Dietary fiber components: relationship to the rate and extent of ruminal digestion

A mathematical model can serve as a useful reference for describing the mechanisms involved in digestion and for discussing the factors that influence the rate and extent of ruminal digestion. Ruminal digestion can be divided into four components: digestion rate, digestion lag, potential extent of digestion, and passage rate. Each component affects the apparent extent of digestion in a distinct manner and is influenced by separate factors. Digestion rate is directly related to apparent extent of digestion. It is not influenced by chemical entities presently being measured, but may be related to the morphological, crystalline, or physical nature of fiber. It may also be influenced by factors that inhibit or stimulate ruman microbial growth and their fiber-degrading enzymes. Digestion lag is inversely related to apparent extent of digestion; however, factors influencing it are poorly defined. The may include factors affecting microbial populations and their attachment to fiber prior to digestion; or the digestion lag may be related to the chemical or physical alteration of fiber that must occur before digestion can begin. The potential extent of digestion is directly related to apparent extent of digestion and is influenced by plant fiber composition, primarily. Lignin, and possibly silica, functions to limit the potential extent of digestion. Rate of passage essentially competes with rate of digestion for fiber particles as they pass through the rumen; therefore it is inversely related to the apparent extent of digestion. Passage rate is associated with feed intake level and particle size, although other factors such as type of diet and animal physiology may be important.     

Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.     

Effects of alpha-galactosidase digestion on lectin staining in human pancreas

Effects of alpha-galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B4 positive acinar cells. beta-Galactosidase digestion following alpha-galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B4 positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple beta-galactosidase digestion as well as sequential digestion with alpha- and beta-galactosidase. However, when alpha-L-fucosidase digestion procedure was inserted between alpha- and beta-galactosidase digestion, UEA-I staining imparted by alpha-galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B4 positive acinar cells. Furthermore, after sequential digestion with alpha-galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B4 positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

典型有机固废厌氧消化微生物研究现状与发展方向

经过人工富集和驯化的兼性和严格厌氧微生物是厌氧消化工艺的核心。不同厌氧消化体系中存在的问题大多可以通过改变微生物群落的代谢活性来得到有效改善。得益于微生物组学检测技术的快速发展,对厌氧消化系统中微生物多样性的认识获得了极大的拓展,同时在微生物类群间、微生物与环境的互作关系研究方面也取得了一系列新的进展。然而,有机固废厌氧消化系统中,各种微生物以及微生物和物质的相互作用构成了更为复杂的代谢网络,所以目前对这些互作关系的解析尚不完善。本文重点关注了厌氧消化过程中的典型菌群互作关系,阐述了典型有机固废厌氧消化系统中存在的问题及微生物在其中发挥的作用,最后,立足于现有组学技术推动的微生物组研究进展,对未来有机固废厌氧消化系统微生物组的研究提出展望。     

Optimization of separation and digestion conditions in immune complexome analysis

Immune complexome analysis is a method for identifying and profiling of antigens in circulating immune complexes (CICs); it involves separation of immune complexes from serum, direct tryptic digestion of these complexes, and protein analysis via nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS). To improve this method, we initially investigated the effects of two factors—the gradient elution program and nano-LC column type (C18-packed, C8-packed, or packed spray capillary column)—on the numbers of peptides and proteins identified. Longer gradient elution times resulted in higher identification capability throughout the range of 25–400 min. Moreover, the packed spray capillary column supported identification of more peptides and proteins than did any other column. In addition, microwave-assisted digestion was compared with conventional digestion, which involved incubation overnight at 37 °C. Microwave-assisted digestion produced more partially digested peptides than did conventional digestion. However, the percentages of miscleaved peptides in all of the identified peptides in microwave-assisted digestion of immune complexes (a protein mixture) were lower than those in the physical stimulation-assisted digestion of a model protein. Microwave-assisted digestion is slightly inferior to, or as effective as, conventional digestion, but it drastically reduces the digestion time.     

Histochemical analysis of the chemical structure of blood group-related carbohydrate chains in serous cells of human submandibular glands using lectin staining and glycosidase digestion

Using lectin staining methods in combination with exo- and endo-glycosidase digestion procedures, we analyzed the chemical structure of different types of blood group-related substances in serous cells of formalin-fixed, paraffin-embedded human submandibular glands. Serous cells produced only H antigen; A and B antigens were not present, and the expression of H antigen is dependent on the secretor status of the tissue donor. Although reactivity with Ulex europaeus agglutinin I (UEA-I) was not markedly reduced by alpha-L-fucosidase digestion, an affinity for peanut agglutinin (PNA) was seen after fucosidase digestion in the cells from secretors. In those from nonsecretors, no PNA reactivity appeared after enzyme digestion. On the other hand, sialidase digestion elicited PNA reactivity in serous cells irrespective of the donor's secretor status. PNA reactivity observed after fucosidase or sialidase digestion was susceptible to endo-alpha-N-acetylgalactosaminidase (endo-GalNAc-dase) digestion. SBA reactivity in UEA-I-negative cells from secretors, or in cells from fetuses and newborn infants, was markedly reduced by beta-galactosidase digestion. After galactosidase digestion, reactivity with Griffonia simplicifolia agglutinin II (GSA-II) appeared in the corresponding cells. This GSA-II reactivity was almost completely eliminated by subsequent beta-N-acetylhexosaminidase digestion. Whereas PNA reactivity in these cells was not reduced by beta-galactosidase treatment, it was significantly diminished by endo-GalNAc-dase digestion. These results suggest that at least two kinds of precursor disaccharides are produced in submandibular serous cells, i.e., SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and PNA-reactive D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine (O-glycosidically linked Type 3 chain or T antigen). Final fucosylation and synthesis of these two types of precursor chain appear to be under the control of the secretor gene.     

不同消化方法对测定生物样品中硒含量的比较研究

本文比较了干法消化、普通湿法消化、高压消化、微波消化对测定富含有机硒的生物样品中硒含量的影响.结果显示,高压及微波消化等密闭消化体系具有显著的优点,适合生物样品中富含有机硒的硒含量的测定.     

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17°C than at 4°C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T₉₀ values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T₉₀ value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.     

Resistance of soil-bound prions to rumen digestion

Before prion uptake and infection can occur in the lower gastrointestinal system, ingested prions are subjected to anaerobic digestion in the rumen of cervids and bovids. The susceptibility of soil-bound prions to rumen digestion has not been evaluated previously. In this study, prions from infectious brain homogenates as well as prions bound to a range of soils and soil minerals were subjected to in vitro rumen digestion, and changes in PrP levels were measured via western blot. Binding to clay appeared to protect noninfectious hamster PrP(c) from complete digestion, while both unbound and soil-bound infectious PrP(Sc) proved highly resistant to rumen digestion. In addition, no change in intracerebral incubation period was observed following active rumen digestion of unbound hamster HY TME prions and HY TME prions bound to a silty clay loam soil. These results demonstrate that both unbound and soil-bound prions readily survive rumen digestion without a reduction in infectivity, further supporting the potential for soil-mediated transmission of chronic wasting disease (CWD) and scrapie in the environment.     

Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin

Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity.     

Effect of Calcium Activated Factor on Properties of Actin and Myosin of Rabbit Skeletal Muscle

The effect of calcium activated factor (CAF) on enzymatic properties of actin and myosin was investigated. SDS polyacrylamide gel electrophoresis revealed that CAF did not degrade actin, but a slight degradation was found in myosin during CAF digestion, which might have been due to contaminated protease (s) in CAF preparation. No influence was found in EDTA ATPase of myosin and polymerization of G-actin during CAF digestion. However, heavy meromyosin (HMM) ATPase activating ability of actin was slightly decreased during CAF digestion. Although CAF digestion slightly decreased the biological activity of myofibrillar proteins, a single sarcomere prepared by CAF digestion is a useful model for studying muscle contraction because of its almost intact contractility.     

Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi.     

利用微波-热效应进行限制性内切核酸酶反应的可行性验证

限制性内切酶催化的酶解反应是分子生物学实验中的常用技术.鉴于酶切反应时间长,有人提出采用微波释放的热效应催化限制性内切酶解反应,以期缩短酶解时间.但微波是否能代替传统酶切反应还需验证.本研究设计了一系列的酶切 试验,验证微波酶切是否有效.结果表明：短时间（2 min）微波炉高火处理未导致DNA降解及内切酶失活.对于质粒的单酶切和部分双酶切,短时间（2 min）微波炉处理可替代水浴加热完成酶解反应.对于部分双酶切体系,微波炉处理酶切不完全,不能替代温水浴.使用微波酶解需严格控制时间,切勿时间过长.     

Effects of alpha-L-fucosidase digestion on lectin staining in human pancreas

We examined the effects of alpha-L-fucosidase digestion on lectin staining in formalin-fixed, paraffin-embedded human pancreatic tissue from individuals of different blood groups. Digestion with the enzyme resulted in apparent diminished intensity of Ulex europaeus agglutinin-I (UEA-I) staining in the acinar cells. In addition to the decreased intensity of UEA-I staining, reactivity with soybean agglutinin (SBA) was increased in the enzyme-susceptible, UEA-I-reactive cells. The intensity of Griffonia simplicifolia agglutinin-II (GSA-II) staining performed after beta-galactosidase digestion in UEA-I-reactive acinar cells was markedly increased by prior treatment with fucosidase. GSA-II staining following sequential digestion with fucosidase and galactosidase was completely abolished by subsequent digestion with beta-N-acetylhexosaminidase. These results therefore substantiate the previous assumption that SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and GSA-II reactive beta-N-acetyl-D-glucosamine imparted following galactosidase digestion represent precursors of H antigen. The present study further demonstrated that intense peanut agglutinin (PNA) staining was imparted after digestion with fucosidase in UEA-I-reactive sites in secretors. In contrast, nonsecretors showed vivid PNA staining that was usually detected throughout the pancreas without prior enzyme digestion. Here, fucosidase digestion had if any little effect on PNA staining. These results suggest that in secretors a terminal trisaccharide, fucosylated D-galactose-(beta 1-3)-N-acetyl-D-galactosamine exhibiting positive PNA reaction after fucosidase digestion, exists in UEA-I-reactive acinar cells. It is assumed that the secretor gene could control the step of final fucosylation of D-galactose-(beta 1-3)-N-acetyl-D-galactosamine in human pancreas.     

中高温污泥厌氧消化系统中微生物群落比较

【目的】结合中温与高温消化两者优势的两相厌氧消化工艺可能是推进污泥厌氧消化发展的重要方向,因此,探究和比较中温和高温污泥厌氧消化系统中微生物群落组成的异同具有重要意义。【方法】利用高通量测序技术检测中温和高温厌氧消化系统中细菌与古菌的16S r RNA基因序列信息和真菌的内转录间隔(ITS)序列信息,利用基因芯片(Geo Chip 5.0)检测病毒和病原菌致病基因的信息,以对比中温和高温条件下微生物群落在物种组成和功能基因层面上的异同。【结果】中温和高温条件下细菌和古菌在群落物种组成上存在显著差异,病毒和病原菌毒性基因也显著不同,而两种系统中真菌群落的物种组成相似且丰度相对较低。中温条件下产甲烷古菌和未分类微生物相对丰度较高,而高温条件下产酸及嗜热菌相对丰度较高,且高温消化后病毒和病原菌毒性基因相对丰度下降。微生物群落结构与COD、TS和VS有着显著相关性。【结论】微生物群落组成和功能基因在中高温的污泥厌氧消化系统中显著不同,从而解释了两个系统功能的差异。微生物群落的形成与进水参数相关,说明微生物对进水条件敏感。     

Kinetic characterization of sequencing grade modified trypsin

Prior to analysis by mass spectrometry, protein samples are often digested. Maximizing the peptide yield from digestion can increase the number of peptides detected and the confidence in protein identification. To determine the optimal conditions for digestion, the Michaelis-Menten kinetic parameters for Promega sequencing grade modified trypsin were measured over a range of temperatures and pHs. The results indicate that an increase in digestion temperature above 37 degrees C, the temperature traditionally used in digestion methods, could offer an increase in peptides detected.     

导电碳颗粒促进污泥厌氧消化及微生物种间电子传递的研究进展

添加导电碳颗粒能够促进厌氧消化过程稳定性、底物降解率以及产沼气品质的同步提高。本文总结了以活性炭和生物炭为代表的导电碳颗粒对城市污泥厌氧消化的影响,探讨了导电碳颗粒促进城市污泥厌氧消化的机理,阐述了导电碳颗粒介导的微生物直接种间电子传递(Directinterspecies electrontransfer,DIET)在强化污泥厌氧消化中的作用机制,分析了复杂厌氧消化体系中微生物DIET互营关系的研究现状,同时对导电碳颗粒的物理化学特性及其对污泥厌氧消化产甲烷的影响进行了分析,最后对未来导电碳颗粒促进城市污泥厌氧消化的研究进行了展望。     

银盾革蜱的中肠上皮变化与血餐消化

用光学显微镜及电子显微镜对不同生理状况下银盾革蜱Dermacentor niveus Neumann雌虫中肠上皮及血餐消化进行了研究.饿蜱的中肠只由一种干细胞组成,脂滴作为饿蜱营养的贮藏形式.非滞育蜱消化分三个阶段,即第一连续消化阶段,减慢消化阶段及第二连续消化阶段.吸血后中肠上皮共观察到四种细胞类型,即替代细胞、分泌细胞、消化细胞及卵赞原细胞.滞育蜱第一连续消化阶段延长.饱血后60天到120天,消化作用几近停止,为停滞消化阶段.卵黄原细胞的超微结构有明显改变.滞育解除后,开始进行减慢消化阶段及第二连续消化阶段.     

Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane

Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction from sucrose-density gradient centrifugation was solubilized by boiling in 1.0% SDC, and directly digested without dilution. During the in-gel digestion of the hippocampal PM proteins separated by SDS-PAGE, 0.1% SDC was added. Before analysis of peptide mixture by liquid chromatography and electrospray mass spectrometry, SDC in the tryptic digests was removed by centrifugation following acidification. Use of 1.0% SDC in solubilization and in-solution digestion of rat PM proteins had led to 77 PM or membrane-associated proteins identified, a more than 2-fold increase over that by use of SDS. The addition of 0.1% SDC to the in-gel digestion of SDS-PAGE-resolved membrane proteins remarkably enhanced the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified. Being a cheaper and more tractable acid-insoluble detergent, SDC could be used at higher concentration in the solubilization and tryptic digestion of proteins including PM proteins with the purpose of enhancing the protein solubility and at the same time making no interference with trypsin activity and subsequent analyses.     

Relative quantitation of transfer RNAs using liquid chromatography mass spectrometry and signature digestion products

Transfer ribonucleic acids (tRNAs) are challenging to identify and quantify from unseparated mixtures. Our lab previously developed the signature digestion approach for identifying tRNAs without specific separation. Here we describe the combination of relative quantification via enzyme-mediated isotope labeling with this signature digestion approach for the relative quantification of tRNAs. These quantitative signature digestion products were characterized using liquid chromatography mass spectrometry (LC-MS), and we find that up to 5-fold changes in tRNA abundance can be quantified from sub-microgram amounts of total tRNA. Quantitative tRNA signature digestion products must (i) incorporate an isotopic label during enzymatic digestion; (ii) have no m/z interferences from other signature digestion products in the sample and (iii) yield a linear response during LC-MS analysis. Under these experimental conditions, the RNase T1, A and U2 signature digestion products that potentially could be used for the relative quantification of Escherichia coli tRNAs were identified, and the linearity and sequence identify of RNase T1 signature digestion products were experimentally confirmed. These RNase T1 quantitative signature digestion products were then used in proof-of-principle experiments to quantify changes arising due to different culturing media to 17 tRNA families. This method enables new experiments where information regarding tRNA identity and changes in abundance are desired.