Description and recognition of regular and distorted secondary structures in proteins using the automated protein structure analysis method

The Automated Protein Structure Analysis (APSA) method, which describes the protein backbone as a smooth line in three‐dimensional space and characterizes it by curvature κ and torsion τ as a function of arc length s, was applied on 77 proteins to determine all secondary structural units via specific κ(s) and τ(s) patterns. A total of 533 α‐helices and 644 β‐strands were recognized by APSA, whereas DSSP gives 536 and 651 units, respectively. Kinks and distortions were quantified and the boundaries (entry and exit) of secondary structures were classified. Similarity between proteins can be easily quantified using APSA, as was demonstrated for the roll architecture of proteins ubiquitin and spinach ferridoxin. A twenty‐by‐twenty comparison of all α domains showed that the curvature‐torsion patterns generated by APSA provide an accurate and meaningful similarity measurement for secondary, super secondary, and tertiary protein structure. APSA is shown to accurately reflect the conformation of the backbone effectively reducing three‐dimensional structure information to two‐dimensional representations that are easy to interpret and understand. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A novel approach to segregate and identify functional loop regions in protein structures using their Ramachandran maps

The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved three‐dimensional structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least twofold higher for unstructured proteins in comparison with ordered proteins. The above results validate the use of MSRP parameter as a tool to identify and investigate functionally active loops and unstructured regions in protein structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

伏曲酵母菌的分类鉴定及来源分析的研究

目的:统计分析酵母菌的来源,为大曲微生物物种及其来源变化提供基础资料.方法:利用Biolog微生物自动分析系统进行精确鉴定,并通过统计分析得出微生物菌种的来源.结果:从伏曲样品中分离纯化出7株酵母菌J-1,2,3,4,5,6,7,利用Biolog微生物自动鉴定系统进行鉴定,确定其分别为白吉利丝孢酵母A、无名假丝酵母菌、汉逊德巴利酵母C、海隐球酵母菌、东方伊萨酵母菌、解脂耶罗威亚酵母菌、皱落假丝酵母菌.这些物种主要来源于原料本身和空气,原料为了人室大曲提供了J-1,2,3三种酵母菌,其比例为25.45%、19.23%和7.5%.结论:空气巾含有大曲中所有的酵母菌的种类,曲房空气中的酵母菌数量和种类均较多.     

Automated mass spectrometric analysis of urinary free catecholamines using on-line solid phase extraction

Analysis of catecholamines (epinephrine, norepinephrine and dopamine) in plasma and urine is used for diagnosis and treatment of catecholamine-producing tumors. Current analytical techniques for catecholamine quantification are laborious, time-consuming and technically demanding. Our aim was to develop an automated on-line solid phase extraction method coupled to high performance liquid chromatography–tandem mass spectrometry (XLC–MS/MS) for the quantification of free catecholamines in urine. Five microlitre urine equivalent was pre-purified by automated on-line solid phase extraction, using phenylboronic acid complexation. Reversed phase (pentafluorophenylpropyl column) chromatography was applied. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Urinary reference intervals were set in 24-h urine collections of 120 healthy subjects. XLC–MS/MS was compared with liquid chromatography with electrochemical detection (HPLC–ECD). Total run-time was 14 min. Intra- and inter-assay analytical variations were <10%. Linearity was excellent (R² > 0.99). Quantification limits were 1.47 nmol/L, 15.8 nmol/L and 11.7 nmol/L for epinephrine, norepinephrine and dopamine, respectively. XLC–MS/MS correlated well with HPLC–ECD (correlation coefficient >0.98). Reference intervals were 1–10 μmol/mol, 10–50 μmol/mol and 60–225 μmol/mol creatinine for epinephrine, norepinephrine and dopamine, respectively. Advantages of the XLC–MS/MS catecholamine method include its high analytical performance by selective PBA affinity and high specificity and sensitivity by unique MS/MS fragmentation.     

The supramolecular organization of collagen VI microfibrils

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.     

Identification of aerobic mesophilic bacilli isolated from board and paper products containing recycled fibres

AIMS: To identify aerobic mesophilic bacteria isolated from coreboard, kitchen roll paper and food packaging boards containing recycled fibres and to create a rapid fingerprint-based database for their identification. METHODS AND RESULTS: A total of 197 isolates and 20 relevant type strains were characterized by automated ribotyping and as far as possible identified by the similarities of their riboprints to the relevant type strains. One strain from each unidentified ribotype, a total of 87 strains, was subjected to partial 16S rDNA sequencing and in most cases also to fatty acid analysis and physiological tests. From the isolates 113 and seven different ribotypes were generated belonging to the genera Bacillus and Paenibacillus, respectively. The dominating species, or closest related to them, were B. simplex (22.8% of isolates), B. licheniformis (18.3%) and B. amyloliquefaciens (12.7%); 5.1% of the isolates were identified as B. cereus, a potential food-borne pathogen. In particular, this species was present in one food packaging board (26.3% of isolates). Based on these results, 40.1% of the isolates and 45.0% of ribotypes were so different from the relevant type strains that they may represent novel species. CONCLUSIONS: All isolates were aerobic spore-formers, indicating that all non-spore-formers were eliminated during the drying stage of the processes. Although many isolates could be affiliated to described species of Bacillus or Paenibacillus, a significant proportion of the isolates could not be identified unambiguously as members of a described species. SIGNIFICANCE AND IMPACT OF THE STUDY: A RiboPrint identification database, composed of 120 composite patters, was established for bacteria originating from the pulp and paper industry. Considering the discrimination power of ribotyping, this database will be extremely useful in future for the reliable and rapid identification of bacteria isolated from pulp and paper industrial sources.     

Assessing genetic diversity in a sugarcane germplasm collection using an automated AFLP analysis

An assessment of genetic diversity within and between Saccharum, Old World Erianthus sect. Ripidium, and North American E.giganteus (S.giganteum) was conducted using Amplified Fragment Length Polymorphism (AFLP^TM) markers. An automated gel scoring system (GelCompar^TM) was successfully used to analyse the complex AFLP patterns obtained in sugarcane and its relatives. Similarity coefficient calculations and clustering revealed a genetic structure for Saccharum and Erianthus sect. Ripidium that was identical to the one previously obtained using other molecular marker types, showing the appropriateness of AFLP markers and the associated automated analysis in assessing genetic diversity in sugarcane. A genetic structure that correlated with cytotype (2n=30, 60, 90) was revealed within the North American species, E. giganteus (S.giganteum). Complex relationships among Saccharum, Erianthus sect. Ripidium, and North American E.giganteus were revealed and are discussed in the light of a similar study which involved RAPD markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

The use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation

Summary Combining stepper-motors for microscope stage movement with a specially designed software program has led to the establishment of an efficient cell tracking system. Cell immobilization, fixed reference points for calibration of target cell positions, and a video recording system complete the cell finder system. Specific cells can be identified (either beforehand or in retrospect), their locations fixed, and subsequent development of the individual cells monitored daily using computer-assisted relocation. In this way, the specific cell type capable of sustained division and regeneration has recently been identified within a low efficiency protoplast system of a recalcitrant species, sugarbeet. These totipotent cells originated from stomatal guard cells. Isolation and purification procedures were then optimized in a directed way to yield millions of guard cell protoplasts (GCPs). Using polyethylene glycol (PEG)-mediated gene transfer and glucuronidase (GUS) activity for transient expression studies proved that GCPs were amenable to transformation. Gene transfer efficiency was high, as was the number of stably transformed plants that can be produced. At present, the optimized procedure yields 600 transgenic individuals per person per year. This number allows for the selection of the best plants with regard to copy number, DNA insert size, gene expression, and field performance. Prospects for future application of the cell finder system will be discussed. Presented as part of the symposium “Early Events in Tissue Culture and Transformation at the Cellular Level” at the 1997 Congress on In Vitro Biology, Washington, DC 14–18 June 1997.     

西藏巨柏核型的图象自动分析与识别的研究

我们应用图象自动分析和识别的原理和方法,建立了植物染色体自动分析CHROMHUK软件系统,并首次对西藏巨柏进行了核型自动分析,抽取了染色体多个特征多数:相对长度、臂比、着丝点指数、相对体密度和次缢痕相对长度.对247个巨柏体细胞进行多参数数据统计和分析,并设置95°.置信判别区域和树分类判别.实现了染色体自动配对和分类,并由计算机直接输出染色体的组型图和核型模式图.分析结果表明:巨柏体细胞的染色体数目为2n=22,按Levan的分类标准,其核型公式为2n=4m(SC)+16m+2sm.据Stabbins分类为1A型.