结果表明,12~168 h时间内,AMPK活性从小到大依次为西门塔尔杂交牛、甘南牦牛、玉树牦牛;西门塔尔杂交牛PRKAA1基因的表达量比甘南牦牛和玉树牦牛略低;西门塔尔杂交牛PRKAA2基因表达量显著小于甘南牦牛和玉树牦牛(P0.05);72~168 h时间内,乳酸含量和游离葡萄糖含量从小到大依次为西门塔尔杂交牛、甘南牦牛、玉树牦牛,p H值、肌糖原含量从大到小依次为西门塔尔杂交牛、甘南牦牛、玉树牦牛;成熟前期ATP、ADP、AMP含量从大到小依次为西门塔尔杂交牛、甘南牦牛、玉树牦牛。高海拔品种牛肉机体PRKAA1和PRKAA2基因表达量高时,AMPK被激活从而活性增加,ATP的浓度水平降低,AMP生成量增加,加速组织内的糖酵解,增加肌肉乳酸含量,降低p H值,进而影响牛肉品质。     

马齿苋多糖对tau蛋白磷酸化影响的研究

目的是研究马齿苋多糖对神经细胞骨架微管结合蛋白-tau蛋白的磷酸化影响。方法通过构建基因表达载体pEGFP- tau-s3和转染表达载体pEGFP- tau-s3到3T3细胞,创造体外神经细胞骨架研究模型;通过共转染pEGFP- tau-s3与pcDNA-GSK-3β到3T3细胞、荧光显微观察和Wstern blotting实验,证明了马齿苋多糖对tau蛋白的磷酸化有影响,可以抑制GSK激酶对tau蛋白的磷酸化。结论马齿苋多糖对神经细胞微管骨架有保护作用     

‘宁紫薯1号’花青素组分鉴定及其对大鼠高脂诱导肥胖的预防效果

【目的】紫甘薯是天然抗氧化物花青素的重要来源。鉴定‘宁紫薯1号’花青素(PSPC)组成成分,探索紫甘薯花青素对高脂诱导肥胖的预防效果并探讨其分子机制。【方法】通过液质联用技术分析紫甘薯新品种‘宁紫薯1号’的花青素组分;采用p H示差法测定制备的样品中总花青素含量。建立因高脂摄入过多导致机体产生营养性肥胖的动物模型,并灌胃不同剂量的紫甘薯花青素比较其预防肥胖效果。每周定时测量大鼠禁食6 h后的体重,通过试剂盒检测大鼠血清样品血糖(Glu)、甘油三酯(TG)、总胆固醇(TC)的含量,采用酶联免疫法检测瘦素(Leptin)含量。通过荧光定量PCR法(RT-PCR)检测下丘脑样品瘦素及其受体的m RNA表达,采用蛋白质免疫印迹法(Western blot)检测瘦素及其下游蛋白的信号表达。【结果】分离鉴定出11种PSPC组分,其中10种为已知的矢车菊素和芍药素酰基化衍生物,峰1为新发现的1种化合物。制备的花青素样品中矢车菊素-3-葡萄糖苷含量为17.4%。体重指标直接表明高剂量花青素组预防肥胖效果最好,为(358.2±20.1)g,中剂量组其次,为(371.6±16.3)g;效果最差的为低剂量组,体重为(384.0±7.2)g。紫甘薯花青素各剂量组大鼠血糖、甘油三酯和总胆固醇的3项血生化指标均趋于正常水平,有效预防肥胖的发生。血清瘦素检测结果显示,高脂组比对照组多释放65.50%;低剂量、中剂量和高剂量紫甘薯花青素组血清瘦素含量分别为(3.13±0.05)、(2.84±0.12)和(2.64±0.06)ng·m L~(-1),与高脂组相比均显著下降。同时不同剂量紫甘薯花青素组下丘脑中瘦素及瘦素受体的m RNA表达均显著高于高脂组。而高剂量紫甘薯花青素组可以调节下丘脑中瘦素信号,从而降低下游腺苷酸活化蛋白激酶α(AMPKα)的磷酸化。【结论】高剂量紫甘薯花青素可以通过调节下丘脑中leptin/AMPKα信号通路有效抑制高脂诱导肥胖的形成。     

野油菜黄单胞菌效应蛋白AvrXccC与拟南芥BIK1在植物体内相互作用

 AvrXccC是野油菜黄单胞菌Xcc8004的一个III型分泌效应蛋白。前期研究发现AvrXccC在拟南芥rar1突变体上发挥毒性功能,并且抑制植物的先天免疫反应。但是,AvrXccC在植物体内的毒性靶标还不清楚。本研究发现AvrXccC与植物体内的蛋白激酶BIK1发生特异的相互作用。然而,在拟南芥原生质体中,AvrXccC 并不能抑制鞭毛蛋白诱导的BIK1迁移,表明AvrXccC不是通过抑制BIK1的磷酸化来发挥功能的。有趣的是,AvrXccC可以在体外直接被BIK1磷酸化,我们推测AvrXccC可能作为BIK1的底物从而干扰了鞭毛蛋白诱导的先天免疫反应。     

去乙酰化酶SIRT1过表达对猪卵巢颗粒细胞中AMPK活性的影响

构建猪SIRT1基因全长编码区(coding region sequence,CDS)的真核表达载体,转染猪原代卵巢颗粒细胞,探讨SIRT1基因过表达对猪卵巢颗粒细胞中AMPK基因的转录及其蛋白活性的影响。测序结果表明,猪SIRT1基因的CDS区全长大小为2 229 bp,与NCBI发布的猪SIRT1基因m RNA序列(EU030283.2)一致;转染p EGFP–C1–SIRT1载体的颗粒细胞中SIRT1的m RNA表达水平和蛋白表达水平极显著高于空载体对照组(P0.01);p EGFP–C1–SIRT1转染组细胞中,AMPK–α1和AMPK–α2基因的m RNA表达量显著高于空载体对照组,且细胞中AMPKαThr172位点的磷酸化水平显著升高(P0.05)。结果表明,体外培养的原代猪卵巢颗粒细胞中的SIRT1基因过表达使AMPK的表达显著增加,影响AMPK的活性,推测SIRT1可能通过AMPK在猪卵巢颗粒细胞凋亡过程中发挥重要的调控作用。