海绵共附生真菌Penicillium chrysogenum次级代谢产物的化学成分及生物活性研究

目的 对分离自西沙隋氏蒂壳海绵共附生真菌Penicilliumchrysogenum的次级代谢产物进行化学成分及其生物活性研究,以期发现结构特异并且活性良好的次级代谢产物。方法 对隋氏蒂壳海绵共附生真菌Penicilliumchrysogenum用真菌2号培养基发酵,发酵后的菌丝体采用溶剂提取、萃取和现代色谱分离纯化手段,再运用现代核磁波谱技术并结合高分辨质谱鉴定化合物结构。基于微阵列技术的表面等离子体共振成像（SPRi）系统,检测化合物与肿瘤相关蛋白的相互作用,并提供化合物与肿瘤相关蛋白的结合动力学数据。结果 通过分离隋氏蒂壳海绵共附生真Penicilliumchrysogenum的菌丝体提取物,从中分离鉴定了7个单体化合物,鉴定结果为conidiogenone（1）、2-acetylquinazolin-4(3H)-one（2）、15β-hydroxyl-(22E,24R)-ergosta-3,5,8,22-tetraen-on（3）、ergosta-4,6,8(14),22-tetraen-3-one（4）、 2-((2E,4E)-hexa-2,4-dienoyl)-5,6-dihydroxy-4,6-dimethylcyclohex-4-ene-1,3-dione（5）、(22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol（6）、2-(1-hydroxyethyl)quinazolin-4(3H)-one（7）。结论 化合物3和6是从Penicillium属内第一次分离得到,化合物4是从真菌Penicilliumchrysogenum中第一次分离得到,化合物5与肿瘤蛋白VEGFR-1、FGFR有亲和作用,KD的数值分别为7.78×10-3和1.44×10-1 μmol/L。     

Biomolecular modification of p(AAm-co-EG/AA) IPNs supports osteoblast adhesion and phenotypic expression

Interpenetrating polymer networks (IPNs) were designed to resist materials fouling caused by non-specific protein adsorption, and indiscriminate cell or bacterial adhesion. These IPNs were thin adherent films ( ~ 20 nm) comprised of acrylamide (AAm), ethylene glycol (EG), and acrylic acid (AA) grafted to either silicon waters or quartz substrates via photoinitiated free radical polymerization. These networks were further modified to promote specific cell adhesion by tethering bioactive groups such as peptides that mimic cell-binding domains found on extracellular matrix molecules. As a specific example of biomolecular surface engineering, peptides from the cell-binding domain of bone sialoprotein were tethered to a p(AAm-co-EG/AA) IPN to control cell behavior at the surface. The networks were characterized by contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy to convey information on IPN wettability, thickness, and chemistry. The surface characterization data supported the theory that the PEG/AA layer formed an IPN with the underlying p(AAm) network, and after graft modification of this IPN with diamino PEG (PEG(NH₂)₂), the PEG(NH₂)₂ chains were enriched at the surface. Rat calvarial osteoblasts attached to Arg-Gly-Asp (RGD) modified IPNs at levels significantly greater than on clean quartz, Arg-Gly-Glu (RGE) modified, or the PEG(NH₂)₂ modified IPN, with or without serum in the media. Cells maintained in media containing 15% fetal bovine serum (FBS) proliferated, exhibited nodule formation, and generated sheets of mineralized extracellular matrix (ECM) with the addition on β-glycerophosphate to the media. Cell adhesion and mineralized ECM formation were specifically dependent on the peptide sequence present at the surface.     

Stress granule formation,disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity

While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)—a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase–deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.

Biomolecular condensates are prevalent in cells and critical for a range of cellular functions, including RNA metabolism, embryonic cell fate specification, and neuronal activity (1–3). While condensates often dynamically exchange components with the surrounding milieu, the overall composition of these cellular structures remains distinct (4). How cells control the specific composition of these condensates remains unclear. Stress granules (SGs), one of the best characterized biomolecular condensates, are RNA–protein assemblies formed in response to a variety of environmental cues (1). While SG composition can vary with the type of stress cue (5), certain common components, such as Ras GTP-activating protein-binding proteins G3BP1/2, are essential for formation of SGs (6, 7). Dysregulation of SG formation and disassembly is implicated in the pathogenesis of diseases, including viral infection, cancer, and neurodegeneration (2, 8–10).SG formation and disassembly are tightly regulated during viral infection, often reflecting cellular translation status (11–14). In the early phase of many viral infections, the presence of double-stranded viral RNAs (vRNAs) activate protein kinase R (PKR), resulting in eIF2α phosphorylation, messenger RNA (mRNA) translation inhibition, and formation of SGs enriched with translation initiation factors such as eIF3b. However, in later infection stages, many viruses instead suppress SG formation or disassemble SGs altogether. The mechanisms underlying this switch, and its physiological function, remain unclear.SG formation and disassembly are regulated by posttranslational modifications of proteins, including those that conjugate simple chemical groups, attach polypeptides, and add nucleotides as in the case of ADP-ribosylation (15–21). ADP-ribosylation refers to the addition of one or more ADP-ribose units onto proteins (22–24). In humans, ADP-ribosylation is accomplished primarily by a family of 17 ADP-ribosyltransferases, commonly known as poly(ADP-ribose) polymerases (PARPs). SG components are specifically ADP-ribosylated, and ADP-ribose polymers [i.e., poly(ADP-ribose) or PAR], five PARPs and two isoforms of the degradative enzyme PAR glycohydrolase (PARG) have been localized to these condensates (17, 25–27). Overexpression of these PARPs and PARG isoforms induces and suppresses SG formation, respectively, while PARG knockdown delays SG disassembly (17, 26). The noncovalent interaction between PAR and proteins facilitates SG targeting (25–27). For example, PAR-mediated targeting regulates TDP-43 localization to SGs and prevents the formation of pathological aggregates in amyotrophic lateral sclerosis (26, 27).The mosquito-borne alphaviruses, which cause a range of diseases from rashes and arthritis to encephalitis, induce SG formation early in infection and later initiate SG disassembly (11, 14, 28, 29). Previous studies have identified the alphaviral nonstructural protein 3 (nsP3), a key factor for virus replication and virulence (30–32), as able to suppress SG formation (28, 33–35). The alphaviral nsP3 is a tripartite protein composed of a highly conserved macrodomain (MD) in the N terminus, a central zinc-binding domain (ZBD), and a C-terminal hypervariable domain (HVD; ref. 30). Recent studies indicate that the HVD, which is of low complexity, directs alphaviral nsP3 binding to host SG proteins (30, 36). For example, the HVD of chikungunya virus (CHIKV) binds the essential SG components G3BP1 and G3BP2 (33, 37). Given that nsP3 expression increases over the course of viral infection, it has been proposed that nsP3 sequesters G3BP1/2, resulting in the suppression of SG formation during the late phase of infection (28, 29, 34).Here, we report that the expression of the G3BP-binding HVD alone does not suppress SG formation; rather, expression of the N-terminal MD alone can trigger the suppression of this biomolecular condensate. The structural integrity of SGs is dependent on ADP-ribosylation (17), and we and others recently found that the viral MD can remove single ADP-ribose groups, and possibly PAR, from ADP-ribosylated proteins (31, 38–40). We therefore hypothesized that MD ADP-ribosylhydrolase activity is required to suppress SG formation across stress conditions, with G3BP1 being a key target substrate. Indeed, we find that MD ADP-ribosylhydrolase activity is critical for disassembling SGs formed by G3BP1 expression and during viral infection. Consistent with this premise, live cell imaging revealed that SGs persist in cells infected with a hydrolase-deficient recombinant CHIKV. ADP-ribosylhydrolase activity is required for altering the composition of biomolecular condensates in nsP3-expressing or virus-infected cells and specifically regulates translation factor localization. Together, these data argue that nsP3 ADP-ribosylhydrolase activity modulates SG formation, disassembly, and composition.     

HBV相关糖基转移酶Glt25D2的核苷酸单糖结合活性分析

目的体外克隆表达人类糖基转移酶Glt25D2,利用Biacore分析系统对其活化单糖及钙离子结合活性进行分析。方法构建Glt25D2原核表达载体pET32a-Glt25D2,转化大肠埃希菌BL21,克隆表达糖基转移酶Glt25D2。离心集菌,制备蛋白样品,进行SDS-PAGE电泳分析;融合蛋白线性梯度洗脱,Ni-NTA柱纯化,后做Western blot鉴定。用纯化后的Glt25D2融合蛋白包被CM5芯片,分别用不同浓度的活性单糖及钙离子灌注芯片,利用Biacore生物分子相互作用分析仪分析Glt25D2的活化单糖及钙离子结合活性。结果体外成功表达人类糖基转移酶Glt25D2融合蛋白。Biacore分析显示,该糖基转移酶与400、200、100、50μg/ml唾液酸的结合活性分别为108、71、50和20 RU,与200、100、50、0μg/ml钙离子的结合活性分别为37、20、10和0 RU。结论人类糖基转移酶Glt25D2重组蛋白具有较强的钙离子及唾液酸结合活性。     

基于生物分子网络探讨桑白皮汤治疗慢性阻塞性肺疾病的作用机制

目的利用生物分子网络探讨桑白皮汤治疗慢性阻塞性肺疾病(COPD)的作用机制。方法通过中药系统药理数据库和分析平台(TCMSP)收集桑白皮汤的化学成分和成分对应的靶点,并利用Cytoscape软件构建中药-成分-靶点网络;通过GeneCards、TTD和DisGeNET数据库获取与COPD相关的靶点,对成分靶点和疾病靶点取交集,利用String和Cytoscape构建交集靶点网络;利用DAVID数据库对交集靶点进行KEGG和GO富集分析。结果筛选得到109个化学成分,230个成分靶点,COPD相关靶点1220个,成分和疾病的交集靶点119个。KEGG分析表明桑白皮汤可能通过PI3K-Akt信号通路、TNF信号通路、MAPK信号通路、NF-κB信号通路、HIF-1信号通路、凋亡相关通路等发挥治疗COPD的作用。GO分析显示桑白皮汤主要涉及炎症反应、细胞增殖、细胞凋亡、RNA转录、基因表达等。结论通过构建生物分子网络,揭示了桑白皮汤治疗COPD的分子机制,为桑白皮汤的后续研究提供了理论依据。     

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.     

石见穿酸性多糖SC4的结构特征和生物活性

目的:研究从石见穿(Salvia chinensis)中得到的酸性多糖SC4的化学结构和生物活性.方法:利用化学和光谱方法分析了SC4的结构特征,并观察了SC4对淋巴细胞增殖的影响以及增强PC12细胞对抗H_2O_2造成的损伤的作用.结果:SC4的分子量为4.5×10~5,由Rha,Xyl,Ser,Gal和GalA组成,摩尔比为1.0:7.0:5.3:1.2:4.2.甲基化和~(13) C NMR进一步确定了SC4中各糖残基的连接方式.SC4能显著促进B淋巴细胞的增殖,增加小鼠脾重,增强PC12细胞对抗H_2O_2造成的氧化损伤的能力.结论:SC4为一多分枝的酸性多糖,其免疫活性的靶细胞为B淋巴细胞,并显著增强PC12细胞对抗H_2O_2造成的损伤的能力.     

~1H磁共振波谱在胶质瘤放疗后复发和放射性脑坏死鉴别中的初步应用

目的　探讨质子磁共振波谱 (1HMRS)对胶质瘤放疗后复发和放射性脑坏死鉴别诊断的价值。方法　 15例有脑部放疗史 ,临床及CT、MRI难以判断为肿瘤复发或放射性脑坏死的患者 ,5例病史明确的放射性脑坏死的患者 ,均行1HMRS检查。结果　前 15例经手术证实 ,14例为胶质瘤 ,1例放射性脑坏死 ,1HMRS诊断正确。 (1) 14例胶质瘤在1HMRS上均表现为明显增高的胆碱(Cho)峰 ,氮乙酰门冬氨酸 (NAA)、肌酸 (Cr)峰下降或消失 ,Cho/Cr比值升高 ,NAA/Cr比值降低 ;12例出现乳酸 (Lac)峰。 (2 )放射性脑坏死表现为 :5例Cho、NAA、Cr下降或消失 ,出现脂质 (Lipid)峰 ;1例Cho、NAA、Cr峰均消失 ,仅表现一较平坦的曲线 ,无Lac峰。结论　1HMRS对胶质瘤放疗后复发和放射性脑坏死的鉴别有重要价值。