Use of recombinant fusion proteins for generation and rapid characterization of monoclonal antibodies. Application to the Kunitz domain of human beta amyloid precursor protein.

Production of peptides by recombinant DNA techniques is an efficient alternative to chemical synthesis of peptides. Proteins and peptides produced by recombinant DNA methods in E. coli are routinely used as antigens for the production of antibodies. However, most small peptides are rapidly degraded within the E. coli cell, and therefore, must initially be expressed as components of larger, more stable fusion proteins. The peptide of interest must be cleaved from the fusion protein, and purified prior to immunization to eliminate epitopes contributed by the fusion partner. We have now established methods for the production and characterization of monoclonal antibodies using partially purified, uncleaved fusion proteins. We have also described a method for efficient production and detection of the fusion protein, an EIA for rapid differential screening of hybridoma supernatants, and a strategy for epitope mapping of the antibodies. These methods have been applied to the production and characterization of monoclonal antibodies specific for a 75-amino-acid internal segment of the Alzheimer amyloid precursor protein, and should be applicable to a wide variety of other peptides and proteins.     

Chromatographic techniques for the determination of putative dietary anticancer compounds in biological fluids

Although a great number of papers demonstrate an association between high intake of fruits and vegetables and reduced risk of certain types of cancer, the epidemiological evidence is not conclusive. The identification and quantification of specific dietary anticancer compounds in plasma, urine and tissues is an important aspect of this research. We surveyed the recent literature for original papers which involved the use of separation techniques for the detection and quantification in biological fluids and tissues of putative anticancer compounds which are present in the diet. The compounds included in this review are flavonoids, phytoestrogens, carotenoids, retinoids, vitamin E and ascorbic acid. The review covers papers published in the last 3 years. For each class of compounds we discuss the sample preparation, chromatographic conditions, and validation of the methods used, in order to identify current trends in the bioanalysis of each class of these substances.     

Cyclophosphamide and related anticancer drugs

This article presents an overview of the methods of bioanalysis of oxazaphosphorines, in particular, cyclophosphamide, ifosfamide, and trofosfamide as well as their metabolites. The metabolism of oxazaphosphorines is complex and leads to a large variety of metabolites and therefore the spectrum of methods used is relatively broad. The various methods used are shown in a table and the particularly important assays are described.     

Stimuli-sensitive hydrogels: ideal carriers for chronobiology and chronotherapy

The development of solid-phase peptide synthesis in the early 1960s and recombinant DNA technology in the early 1970s boosted the scientific interest of utilizing proteins and peptides as potential therapeutic agents to battle poorly controlled diseases. While there has been rapid progress in the development and synthesis of new proteins and peptides as potential therapeutic agents, the formulation and development of the associated delivery systems is lacking. The development of delivery systems is equally important due to the problems of stability, low bioavailability and short half-life of proteins and peptides. The main problem in this field is that low stability leads to low bioavailability. In this review we draw attention to chrono-pharmacological drug-delivery systems, which can be used to match the delivery of therapeutic agents with the biological rhythm. They are very important especially in endocrinology and in vaccine therapy. We show that the treatment of hypopituitary dwarfism by administration of human growth-hormone-releasing hormone (GHRH) is more effective when GHRH is administered in a pulsatile manner that exhibits a period characteristic of the patient's circadian rhythm. Here we examine how to design novel chrono-pharmacological drug-delivery systems that should be able to release the therapeutic agents at predetermined intervals.     

Stimuli-sensitive hydrogels: ideal carriers for chronobiology and chronotherapy

The development of solid-phase peptide synthesis in the early 1960s and recombinant DNA technology in the early 1970s boosted the scientific interest of utilizing proteins and peptides as potential therapeutic agents to battle poorly controlled diseases. While there has been rapid progress in the development and synthesis of new proteins and peptides as potential therapeutic agents, the formulation and development of the associated delivery systems is lacking. The development of delivery systems is equally important due to the problems of stability, low bioavailability and short half-life of proteins and peptides. The main problem in this field is that low stability leads to low bioavailability. In this review we draw attention to chrono-pharmacological drug-delivery systems, which can be used to match the delivery of therapeutic agents with the biological rhythm. They are very important especially in endocrinology and in vaccine therapy. We show that the treatment of hypopituitary dwarfism by administration of human growth-hormone-releasing hormone (GHRH) is more effective when GHRH is administered in a pulsatile manner that exhibits a period characteristic of the patient's circadian rhythm. Here we examine how to design novel chrono-pharmacological drug-delivery systems that should be able to release the therapeutic agents at predetermined intervals.     

Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen

The Gram-negative pathogen Pseudomonas aeruginosa encodes multiple protein export systems, the substrates of which contain export signals such as N-terminal signal peptides. Here we report the first genome-wide computational and laboratory screen for N-terminal signal peptides in this important opportunistic pathogen. The computational identification of signal peptides was based on a consensus between multiple predictive tools and showed that 38% of the P. aeruginosa PAO1 proteome was predicted to encode exported proteins, most of which utilize cleavable type I signal peptides or uncleavable transmembrane helices. In addition, known and novel lipoproteins (type II), twin arginine transporter (TAT), and prepilin peptidase substrates (type IV) were also identified. A laboratory-based screen using the alkaline phosphatase (PhoA) fusion method was then used to test our predictions. In total, 310 nonredundant PhoA fusions were successfully identified, 296 of which possess a predicted export signal. Analysis of the PhoA fusion proteins lacking an export signal revealed that three proteins have alternate translation start sites that encode signal peptides, two proteins may use an unknown export signal, and the remaining nine proteins are likely cytoplasmic proteins and represent false positives associated with the PhoA screen. Our approach to identify exported proteins illustrates how computational and laboratory-based methods are complementary, where computational analyses provide a large number of accurate predictions while laboratory methods both confirm predictions and reveal unique cases meriting further analysis.     

Immunological identification and characterization of individual food allergens.

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Capillary electrophoretic separation of proteins and peptides using Pluronic liquid crystals and surface-modified capillaries

Separation of model mixtures of peptides/proteins carried out in a hydrophilically coated capillary in 10 mmol/l Tris and 75 mmol/l phosphate buffer containing 7.5% (w/w) Pluronic F127 copolymer (apparent pH 2.9) revealed that the separation is predominantly driven by the charge/mass ratio with little or no sieving effect. Using a coated capillary helped to remove current fluctuations that are observed in the fused-silica capillaries in the presence of the Pluronic copolymer. With peptides bearing distinct positive charge (polylysine of Mr around 3300) molecular sieving helps more detailed separation of individual species. Polyamino acids carrying negative charge can be brought to the detector window in the reversed polarity mode, however, no detailed separation of the individual species involved was observed under the conditions used. With a naturally occurring mixture of collagen fragments released by CNBr treatment of the protein the sequence of emerging peptides (positive polarity mode) with no relation to the rel. mol. mass could be revealed. It is concluded that separation of proteins/peptides in the presence of Pluronic in the background electrolyte occur on the charge/mass ratio basis with molecular sieving effects acting as a secondary partition mechanism.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure-activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Molecular mechanisms of class I major histocompatibility complex antigen processing and presentation

The presentation of antigenic peptides by class I major histocompatibility complex molecules plays a central role in the cellular immune response, since immune surveillance for detection of viral infections or malignant transformations is achieved by CD8+ T lymphocytes which inspect peptides, derived from intracellular proteins, bind to class I molecules on the surface of most cells. The transporter associated with antigen processing selectively translocates cytoplasmically derived peptides of appropriate sequence and length into the lumen of the endoplasmic reticulum where they associate with newly synthesized class I molecules. The translocated peptides are generated by multicatalytic and multisubunit proteasomes which degrade cytoplasmic proteins in a ATP-ubiquitin-dependent manner. This review discusses our current molecular understanding of class I antigen processing and presentation.     

A combinatorial approach to the selective capture of circulating malignant epithelial cells by peptide ligands

Early detection is critical in the administration of definitive and curative therapy of cancer. However, current detection methods are ineffective at identifying the presence of circulating metastatic cancer cells in the blood because they typically sample only a relatively small volume of blood. One strategy for sampling larger blood volumes would be to capture circulating cells in vivo over an extended period of time. The development of such a method would be substantially facilitated by the identification of peptide ligands that bind selectively to metastatic cancer cells in the blood with high affinity. To identify such ligands a combinatorial peptide library was synthesized on polyethylene acrylamide (PEGA) resin and screened for binding to malignant epithelial cells. Using Biacore, cell binding assays were performed to demonstrate that peptides selected from PEGA bead screen can bind selectively to malignant epithelial cancer cells and not to circulating leukocytes under physiologic shear stress conditions. One peptide, with the sequence QMARIPKRLARH, was used to demonstrate selective labeling of malignant epithelial cells spiked in whole blood. When immobilized on appropriate surfaces, these peptides could be used in both in vivo and ex vivo cell separation devices to efficiently and selectively capture metastatic epithelial cancer cells from flowing blood.     

The potential of proteomics and peptidomics for allergy and asthma research

Progress in the field of proteomics, the branch of biology that studies the full set of proteins derived from a given genome, is moving fast. Two-dimensional gel electrophoresis (2DG) separation of complex protein mixtures and the subsequent analysis of isolated protein spots by mass spectrometry allow fast and accurate identification of proteins. The comparison of spots from different samples separated on customized 2D gels allows the detection of punctual differences in their mobility and facilitates tracing back differences in protein expression, presence of isoforms, splice variants and posttranslational modifications by mass spectrometry. In spite of significant analytical challenges owing to the high complexity of the proteome and the challenge deriving from the necessity to process huge amounts of raw data generated by mass spectrometric profiling, proteomics has evolved to an indispensable tool in life sciences. A restricted window of the proteome that consists of peptides and small proteins not easily manageable by conventional gel electrophoresis prompted the development of separation methods based on liquid chromatography. This new research field termed peptidomics already contributed, together with proteomics to enlarge our knowledge about biological processes and supported by sophisticated bioinformatics tools, to the discovery of new diagnostic and therapeutic targets. The technological capabilities of biophysical separation, mass spectrometry and bioinformatics form the basis of discovery programs that aim at mining the proteome starting from microgram amounts of protein extracts derived from body fluids and tissues. Proteomics and peptidomics have a great potential to speed up allergy and asthma research, where disease- and tissue-specific samples are easy to obtain.     

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid-liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity. High-performance liquid chromatography (HPLC) is the favored method for the bioanalysis of TOPO-2 inhibitors. UV or diode array detection is generally employed for early pharmacokinetic studies, while fluorescence or electrochemical detection is used more frequently for analytes with fluorescent or oxidative-reductive properties. For analyses requiring highly sensitive and/or specific detection, electrospray mass spectrometry (ESI-MS or ESI-MS-MS) provides a suitable alternative. A comprehensive compilation of the HPLC techniques currently used to separate TOPO-2 inhibitors will aid the future development of analytical methods for new TOPO-2 inhibitors.     

Molecular Biology and DNA Microarray Technology for Microbial Quality Monitoring of Water

Public concern over polluted water is a major environmental issue worldwide. Microbial contamination of water arguably represents the most significant risk to human health on a global scale. An important challenge in modern water microbial quality monitoring is the rapid, specific, and sensitive detection of microbial indicators and waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. Rapid microbiological analyses and detection of rare events in water systems are important challenges in water safety assessment since culture methods present serious limitations from both quantitative and qualitative points of view. To circumvent lengthy culture methods, newer enzymatic, immunological, and genetic methods are being developed as an alternative. DNA microarray technology is a new and promising tool that allows the detection of several hundred or even thousands DNA sequences simultaneously. Recent advances in sample processing and DNA microarray technologies provide new perspectives to assess microbial water quality.

The aims of this review are to (1) summarize what is currently known about microbial indicators, (2) describe the most important waterborne pathogens, (3) present molecular methods used to monitor the presence of pathogens in water, and (4) show the potential of DNA microarrays in water quality monitoring.     

Molecular biology and DNA microarray technology for microbial quality monitoring of water

Public concern over polluted water is a major environmental issue worldwide. Microbial contamination of water arguably represents the most significant risk to human health on a global scale. An important challenge in modern water microbial quality monitoring is the rapid, specific, and sensitive detection of microbial indicators and waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. Rapid microbiological analyses and detection of rare events in water systems are important challenges in water safety assessment since culture methods present serious limitations from both quantitative and qualitative points of view. To circumvent lengthy culture methods, newer enzymatic, immunological, and genetic methods are being developed as an alternative. DNA microarray technology is a new and promising tool that allows the detection of several hundred or even thousands DNA sequences simultaneously. Recent advances in sample processing and DNA microarray technologies provide new perspectives to assess microbial water quality. The aims of this review are to (1) summarize what is currently known about microbial indicators, (2) describe the most important waterborne pathogens, (3) present molecular methods used to monitor the presence of pathogens in water, and (4) show the potential of DNA microarrays in water quality monitoring.     

Exocytosis and proteomic analysis of the vesicle content of granular hemocytes from a crayfish

The circulating blood cells (hemocytes) of invertebrates are important in cellular immune reactions and to deliver immune factors synthesized in these cells to the external milieu. Previously, we have shown that release of vesicle contents is involved in a regulated exocytosis and here we show which proteins in the vesicles are most abundant and which are released by triggering of exocytosis using a calcium ionophore, lipopolysaccharides–peptidoglycan and peroxinectin, a cell adhesion and degranulation factor from the hemocytes. The ionophore caused release of nine proteins and six of them were characterized and found to be a masquerade-like protein, a masquerade-like serine proteinase, a mannose receptor protein, a vitelline membrane outer layer protein I, and two anti-microbial peptides. The released protein band with a mass of 76 kDa is more likely pro-phenoloxidase and/or peroxinectin. When peroxinectin was used as a trigger of exocytosis, seven proteins could be identified and for the lipopolysaccharides–peptidoglycan six proteins could be identified and all of them were also released by the ionophore treatment. Interestingly, several anti-microbial peptides were the most abundant proteins and were efficiently released by all treatments as were two masquerade-like proteins one of which is functioning as an opsonic protein.     

Non-covalent labeling of human serum albumin with indocyanine green: a study by capillary electrophoresis with diode laser-induced fluorescence detection.

Indocyanine green (ICG) is a negatively charged, water-soluble, tricarbocyanine dye used primarily for medical imaging. ICG is only weakly fluorescent in the near-infrared region in its free (unbound) state in dilute aqueous solution. However, when non-covalently bound to protein, its fluorescence is greatly enhanced, making it a candidate for diode laser-induced fluorescence (diode-LIF) detection of proteins in capillary electrophoresis (CE). This paper investigates the suitability of ICG as a fluorescent label for the separation and detection of human serum albumin (HSA) by CE with diode-LIF detection. Specifically, we have considered the separation conditions necessary to resolve free ICG from ICG-HSA complexes; the limits of detection for free and HSA-bound ICG; the stability of aqueous ICG and ICG-HSA solutions over time; and the stoichiometry of the ICG-HSA complex.     

Triple helical collagen-like peptides: engineering and applications in matrix biology

Collagen, a large insoluble protein with a characteristic triple helical structure, is found as the most prominent component of extracellular matrix. The functions of collagen are not limited to providing mechanical strength to various tissues and organs as a structural protein, as it has been pointed out that collagen exhibits various biological functions through specific interactions with other macromolecules. However, the use of native triple helical collagen is often troublesome because of its insolubility and gelating properties. Instead, triple helical collagen-like peptides have been designed and are used as collagen surrogates in studies on collagen structure, stability, and biological functions including binding to other proteins and cultured cells. This article reviews recent progress in peptide design, synthesis, and the applications of collagen-like peptides in current matrix biology, while emphasizing the advantages of the peptide-based strategy.     

Separation methods for anthraquinone related anti-cancer drugs

The quinoid anthracycline-related anti-cancer agents represent an important group of anti-tumour drugs with a wide spectrum of activity. We review here some of the separation techniques used for the analysis of anthracyclines and related compounds. In this review we have covered a range of compounds from the early anthracycline antibiotics such as doxorubicin to the more recent anthracenediones and anthrapyrazoles such as mitoxantrone and losoxantrone, respectively. We also include novel compounds such as AQ4N and C1311, both awaiting clinical trial. Separations of the anthraquinone related anti-cancer agents are predominantly by HPLC. These separation techniques have been used for a variety of applications including drug stability, protein binding and therapeutic drug monitoring as well as detailed pharmacokinetic and metabolic studies. Pharmacokinetics, and therefore drug analysis, plays a central role in both the development of new agents and also leads to a better understanding of clinically established agents in this class. Sample preparation and extraction methods including solid-phase and liquid-liquid extraction have also been highlighted. Many anthraquinone related compounds are highly coloured and fluoresce. They are suitable for a range of detection methods including UV-Vis, electrochemical and fluorescence. The methods described are used for sometimes complex separations that are needed for the evaluation of such compounds in biological samples.