This chapter reviews the current status of research on investigations of the free radical chemistry of green tea and its constituent polyphenols (GTP). It is based on the use of electron paramagnetic resonance (EPR) spectroscopy, and also includes a section on practical aspects of the technique, which should be of value to readers who are unfamiliar with the detailed operation of EPR. The free radical chemistry of GTP is important, because many of their antioxidant functions involve reactions with O(2)-derived free radicals, and the products of such reactions are themselves generally free radicals. The stability of these products and their abilities to participate in subsequent reactions may have considerable bearing on their biological function. These are also discussed briefly along with the authors' views of future investigations which would appear to be valuable for this topic.
Recent genetic discoveries and related developments in genomic techniques have led to the commercialization of novel diagnostic platforms for studying disease or gauging therapeutic outcomes in individual patients. This newly emerging field is called "personalized medicine," and uses the patient's genetic composition to tailor strategies for patient-specific disease detection, treatment, or prevention. Personalized diagnostic tests are used to detect patient-to-patient variations in gene or protein expression levels, which act as indicators for drug treatments or disease prognosis. In turn, medical professionals can better answer questions such as: "Who should be treated with which drug?" and "How should the treatment be administered?" The regulations governing personalized medicine can be complicated because they encompass in vitro diagnostic systems and laboratory tests as well as methods of disease treatment and patient care. Industry, academia, medicine, and the Food and Drug Administration (FDA) are all involved in the cultivation of the field: substantial collaborations between drug developers and regulatory authorities are required to consider and shape emerging regulations as personalized drug strategies mature. Some of the regulatory issues identified by industry and the FDA about personalized medicine and personalized diagnostics will be addressed. In addition, relevant collaborations, advances, and current and draft regulatory guidances will be discussed with respect to the future of personalized medicine.
Network motifs and modelling paradigms are attracting increasing attention as modelling tools in drug design and development, and in regenerative medicine. There is a gradual but inexorable convergence between these hitherto disparate disciplines. This review summarizes some very recent work in these areas, leading to an understanding of the complementary roles networks play and factors driving this convergence:network paradigms can be excellent ways of modelling and understanding drug molecules and their action,an understanding of the robustness and vulnerabilities of biological targets may improve the efficacy of drug design and discovery,drug design has an increasingly large role to play in directing stem cell properties,stem cell regulatory networks can be modelled in useful ways using network models at a suitable level of scale, andthe network tools of drug design are also very useful for the design of biomaterials used in regenerative medicine.
Carotenoids are a group of pigments that are essential to human diets. An increasing interest in carotenoids as a nutritional source of vitamin A and health-promoting compounds has prompted the recent progress in metabolic engineering of carotenogenesis in food crops. Current strategies have been mainly focused on manipulating genes encoding carotenogeic enzymes. In many cases, it is difficult to reach the desired levels of carotenoid enhancement. In this chapter, we briefly summarize the recent progress on our understanding of carotenoid biosynthesis. We describe the isolation of a novel gene, the Or gene, from a high-beta-carotene orange cauliflower mutant. The Or gene encodes a plastid-targeted protein containing a cysteine-rich zinc finger domain and appears to be plant-specific. The insertion of a copia-like LTR retrotransponson in the Or gene confers high levels of carotenoid accumulation in the normally low-pigmented tissues. Rather than directly regulating carotenoid biosynthesis, the Or gene controls carotenoid accumulation by inducing the formation of chromoplasts, which provide a metabolic sink to sequester and deposit carotenoids. Examination of the Or transgenic potato tubers confirms that the Or-induced carotenoid accumulation is associated with the formation of a metabolic sink. Thus, the Or gene offers a new molecular tool to complement current approaches for nutritional enhancement in agriculturally important crops.
The increasing use of gene expression microarrays, and depositing of the resulting data into public repositories, means that more investigators are interested in using the technology either directly or through meta analysis of the publicly available data. The tools available for data analysis have generally been developed for use by experts in the field, making them difficult to use by the general research community. For those interested in entering the field, especially those without a background in statistics, it is difficult to understand why experimental results can be so variable. The purpose of this review is to go through the workflow of a typical microarray experiment, to show that decisions made at each step, from choice of platform through statistical analysis methods to biological interpretation, are all sources of this variability.
Papaya with resistance to papaya ringspot virus (PRSV) is the first genetically modified tree and fruit crop and also the first transgenic crop developed by a public institution that has been commercialized. This chapter reviews the different transformation systems used for papaya and recent advances in the use of transgenic technology to introduce important quality and horticultural traits in papaya. These include the development of the following traits in papaya: resistance to PRSV, mites and Phytophthora, delayed ripening trait or long shelf life by inhibiting ethylene production or reducing loss of firmness, and tolerance or resistance to herbicide and aluminum toxicity. The use of papaya to produce vaccine against tuberculosis and cysticercosis, an infectious animal disease, has also been explored. Because of the economic importance of papaya, there are several collaborative and independent efforts to develop PRSV transgenic papaya technology in 14 countries. This chapter further reviews the strategies and constraints in the adoption of the technology and biosafety to the environment and food safety. Constraints to adoption include public perception, strict and expensive regulatory procedures and intellectual property issues.
Herpesviruses are important human pathogens that can cause mild to severe lifelong infections with high morbidity in susceptible adults. Moreover, Herpes simplex virus (HSV) type 2, for example, has been reported to be responsible for increased transmission and disease progression of human immunodeficiency virus (HIV). Therefore, the discovery of novel anti-HSV drugs deserves great efforts. Herbal medicinal products have been used as source of putative candidate drugs in many diseases. However, in case of viral diseases the development of antivirals from natural source is less explored probably because within the virus there are few specific targets where the small molecules can interact to inhibit or kill the virus. The currently available antiherpes drugs are nucleoside analogs that did not cure the lifelong or recurrent infections and the use of these drugs often lead to the development of viral resistance coupled with the problem of side effects, recurrence and viral latency. However a wide array of herbal products, used by diverse medicinal systems throughout the world, showed high level of antiherpesvirus activities and many of them have complementary and overlapping mechanism of action, either by inhibiting viral replication, or viral genome synthesis. This chapter will summarize some of the promising herbal extracts and purified compounds isolated from the herbal sources by several laboratories. Cases with proven in vitro and documented in vivo activities, along with their structure-activity relationship against herpesviruses are discussed.
Critical review of published literature may be necessary during several stages of biotechnology product development. The reviewer should develop a standardized method for reviewing and comparing published papers on a given topic and should be aware of common errors found in published papers.
In 1901, a unique bacterium was isolated as a pathogen of the sotto disease of the silkmoth larvae, and later in 1915, the organism was described as Bacillus thuringiensis. Since the discovery, this bacterium has widely attracted attention of not only insect pathologists but many other scientists who are interested in strong and specific insecticidal activity associated with inclusion bodies of B. thuringiensis. This has led to the recent worldwide development of B. thuringiensis-based microbial insecticides and insect-resistant transgenic plants, as well as the epoch-making discovery of parasporin, a cancer cell-specific cytotoxin. In the review, we introduce a detection study of interaction between inclusion proteins of B. thuringiensis and brush border membrane of insects using surface plasmon resonance-based biosensor, and then identification and cloning of parasporin-4, a latest cancer cell-killing protein produced by B. thuringiensis A1470 strain. Inclusion bodies of the parasporin-4 produced by recombinant Escherichia coli were solubilized and activated with a new method and purified by an anion-exchange chromatography. At last the characterization of the recombinant parasporin-4 was shown.
Three-dimensional structure of a protein molecule is primarily determined by its amino acid sequence, and thus the elucidation of general rules embedded in amino acid sequences is of great importance in protein science and engineering. To extract valuable information from sequences, we propose an analytical method in which a protein sequence is considered to be constructed by serial superimpositions of short amino acid sequences of n amino acid sets, especially triplets (3-aa sets). Using the comprehensive nonredundant protein database, we first examined "availability'' of all possible combinatorial sets of 8,000 triplet species. Availability score was mathematically defined as an indicator for the relative 'preference'' or "avoidance'' for a given short constituent sequence to be used in protein chain. Availability scores of real proteins were clearly biased against those of randomly generated proteins. We found many triplet species that occurred in the database more than expected or less than expected. Such bias was extended to longer sets, and we found that some species of pentats (5-aa sets) that occurred reasonably frequently in the randomly generated protein population did not occur at all in any real proteins known today. Availability score was dependent on species, potentially serving as a phylogenetic indicator. Furthermore, we suggest possibilities of various biotechnological applications of characteristic short sequences such as human-specific and pathogen-specific short sequences obtained from availability analysis. Availability score was also dependent on secondary structures, potentially serving as a structural indicator. Availability analysis on triplets may be combined with a comprehensive data collection on the phi and psi peptide-bond angles of the amino acid at the center of each triplet, i.e., a collection of Ramachandran plots for each triplet. These triplet characters, together with other physicochemical data, will provide us with basic information between protein sequence and structure, by which structure prediction and engineering may be greatly facilitated. Availability analysis may also be useful in identifying word processing units in amino acid sequences based on an analogy to natural languages. Together with other approaches, availability analysis will elucidate general rules hidden in the primary sequences and eventually contributes to rebuilding the paradigm of protein science.
Protein arginine methylation is a rapidly growing field of biomedical research that holds great promise for extending our understanding of developmental and pathological processes. Less than ten years ago, fewer than two dozen proteins were verified to contain methylarginine. Currently, however, hundreds of methylarginine proteins have been detected and many have been confirmed by mass spectrometry and other proteomic and molecular techniques. Several of these proteins are products of disease genes or are implicated in disease processes by recent experimental or clinical observations. The purpose of this chapter is twofold; (1) to re-examine the role of protein arginine methylation placed within the context of cell growth and differentiation, as well as within the rich variety of cellular metabolic methylation pathways and (2) to review the implications of recent advances in protein methylarginine detection and the analysis of protein methylarginine function for our understanding of human disease.
Proteins very rarely act in isolation. Biomolecular interactions are central to all biological functions. In human, for example, interference with biomolecular networks often lead to disease. Protein-protein and protein-metabolite interactions have traditionally been studied one by one. Recently, significant progresses have been made in adapting suitable tools for the global analysis of biomolecular interactions. Here we review this suite of powerful technologies that enable an exponentially growing number of large-scale interaction datasets. These new technologies have already contributed to a more comprehensive cartography of several pathways relevant to human pathologies, offering a broader choice for therapeutic targets. Genome-wide scale analyses in model organisms reveal general organizational principles of eukaryotic proteomes. We also review the biochemical approaches that have been used in the past on a smaller scale for the quantification of the binding constant and the thermodynamics parameters governing biomolecular interaction. The adaptation of these technologies to the large-scale measurement of biomolecular interactions in (semi-)quantitative terms represents an important challenge.
The volume and complexity of genomic sequence data, and the additional experimental data required for annotation of the genomic context, pose a major challenge for display and access for biomedical researchers. Genome browsers organize this data and make it available in various ways to extract useful information to advance research projects. The UCSC Genome Browser is one of these resources. The official sequence data for a given species forms the framework to display many other types of data such as expression, variation, cross-species comparisons, and more. Visual representations of the data are available for exploration. Data can be queried with sequences. Complex database queries are also easily achieved with the Table Browser interface. Associated tools permit additional query types or access to additional data sources such as images of in situ localizations. Support for solving researcher's issues is provided with active discussion mailing lists and by providing updated training materials. The UCSC Genome Browser provides a source of deep support for a wide range of biomedical molecular research (http://genome.ucsc.edu).
Therapeutic proteins in general induce an immune response, especially when administered as multiple doses over prolonged periods. Non-human therapeutic proteins such as asparaginase and streptokinase induce antibodies by the classical immune reaction and their primary immunogenic factor is the degree of non-self. Human therapeutic proteins such as the interferons and GM-CSF breakdown immune tolerance and protein aggregation is their main factor inducing antibodies. Many other factors influence the level of immunogenicity of proteins, such as storage conditions,contaminants or impurities in the preparation, downstream processing, dose and length of treatment, as well as route of administration, appropriate formulation and disease status and concomitant treatment of patients. Clinical manifestations of antibodies directed against the protein include loss of efficacy, cross neutralization of endogenous proteins and general immune system effects, such as anaphylaxis or serum sickness.
Conventional cell-based assays for seven-transmembrane receptors, also known as G protein-coupled receptors, rely on the coupling of the ligand-bound receptor to heterotrimeric G proteins. New assay methods have become available that are not based on G protein activation, but that apply the molecular mechanism underlying the attenuation of G protein signaling mediated by beta-arrestin. beta-arrestin is a cytoplasmic protein that targets receptors to clathrin-coated endocytotic vesicles for degradation or recycling. This process has been visualized and quantified in high-content imaging assays using receptor- or beta-arrestin-chimeras with green fluorescent protein. Other assay methods use bioluminescence resonance energy transfer, enzyme fragment complementation, or a protease-activated transcriptional reporter gene, to measure receptor-beta-arrestin proximity. beta-arrestin recruitment assays have been applied successfully for receptors coupling to Galpha(q), Galpha(s) and Galpha(i) proteins, thus providing a generic assay platform for drug discovery on G protein-coupled receptors. The best understood signal transduction pathway elicited by the seven-transmembrane Frizzled receptors does not involve G proteins. The activation of Frizzleds by their cognate ligands of the Wnt family recruits the phosphoprotein dishevelled. Dishevelled regulates a protein complex involved in the destruction of beta-catenin. Activation of Frizzled blocks degradation of beta-catenin, which translocates to the nucleus to activate transcription of Wnt-responsive genes. The cytoplasm-to-nuclear translocation of beta-catenin forms the basis of several high-content assays to measure Wnt/Frizzled signal transduction. Interestingly, Frizzled receptors have recently been shown to internalize and to recruit beta-arrestin. This suggests that beta-arrestin recruitment assays may be applied for drug discovery on seven-transmembrane receptors beyond G protein-coupled receptors.
The practice of cell culture has been virtually unchanged for 100 years. Until recently, life scientists have had to content themselves with two-dimensional cell culture technology. Clearly, living creatures are not constructed in two dimensions and thus it has become widely recognized that in vitro culture systems must become three dimensional to correctly model in vivo biology. Attempts to modify conventional 2-D culture technology to accommodate 3-D cell growth such as embedding cells in extracellular matrix have demonstrated the superiority of concept. Nevertheless, there are serious drawbacks to this approach including limited mass transport and lack of scalability. Recently, a new cell culture technology developed at NASA to study the effects of microgravity on cells has emerged to solve many of the problems of 3-D cell culture. The technology, the Rotating Wall Vessel (RWV) is a single axis clinostat consisting of a fluid-filled, cylindrical, horizontally rotating culture vessel. Cells placed in this environment are suspended by the resolution of the gravitational, centrifugal and Coriolis forces with extremely low mechanical shear. These conditions, which have been called "low shear modeled microgravity", enable cells to assemble into tissue-like aggregates with high mass transport of nutrients, oxygen and wastes. Examples of the use of the RWV for basic cell biology research and tissue engineering applications are discussed.
The concept of curing diseases at the genetic level was already introduced in the 1970s, but only the evolution of molecular biology and tools for genetic manipulation brought the idea into labs and clinics during the last 16 years. Viral and non-viral vectors and delivery systems were developed to transfer therapeutic genes into the target cells. In the case of non-viral approaches plasmid DNA has become a very promising gene delivery vector because it can easily be genetically manipulated and produced by cultivation of plasmid harbouring Escherichia coli and subsequent downstream processing, thus making production easy in comparison to other gene delivery vectors. Another advantage in using plasmid DNA is the low risk of immunogenic reactions and oncogen activation that can arise while using viral vectors. This review describes the recent development in plasmid manufacturing ranging from bacterial cultivation in batch and fedbatch mode to produce plasmid-bearing E. coli over cell lysis and subsequent purification to storage, application, and process and quality control.
Vaccination is one of the most efficient ways to eradicate some infectious diseases in humans and animals. The material traditionally used as vaccines is attenuated or inactivated pathogens. This approach is sometimes limited by the fact that the material for vaccination is not efficient, not available, or generating deleterious side effects. A possible theoretical alternative is the use of recombinant proteins from the pathogens. This implies that the proteins having the capacity to vaccinate have been identified and that they can be produced in sufficient quantity at a low cost. Genetically modified organisms harboring pathogen genes can fulfil these conditions. Microorganisms, animal cells as well as transgenic plants and animals can be the source of recombinant vaccines. Each of these systems that are all getting improved has advantages and limits. Adjuvants must generally be added to the recombinant proteins to enhance their vaccinating capacity. This implies that the proteins used to vaccinate have been purified to avoid any immunization against the contaminants. The efficiency of a recombinant vaccine is poorly predictable. Multiple proteins and various modes of administration must therefore be empirically evaluated on a case-by-case basis. The structure of the recombinant proteins, the composition of the adjuvants and the mode of administration of the vaccines have a strong and not fully predictable impact on the immune response as well as the protection level against pathogens. Recombinant proteins can theoretically also be used as carriers for epitopes from other pathogens. The increasing knowledge of pathogen genomes and the availability of efficient systems to prepare large amounts of recombinant proteins greatly facilitate the potential use of recombinant proteins as vaccines. The present review is a critical analysis of the state of the art in this field.
Ganoderma lucidum is a wood-degrading basidiomycete with numerous pharmacological effects. Since the mushroom is very rare in nature, artificial cultivation of fruiting bodies has been known on wood logs and on sawdust in plastic bags or bottles. Biotechnological cultivation of G. lucidum mycelia in bioreactors has also been established, both on solid substrates and in liquid media by submerged cultivation of fungal biomass. The most important pharmacologically active constituents of G. lucidum are triterpenoids and polysaccharides. Triterpenoids have been reported to possess hepatoprotective, anti-hypertensive, hypocholesterolemic and anti-histaminic effects, anti-tumor and anti-engiogenic activity, effects on platelet aggregation and complement inhibition. Polysaccharides, especially beta-d-glucans, have been known to possess anti-tumor effects through immunomodulation and anti-angiogenesis. In addition, polysaccharides have a protective effect against free radicals and reduce cell damage caused by mutagens.
