Showing papers in "Advances in Biochemical Engineering \/ Biotechnology in 2008"

Probiotics, Prebiotics, and Synbiotics

Abstract: According to the German definition, probiotics are defined viable microorganisms, sufficient amounts of which reach the intestine in an active state and thus exert positive health effects. Numerous probiotic microorganisms (e.g. Lactobacillus rhamnosus GG, L. reuteri, bifidobacteria and certain strains of L. casei or the L. acidophilus-group) are used in probiotic food, particularly fermented milk products, or have been investigated—as well as Escherichia coli strain Nissle 1917, certain enterococci (Enterococcus faecium SF68) and the probiotic yeast Saccharomyces boulardii—with regard to their medicinal use. Among the numerous purported health benefits attributed to probiotic bacteria, the (transient) modulation of the intestinal microflora of the host and the capacity to interact with the immune system directly or mediated by the autochthonous microflora, are basic mechanisms. They are supported by an increasing number of in vitro and in vivo experiments using conventional and molecular biologic methods. In addition to these, a limited number of randomized, well-controlled human intervention trials have been reported.

Production of Secondary Metabolites Using Plant Cell Cultures

Abstract: Plant cell cultures represent a potential source of valuable secondary metabolites which can be used as food additives, nutraceuticals, and pharmaceuticals The synthesis of phytochemicals by the cell cultures in contrast to these in plants is independent of environmental conditions and quality fluctuations In many cases, the chemical synthesis of metabolites is not possible or economically feasible Moreover, the natural food additives are better accepted by consumers in contrast to those which are artificially produced

Computational methods for predicting protein-protein interactions

Abstract: Protein-protein interactions (PPIs) play a critical role in many cellular functions. A number of experimental techniques have been applied to discover PPIs; however, these techniques are expensive in terms of time, money, and expertise. There are also large discrepancies between the PPI data collected by the same or different techniques in the same organism. We therefore turn to computational techniques for the prediction of PPIs. Computational techniques have been applied to the collection, indexing, validation, analysis, and extrapolation of PPI data. This chapter will focus on computational prediction of PPI, reviewing a number of techniques including PIPE, developed in our own laboratory. For comparison, the conventional large-scale approaches to predict PPIs are also briefly discussed. The chapter concludes with a discussion of the limitations of both experimental and computational methods of determining PPIs.

Improvement of Saccharomyces yeast strains used in brewing, wine making and baking.

Abstract: Yeast was the first microorganism domesticated by mankind. Indeed, the production of bread and alcoholic beverages such as beer and wine dates from antiquity, even though the fact that the origin of alcoholic fermentation is a microorganism was not known until the nineteenth century. The use of starter cultures in yeast industries became a common practice after methods for the isolation of pure yeast strains were developed. Moreover, effort has been undertaken to improve these strains, first by classical genetic methods and later by genetic engineering. In general, yeast strain development has aimed at improving the velocity and efficiency of the respective production process and the quality of the final products. This review highlights the achievements in genetic engineering of Saccharomyces yeast strains applied in food and beverage industry.

Filamentous fungi for production of food additives and processing aids.

Abstract: Filamentous fungi are metabolically versatile organisms with a very wide distribution in nature. They exist in association with other species, e.g. as lichens or mycorrhiza, as pathogens of animals and plants or as free-living species. Many are regarded as nature's primary degraders because they secrete a wide variety of hydrolytic enzymes that degrade waste organic materials. Many species produce secondary metabolites such as polyketides or peptides and an increasing range of fungal species is exploited commercially as sources of enzymes and metabolites for food or pharmaceutical applications. The recent availability of fungal genome sequences has provided a major opportunity to explore and further exploit fungi as sources of enzymes and metabolites. In this review chapter we focus on the use of fungi in the production of food additives but take a largely pre-genomic, albeit a mainly molecular, view of the topic.

Advanced technologies for studies on protein interactomes.

Abstract: One of the key challenges of biology in the post-genomic era is to assign function to the many genesrevealed by large-scale sequencing programmes, since only a small fraction of gene function can bedirectly inferred from the coding sequence. Identifying interactions between proteins is a substantialpart in understanding their function. The main technologies for investigating protein–protein interactionsand assigning functions to proteins include direct detection intermolecular interactions through proteinmicroarray, yeast two-hybrid system, mass spectrometry fluorescent techniques to visualize protein complexesor pull-down assays, as well as technologies detecting functional interactions between genes, such as RNAiknock down or functional screening of cDNA libraries. Over recent years, considerable advances have beenmade in the above techniques. In this review, we discuss some recent developments and their impact on thegene function annotation.

Large-scale analysis of protein-protein interactions using cellulose-bound peptide arrays.

Abstract: Peptide arrays for screening large numbers of peptide fragments and probing with large numbers ofsamples is discussed.

Plant biotechnology: transgenic crops.

Abstract: Transgenesis is an important adjunct to classical plant breeding, in that it allows the targeted manipulation of specific characters using genes from a range of sources. The current status of crop transformation is reviewed, including methods of gene transfer, the selection of transformed plants and control of transgene expression. The application of genetic modification technology to specific traits is then discussed, including input traits relating to crop production (herbicide tolerance and resistance to insects, pathogens and abiotic stresses) and output traits relating to the composition and quality of the harvested organs. The latter include improving the nutritional quality for consumers as well as the improvement of functional properties for food processing.

Lab-on-a-chip in vitro compartmentalization technologies for protein studies.

Abstract: In vitro compartmentalization (IVC) is a powerful tool for studying protein–proteinreactions, due to its high capacity and the versatility of droplet technologies. IVC bridges thegap between chemistry and biology as it enables the incorporation of unnatural amino acids with modificationsinto biological systems, through protein transcription and translation reactions, in a cell-likemicrodrop environment. The quest for the ultimate chip for protein studies using IVC is the drivefor the development of various microfluidic droplet technologies to enable these unusual biochemicalreactions to occur. These techniques have been shown to generate precise microdrops with a controlledsize. Various chemical and physical phenomena have been utilized for on-chip manipulation to allowthe droplets to be generated, fused, and split. Coupled with detection techniques, droplets can besorted and selected. These capabilities allow directed protein evolution to be carried out on a microchip.With further technological development of the detection module, factors such as addressable storage,transport and interfacing technologies, could be integrated and thus provide platforms for proteinstudies with high efficiency and accuracy that conventional laboratories cannot achieve.

Identification of Protein–Protein Interactions by Mass Spectrometry Coupled Techniques

Abstract: The use of mass spectrometry in protein identification has revolutionized the field of proteomicsCoupled to various affinity purification techniques, mass spectrometry is used to identify protein–proteininteractions This chapter looks at the use of these affinity purification techniques in the identificationof protein interactions Various tags are used to purify protein complexes including tandem affinity purificationThe FLAG tag is another commonly used tag which is a small tag that tends not to interfere with theprotein function These different affinity purification methods are used to purify proteins that are furtheridentified by either ESI-MS or MALDI-MS

Food and agricultural biotechnology: a summary and analysis of ethical concerns.

Abstract: The range of social and ethical concerns that have been raised in connection with food and agricultural biotechnology is exceedingly broad. Many of these deal with risks and possible outcomes that are not unique to crops or animals developed using recombinant DNA. Food safety, animal welfare, socio-economic and environmental impacts, as well as shifts in power relations or access to technology raise concerns that might be generalized to many technologies. These aspects of the controversy over biotechnology are analyzed below as elements of general technological ethics, and key norms or values pertinent to each of these categories are specified in some detail. However, a number of special concerns unique to the use of rDNA in manipulating plant and animal genomes have been raised, and these are reviewed as well. The chapter concludes by reviewing two broad policy strategies for responding to the issues, one involving labels and consumer consent, the other applying the precautionary principle.

Stem cells and scaffolds for vascularizing engineered tissue constructs.

Abstract: The clinical impact of tissue engineering depends upon our ability to direct cells to form tissues with characteristic structural and mechanical properties from the molecular level up to organized tissue. Induction and creation of functional vascular networks has been one of the main goals of tissue engineering either in vitro, for the transplantation of prevascularized constructs, or in vivo, for cellular organization within the implantation site. In most cases, tissue engineering attempts to recapitulate certain aspects of normal development in order to stimulate cell differentiation and functional tissue assembly. The induction of tissue growth generally involves the use of biodegradable and bioactive materials designed, ideally, to provide a mechanical, physical, and biochemical template for tissue regeneration. Human embryonic stem cells (hESCs), derived from the inner cell mass of a developing blastocyst, are capable of differentiating into all cell types of the body. Specifically, hESCs have the capability to differentiate and form blood vessels de novo in a process called vasculogenesis. Human ESC-derived endothelial progenitor cells (EPCs) and endothelial cells have substantial potential for microvessel formation, in vitro and in vivo. Human adult EPCs are being isolated to understand the fundamental biology of how these cells are regulated as a population and to explore whether these cells can be differentiated and reimplanted as a cellular therapy in order to arrest or even reverse damaged vasculature. This chapter focuses on advances made toward the generation and engineering of functional vascular tissue, focusing on both the scaffolds – the synthetic and biopolymer materials – and the cell sources – hESCs and hEPCs.

Cardiac systems biology and parameter sensitivity analysis: intracellular Ca2+ regulatory mechanisms in mouse ventricular myocytes.

Abstract: Intracellular Ca2+ dynamics of cardiac myocytes are regulated by complexmechanisms of a variety of ion channels, transporters, and exchangers. Alterations of these Ca2+regulatory components might lead to development of cardiac diseases. To investigate the regulatory mechanismsand hidden Ca2+ dynamics we use integrative systems analysis. Herein, we brieflysummarize cardiac systems biology and, within the context of cardiac systems biology, identify the functionalrole of key Ca2+ regulatory proteins and their influence on intracellular Ca2+dynamics (i.e., Ca2+ transient, SR Ca2+ content, CICRgain, half-decay time) using parameter sensitivity analysis based on an experimentally validated mathematicalmodel of mouse ventricular myocytes. In addition, we analyze the influence of the pacing period (frequency)of a stimulus current since most of the Ca2+ regulatory proteins react withdifferent timescales. Throughout the parameter sensitivity analysis, we found that alteration of SERCAor LTCC has a more significant effect on the Ca2+ dynamics than that ofRyR or NCX. In particular, for the 70% down-regulation of LTCC, the Ca2+ influxthrough LTCC failed to initialize the SR Ca2+ release and thereby the intracellularCa2+ dynamics was dramatically changed. We also found that the pacing periodhas a significant effect on the half-decay time of the Ca2+ transients.These findings provide us with new insights into the pathophysiology of cardiac failure as well as the developmentof new therapeutic strategies.

Antibody microarrays as an experimental platform for the analysis of signal transduction networks.

Abstract: A significant bottleneck for the time-resolved and quantitative description of signaling networksis the limited sample capacity and sensitivity of existing methods. Recently, antibody microarrays haveemerged as a promising experimental platform for the quantitative and comprehensive determination of proteinabundance and protein phosphorylation. This review summarizes the development of microarray applicationsinvolving antibody-based capture of target proteins with a focus on quantitative applications. Technicalaspects regarding the production of antibody microarrays, identification of suitable detection and captureantibody pairs, signal detection methods, detection limit, and data analysis are discussed in detail.

Using aptamers to study protein-protein interactions

Abstract: The emerging science of systems biology focuses on the systematic study of complex interactions inwhole biological systems. A systemic, or integrative, methodology is employed as the chief means ofdiscovering new properties and understanding the aggregate of processes that occur in a biologicalsystem. Accordingly, the Human Genome Project has provided a complete map of genes and resultant proteinscorresponding to their function. Protein–protein interactions are important pieces of this biologicaltapestry, and understanding how they work cooperatively in a cell will result in a better understandingof the whole organism. To accomplish this objective, we report the use of DNA/RNA aptamers as a noveltool for the study and elucidation of protein–protein interactions, both in vivo and in vitro.

Investigating protein-protein interactions by far-Westerns.

Abstract: The identification of protein interaction partners can often elucidate the function of the protein under investigation based on the "guilty by association" concept. Furthermore, the binding event between two proteins can be used as a functional assay when no such assay is available. Despite the large number of advanced techniques that are currently available for studying protein-protein interactions, far-Westerns or blot overlays are still very commonly used in the average laboratory setting due to their powerfulness. This is due to the simplicity and clarity in the results that they produce. Here, the details and mechanics of far-Westerns are discussed to help the reader choose amongst the different variations that exist depending on the question being investigated and the materials available to them. Some examples involving unique questions are also discussed in order to educate the reader on the versatility of far-Westerns. Finally, a troubleshooting section provides the reader with an understanding of the common problems that can be encountered and how these problems can be circumvented.

Protein Interactions: Analysis Using Allele Libraries

Abstract: Interaction defective alleles (IDAs) are alleles that contain mutations affecting their ability tointeract with their wild type binding partners. The locations of the mutations may lead to the identificationof protein interaction domains and interaction interfaces. IDAs may also distinguish different bindinginterfaces of multidomain proteins that are part of large complexes, thus shedding light on large proteinstructures that have yet to be determined. IDAs may also be used in conjunction with RNAi to dissect proteininteraction networks. Here, the wild type allele is knocked down and replaced with an IDA that has lostthe ability to interact with a specific binding partner. As a result, interactions are disruptedrather than knocking out the entire gene. Thus, IDAs have the potential to be extremely valuable toolsin protein interaction network analysis. IDAs can be isolated by reverse two-hybrid analysis, which wasdemonstrated over a decade ago, but high background levels caused by truncated IDAs have preventedits widespread adoption. We recently described a novel method for full-length allele library generationthat eliminates this background and increases the efficiency of the reverse two-hybrid protocol (and IDAisolation) significantly. Here we discuss our strategy for allele library generation, the potential usesof IDAs as outlined above, and additional applications of allele libraries.