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The current focus of many functional genomic studies is on the elucidation of gene regulatory networks. The functional analyses of transcription factors and their DNA-binding sites, in conjunction with genome-wide expression profiling, are crucial in understanding of gene regulatory networks. This paper describes an efficient and easy method for characterizing the DNA-binding sequence specificity of novel plant transcription factors. This new method is based on the fusion of a DNA-binding protein (DBP) to 6×His-tagged cellulase D (CELD), which serves both as a means for affinity purification of DBP-DNA complex in the selection of binding sites from a pool of biotinylated random-sequence oligonucleotides and as a reporter for measurement of DNA-binding activity. Thus, it eliminates the use of radioactivity and gel electrophoresis techniques currently used for purification of DBP-DNA complexes and assays of DNA-binding activity. The effectiveness of this method was demonstrated by the success of simultaneous selection of the binding sites of nine plant DBPs from four superfamilies (AP2, bHLH, NAC and MYB). The high-throughput capacity of CELD-based DNA-binding assays allows the quantitative analysis of the binding sequence specificity from a large number of DBP-selected oligonucleotides. The binding sequence specificity of three novel transcription factors (rice OsbHLH66, wheat TaNAC69 and TaMYB80), determined with this method, is presented. This new method provides the capacity of high-throughput analysis on the DNA-binding sequence specificity of a large number of putative transcription factors, predicted on the basis of conserved DNA-binding domains. [ABSTRACT FROM AUTHOR]

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterium and a highly infectious pathogen. The disease is a widespread zoonosis and is endemic throughout the world. An easy aerosol dissemination, environmental persistence, and high infectivity make the bacterium a serious threat for humans and animals. Lipopolysaccharide is considered one of the major factors of virulence expression and infection of the bacterium. Detailed glycomic studies enabled to better understand structural and functional peculiarities of this biopolymer and its role in pathogenesis and immunity of Q fever. Recent proteomic studies of C. burnetii have brought new approaches in accurate detection of the infectious agent and offered new insights into the inter- or intra-species relatedness. Thus, structure/function relationship studies are currently of utmost importance in the field. This paper will focus on glycomic and proteomic approaches providing information on unique glycan and protein species of the microorganism as the candidate molecules for the use in detection/diagnosis, therapy, and prophylaxis. [ABSTRACT FROM AUTHOR]

Proteomic tools—in particular, mass spectrometry (MS)—have advanced significantly in recent years, and the identification of proteins within complex mixtures is now a routine procedure. Quantitative methods of analysis are less well advanced and continue to develop. These include the use of stable isotope ratio approaches, isotopically labeled peptide standards, and nonlabeling methods. This paper summarizes the use of MS as a proteomics tool to identify and semiquantify proteins and their modified forms by using examples of relevance to the Maillard reaction. Finally, some challenges for the future are presented. [ABSTRACT FROM AUTHOR]

The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell- free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target. [ABSTRACT FROM AUTHOR]

It was found that the DEAE-cellulose-treated UDP-Glc:protein transglucosylase I catalyzing the first step (reaction 1) in the formation of α-glucan bound to protein in potato tuber is not only specific for the glucosyl donor but also for the endogenous acceptor. A single radioactive 38-kDa macromolecular component appeared during denaturing polyacrytamide get electrophoresis of reaction 1 product. The labeled componem is probably the polypeptide subunit of the endogenous acceptor which is being glucosytated. The radioactivity incorporated in reaction 1 product was isolated from a protease digest as a low-molecular-mass glucopeptide fraction. A β-elimination reaction carried out in the presence of a reducing agent demonstrated that only one glucosyl moiety is transferred from UDP-Glc to the ammoacyl residue, thus forming an O-glucosidic linkage. ³H-labeled sodium borohydride showed that serine and threonine are involved in the peptide bond to glucose. Ion-exchange chromatography on DEAE-cellulose, affinity chromatography on concanavalin-A-Sepharose, gel filtration on Sephacryl S-300 and sucrose density gradient centrifugation failed to separate the enzyme catalyzing reaction 1 from the endogenous acceptor. [ABSTRACT FROM AUTHOR]

Proteomic analyses of the excretory/secretory proteins from the RH strain of Toxoplasma gondii have been performed to understand their functions in the host-parasite interaction. A total of 34 proteins were identified from LC/MS/MS analysis and their abundance was estimated by spectral counting methods. Among them, 8 species of micronemal proteins (MICs), 2 species of rhoptry proteins (ROPs), and 6 species of dense granular proteins (GRAs) were confirmed. Besides these, 18 species of protein were newly identified, and their cellular functions were estimated from sequence analysis. The three most abundant of the 34 identified extractor/secretory proteins GRA1, GRA7 and GRA2 were confirmed to be highly expressed in T. gondii using the spectral count method. This phenomenon is another demonstration of the importance of GRA proteins for the penetration and survival of T. gondii. [ABSTRACT FROM AUTHOR]

The article examines the isolation and characterization of a radiation-resistant bacterium from a hot spring in Iran. During which the water samples were irradiated by 22 KGy radiations, the researchers observed that only one of the 30 pure colonies survived. The survived bacterium, with an optimum growth of 30 to 37 degree Celsius, showed promising characteristics. Radio-resistancy were then compared with E. coli and B. megaterium which were inactivated in 2 KGy and 5.9 KGy. Its proteomic map was also examined to reveal the mechanisms of the extreme radio-resistancy and DNA repair system.

Students of biochemistry and related biosciences are urged to solve problems where kinetic parameters are calculated from initial rates obtained at different substrate concentrations. Troubles begin when they go to the laboratory to perform kinetic experiments and realize that usual laboratory instruments do not measure initial rates but only substrate or product concentrations as a function of reaction time. To overcome this problem we present a methodology which uses the integrated form of Michaelis-Menten equation. The method presented has a theoretical and pedagogic basis which is not as arbitrary as other approaches. Here we present and describe the methodology for analyzing time course data together with some examples of the essential computer procedures to implement these analyses. To simplify the understanding of this methodology the experimental examples are confined to linear inhibitions and experimental points utilized are the same from which the initial rates are determined. [ABSTRACT FROM AUTHOR]

New technologies enable acquisition of large data-sets containing genomic, proteomic and metabolic information that describe the state of a cell. These data-sets call for systematic methods enabling relevant information about the inner workings of the cell to be extracted. One important issue at hand is the understanding of the functional interactions between genes, proteins and metabolites. We here present a method for identifying the dynamic interactions between biochemical components within the cell, in the vicinity of a steady-state. Key features of the proposed method are that it can deal with data obtained under perturbations of any system parameter, not only concentrations of specific components, and that the direct effect of the perturbations does not need to be known. This is important as concentration perturbations are often difficult to perform in biochemical systems and the specific effects of general type perturbations are usually highly uncertain, or unknown. The basis of the method is a linear least-squares estimation, using time-series measurements of concentrations and expression profiles, in which system states and parameter perturbations are estimated simultaneously. An important side-effect of also employing estimation of the parameter perturbations is that knowledge of the system's steady-state concentrations, or activities, is not required and that deviations from steady-state prior to the perturbation can be dealt with. Time derivatives are computed using a zero-order hold discretization, shown to yield significant improvements over the widely used Euler approximation. We also show how network interactions with dynamics that are too fast to be captured within the available sampling time can be determined and excluded from the network identification. Known and unknown moiety conservation relationships can be processed in the same manner. The method requires that the number of samples equals at least the number of network components and, hence, is at present restricted to relatively small-scale networks. We demonstrate herein the performance of the method on two small-scalein silicogenetic networks. [ABSTRACT FROM AUTHOR]

During the second half of the 20th century, biochemistry and subsequently molecular biology blossomed into the core upon which all biological and biomedical sciences now depend. A major part of these closely related disciplines has been the study of the structure and function of proteins and the diverse biological functions that they perform. Early experimentation necessarily focused on individual entities, selected mainly for their activities, but as technology improved there developed a tendency to look at proteins as larger, interactive groups or clusters. Spurred by the recent exponential production of genomic sequence data for a rapidly increasing number of species, protein chemistry has now evolved into a new discipline, proteomics. In addition to embracing the methods and approaches that have served protein scientists well in the past, it includes, and is perhaps best defined by, high-throughput analyses based in large part on 2D gel electrophoresis, MALDI and ESI mass spectrometry and combinatorial arrays. Proteomic targets include the identification of all genome products and a mapping of their interactions and expression profiles. These hold great promise for the identification of disease markers and drug targets, but are not without their challenges and pitfalls. IUBMB Life, 57: 267–272, 2005 [ABSTRACT FROM AUTHOR]

We initiated a proteomics-based approach to identify root proteins affected by salinity in pea (Pisum sativum cv. Cutlass). Salinity stress was imposed either on 2-wk old pea plants by watering with salt water over 6 wk or by germinating and growing pea seeds for 7 days in Petri dishes. Concentrations of NaC1 above 75 mM had significant negative effects on growth and development of peas in both systems. Salinity-induced root proteome-level changes in pea were investigated by 2-D electrophoresis of proteins from control, 75 and 150 mM NaCl-treated plants and seedlings. The majority of the protein spots visualised showed reproducible abundance in root protein extracts from whole plants and seedlings. Of these proteins, 35 spots that exhibited significant changes in abundance due to NaC1 treatment were selected for identification using ESI-Q-TOF MS/MS. The identities of these proteins, which include pathogenesis-related (PR) 10 proteins, antioxidant enzymes such as superoxide dismutase (SOD) as well as nucleoside diphosphate kinase (NDPK) are presented, and the roles of some of them in mediating responses of pea to salinity are discussed. This is the first report of salinity-induced changes in the root proteome of pea that suggests a potential role for PR10 proteins in salinity stress responses. Our findings also suggest the possible existence of a novel signal transduction pathway involving SOD, H2O2, NDPK and PR10 proteins with a potentially crucial role in abiotic stress responses. [ABSTRACT FROM AUTHOR]

Understanding the basic principles of structural biology is becoming a major subject of study in most undergraduate level programs in biology. In the genomic and proteomic age, it is becoming indispensable for biology students to master concepts related to the sequence and structure of proteins in order to develop skills that may be useful in a wide range of applications. Within this context, this article shows a scheme for teaching structural biology based on hands-on computer approaches, including computational genomics, and structural bioinformatics tools. The students learn to use most of the leading computer programs available for the complete path that goes from sequence to structure and eventually function. During the course, they are taught to build models of proteins based on sequence and structure information. The students are also provided with a critical point of view on automatic procedures and learn to discern between likely and unlikely structures of their final models. [ABSTRACT FROM AUTHOR]

We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins. [ABSTRACT FROM AUTHOR]

In an effort to explain the mechanism of inhibition of human lymphocyte transformation by human alpha-foetoprotein (HAFP), we sought for, and failed to find, evidence of physical association between HAFP and phytomitogens or antihuman thymocyte antiserum. In addition, 20–40 fold increases in mitogen dose do not reverse the inhibition of lymphocyte transformation by a constant dose of HAFP. The presence of HAFP does not interfere with the attachment of 125I-labelled phytohaemagglutinin to the lymphocyte surface. When analyzed by 2-dimensional crossed immunoelectrophoresis, HAFP isolated from the body fluids of hepatoma patients displays electrophoretic heterogeneity, and demonstrates three charged species of HAFP designated as HAFP-1 (the most cathodal), HAFP-2, and HAFP-3 (the most anodal). The potency of hepatoma-HAFP isolates in inhibiting lymphocyte transformation can be positively correlated with the ratio HAFP-3:HAFP-1 in each preparation. Passage of hepatoma HAFP isolates over CM-cellulose allows the isolation of two distinct HAFP fractions, one at pH 4.95, and the other at pH 5.24. The pH 4.95 CM-cellulose isolate is enriched in the more electronegative HAFP species (HAFP-3) and is, on the average, two times more potent than the pH 5.24 CM-cellulose HAFP isolate. The latter, by comparison with native HAFP, is enriched in the more electropositive HAFP species (HAFP-1). The structural basis for the charge differences which govern the biological potency of HAFP as a modulator of lymphocyte responses is unknown, but it is independent of HAFP sialic acid content. Inhibition of lymphocyte transformation by HAFP cannot be explained by simple competition between the lymphocyte membrane and HAFP for the mitogen combining sites. [ABSTRACT FROM AUTHOR]