ABSTRACT
Endocrine tissues like the pituitary, hypothalamus and islets of Langerhans are rich in bioactive peptides. These are used for intercellular signalling and are involved in regulation of almost all physiological processes. Peptidomics is the comprehensive analysis of peptides in tissues, fluids and cells. Peptidomics applied to (neuro-)endocrine tissues aims therefore to identify as many bioactive peptides as possible. Peptidomics of (neuro-)endocrine tissues requires an integrated approach that consists of careful sample handling, peptide separation techniques, mass spectrometry and bioinformatics. Here we describe the methods for isolation and dissection of endocrine tissues, the extraction of bioactive peptides and further sample handling and identification of peptides by mass spectrometry and hyphenated techniques. We also present a straightforward method for the comparison of relative levels of bioactive peptides in these endocrine tissues under varying physiological conditions. The latter helps to elucidate functions of the bioactive peptides.
Subject(s)
Endocrine Glands/chemistry , Neuropeptides/analysis , Animals , Brain Chemistry , Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , Mice , Mice, Inbred C57BL , Neuropeptides/geneticsABSTRACT
The migration of cells and the extension of cellular processes along pathways to their defined destinations are crucial in the development of higher organisms. Caenorhabditis elegans unc-53 plays an important role in cell migration and the outgrowth of cellular processes such as axons. To gain further insight into the biological function of unc53H2, a recently identified mammalian homologue of unc-53, we have generated mice carrying a mutation of unc53H2 and provide evidence that unc53H2 is involved in neuronal development and, more specifically, the development of different sensory systems. The unc53H2 hypomorphic mouse showed a general impaired acuity of several sensory systems (olfactory, auditory, visual and pain sensation) which in case of the visual system was corroborated by the morphological observation of hypoplasia of the optic nerve. We hypothesize that in analogy with its C. elegans homologue, unc53H2 may play a role in the processes of cellular outgrowth and migration.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Embryonic and Fetal Development/physiology , Gene Expression Regulation, Developmental , Genotype , Microfilament Proteins/physiology , Sensation Disorders/genetics , Sequence Homology , Animals , Behavior, Animal , Blotting, Northern/methods , Caenorhabditis elegans Proteins/genetics , Cloning, Molecular , Embryo, Mammalian , Exploratory Behavior/physiology , Female , Humans , In Situ Hybridization/methods , Mice , Mice, Inbred Strains , Mice, Mutant Strains , Microfilament Proteins/genetics , Motor Activity/genetics , Mutation , Optic Disk/growth & development , Optic Disk/pathology , Optic Nerve/growth & development , Optic Nerve/pathology , Pain/genetics , Pain Measurement/methods , Pregnancy , Psychomotor Performance/physiology , RNA, Messenger/biosynthesis , Reaction Time/genetics , Reflex, Startle/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Rotarod Performance Test/methodsABSTRACT
Antisense oligonucleotides (AOs) are widely used as tools for inhibiting gene expression in the mammalian central nervous system. Successful gene suppression has been reported for different targets such as neurotransmitter receptors, neuropeptides, ion channels, trophic factors, cytokines, transporters, and others. This illustrates their potential for studying the expression and function of a wide range of proteins. AOs may even find therapeutic applications and provide an attractive strategy for intervention in diseases of the central nervous system (CNS). However, a lack of effectiveness and/or specificity could be a major drawback for research or clinical applications. Here we provide a critical overview of the literature from the past decade on AOs for the study of G-protein-coupled receptors (GPCRs). The following aspects will be considered: mechanisms by which AOs exert their effects, types of animal model system used, detection of antisense action, effects of AO design and delivery characteristics, non-antisense effects and toxicological properties, controls used in antisense studies to assess specificity, and our results (failures and successes). Although the start codon of the mRNA is the most popular region (46%) to target by AOs, targeting the coding region of GPCRs is almost as common (41%). Moreover, AOs directed to the coding region of the GPCR mRNA induce the highest reductions in receptor levels. To resist degradation by nucleases, the modified phosphorothioate AO (S-AO) is the most widely used and effective oligonucleotide. However, the end-capped phosphorothioate AOs (ECS-AOs) are increasingly used due to possible toxic and non-specific effects of the S-AO. Other parameters affecting the activity of a GPCR-targeting AO are the length (mostly an 18-, 20- or 21-mer) and the GC-content (mostly varying from 30 to 80%). Interestingly, one-third of the AOs successfully targeting GPCRs possess a GC/AT ratio of 61-70%. AO-induced reductions in GPCR expression levels and function range typically from 21 to 40% and 41 to 50%, respectively. In contrast to many antisense reviews, we therefore conclude that the functional activity of a GPCR after AO treatment correlates mostly with the density of the target receptors (maximum factor 2). However, AOs are no simple tools for experimental use in vivo. Despite successful results in GPCR research, no general guidelines exist for designing a GPCR-targeting AO or, in general, for setting up a GPCR antisense experiment. It seems that the correct choice of a GPCR targeting AO can only be ascertained empirically. This disadvantage of antisense approaches results mostly from incomplete knowledge about the internalisation and mechanism of action of AOs. Together with non-specific effects of AOs and the difficulties of assessing target specificity, this makes the use of AOs a complex approach from which conclusions must be drawn with caution. Further antisense research has to be carried out to ensure the adequate use of AOs for studying GPCR function and to develop antisense as a valuable therapeutic modality.
Subject(s)
Brain/drug effects , GTP-Binding Proteins/metabolism , Gene Expression Regulation/drug effects , Oligonucleotides, Antisense/pharmacology , Receptors, Cell Surface/antagonists & inhibitors , Animals , Brain/metabolism , Dose-Response Relationship, Drug , Humans , Models, Animal , Oligonucleotides, Antisense/genetics , Receptors, Cell Surface/biosynthesisABSTRACT
We studied the effects in rats of a 6-day intracerebroventricular (i.c.v) infusion of four different end-capped phosphorothioate-modified antisense oligonucleotides (AOs), specifically targeting different regions of the 5-hydroxytryptamine2A (5-HT2A) receptor mRNA, on central 5-HT2A receptor expression and 5-HT2A receptor-mediated behaviours. Only one of the AOs (sequence 4), directed against the 5'-untranslated region (from + 557 to + 577), specifically affected central 5-HT2A receptor expression and receptor-mediated behaviour. This AO (sequence 4) reduced binding of the 5-HT2A agonist 1-(2,5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane ([125I]DOI) up to 25% in cortical areas, as measured by quantitative autoradiography. Cortical binding of the antagonist [3H]ketanserin was not affected. As the specific AO treatment presumably affects the synthesis of new receptor, we hypothesize that this newly synthesized receptor represents the major part of the functionally active, G protein coupled receptor. A 5-day infusion of AO (sequence 4) resulted in profound inhibition of the head-twitch response (HTR) to 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast, treatment with vehicle, sense oligonucleotides (SOs) and other AOs (sequences 1, 2 and 3) caused an increased DOM-induced HTR as well as a spontaneous HTR. The latter was abolished by treatment with the 5-HT2 receptor antagonist, ritanserin. Systematic investigation of the surgical and infusion procedures revealed that the enhanced HTR already appeared following drilling of the skull. This wounding can probably damage the blood-brain barrier and cause a stress-induced increase in serotonergic transmission. AO (sequence 4) treatment also abolished the spontaneous HTR. AO (sequence 4) treatment allowed the identification of specific central 5-HT2A receptor-mediated behaviours in the complex serotonergic syndrome induced by tryptamine in rats. Only bilateral convulsions and body tremors were significantly inhibited. The backward locomotion, hunched back and Straub tail were not affected, nor was cyanosis, an index of vasoconstriction induced by peripheral 5-HT2A receptor activation. Labelling of central 5-HT2C receptors by [3H]mesulergine, and 5-HT2C receptor-mediated anxiety were not attenuated by AO or SO treatment. Rats treated with AO (sequence 4) showed increased locomotor activity and a strong reactivity towards touching. We hypothesize that the down-regulation of functional 5-HT2A receptors may shift the balance between various 5-HT receptor subtypes. Our analysis of the behavioural consequences of AO treatment and the use of different AOs and SOs has shown that specific receptor-mediated behaviour can be identified.
Subject(s)
Brain/drug effects , Oligonucleotides, Antisense/administration & dosage , RNA, Messenger/antagonists & inhibitors , Receptors, Serotonin/genetics , Serotonin Syndrome/physiopathology , Animals , Autoradiography , Behavior, Animal/drug effects , Brain/metabolism , Coloring Agents/pharmacology , Drug Antagonism , Evans Blue/pharmacology , Injections, Intraventricular , Male , Maze Learning/drug effects , Motor Activity/drug effects , Rats , Rats, Wistar , Receptor, Serotonin, 5-HT2A , Receptors, Serotonin/drug effects , Receptors, Serotonin/metabolism , Seizures/chemically induced , Seizures/drug therapy , Seizures/prevention & control , Serotonin Antagonists/pharmacology , Serotonin Syndrome/chemically induced , Serotonin Syndrome/drug therapy , Sulfur Radioisotopes , TryptaminesABSTRACT
The 5-HT(2A) and 5-HT(2C) receptors belong to the G-protein-coupled receptor (GPCR) superfamily. GPCRs transduce extracellular signals to the interior of cells through their interaction with G-proteins. The 5-HT(2A) and 5-HT(2C) receptors mediate effects of a large variety of compounds affecting depression, schizophrenia, anxiety, hallucinations, dysthymia, sleep patterns, feeding behaviour and neuro-endocrine functions. Binding of such compounds to either 5-HT(2) receptor subtype induces processes that regulate receptor sensitivity. In contrast to most other receptors, chronic blockade of 5-HT(2A) and 5-HT(2C) receptors leads not to an up- but to a (paradoxical) down-regulation. This review deals with published data involving such non-classical regulation of 5-HT(2A) and 5-HT(2C) receptors obtained from in vivo and in vitro studies. The underlying regulatory processes of the agonist-induced regulation of 5-HT(2A) and 5-HT(2C) receptors, commonly thought to be desensitisation and resensitisation, are discussed. The atypical down-regulation of both 5-HT(2) receptor subtypes by antidepressants, antipsychotics and 5-HT(2) antagonists is reviewed. The possible mechanisms of this paradoxical down-regulation are discussed, and a new hypothesis on possible heterologous regulation of 5-HT(2A) receptors is proposed.
Subject(s)
Receptors, Serotonin/physiology , Animals , Antidepressive Agents/pharmacology , Antipsychotic Agents/pharmacology , GTP-Binding Proteins/physiology , Humans , Receptor, Serotonin, 5-HT2A , Receptor, Serotonin, 5-HT2C , Receptors, Serotonin/drug effects , Serotonin Antagonists/pharmacology , Serotonin Receptor Agonists/pharmacologyABSTRACT
So far, antifungal drug discovery seems to have benefited little from the enormous advances in the field of genomics in the last decade. Although it has become clear that traditional drug screening is not delivering the long-awaited novel potent antifungals, little has been reported on efforts to use novel genome-based methodologies in the quest for new drugs acting on human pathogenic fungi. Although the market for a novel systemic and even topical broad-spectrum antifungal appears considerable, many large pharmaceutical companies have decided to scale back their activities in antifungal drug discovery. Here we report on some of the recent advances in genomics-based technologies that will allow us not only to identify and validate novel drug targets but hopefully also to discover active therapeutic agents. Novel drug targets have already been found by 'en masse' gene inactivation strategies (e.g. using antisense RNA inhibition). In addition, genome expression profiling using DNA microarrays helps to assign gene function but also to understand better the mechanism of action of known drugs (e.g. itraconazole) and to elucidate how new drug candidates work. No doubt, we have a long way to go just to catch up with the advances made in other therapeutic areas, but all tools are at hand to derive practical benefits from the genomics revolution. The next few years should prove a very exciting time in the history of antifungal drug discovery.
Subject(s)
Antifungal Agents/administration & dosage , Drug Delivery Systems/methods , Genomics/methods , Animals , Gene Expression Profiling/methods , Humans , Technology, Pharmaceutical/methodsABSTRACT
Perfect drugs are potent, specific and nontoxic. Many compounds fail because of unexpected toxicity and lack of efficacy in later stages of clinical development. Therefore, more complete knowledge and understanding of the properties of a drug is needed at an earlier stage of drug development. DNA microarrays can yield gene expression profiles from cells or tissues treated with a compound. Such "expression fingerprints" are used in drug discovery for drug target identification and validation and for elucidating the mode of action of novel compounds during lead identification and optimization. Moreover, during drug development, DNA microarrays help in the discovery of new diagnostic and prognostic biomarkers, as well as in the prediction of resistance and toxic side effects. This review aims to assess to what extent the promise of gene expression profiling has already materialized for the different stages of drug discovery and development. (c) 2002 Prous Science. All rights reserved.
ABSTRACT
Almost all eukaryotic mRNAs are capped at their 5'-terminus. Capping is crucial for stability, processing, nuclear export and efficient translation of mRNA. We studied the phenotypic effects elicited by depleting a Candida albicans strain of mRNA 5'-guanylyltransferase (mRNA capping enzyme; CGT1). Construction of a Cgt1-deficient mutant was achieved by URA-blaster-mediated genetic disruption of one allele of the CGT1 gene, which was localized on chromosome III. The resulting heterozygous mutant exhibited an aberrant colony morphology resembling the 'irregular wrinkle' phenotype typically obtained from a normal C. albicans strain upon mild UV treatment. Its level of CGT1 mRNA was reduced two- to fivefold compared to the parental strain. Proteome analysis revealed a large number of differentially expressed proteins confirming the expected pleiotropic effect of CGT1 disruption. The disrupted strain was significantly more resistant to hygromycin B, an antibiotic which decreases translational fidelity, and showed increased resistance to heat stress. Proteome analysis revealed a 50-fold overexpression of Ef-1alphap and a more than sevenfold overexpression of the cell-wall heat-shock protein Ssa2p. Compared to a reference strain, the cgt1/CGT1 heterozygote was equally virulent for mice and guinea pigs when tested in an intravenous infection model of disseminated candidiasis.