MicroRNA-298 determines the radio-resistance of colorectal cancer cells by directly targeting human dual-specificity tyrosine(Y)-regulated kinase 1A

BACKGROUND Radiotherapy stands as a promising therapeutic modality for colorectal cancer(CRC);yet,the formidable challenge posed by radio-resistance significantly undermines its efficacy in achieving CRC remission.AIM To elucidate the role played by microRNA-298(miR-298)in CRC radio-resistance.METHODS To establish a radio-resistant CRC cell line,HT-29 cells underwent exposure to 5 gray ionizing radiation that was followed by a 7-d recovery period.The quantification of miR-298 levels within CRC cells was conducted through quantitative RT-PCR,and protein expression determination was realized through Western blotting.Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferation by clonogenic assay.Radio-induced apoptosis was discerned through flow cytometry analysis.RESULTS We observed a marked upregulation of miR-298 in radio-resistant CRC cells.MiR-298 emerged as a key determinant of cell survival following radiation exposure,as its overexpression led to a notable reduction in radiation-induced apoptosis.Intriguingly,miR-298 expression exhibited a strong correlation with CRC cell viability.Further investigation unveiled human dual-specificity tyrosine(Y)-regulated kinase 1A(DYRK1A)as miR-298’s direct target.CONCLUSION Taken together,our findings underline the role played by miR-298 in bolstering radio-resistance in CRC cells by means of DYRK1A downregulation,thereby positioning miR-298 as a promising candidate for mitigating radioresistance in CRC.

Levetiracetam induces tyrosine kinase receptor B expression in SH-SY5Y cells

Tyrosine kinase receptor B （TrkB） plays an important role in long-term potentiation and memory formation.The present study used all-trans retinoic acid to induce TrkB expression in SH-SY5Y cells,and observed the effects of levetiracetam （LEV） on TrkB expression.Following exposure to 10,50,and 100 μg/mL LEV,the number of TrkB-positive cells,and average absorbance value were increased.Results demonstrated that LEV can induce TrkB expression in SH-SY5Y cells.

大鼠孤束核内TH能神经元和NPY能神经支配

目的和方法：本文用双重免疫染色包埋前免疫电镜法，在光镜和电镜水平，观察了大鼠孤束核内ＮＰＹ神经元分布及ＮＰＹ神经末稍与儿茶酚胺能神经元之间的相互关系；结果：在大鼠孤束核嘴尾侧全区内见到中型的圆形或卵圆形的ＮＰＹ阳性神经元，ＮＰＹ阳性神经纤维集中分布于孤束核的内侧部及背侧周边区。在孤束核的内侧部可见到蓝绿色的ＴＨ阳性神经元和棕色的ＮＰＹ阳性神经元及阳性神经纤维，其中的梢嘴侧平面的孤束核背内侧一部分神经元同时染成蓝绿色和棕色。ＴＨ阳性神经元细胞质内有棒状或卵圆形的电子密度较高的反应产物，ＮＰＹ阳性神经末梢内见到电子密度较高的ＤＡＢ反应产物；

神经肽Y在杏仁内侧核引起的心血管效应及外周血中儿茶酚胺的变化

将神经肽Ｙ（ＮＰＹ）（５０ｐｍｏｌ）注入大鼠的杏仁内侧核，观察由此引起的血压和心率的变化。在此变化达到最大值时，用ＨＰＬＣ－ＥＤ法检测外周血中儿茶酚胺的变化。结果表明：ＮＰＹ能显著降低血压，这时外周血中去甲肾上腺素（ＮＥ）明显下降。提示ＮＰＹ在杏仁内侧核引起的心血管效应与外周血中ＮＥ有密切的关系。

Is X-linked methyl-CpG binding protein 2 a new target for the treatment of Parkinson's disease?

X-linked methyl-CpG binding protein 2 mutations can induce symptoms similar to those of Parkinson’s disease and dopamine metabolism disorders, but the specific role of X-linked methyl-CpG binding protein 2 in the pathogenesis of Parkinson’s disease remains unknown. In the present study, we used 6-hydroxydopamine-induced human neuroblastoma cell （SH-SY5Y cells） injury as a cell model of Parkinson’s disease. The 6-hydroxydopamine （50 μmol/L） treatment decreased protein levels for both X-linked methyl-CpG binding protein 2 and tyrosine hydroxylase in these cells, and led to cell death. However, overexpression of X-linked methyl-CpG binding protein 2 was able to ameliorate the effects of 6-hydroxydopamine, it reduced 6-hydroxydopamine-induced apoptosis, and increased the levels of tyrosine hydroxylase in SH-SY5Y cells. These findings suggesting that X-linked methyl-CpG binding protein 2 may be a potential therapeutic target for the treatment of Parkinson’s disease.

Light-controlled phosphorylation in the TrkA-Y785 site by photosensitive UAAs activates the MAPK/ERK signaling pathway

Background:This paper aims to establish a light-controlled phosphorylation detection method at the Y785 site of tropomyosin receptor kinase A(TrkA)receptor in mammalian cells by using genetic code expansion technology and detecting the effects of optical activation of this site on the downstream MAPK/ERK pathway.The study is based on the current situation that the regulatory mechanism of TrkA phosphorylation has not been fully elucidated.Methods:Two photosensitive unnatural amino acids,p-azido-L-phenylalanine(AzF)and photo-caged tyrosine(ONB)were introduced into the TrkA-Y785 site by genetic code expansion technology and site-directed mutagenesis.Western blotting and laser confocal imaging were conducted to analyze the effects of this site on activating the MAPK/ERK pathway and nerve cell differentiation before and after photostimulation.Results:Our results supplemented the light-controlled results of the TrkA-Y785 site based on our previous research and verified that Y785 also makes important contributions in regulating the MAPK/ERK pathway.Conclusion:This study demonstrated the significant contributions of the TrkAY785 site in regulating the ERK pathway by precisely controlling the phosphorylation state of a single tyrosine site.

Solid-Phase Enzymatic Peptide Synthesis to Produce an Antioxidant Dipeptide

Peptide bond synthesis is favorable to the production of bioactive small peptides. However, the abuse of toxic reagents remains an issue for chemical synthesis method, whereas the low product yield and purity limit the widespread use of enzymatic method. In this study, a new solid-phase enzymatic peptide synthesis(SPEPS) strategy was developed to produce an antioxidant tyrosine-alanine dipeptide(Tyr-Ala) by using recombinant carboxypeptidase Y(CPY) as the catalyst. The general SPEPS procedure involves three steps. First, the N-protected acyl donor was covalently attached to solid resin. Second,the peptide bond was condensed between the acyl donor and the nucleophile under the catalysis of CPY. Finally, one-step cleavage was performed to remove the protecting group and cleave the peptides from solid resin. Upon the optimization of reaction conditions, 77.92%(±2.723%) yield of Tyr-Ala with high product purity of 90.971%(±2.695%) was obtained.In addition, the antioxidant activity of Tyr-Ala was determined by ABTS method, indicating that the synthesized Tyr-Ala obtained by SPEPS showed a superior antioxidant capability compared with commercial glutathione.

Synaptic non-GluN2B-containing NMDA receptors regulate tyrosine phosphorylation of GluN2B 1472 tyrosine site in rat brain slices

Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases（Src and Fyn）or phosphatases（PTPαand STEP）are involved in regulating the phosphorylation of NMDARs.In this study,we used immunoblotting to investigate the role of an NMDAR subpopulation on the phosphorylation level of the GluN2B subunit at the Y1336 and Y1472sites in rat brain slices after NMDA treatment.We found that NMDA stimulation dramatically decreased the phosphorylation level of GluN2B at Y1472 in a dose-and time-dependent manner,but not at Y1336.Extrasynaptic NMDAR activation did not reduce the phosphorylation of GluN2B at Y1472.In addition,ifenprodil,a selective antagonist of GluN2Bcontaining NMDARs,did not abolish the decreased phosphorylation of GluN2B at Y1472 triggered by NMDA.These results suggest that the activation of synaptic GluN2A-containing NMDARs is required for the decreased phosphorylation of GluN2B at Y1472that is induced by NMDA treatment in rat brain slices.

番石榴叶三萜化合物乌苏酸对3T3-L1前脂肪细胞增殖、分化及胰岛素抵抗的影响

目的:探讨番石榴叶三萜化合物乌苏酸对3T3-L1前脂肪细胞增殖、分化及胰岛素抵抗的影响及其机制。方法:培养3T3-L1前脂肪细胞,给予不同药物作用48 h后,采用MTT法检测对细胞增殖的影响;采用油红-O染色法对细胞分化的影响。以地塞米松诱导分化成熟的脂肪细胞,建立胰岛素抵抗模型,给予相应的药物后干预48 h。采用GOD-POD法、比色法和ELISA法检测细胞培养上清液中葡萄糖、游离脂肪酸含量及脂联素水平;Western blotting分析脂肪细胞中PPARγ和PTP1B的表达。结果:与溶媒对照组比较,30、100μmol/L乌苏酸能显著促进3T3-L1前脂肪细胞增殖及分化(P<0.05或P<0.01)。乌苏酸在30μmol/L时即可显著增加胰岛素抵抗脂肪细胞葡萄糖消耗,同时能明显减少胰岛素抵抗脂肪细胞游离脂肪酸的产生(P<0.05),也能显著促进脂联素分泌(P<0.05);在100μmol/L时上调PPARγ蛋白表达(P<0.05),但对PTP1B蛋白表达无明显影响(P>0.05)。结论:乌苏酸促进3T3-L1前脂肪细胞增殖和分化,增加脂肪细胞葡萄糖摄取,抑制游离脂肪酸产生,促进脂联素分泌,对脂肪细胞胰岛素抵抗有明显的改善作用,其机制可能与上调PPARγ蛋白表达、提高胰岛素敏感性有关。

Laminar Distribution of Neurochemically-Identified Interneurons and Cellular Co-expression of Molecular Markers in Epileptic Human Cortex

Inhibitory GABAergic interneurons are fundamental elements of cortical circuits and play critical roles in shaping network activity. Dysfunction of interneurons can lead to various brain disorders, including epilepsy,schizophrenia, and anxiety. Based on the electrophysiological properties, cell morphology, and molecular identity,interneurons could be classified into various subgroups. In this study, we investigated the density and laminar distribution of different interneuron types and the coexpression of molecular markers in epileptic human cortex.We found that parvalbumin(PV) and somatostatin(SST)neurons were distributed in all cortical layers except layer I, while tyrosine hydroxylase(TH) and neuropeptide Y(NPY) were abundant in the deep layers and white matter.Cholecystokinin(CCK) neurons showed a high density in layers IV and VI. Neurons with these markers constituted*7.2%(PV), 2.6%(SST), 0.5%(TH), 0.5%(NPY), and4.4%(CCK) of the gray-matter neuron population. Doubleand triple-labeling revealed that NPY neurons were also SST-immunoreactive(97.7%), and TH neurons were more likely to express SST(34.2%) than PV(14.6%). A subpopulation of CCK neurons(28.0%) also expressed PV, but none contained SST. Together, these results revealed the density and distribution patterns of different interneuron populations and the overlap between molecular markers in epileptic human cortex.

cDNA-RDA技术分离新的肝硬化相关基因片段

目的 :分离在肝硬化组织上调表达的新基因 ,以阐述肝硬化发生的分子机制。方法 :利用优化的代表性差异分析方法分析正常肝脏及肝硬化组织表达基因的差异。两轮杂交后的产物克隆到T载体中 ,斑点杂交筛选阳性克隆以获得差异表达基因片段 ,并进一步以半定量RT PCR鉴定。结果 :上调表达的克隆包括与一些序列和功能完全未知的基因同源的cDNA以及未曾报道过的在肝脏病变中发生表达变化的已知基因———钙结合酪氨酸磷酸化调节蛋白、雌激素应答B盒蛋白等。结论 :利用优化的代表性差异分析方法成功分离获得在肝硬化组织中上调表达的基因片段 ,其中一些新基因的克隆及其在肝硬化发生中的作用有待进一步研究。