The usefulness of radiotracers to make the body biochemically transparent

Summary. Radioactive isotopes are uniquely applicable to observe reactions or circuits of reactions at the molecular level without disturbing the system being studied. The advent of molecular imaging modalities, particularly positron emission tomography (PET), is a major breakthrough for the visualisation and quantitative assessment of cellular and molecular processes occurring in living tissues. The recent development of animal PET scanners that offers 2-mm resolution and is tailored to laboratory rodent models, has made a further great impact on in vivo biochemistry. With these live-imaging modalities at hand, radiotracer-based technologies allow to look directly at biochemical distribution and interaction processes. Tremendous progress made in radiotracer chemistry, primarily in carbon-11 and fluorine-18 radiochemistry, and in the design of imaging devices strengthens the usefulness of radiotracers in nuclear medicine and drug research and development and opens exciting opportunities for new applications, e.g., in food science.     

Histidine biosynthesis in plants

Summary. The study of histidine metabolism has never been at the forefront of interest in plant systems despite the significant role that the analysis of this pathway has played in development of the field of molecular genetics in microbes. With the advent of methods to analyze plant gene function by complementation of microbial auxotrophic mutants and the complete analysis of plant genome sequences, strides have been made in deciphering the histidine pathway in plants. The studies point to a complex evolutionary origin of genes for histidine biosynthesis. Gene regulation studies have indicated novel regulatory networks involving histidine. In addition, physiological studies have indicated novel functions for histidine in plants as chelators and transporters of metal ions. Recent investigations have revealed intriguing connections of histidine in plant reproduction. The exciting new information suggests that the study of plant histidine biosynthesis has finally begun to flower.     

Using cellular automata images and pseudo amino acid composition to predict protein subcellular location

Summary. The avalanche of newly found protein sequences in the post-genomic era has motivated and challenged us to develop an automated method that can rapidly and accurately predict the localization of an uncharacterized protein in cells because the knowledge thus obtained can greatly speed up the process in finding its biological functions. However, it is very difficult to establish such a desired predictor by acquiring the key statistical information buried in a pile of extremely complicated and highly variable sequences. In this paper, based on the concept of the pseudo amino acid composition (Chou, K. C. PROTEINS: Structure, Function, and Genetics, 2001, 43: 246–255), the approach of cellular automata image is introduced to cope with this problem. Many important features, which are originally hidden in the long amino acid sequences, can be clearly displayed through their cellular automata images. One of the remarkable merits by doing so is that many image recognition tools can be straightforwardly applied to the target aimed here. High success rates were observed through the self-consistency, jackknife, and independent dataset tests, respectively.     

Classifying G protein-coupled receptors and nuclear receptors on the basis of protein power spectrum from fast Fourier transform

Summary. As the potential drug targets, G-protein coupled receptors (GPCRs) and nuclear receptors (NRs) are the focuses in pharmaceutical research. It is of great practical significance to develop an automated and reliable method to facilitate the identification of novel receptors. In this study, a method of fast Fourier transform-based support vector machine was proposed to classify GPCRs and NRs from the hydrophobicity of proteins. The models for all the GPCR families and NR subfamilies were trained and validated using jackknife test and the results thus obtained are quite promising. Meanwhile, the performance of the method was evaluated on GPCR and NR independent datasets with good performance. The good results indicate the applicability of the method. Two web servers implementing the prediction are available at and .     

Hallmarks of protein oxidative damage in neurodegenerative diseases: focus on Alzheimer’s disease

Summary. The pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, has been linked to a condition of oxidative and nitrosative stress, arising from the imbalance between increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and antioxidant defences or efficiency of repair or removal systems. The effects of free radicals are expressed by the accumulation of oxidative damage to biomolecules: nucleic acids, lipids and proteins. In this review we focused our attention on the large body of evidence of oxidative damage to protein in Alzheimer’s disease brain and peripheral cells as well as in their role in signalling pathways. The progress in the understanding of the molecular alterations underlying Alzheimer’s disease will be useful in developing successful preventive and therapeutic strategies, since available drugs can only temporarily stabilize the disease, but are not able to block the neurodegenerative process.     

Detecting oxidative post-translational modifications in proteins

Summary. Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).     

Recombinant human alpha-1 proteinase inhibitor: towards therapeutic use

Summary. Human alpha-1-proteinase inhibitor is a well-characterized protease inhibitor with a wide spectrum of anti-protease activity. Its major physiological role is inhibition of neutrophil elastase in the lungs, and its deficiency is associated with progressive ultimately fatal emphysema. Currently in the US, only plasma-derived human alpha-1-proteinase inhibitor is available for augmentation therapy, which appears to be insufficient to meet the anticipated clinical demand. Moreover, despite effective viral clearance steps in the manufacturing process, the potential risk of contamination with new and unknown pathogens still exists. In response, multiple efforts to develop recombinant versions of human alpha-1-proteinase inhibitor, as an alternative to the plasma-derived protein, have been reported. Over the last two decades, various systems have been used to express the human gene for alpha-1-proteinase inhibitor. This paper reviews the recombinant versions of human alpha-1-proteinase inhibitor produced in various hosts, considers current major safety and efficacy issues regarding recombinant glycoproteins as potential therapeutics, and the factors that are impeding progress in this area¹. ¹ The opinions expressed in this paper reflect the authors’ personal views, based on published data and the information available from the public domains, and have no relation to the official statements, if any, held by the US FDA, National Institutes of Health, or the US Department of Health and Human Services. FDA official recommendations for plasma protein therapeutics and recombinant proteins regarding safety, purity, and potency of new drugs and biologics produced by recombinant DNA technology are referred below as the US FDA Guidances.     

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary. The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.     

The renal type H+/peptide symporter PEPT2: structure-affinity relationships

Summary. The H⁺/peptide cotransporter PEPT2 is expressed in a variety of organs including kidney, lung, brain, mammary gland, and eye. PEPT2 substrates are di- and tripeptides as well as peptidomimetics, such as β-lactam antibiotics. Due to the presence of PEPT2 at the bronchial epithelium, the aerosolic administration of peptide-like drugs might play a major role in future treatment of various pulmonary and systemic diseases. Moreover, PEPT2 has a significant influence on the in vivo disposition and half-life time of peptide-like drugs within the body, particularly in kidney and brain. PEPT2 is known to have similar but not identical structural requirements for substrate recognition and transport compared to PEPT1, its intestinal counterpart. In this review we compiled available affinity constants of 352 compounds, measured at different mammalian tissues and expression systems and compare the data whenever possible with those of PEPT1.     

Protein tyrosine nitration in hydrophilic and hydrophobic environments

Summary. In this review we address current concepts on the biological occurrence, levels and consequences of protein tyrosine nitration in biological systems. We focused on mechanistic aspects, emphasizing on the free radical mechanisms of protein 3-nitrotyrosine formation and critically analyzed the restrictions for obtaining large tyrosine nitration yields in vivo, mainly due to the presence of strong reducing systems (e.g. glutathione) that can potently inhibit at different levels the nitration process. Evidence is provided to show that the existence of metal-catalyzed processes, the assistance of nitric oxide-dependent nitration steps and the facilitation by hydrophobic environments, provide individually and/or in combination, feasible scenarios for nitration in complex biological milieux. Recent studies using hydrophobic tyrosine analogs and tyrosine-containing peptides have revealed that factors controlling nitration in hydrophobic environments such as biomembranes and lipoproteins can differ to those in aqueous compartments. In particular, exclusion of key soluble reductants from the lipid phase will more easily allow nitration and lipid-derived radicals are suggested as important mediators of the one-electron oxidation of tyrosine to tyrosyl radical in proteins associated to hydrophobic environments. Development and testing of hydrophilic and hydrophobic probes that can compete with endogenous constituents for the nitrating intermediates provide tools to unravel nitration mechanisms in vitro and in vivo; additionally, they could also serve to play cellular and tissue protective functions against the toxic effects of protein tyrosine nitration.     

Corticotropin-releasing factor, vasopressin and receptor systems in depression and anxiety

Summary. Affective disorders tend to be chronic and life-threatening diseases: suicide is estimated to be the cause of death in 10–15% of individuals with major depressive disorders. Major depression is one of the most prevalent and costly brain diseases with up to 20% of the worldwide population suffering from moderate to severe forms of the disease. Only 50% of individuals with depression show full remission in response to currently available antidepressant drug therapies which are based on serendipitous discoveries made in the 1950s. Previously underestimated, other severe depression-associated deleterious health-related effects have increasingly been recognized. Epidemiological studies have provided substantial evidence that patients with depression have a 2–4-fold increased risk both of developing cardiovascular disease and of mortality after experiencing a myocardial infarction. The majority of patients suffering from affective disorders have measurable shifts in their stress hormone regulation as reflected by elevated secretion of central and peripheral stress hormones or by altered hormonal responses to neuroendocrine challenge tests. In recent years, these alterations have increasingly been translated into testable hypotheses addressing the pathogenesis of illness. Refined molecular technologies and the creation of genetically engineered mice have allowed to specifically target individual genes involved in regulation of corticotropin releasing factor (CRF) and vasopressin (AVP) system elements. The cumulative evidence makes a strong case implicating dysfunction of these systems in the etiology and pathogenesis of depression and pathological anxiety. Translation of these advances into novel therapeutic strategies has already been started.     

Chlorpromazine,clozapine and olanzapine inhibit anionic amino acid transport in cultured human fibroblasts

Summary. We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X⁻ _AG (EAAT transporters) in cultured human fibroblasts. With 30 μM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (>60%), and referable to a decrease in V_max. Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X⁻ _AG since transport systems A and y⁺ are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients.     

Protein-bound advanced glycation endproducts (AGEs) as bioactive amino acid derivatives in foods

Summary. The Maillard reaction or nonenzymatic browning is of outstanding importance for the formation of flavour and colour of heated foods. Corresponding reactions, also referred to as “glycation”, are known from biological systems, where the formation of advanced glycation endproducts (AGEs) shall play an important pathophysiological role in diabetes and uremia. In this review, pathways leading to the formation of individual protein-bound lysine and arginine derivatives in foods are described and nutritional consequences resulting from this posttranslational modifications of food proteins are discussed.     

Predicting secretory protein signal sequence cleavage sites by fusing the marks of global alignments

Summary. A newly synthesized secretory protein in cells bears a special sequence, called signal peptide or sequence, which plays the role of “address tag” in guiding the protein to wherever it is needed. Such a unique function of signal sequences has stimulated novel strategies for drug design or reprogramming cells for gene therapy. To realize these new ideas and plans, however, it is important to develop an automated method for fast and accurately identifying the signal sequences or their cleavage sites. In this paper, a new method is developed for predicting the signal sequence of a query secretory protein by fusing the results from a series of global alignments through a voting system. The very high success rates thus obtained suggest that the novel approach is very promising, and that the new method may become a useful vehicle in identifying signal sequence, or at least serve as a complementary tool to the existing algorithms of this field.     

Cinnamomin: separation,crystallization and preliminary X-ray diffraction study

Cinnamomin from Cinnamonum camphora seeds, a type II ribosome-inactivating protein that interferes with protein biosynthesis in mammalian cells, can induce the apoptosis of carcinoma cells and be used as an insecticide. A rapid and improved method has been developed for the extraction and purification of cinnamomin from camphora seed. Purification of cinnamomin is achieved with two successive steps of hydrophobic interaction chromatography carried out on a fast protein liquid chromatography (FPLC) system. Crystals suitable for X-ray diffraction analysis were obtained by vapor diffusion method. A complete data set at 2.8 A resolution has been collected. Data indexation and refinement indicate that the crystal is orthorhombic with space group P2(1)2(1)2(1) and unit cell dimensions a = 52.39 A, b = 126.33 A, c = 161.45 A. There are two molecules per asymmetric unit. Initial phasing by molecular replacement method yielded a solution, which will contribute to the structure determination. A molecular model will further the understanding of the mechanism of cinnamomin function. The latter will be combined with bio-informatics to facilitate the medical and other applications of cinnamomin.     

Metabolite analysis in positron emission tomography studies: examples from food sciences

Summary. Substances of various chemical structures can be labelled with appropriate positron emitting isotopes and applied as tracer compounds in PET examinations. Using dynamic data acquisition protocols, time-activity curves of radioactivity uptake in organs can be derived and the measurements of tissue tracer concentrations can be translated into quantitative values of tissue function. However, analysis of metabolites of these tracers regarding their nature and distribution in the living organism is an essential need for the quantitative analysis of PET measurements. In addition, metabolite analysis contributes to the interpretation of the images obtained as well as to the identification of pathological changes in metabolic pathways. This paper reports on representative examples of radiolabelled compounds which might be of importance in food science (e.g., amino acids, polyphenols, and model compounds for advanced glycation end products (AGEs)). Typical procedures of analysis (radio-HPLC, radio-TLC) including pre-analytical sample preparation are described. Specific challenges of the method, e.g., trace amounts of radiolabelled compounds and the influence of the often very short half-lives of positron-emitting nuclides used are highlighted. Representative results of analyses of plasma, urine, and tissue samples are presented and discussed in terms of the metabolic fate of the tracers.     

D-Aspartic acid and L-amino acids in the neural system of the amphioxus <Emphasis Type="Italic">Branchiostoma lanceolatum</Emphasis>

Summary. The lancelet (amphioxus), a cephalochordate, is the closest invertebrate relative to vertebrates, with a simple vertebrate-like body plan and a prototypical genome. We have determined D-aspartic acid (D-Asp) and major free L-amino acids (L-AAs) content in the nervous system (neural tube) of the European amphioxus Branchiostoma lanceolatum, and have compared these values with those of molluscs and human brain. The B. lanceolatum neural tube contains relatively high amounts of L-Glu, L-Asp, L-Ala and L-Gly. Thus, the amphioxus neural tube has in common with the molluscan and human nervous systems the presence of appreciable amounts of L-Glu and L-Asp, which suggests that they are the most common neurotransmitters among these phylogenetically distant animal groups. The relatively high concentration of L-Ala in amphioxus is consistent with that found in molluscs and the low concentration of taurine is consistent with that described in the human brain. The D-Asp concentration, very high in the molluscan nervous system, was rather low in amphioxus, although a little higher than the extremely low amounts observed in the human brain. Our data on free amino acids composition is in agreement with the intermediate phylogenetic position of cephalochordates, in terms of the evolutionary transition from simple to complex neural systems.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

Brucellosis, botflies, and brainworms: the impact of edge habitats on pathogen transmission and species extinction

Ecological interactions between species that prefer different habitat types but come into contact in edge regions at the interfaces between habitat types are modeled via reaction-diffusion systems. The primary sort of interaction described by the models is competition mediated by pathogen transmission. The models are somewhat novel because the spatial domains for the variables describing the population densities of the interacting species overlap but do not coincide. Conditions implying coexistence of the two species or the extinction of one species are derived. The conditions involve the principal eigenvalues of elliptic operators arising from linearizations of the model system around equilibria with only one species present. The conditions for persistence or extinction are made explicit in terms of the parameters of the system and the geometry of the underlying spatial domains via estimates of the principal eigenvalues. The implications of the models with respect to conservation and refuge design are discussed. Received: 10 June 1999 / Revised version: 7 July 2000 / Published online: 20 December 2000