稳定同位素标记的色氨酸合成过程中脱色的研究

脱色是色氨酸合成过程中的关键步骤之一,由于色氨酸在水中的溶解度较低,活性炭和膜脱色并不十分理想。采用溶剂与树脂相结合的脱色方法,可使溶液的吸光度从0.986降到0.005,收率大于80%,产品的纯度达到98.6%。     

哺乳动物消化道内食物通过速率的测定方法

通过速率是消化道功能的一个重要量度,通常采用平均滞留时间来描述食物的通过速率。测定平均滞留时间的方法很多,实验条件不同,所采用的测定及计算方法也不同。本文对主要测定方法进行了介绍。     

Rapid Preparation of Stable Isotope Labeled Peptides that Bind to Target Proteins by a Phage Library System

We have developed a system for directly isolating foreign peptides displayed on the N-terminus of the major coat protein of bacteriophage M13. The phage particle in this system is formed as a mixture of wild type and modified coat proteins. The N-terminal segment of the modified coat protein was mutated for chemical cleavage, in order to obtain the displayed peptide from the major coat protein. Using ¹³C, ¹⁵N- labeled medium, we introduced stable isotopes, ¹³C and/or ¹⁵N, into the coat proteins. The NMR spectra for the cleaved peptides from the phage particles could be recorded within a few days after the selection of the phage clone.     

Synthesis of Stable Isotope Labeled Analogs of the Anti-Hepatitis C Virus Nucleotide Prodrugs PSI-7977 and PSI-352938

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent ¹³CD₃MgI to the corresponding 2 ′-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 ′-C-(trideuterated-¹³C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones.     

The Effect of Coprophagy on the Digesta Passage through the Gut of the Daurian Pika Ochotona dauurica (Ochotonidae,Lagomorpha)

Biology Bulletin - Fibrous food passage through the digestive tract of Daurian pika (Ochtona dauurica) was studied by means of multiple dose marking of food by neutral plastic markers. Contrary to...     

大熊猫胃肠道中消化酶活力的分析

为了研究大熊猫对食物的化学性消化特点和机制,测定了9只大熊猫唾液和3只大熊猫胃肠道中主要消化酶的活力,并与其他动物进行了比较.结果显示,大熊猫唾液呈碱性,蛋白酶和淀粉酶等消化酶活力低;肠道中淀粉酶活力高,而脂肪酶活力明显低于棕熊.大熊猫小肠粘膜中存在显著量的蔗糖酶、乳糖酶和麦芽糖酶活力.另外,在1只大熊猫胃和直肠液中检测到了少量纤维素酶活力.研究结果提示,大熊猫唾液直接参与食物消化的作用可能很弱;大熊猫对淀粉类食物有很好的消化能力,但对脂肪类食物消化能力相对不高.大熊猫胃肠道消化酶的活力特点适应其消化天然食物中的营养物质.     

Prion Remains Infectious after Passage through Digestive System of American Crows (Corvus brachyrhynchos)

Avian scavengers, such as American crows (Corvus brachyrhynchos), have potential to translocate infectious agents (prions) of transmissible spongiform encephalopathy (TSE) diseases including chronic wasting disease, scrapie, and bovine spongiform encephalopathy. We inoculated mice with fecal extracts obtained from 20 American crows that were force-fed material infected with RML-strain scrapie prions. These mice all evinced severe neurological dysfunction 196–231 d postinoculation ( = 198; 95% CI: 210–216) and tested positive for prion disease. Our results suggest a large proportion of crows that consume prion-positive tissue are capable of passing infectious prions in their feces ( = 1.0; 95% CI: 0.8–1.0). Therefore, this common, migratory North American scavenger could play a role in the geographic spread of TSE diseases.     

Intrinsic Innervation of the Chicken Lower Digestive Tract

We have studied the different components of the enteric nervous system in the rectum and cloaca of the chicken by means of hystochemical and immunohistochemical techniques. We found cholinergic neuronal bodies as well as nervous fibers, which constitute part of the Meissner and Auerbach plexuses. We also observed plentiful catecholaminergic fibers in both plexuses, though there were no catecholaminergic neuronal bodies. With respect to the Vasoactive Intestinal Peptide (VIP) and substance P (SP) positive peptidergic innervation, only positive fibers were found, which were less abundant than in the other zones of the gastrointestinal tract. The optic microscopy results were confirmed by electron microscopy.     

Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

Background

Metabolic phenotyping has become an important ‘bird''s-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems.

Methodology/Principal Findings

The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 ¹H and ¹³C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each ¹³C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient ¹³C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts.

Conclusions/Significance

Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of ¹³C atoms of given metabolites on development-dependent changes in the 56 identified ¹³C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism.     

白胸苦恶鸟消化管的组织学观察

采用石蜡切片技术对白胸苦恶鸟(Amaurornis phoenicurus)的消化道进行了组织学观察.结果表明,食管皱襞发达,黏膜上皮为复层扁平上皮,食管腺发达,颈段多于胸段,黏膜肌层为一层纵肌,厚约0.06 ～0.26 mm.肌层为一层厚约0.19～0.27mm的环肌.腺胃被覆单层柱状上皮,固有层内有单管腺和复管腺两种腺体,单管腺仅深约0.11 ～0.20 mm;复管腺厚约1.19～1.26 mm,占管壁的77.8％ ～80.4％.肌胃的类角质层发达,厚约0.16～0.24 mm.肌胃腺呈管状,与类角质层突起形成皱襞.肌层发达,由内环外纵两层平滑肌构成.肠绒毛无分支和中央乳糜管,十二指肠绒毛长而密集,空肠绒毛呈细长指状,直肠绒毛长且呈叶状.十二指肠与直肠肠绒毛内有大量致密淋巴小结,盲肠绒毛短,肠腺少.     

商城肥鲵消化道的解剖学观察

该文首次报道了商城肥鲵消化道各部分的形态结构、长度及其组织学结构。结果表明,除口腔外,消化道管壁由粘膜层、粘膜下层、肌层和浆膜层构成,消化道各部分的差别主要在粘膜层和肌层食道粘膜为复层柱状上皮,食完纵肌层,胃、肠粘膜上皮为单层柱状上皮,胃、肠肌层由内环外纵两层平滑肌组成。粘膜上皮的皱褶程度、腺体分布情况、肌层的相对厚度等在消化道各部分也存在差异。     

Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics

Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which ¹⁸O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.Protein phosphorylation regulates almost all aspects of cell life, such as cell cycle, migration, and apoptosis (1), and deregulation of protein phosphorylation is one of the most frequent causes or consequences of human diseases including cancers, diabetes, and immune disorders (2). Up till now, however, known substrates are far from saturation for the majority of protein kinases (3); thus, mapping comprehensive kinase-substrate relationships is essential to understanding biological mechanisms and uncovering new drug targets (4).Accompanied with advances of high-speed and high-resolution mass spectrometry, the technique of kinase substrate screening using proteomic strategy is quickly evolving (5–7). Mass spectrometry has been extensively used for kinase-substrate interaction mapping (8) and global phosphorylation profiling (9). Although thousands of phosphorylation sites have been detected, complex phosphorylation cascade and crosstalk between pathways make it difficult for large-scale phosphoproteomics to reveal direct relationships between protein kinases and their substrates (10, 11). Extensive statistics, bioinformatics, and downstream biochemical assays are mandatory for the substrate verification (12, 13). Another strategy uses purified, active kinases to phosphorylate cell extracts in vitro, followed by mass spectrometric analysis to identify phosphoproteins. This approach inevitably faces the major challenge of separating real sites phosphorylated by target kinase and the phosphorylation triggered by endogenous kinases from cell lysates (14). Analog-sensitive kinase allele (15) overcomes the issue by utilizing the engineered kinase that can exclusively take a bulky-ATP analog under the reaction condition. Analog-sensitive kinase allele has been coupled with γ-thiophosphate analog ATP to facilitate the mass spectrometric analysis (16–18).We have introduced kinase assay-linked phosphoproteomics (KALIP)¹ to link the in vitro substrate identification and physiological phosphorylation events together in a high throughput manner (19, 20). The strategy, however, has only been applied to identify direct substrates of tyrosine kinases. In this study, we expanded the application of KALIP to serine/threonine kinases by introducing a quantitative strategy termed Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics (siKALIP). The method was applied to identify direct substrates of extracellular signal-regulated kinase 1 (ERK1), a serine/threonine kinase acting as an essential component of the Mitogen-activated protein kinase (MAPK) signal transduction pathway (21). A defect in the MAP/ERK pathway causes uncontrolled growth, which likely leads to cancer (22) and other diseases (23–25). ERK1 can be activated by growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and nerve growth factor (NGF) (26). Upon stimulation, ERK1 phosphorylates hundreds of substrates in various cellular compartments including cytoplasm, nucleus, and membrane (27). Among 38 ERK1 direct substrates identified by siKALIP, more than one third are previously discovered by classical molecular biology approaches, highlighting high specificity and sensitivity of the strategy. The results also support the hypothesis that ERK1 plays complex roles in multiple pathways that are essential for the cell growth regulation.     

泽陆蛙消化道组织学观察

采用解剖学和组织学的方法对泽陆蛙消化道各部分的形态和组织学结构进行了观察。泽陆蛙的消化道分为口腔、咽、食道、胃、十二指肠、回肠和大肠。各管壁都由黏膜层、黏膜下层、肌层和外膜组成。食道黏膜层有皱褶和纤毛,无杯状细胞;胃黏膜层有杯状细胞和胃腺,黏膜下层有血管分布;小肠具绒毛、杯状细胞和肠腺,大肠皱褶少,杯状细胞也少。