绣球菌降血糖作用及活性成分分析

绣球菌是一种珍稀食药用真菌,本文对其醇提取物的降血糖作用及活性成分进行了研究.高脂高糖饲料联合链脲佐菌素造模建立了 Ⅱ型糖尿病小鼠模型,设置空白对照组、模型组、绣球菌提取物高、中、低3个剂量组(300、200和100mg/kg),连续给予相应药物3周,对各组小鼠的体重、血糖、血脂和胰腺进行组织病理学研究.结果显示:与模...     

污水土地生态处理脱氮技术的中型试验研究

地沟式污水土地生态处理工艺,是自然生态净化与人工工艺相结合的小规模污水处理回用技术。它是采用土壤毛细管浸润扩散原理的浅型土壤处理技术,在人工可控条件下,将污水科学、合理地投配到设计定型的装置内,利用污水的能量,把其所携带的污染物,通过人工基质(土壤、砂、碎石等,填料-水-微生物-植物系统)的物质循环和能量流动,逐级降解;在不同的污染负荷、水力负荷下,完成一系列物理、化学、物理化学和微生物化学、生物化学的反应。通过以贵州典型的黄壤土为主配比的人工土作为处理系统填料的现场中型试验,探讨地沟式污水土地处理系统的脱氮效果及其影响因素。地沟式污水土地生态系统对氨氮和总氮去除效果良好,去除率分别达到84 .7%和70 .7% ,出水氨氮(14 .0 mg/L )和总氮(2 4 .7mg/L ) ,达到建设部颁发的生活杂用水水质标准。对处理系统微生物数量及分布的研究表明:处理系统中氮转化细菌丰富,氨化细菌为10 3～10 6 cfu MPN/g(土壤) (cfu:形成菌落数:MPN:最大可能数量) ,亚硝化菌为10 3～10 6 MPN/g(土壤) ,硝化菌10 4～10 6 MPN/g(土壤) ,反硝化细菌为10 3～10 6 MPN/g(土壤)。由硝化/反硝化实现生物脱氮是土地生态处理系统去除总氮的主要途径;建立土壤、土壤微生物、土壤植被环境以促进硝化作用是提高总     

温带半干旱地区一年生植物种子的萌发特性

在实验室条件下研究了中国温带半干旱地区科尔沁沙地的23种1年生植物的种子萌发特性（新采集种子、冷藏和干藏种子）.大籽蒿、虎尾草、冠芒草、沙蓬和地锦的新种子萌发率达90%左右,11种植物新种子萌发率均低于70%,说明这些植物的新种子具有或多或少的休眠属性.经过150d的冷干藏后,大籽蒿、虎尾草萌发率保持在90%以上,说明这两种植物完全没有休眠机制;冠芒草、沙蓬和地锦的种子萌发率下降较多,可能是储藏的环境条件导致的2次休眠现象;冷藏和干藏处理均能使绿珠藜、毛马唐、细叶益母草、雾冰藜、金狗尾草、苋菜、马齿苋、碱地肤和水稗草的种子在生长季开始时完成生理后熟,萌发率达到80%以上;干藏有利于促进毛马唐、细叶益母草、马齿苋和鹤虱的种子成熟,冷藏有利于促进绿珠藜和金狗尾草的种子成熟;黄蒿、灰绿藜、画眉草和烛台虫实在不同处理下的萌发率都比较低,说明种子内在生理休眠作用较强,具有减少种子一次性萌发数量的风险分摊策略.大多数1年生植物均能在较短时间内达到最终萌发率的90%,表现出迅速萌发的特性;黄蒿、灰绿藜、碱地肤和沙蓬种子则在不同处理中表现出延长萌发时间的策略来适应半干旱地区不确定的环境条件.最后,探讨了几种主要1年生植物的种子萌发对策与其对环境适应机制之间的关系.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at ?30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.     

Quantitative Metaproteomics and Activity-based Protein Profiling of Patient Fecal Microbiome Identifies Host and Microbial Serine-type Endopeptidase Activity Associated With Ulcerative Colitis

The gut microbiota plays an important yet incompletely understood role in the induction and propagation of ulcerative colitis (UC). Organism-level efforts to identify UC-associated microbes have revealed the importance of community structure, but less is known about the molecular effectors of disease. We performed 16S rRNA gene sequencing in parallel with label-free data-dependent LC-MS/MS proteomics to characterize the stool microbiomes of healthy (n = 8) and UC (n = 10) patients. Comparisons of taxonomic composition between techniques revealed major differences in community structure partially attributable to the additional detection of host, fungal, viral, and food peptides by metaproteomics. Differential expression analysis of metaproteomic data identified 176 significantly enriched protein groups between healthy and UC patients. Gene ontology analysis revealed several enriched functions with serine-type endopeptidase activity overrepresented in UC patients. Using a biotinylated fluorophosphonate probe and streptavidin-based enrichment, we show that serine endopeptidases are active in patient fecal samples and that additional putative serine hydrolases are detectable by this approach compared with unenriched profiling. Finally, as metaproteomic databases expand, they are expected to asymptotically approach completeness. Using ComPIL and de novo peptide sequencing, we estimate the size of the probable peptide space unidentified (“dark peptidome”) by our large database approach to establish a rough benchmark for database sufficiency. Despite high variability inherent in patient samples, our analysis yielded a catalog of differentially enriched proteins between healthy and UC fecal proteomes. This catalog provides a clinically relevant jumping-off point for further molecular-level studies aimed at identifying the microbial underpinnings of UC.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.