Small‐Molecule Inhibitors of the Tumor Suppressor Fhit

The tumor suppressor Fhit and its substrate diadenosine triphosphate (Ap₃A) are important factors in cancer development and progression. Fhit has Ap₃A hydrolase activity and cleaves Ap₃A into adenosine monophosphate (AMP) and adenosine diphosphate (ADP); this is believed to terminate Fhit‐mediated signaling. How the catalytic activity of Fhit is regulated and how the Fhit ? Ap₃A complex might exert its growth‐suppressive function remain to be discovered. Small‐molecule inhibitors of the enzymatic activity of Fhit would provide valuable tools for the elucidation of its tumor‐suppressive functions. Here we describe the development of a high‐throughput screen for the identification of such small‐molecule inhibitors of Fhit. Two clusters of inhibitors that decreased the activity of Fhit by at least 90 % were identified. Several derivatives were synthesized and exhibited in vitro IC₅₀ values in the nanomolar range.     

Expression Pattern of Purinergic Signaling Components in Colorectal Cancer Cells and Differential Cellular Outcomes Induced by Extracellular ATP and Adenosine

The purine nucleotide adenosine triphosphate (ATP) is known for its fundamental role in cellular bioenergetics. However, in the last decades, different works have described emerging functions for ATP, such as that of a danger signaling molecule acting in the extracellular space on both tumor and stromal compartments. Beside its role in immune cell signaling, several studies have shown that high concentrations of extracellular ATP can directly or indirectly act on cancer cells. Accordingly, it has been reported that purinergic receptors are widely expressed in tumor cells. However, their expression pattern is often associated with contradictory cellular outcomes. In this work, we first investigated gene expression profiles through “RNA-Sequencing” (RNA Seq) technology in four colorectal cancer (CRC) cell lines (HT29, LS513, LS174T, HCT116). Our results demonstrate that CRC cells mostly express the A2B, P2X4, P2Y1, P2Y2 and P2Y11 purinergic receptors. Among these, the P2Y1 and P2Y2 coding genes are markedly overexpressed in all CRC cells compared to the HCEC-1CT normal-like colonic cells. We then explored the cellular outcomes induced by extracellular ATP and adenosine. Our results show that in terms of cell death induction extracellular ATP is consistently more active than adenosine against CRC, while neither compound affected normal-like colonic cell survival. Intriguingly, while for the P2Y2 receptor pharmacological inhibition completely abolished the rise in cytoplasmic Ca²⁺ observed after ATP exposure in all CRC cell lines, Ca²⁺ mobilization only impacted the cellular outcome for HT29. In contrast, non-selective phosphodiesterase inhibition completely abolished the effects of extracellular ATP on CRC cells, suggesting that cAMP and/or cGMP levels might determine cellular outcome. Altogether, our study provides novel insights into the characterization of purinergic signaling in CRC.     

非天然碱基基因密码扩展 DNA 数据存储的机遇与挑战

目前,传统的存储技术主要将硅基材料作为存储介质,但全球现有的硅资源却无法满足日益增长的数据存储需求。随着数据时代的发展,存储技术创新面临新的挑战。在自然界,DNA 分子储存着丰富的遗传信息。从化学生物学的角度分析,将 DNA 分子作为介质进行数据信息存储有望为存储技术的创新提供一个新机遇,而非天然碱基核苷酸可以扩充遗传字母表,增加 DNA 存储容量,但在其实际应用方面,目前还有很多问题待解决。该文综述了 DNA 存储技术的研究进展,对当前 DNA 存储现状、待解决的技术难题与发展前景等进行了分析;并在此基础上,介绍了非天然碱基对(UBPs)作为合成生物学的一个新方向在 DNA 信息存储领域的潜在优势和技术挑战。     

配方奶粉中核苷酸含量高效液相色谱测定

研究建立了定量测定配方奶粉中5种核苷一磷酸(钠盐)的高效液相色谱方法。通过添加适量乙酸、离心浓缩、调节pH等技术方法,快捷有效地去除奶粉中影响核苷酸含量定量测量的蛋白质、脂肪及其他杂质成分,使用核苷酸国家级标准物质,采用外标法对样品中的核苷酸含量进行了定量测量。结果表明:该方法检出限低,准确度在90.8%～99.3%之间,方法的重复性限为1.43,再现性限为2.44。该方法准确度高,重复性好,可行性强,易于掌握,并且快捷、成本低,为奶粉中核苷酸含量的定量测量及相关产品的质量技术监督检验提供了方法学依据。     

外源性核苷酸对乙醇致发育毒性的体外干预作用

目的：探讨外源性核苷酸对乙醇致发育毒性的干预作用。方法：采用体外全胚胎培养模型,将胎龄8.5d的ICR小鼠胚胎以4.0g/L乙醇染毒,然后用不同剂量(0.032、0.80、20.0mg/L)的外源性核苷酸干预。培养48h后对小鼠胚胎生长发育和组织器官形态分化进行评分。结果：0.80mg/L核苷酸干预效果最为明显,可使胚胎颅臀长、头长增大,卵黄囊血管分化情况得到改善,并对乙醇所致的体位、心脏、后神经管、后脑、中脑、前脑、听觉、腮弓及前肢芽分化抑制均有改善。0.032mg/L和20.0mg/L剂量组的核苷酸干预也有一定的保护作用,但效果均不及0.80mg/L剂量组。结论：适量补充外源性核苷酸可能对乙醇致发育毒性有拮抗作用。     

三重四级杆串联质谱法测定乳粉类产品中 5种核苷酸的含量

目的建立超高效液相色谱-三重四级杆串联质谱法测定乳粉类产品中5种核苷酸,包括胞嘧啶核苷酸(cytidine monophosphate,CMP)、尿嘧啶核苷酸(uridine monophosphate,UMP)、腺嘌呤核苷酸(adenosine monophosphate,AMP)、鸟嘌呤核苷酸(guanosinemonophosphate,GMP)、次黄嘌呤核苷酸(inosinemonophosphate,IMP)含量的分析方法。方法样品经温水溶解后,经1%(V:V)甲酸沉淀蛋白离心后,分别通过固相萃取SPE法和超滤离心法有效净化其他干扰成分,从而进行前处理方法评价;采用Waters ACQUITY UPLC HSS T3(2.1mm×50 mm,1.8μm)为分析柱,0.1%(V:V)甲酸水溶液和乙腈作为流动相,等度洗脱分离。质谱采用正离子模式,多反应监测模式(MRM)进行检测,对纯水配制标准溶液和阴性基质溶液配制标准溶液进行比较,外标法定量分析。结果 5种核苷酸在4.5 min内达到基线分离,AMP、IMP和GMP在10~1000.0 ng/mL浓度范围内线性关系良好,CMP和UMP分别在25~1000 ng/mL和100~1000 ng/mL浓度范围内线性关系良好,相关系数均在0.9990以上,方法检出限分别为UMP 5 mg/kg、CMP 1 mg/kg、AMP0.5 mg/kg、IMP 0.5 mg/kg、GMP 0.1 mg/kg。在10.0、50.0、100.0mg/kg加标水平下的超滤离心法的平均回收率在73.6%~110.9%之间,相对标准偏差(RSD)为3.56%~15.89%;SPE法的平均回收率在86.4%~109.5%之间,相对标准偏差(RSD)为2.32%~14.45%。结论该方法灵敏度高,可以快速准确鉴别并定量乳粉产品中的核苷酸。     

Stereospecific formation of a ternary complex of (S)-α,β-fluoromethylene-dATP with DNA pol β

The influence of water: crystallization of (R/S)-α,β-CHF-dATP with the preorganized pol β-DNA complex shows that (S)-α,β-CHF-dATP is preferentially bound to the active site with the C=F fluorine proximal to a structural water bound to Asp276.     

Insight into the rescue of oxidized soluble guanylate cyclase by the activator cinaciguat

Nitric oxide (NO) signaling mediates many important physiological processes through the receptor soluble guanylate cyclase (sGC). Under disease conditions sGC heme can be oxidized resulting in NO insensitivity. Here, we show that the therapeutic compound cinaciguat (Cin) rescues dysfunctional sGC by direct displacement of the oxidized heme.