Presenilin 1 independently regulates beta-catenin stability and transcriptional activity.

Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.

Localisation of endothelin like immunoreactivity in adult and developing human gut.

The distribution of immunoreactivity for the potent vasoconstrictor endothelin-1 was studied in adult and developing human gut using antisera to endothelin-1 (1-21) and the C terminus of big endothelin-1. The coexistence of these peptides with other neuropeptides was investigated using comparative immunocytochemistry. Endothelin-1 like immunoreactivity was detected in extracts of adult (range 20-60 fmol/g wet weight) and fetal (33 fmol/g) gastrointestinal tract and was shown by chromatography to be the predominant isoform of endothelin present in both. It was localised by immunocytochemistry to ganglion cells in the submucous and myenteric plexuses and to scattered nerves, whereas big endothelin-1 like immunoreactivity was found in the submucous plexus only. Colocalisation studies showed immunoreactivity for both endothelin-1 and vasoactive intestinal peptide in the same ganglion cells of the submucous plexus. Although endothelin-1 immunoreactivity was not detected by immunocytochemistry in the fetal human gut until the 32nd week of gestation, big endothelin-1 was found as early as 11 weeks in the developing neural structures and epithelial cells. The latter were shown to be endocrine cells by their immunoreactivity for chromogranin. Our results indicate that endothelin is a neuropeptide found in adult human gut which shows transient expression in endocrine cells during development.

Oncoprotein immunoreactivity in human endocrine tumours.

Abnormally expressed oncogenes are implicated in neoplastic transformation. We have investigated a series of endocrine tumours using immunocytochemistry as a first screening tool to detect oncogene expression. Paraffin sections of 44 pulmonary small cell carcinomas, 15 pulmonary atypical carcinoids, 12 bronchial carcinoids, 28 medullary thyroid carcinomas, 27 phaeochromocytomas, and 17 insulinomas were immunostained with antibodies to c-erbB-2, c-myc, L-myc, and N-myc. Diffuse immunoreactivity was detectable for c-erbB-2 in 8 out of 44 (18 per cent) pulmonary small cell carcinomas, 3 out of 15 (20 per cent) pulmonary atypical carcinoids, and 6 out of 27 (22 per cent) phaeochromocytomas; for c-myc in 18 out of 44 (41 per cent) pulmonary small cell carcinomas and 5 out of 15 (33 per cent) pulmonary atypical carcinoids; for N-myc in 6 out of 28 (21 per cent) medullary thyroid carcinomas; and for L-myc in 4 out of 27 (15 per cent) phaeochromocytomas. There was considerable intratumoral and intertumoral heterogeneity and, in each tumour group, no relationship was found between tumour pattern, mitotic index, and oncoprotein immunoreactivity. These results suggest that oncogene products are present in a proportion of endocrine tumours, and that specific oncoproteins seem to be related to tumour type but not to other histopathological findings. Thus, oncoprotein detection may be a useful tool for identifying subsets of endocrine tumours that are not otherwise recognizable morphologically.

The production and characterisation of monoclonal antibodies to myc, c-erbB-2 and EFG-receptor using a synthetic peptide approach.

Monoclonal antibodies to myc, c-erbB-2 and epidermal growth factor-receptor (EGF-R) were raised using a synthetic peptide approach. The antibodies were characterised by ELISA, immunoblotting, immunoprecipitation and immunocytochemical procedures against cognate peptide and native proteins. All of the monoclonal antibodies detected peptide-blockable bands of appropriate molecular weight (myc-p62/66 kDa, c-erbB-2-185kDa; EGF-R-150/170 kDa) on immunoblots. The monoclonal antibodies to c-erbB-2 and EGF-R immunostained subpopulations of tumour cells on sections of formalin-fixed, paraffin wax embedded human infiltrating and invasive ductal carcinomas of breast. Intense blood cell staining was observed with the EGF-R antibody. This staining was shown to be peptide blockable and may reveal a true localisation for the EGF-receptor protein, a closely-related (erbB) protein or a degradation product. The monoclonal antibody to a common peptide from the myc protein family was epitope scanned using a modification of the Geysen pin technique. Hexapeptide sequence Ala-Pro-Ser-Glu-Asp-Ile was found to be bound most strongly by the myc monoclonal antibody, and amino acids Pro2 and Glu4 were found to be essential for antibody binding. The use of synthetic peptides for the production of monoclonal antibodies with predetermined specificity, which may be precisely identified using the epitope scanning technique, is discussed.

Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia.

The localization of endothelin 1 mRNA and endothelin-like immunoreactivity was investigated in samples of neurologically normal nervous system tissue from 10 adults by using in situ hybridization and immunocytochemistry. Tissue sections of spinal cord and dorsal root ganglia were hybridized with an 35S-radiolabeled endothelin 1 complementary RNA probe. Autoradiograms showed labeled neurons in the spinal cord (laminae IV-VI and many motoneurons) and numerous small and large neurons in the dorsal root ganglia. Endothelin 1 transcripts were also found in association with the endothelial layer of some blood vessels in the white matter of the spinal cord. A similar distribution of immunoreactivity was seen using three antisera to endothelin 1, but fewer cells were immunostained than were labeled after the hybridization. Two other endothelin 1 antisera immunostained the endothelial lining of blood vessels in the spinal cord white matter but not neurons. In the ganglia, endothelin 1 transcripts were localized to all cells expressing beta-preprotachykinin and most expressing calcitonin gene-related peptide mRNAs; in the majority of the motoneurons, coexistence of endothelin 1 and calcitonin gene-related peptide mRNAs was noted. There was a similar pattern of coexistence for respective peptide immunoreactivities in the ganglia and spinal cord. The expression of endothelin 1 mRNA in distinct neuronal cell types suggests that this peptide plays a part in neural transmission/modulation and adds a further dimension to the known vascular-related actions of endothelin 1.

N-myc gene expression and oncoprotein characterisation in medulloblastoma.

Although medulloblastoma and neuroblastoma share many common biological, histological and immunological features, the frequency of N-myc amplification differs markedly between the two tumours. In this study, Southern blot analysis revealed that the N-myc gene was not amplified in any of the nine medulloblastoma samples analysed. In contrast, over-expression of the gene was found in six of 11 samples as determined by immunocytochemistry and/or Western blot analysis, using an antiserum raised against a synthetic peptide representing a sequence unique to the N-myc gene product. The specificity of this reagent was demonstrated by studies on a variety of cell lines expressing N-myc and/or c-myc oncoproteins. Of the 12 medulloblastoma samples collected over a two-year period and analysed in the course of this project, a trend towards longer disease-free survival was noted in the patients having low levels of the N-myc protein in their tumour.

Ultrastructure and electron immunocytochemistry of insulin-producing B-cell tumors from transgenic mice: comparison with counterpart human tumors.

This paper reports on an ultrastructural and electron-microscopic immunocytochemical study of pancreatic B cells from normal mice, pancreatic B cells and derivative tumors from transgenic mice, and tissue from human pancreatic B-cell tumors. In normal and neoplastic B cells from both species, typical immature and mature beta-granules (with spherical cores of variable density) were observed, whereas typical beta-granules with a crystalloid core were only present in human B cells (normal and tumor). A small number of atypical granules were found in distinct neoplastic cells which contained no typical beta-granules. The atypical granules were smaller (100-200 nm diameter) than typical beta-granules (250-450 nm diameter) seen in other cells. Immunoreactivity for proinsulin was localized only to immature granules, whereas insulin and C-peptide immunoreactivities were demonstrated in atypical, immature, and mature granules. In transgenic mouse and human B-cell tumors, insulin immunoreactivity was consistently weaker than the immunostaining for C-peptide. An intragranular, topographic segregation of immunoreactive C-peptide was observed in a population of transgenic tumor cells. Our results showed similarities in antigenic distribution and only slight differences in morphology between human and mouse B cells. Therefore, the transgenic mouse system may prove to be an effective model for studying mammalian B-cell tumorigenesis.

Multiple peptide production and presence of general neuroendocrine markers detected in 12 cases of human phaeochromocytoma and in mammalian adrenal glands.

In this study, antibodies to a range of markers of neuroendocrine differentiation were evaluated for their use in the histopathological assessment and characterisation of phaeochromocytomas. Routinely processed wax blocks from eleven adrenal phaeochromocytomas (10 benign and 1 malignant) and one benign phaeochromocytoma of the urinary bladder were investigated. In addition to these tumours, normal human, cat and piglet adrenal glands were examined. In the phaeochromocytomas, immunostaining was obtained with 21 of the 25 antisera used. Of the general neuroendocrine markers, neuron-specific enolase was found in all tumours, and chromogranin and protein gene-product 9.5 in most of the cases. A range of regulatory peptide immunoreactivities could be demonstrated, such as enkephalin, neuropeptide tyrosine (NPY), 7B2, galanin and vasoactive intestinal polypeptide (VIP). In addition, two peptides were found which have not been reported previously in these tumours, peptide histidine methionine (PHM) and the cryptic fragment of the precursor encoding VIP. Co-localisation studies revealed that peptides derived from the same precursor or peptide family were found in the same tumour cells (e.g. VIP and PHM, NPY and its C-flanking peptide CPON). In the normal adrenal medulla, all the peptides previously reported to be present could be demonstrated immunocytochemically. Galanin was present in a subpopulation of cells also immunoreactive for enkephalin. Neuropeptide tyrosine and CPON were demonstrated in another subpopulation. Occasionally, cells were found to contain all four antigen immunoreactivities. Using antisera to enzymes involved in catecholamine synthesis, galanin was found to be present in noradrenaline-containing cells. The study demonstrates the presence of various antigens in chromaffin tissue of the adrenal gland. A range of substances can also be identified immunocytochemically in phaeochromocytoma tissue, using routinely-processed material.

Extraadrenal paragangliomas. An immunocytochemical and ultrastructural report.

Although the majority of extraadrenal paragangliomas are nonfunctional, some of these tumors are associated with hormone production and clinical symptoms, notably hypertension. The authors have investigated 22 paragangliomas, five of which were diagnosed as clinically functional in a light microscopic immunocytochemical and electron microscopic study (nine cases). Histologically, all the paragangliomas exhibited similar features, with a "Zellballen" pattern of polygonal cells. All 22 cases were strongly immunoreactive to protein gene product 9.5 (PGP 9.5) antisera and moderately reactive to antineuron-specific enolase (NSE) sera. Ten cases (five functional) were focally immunoreactive to antichromogranin sera. Seven cases (four functional) were immunoreactive to neuropeptide Y and enkephalin antisera, and six (five functional) to tyrosine hydroxylase antisera. The clinically functional tumors expressed at least two of the antigens, enkephalin, neuropeptide Y, or tyrosine hydroxylase, whereas none of the 17 nonfunctional possessed more than one of these. Electron microscopic study revealed cells from all the nine cases studied to contain secretory granules. Granule sizes ranged from 100 to 280 nm and the morphologic examination of the secretory granules generally showed a dense core with a membrane-bound halo of variable size. Secretory granules were observed in the five functional cases and these were larger (220-280 nm) than those seen in the nonfunctional tumor cells (100-180 nm). Also, tumor cells from the functional cases contained numerous dilated mitochondrial profiles.

Expression of the C-terminal flanking peptide of human progastrin in human gastroduodenal mucosa, G-cell hyperplasia and islet cell tumours producing gastrin.

Three antisera to the C-terminally extended form of gastrin or the C-terminal flanking peptide of progastrin were used in an attempt to investigate the post-translational processing of progastrin at the cellular level by light and electron microscopical immunocytochemistry. In the normal human gastric antrum, the G-cell secretory granules were found to contain both gastrin and the C-terminal progastrin determinants (progastrin 87-93, 87-95 and 93-101). Immunostaining of serial sections at the light microscopical level revealed that duodenal gastrin-containing cells also express the C-terminal progastrin determinants, as well as gastrin-34. In foetal tissue, cells containing C-terminal gastrin and the C-flanking peptide of progastrin were first seen at 8 weeks of gestation, in the duodenum. They were not found in the stomach until the 11th week. In hyperplastic G-cells and in gastrin-producing tumour cells, the level of C-terminal peptide immunoreactivity was variable and often lower than that seen in normal antrum and only minimal immunoreactivity could be detected using electron immunocytochemistry. This was interpreted as representing altered post-translational processing of progastrin in modified G-cells.

Transgenic mouse model: a new approach for the investigation of endocrine pancreatic B-cell growth.

The transformation and adaptation of pancreatic insulin-producing (B) cells has been studied in a transgenic mouse model using a panel of antisera recognising peptides and general neuroendocrine markers at both light and electron microscopical levels. Stages of tumour genesis in the transgenic mouse model from hyperplasia to neoplasia, have been compared with human B-cell tumours. A normal complement of peptide containing cells was seen in the transgenic mouse pancreas, but cells containing pro-insulin-derived peptides became more numerous as hyperplasia commenced. The transgenic mouse tumours were composed of B cells, although 30-35% of the tumours were also found to contain PP cells--a finding which is directly comparable with that in human insulin-producing tumours. NSE, 7B2 and chromogranin immunoreactivities were found in most cells from all the tumours examined. Antisera to PGP 9.5, a novel marker for elements of the neuroendocrine system, were found to stain hyperplastic and neoplastic B-cells intensely. In contrast, normal mouse B-cells did not show PGP 9.5 immunoreactivity thus it appears that PGP 9.5 is differentially expressed in transformed and/or growing mouse B-cells and hence may be used as an indicator in studies of early tumour growth.

FMRFamide-like immunoreactivity and arylamidase activity in turbellarians and nemerteans--evidence for a novel neurovascular coordinating system in nemerteans.

The tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) has been immunolocalized in the nervous systems of seven species of Turbellaria and four species of Nemertea. The 11 species represent all the major turbellarian and nemertean taxa, and illustrate most of the various life styles found in these animals. The FMRFamide-like reactivity coincides with histochemically demonstrable arylamidase activity in the nervous systems. It is suggested that the FMRFamide-like reactivity demonstrates the presence in these lower invertebrates of one or more biologically active peptides, analogous to those of higher invertebrates and chordates and acting as putative neurotransmitters and coordinators of growth, maturation and muscular activities. The arylamidases occurring with the peptides are probably an integral part of these peptide-mediated control systems. The nemertean vascular system is especially rich in arylamidases and is believed to be concerned primarily with peptidergic control of bodily functions, rather than with transport of metabolites.

Immunoglobulin E-dependent stimulation of human alveolar macrophages: significance in type 1 hypersensitivity.

Human alveolar macrophages were obtained during diagnostic bronchoalveolar lavage. Cells were cultured, and morphological examination (including electron microscopy) revealed that not more than 5% of the cultured cells were identifiable as cells other than alveolar macrophages. The cells were sensitized with human myeloma immunoglobulin E. and then challenged with anti-immunoglobulin E anti-sera. The experiments employed a highly specific monoclonal antibody and three affinity purified reagents. The formation of immunoglobulin E/anti-immunoglobulin E complexes facilitated release from alveolar macrophages of leukotriene B4, prostaglandin F2 alpha, thromboxane B2 and the lysosomal hydrolase N-acetyl-beta-D-glucosaminidase. There was no release of active oxygen species, with this stimulus, as measured by lucigenin chemiluminescence. Immunoglobulin E receptors were identified histochemically on the surface of human alveolar macrophages, and were visualized as conjugates with colloidal gold by electron microscopy. These results support the view that human alveolar macrophages may contribute to type 1 hypersensitivity reactions in the lung.

Ultrastructural evidence for the coexistence of calcitonin gene-related peptide and substance P in secretory vesicles of peripheral nerves in the guinea pig.

Using double immunogold staining procedures, calcitonin gene-related peptide (CGRP)-like and substance P (SP)-like immunoreactivities were localized at the ultrastructural level to guinea pig trigeminal ganglia, dorsal root ganglia and peripheral nerve fibres associated with the vascular system. CGRP-like and SP-like immunoreactivities were found consistently in large granular secretory vesicles (70-100 nm in diameter), and both peptide immunoreactivities were co-localized to the same vesicle in both sensory ganglion cells and within axons and their terminals in the adventitia and adventitial-medial border of the superior mesenteric artery. These results suggest that CGRP and SP are co-stored and may be released together from peripheral axons in the guinea pig.

Radioimmunoassay of alpha rat atrial natriuretic peptide.

Specific antibodies to alpha 1-28 atrial natriuretic peptide have been raised and used for radioimmunoassay of tissue extracts and for light and electron microscopic immunocytochemistry. The radioimmunoassay has been used to quantitate ANP-immunoreactivity in normal rat heart and hypothalamus and immunocytochemistry to demonstrate its localisation in specific tissue structures. Gel chromatography and high pressure liquid chromatography confirm that the majority of ANP-like immunoreactivity in the atria exists as high molecular weight forms. Rat hypothalamus contains immunoreactive ANP; the concentration per gram of tissue being 2-4 thousand fold less than that of the cardiac atria. The supraoptic region of the hypothalamus did not have a significantly different concentration of ANP-like immunoreactivity from the hypothalamic region as a whole. Immunocytochemical staining with ANP antiserum revealed the cardiac ANP-immunoreactivity to be concentrated around the nuclear poles within the cytoplasm of atrial muscle cells. Electron microscopic study of atrial cells stained with the immunogold technique confirmed the localisation of the ANP immunoreactivity to electron-dense secretory granules. The highest density of regional immunoreactive staining was in the subepicardial area, the lowest in the interatrial septum. The finding that the highest quantities of ANP immunoreactivity occur in areas subjected to the greatest distensional forces supports the hypothesis that atrial stretch is a stimulus to the release of this peptide from cells.

Immunocytochemical characterization of 10 pancreatic tumours, associated with the glucagonoma syndrome, using antibodies to separate regions of the pro-glucagon molecule and other neuroendocrine markers.

Histological diagnosis of neuroendocrine tumours can be hampered by their lack of peptide or amine immunoreactivity. In order to assess the usefulness of a range of specific and general markers of neuroendocrine differentiation, 10 pancreatic endocrine tumours, associated with high levels of circulating glucagon, were studied using histology, histochemistry, immunocytochemistry and electron microscopy. All cases showed immunoreactivity for one or other of the peptides derived from pro-glucagon, although only seven were found to contain immunoreactive pancreatic glucagon. The presence of secretory granules in eight of the tumours was demonstrated by electron microscopy, argyrophilia or chromogranin immunoreactivity. Not only was neuron specific enolase positively immunostained in all the tumours, thereby revealing their neuroendocrine nature, but also the intensity of the immunostain was higher in four of the five malignant ones than in the rest of the cases. Pancreatic polypeptide was present in non-glucagon cells in six out of 10 cases. Our results emphasize the importance of the use, not only of general histochemical and immunocytochemical tests but also antibodies to all possible derivatives of the precursor form of the active tumour product in the diagnosis of possible endocrine tumours. In this way, any abnormal molecular forms of the peptide synthesized by tumour cells with altered synthetic and secretory mechanisms may be detected.

Somatostatin-containing D cells exhibit immunoreactivity for rat somatostatin cryptic peptide in six mammalian species. An electron-microscopical study.

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 SS-28-(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.

Calcitonin gene-related peptide-immunoreactive nerve fibres in the small intestine of the guinea-pig: electron-microscopic immunocytochemistry.

Calcitonin gene-related peptide (CGRP)-containing nerve fibres were identified by pre- and post-embedding electron-microscopic immunocytochemistry in the guinea-pig small intestine. Immunoreactive nerve processes were numerous in the mucosa and submucosa, especially in the connective tissue among the crypts of Lieberkühn. In some cases they were found in close apposition to epithelial cells. Many of the labelled nerve fibres were observed around blood vessels, especially arterioles. In the inner circular muscle layer, the immunoreactive nerve processes were found in close association (sometimes less than 40 nm) to smooth muscle cells. CGRP-positive terminals contained a predominance of electron-lucent synaptic vesicles (35-40 nm in diameter) together with a few large granular vesicles (80-120 nm in diameter). Post-embedding immunostaining, using the immunogold procedure, localized CGRP-immunoreactivity in large granular vesicles, 80-92 nm in diameter. These ultrastructural observations confirm that CGRP-containing nerve fibres exist in the small intestine and suggest that they may participate in the regulation of the smooth muscle activity, mucosal cell secretion and blood flow and, by analogy with other systems, a sensory role also seems likely.

Medullary carcinoma of the thyroid. An immunocytochemical and histochemical study of 25 cases using eight separate markers.

The current study was undertaken on 25 cases of thyroid medullary carcinoma to compare the diagnostic value of calcitonin with other peptides including PDN-21, the C-terminal flanking peptide of human calcitonin within the calcitonin precursor, and calcitonin gene-related peptide, CGRP. Antiserum raised to chromogranin, an acidic protein of 68,000 daltons, was also used to compare its diagnostic value as a general marker for neuroendocrine neoplasia with neuron-specific enolase (NSE) and Grimelius' argyrophil silver staining. Immunocytochemistry was performed using the peroxidase-antiperoxidase method at the light microscopic level and the immunogold staining procedure at the ultrastructural level. All tumors were reactive to calcitonin and CGRP antisera, whereas PDN-21 was present in 23 cases. It was also found that these peptides were colocalized in the majority of C-cells. The intensity and specificity of CGRP and PDN-21 immunoreaction was comparable to and in some cases even better than that obtained with calcitonin antiserum. In the majority of tumors, somatostatin and bombesin immunoreactivity was either absent, weak, or variable in intensity and distribution. The current study thus demonstrates that together with calcitonin, PDN and, in particular, CGRP antisera may be applied to corroborate immunocytochemical diagnosis in medullary carcinoma of the thyroid. With regard to general neuroendocrine markers, Grimelius' and chromogranin provided the most consistent results. NSE isoenzyme immunoreactivity, on the other hand, was more variable, probably reflecting the metabolic state of the tumor cells.