Immunogold staining procedure for the localisation of regulatory peptides

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.     

Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides

Recent studies have suggested that the morphological characteristics of secretory granules contained within endocrine cells and nerves may be determined largely by their chemical composition. The use of the immunogold staining (IGS) method, which is based on the adsorption of colloidal gold to immunoglobulins, has been used in our laboratory to demonstrate a wide range of intracellular antigens at both the light and electron microscope levels. In this study we have applied a modification of the IGS method for the simultaneous detection of two separate antigens in a single tissue section, using a variety of region-specific antisera to different peptides. Peptide antisera were raised in rabbits or in guinea pigs and these were applied simultaneously or sequentially to grid-mounted ultrathin tissue sections. Antigenic sites were visualized at the electron microscope level using antisera raised in goats, adsorbed to gold particles of 12, 20, or 40 nm. Using this technique we have attempted to investigate the coexistence of multiple antigens in single tissue sections, in particular in single granules; the topographic distribution of molecular forms within one single granule or granule population; the heterogeneity of peptidergic neurons and also the heterogeneity of peptide content in morphologically similar granules. The double immunogold staining procedures described here have proved to be extremely effective for the simultaneous ultrastructural localization of two antigens (peptide-peptide; peptide-propeptide) on a single tissue section. The further development of this technique may provide useful information on neuroendocrine cell dynamics in normal and diseased states.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

Somatostatin-containing D cells exhibit immunoreactivity for rat somatostatin cryptic peptide in six mammalian species

Summary Antisera raised against rat somatostatin cryptic peptide (RSCP; corresponding to amino acids 63–77 of rat pro-somatostatin), somatostatin-28-(1–12) and somatostatin-28-(17–28) were used to compare the morphological distribution of these pro-somatostatin-derived sequences within the gastroenteropancreatic system of six mammalian species, including man. Using the immunogold staining procedure, RSCP, SS28-(1–12) and SS28-(17–28) immunoreactivity was found to be present in all the D cells of the tissues investigated. Extra-islet RSCP and SS28-(1–12) immunoreactive cells were also identified in some species. RSCP, SS28-(1–12) and SS28-(17–28) immunoreactivities were also present in a single case of human duodenal somatostatinoma. Immunostaining of serial ultrathin sections from all specimens in this study revealed that RSCP and both somatostatin immunoreactivities were co-localised in a majority of the reactive cells. Corroborative evidence was obtained by double immunogold staining which further showed that RSCP, SS28-(1–12) and SS28-(17–28) immunoreactivities were co-localised to individual secretory granules in D type cells, both normal and tumour. RSCP and SS28-(17–28) immunoreactivities were invariably co-localised, whereas SS28-(1–12) immunoreactivity was restricted to a sub-population of secretory granules.Our findings suggest that RSCP immunoreactivity is conserved in a number of mammalian species and is stored in each secretory granule type. Consequently, detection of the RSCP sequence may serve as a useful marker for somatostatin-producing systems throughout the diffuse neuroendocrine system.     

Effect of sex and bright coloration on survival and predator‐induced wing damage in an aposematic lantern fly with startle display

1. Aposematic coloration in prey promotes its survival by conspicuously advertising unpalatability to predators. Although classical examples of aposematic signals involve constant presentation of a signal at a distance, some animals suddenly display warning colours only when they are attacked. 2. Characteristics of body parts suddenly displayed, such as conspicuous coloration or eyespot pattern, may increase the survival of the prey by startling the predator, and/or by signalling unpalatability to the predators at the moment of attack. 3. The adaptive value of such colour patterns suddenly displayed by unpalatable prey has not been studied. We experimentally blackened the red patch in the conspicuous red–white–black hindwing pattern displayed by an unpalatable insect Lycorma delicatula White (Hemiptera: Fulgoridae) in response to predator's attack. 4. There was no evidence that the presence of the red patch increased prey survival over several weeks. We hypothesise that predators generalised from the red–white–black patches on the hindwings of unpalatable L. delicatula to any similar wing display as a signal of unpalatability. Because a higher proportion of males than females stay put at their resting sites, displaying their wings in response to repeated attacks by predators, wing damage was more frequent in males than in females. 5. To our knowledge, this is the first experimental test of an adaptive role of aposematic signals presented by unpalatable prey during sudden displays triggered by direct predatory attack.