Cloning and expression of neuron-specific enolase in the corpus luteum of dairy goats

Neuron-specific enolase (NSE) is the key molecular marker for diffuse neuroendocrine system (DNES) cells, its expression in the pregnant corpus luteum (CL) of dairy goats was studied by the immunofluorescence method and the ultra structural characteristics of luteal cells were detected by the electron microscopy to identify the existence of DNES cells in the pregnant CL of dairy goats. Besides, the coding sequence of dairy goats NSE gene was cloned and its biological information was analyzed. Results revealed that NSE immunopositive cells exhibited widespread cytoplasmic staining throughout the whole pregnant CL. In addition, these cells showed typical characteristics of DNES cells in the electron microscopy. These results suggested that many DNES cells exist in the pregnant CL of dairy goats. Meanwhile, we identified the coding sequence of dairy goats NSE (GenBank Accession No. JN887466). Its nucleotide sequence homology was found to be 97.9%, 89.3%, 90% and 92.6%, respectively, compared with that of Bos taurus, Rattus norvegicus, Mus musculus and Homo sapiens, while the amino acid sequence homology was 99.1%, 97%, 97.2% and 98.2% respectively. These results first showed that the functional amino acids coded by the NSE gene were highly conserved in Caprine, B. taurus, R. norvegicus, M. musculus and H. sapiens. It was implied that the gene NSE in dairy goats had close homology to that of NSE of other species. Our findings demonstrated the possible existence of DNES cells in pregnant CL, providing new clue for further understanding of interactions between the neuroendocrine and reproductive systems. Characterization of gene sequence of dairy goats NSE will enable us to synthesize interference RNA for further study on the role of NSE on the formation, function and apoptosis of pregnant CL in dairy goats.     

Embryology of the diffuse neuroendocrine system and its relationship to the common peptides.

The diffuse neuroendocrine system is constituted by the cells, now more than 40 in number, of the central and peripheral divisions of the amine precursor uptake and decarboxylation (APUD) series. At one time presumed to be derived from a common "neural" ancestor, all are now deemed to be "neuroendocrine-programmed," arising either in the embryonic epiblast itself or in one of its principal descendants. The APUD cells produce more than 35 physiologically active peptides and a small number of equally active amines. Within the last 3 years, 17 of these peptides have been identified jointly in endocrine cells and in neuronal cell bodies or processes. Sharing in this way a neural and an endocrine location and site of production, they are called the "common peptides." The diffuse neuroendocrine system is to be regarded as a third division of the nervous system, whose products suppress, amplify, or modulate the activities of the other two divisions. The relationship of its products to the cells and processes of these two divisions is currently the object of intensive inquiry.     

Cytochemical evidence for the neural crest origin of mammalian ultimobranchial C cells

Summary The cells of the neural crest have APUD properties at an early stage of devel opment (72 hours in the chick embryo). The FIF procedure provides a cytochemical means for their distinction.Using mouse embryos from mothers injected, intraperitoneally, 1 hr before removal, with l-DOPA (100 mg/kg), the peripheral stream of neural crest cells was clearly identifiable at the 7-somite stage (7–8 days). At the 10-somite stage (8–9 days) the cells were observed to invade the lateral processes of the foregut, and the foregut itself. A particularly high concentration of fluorescent APUD cells was observed in the anterior portion of the IVth pharyngeal pouch, destined to become the ultimobranchial body.At the 14-somite stage (11–12 days) the developing ultimobranchial body still contains fluorescent cells of neural crest origin.The implications of these findings on the question of the origin of the entire APUD series of endocrine polypeptide cells is discussed.     

The neuroendocrine phenotype,cellular plasticity,and the search for genetic switches: redefining the diffuse neuroendocrine system

The term neuroendocrine has been used to define cells that secrete their products in a regulated manner, in response to a specific stimulus. The neuroendocrine system includes neurons and endocrine cells sharing a common phenotypic program characterized by the expression of markers such as neuropeptides, chromogranins, neuropeptide processing enzymes SPC2 and SPC3 (subtilase-like pro-protein convertases) or dense core secretory granules. Various theories such as the APUD (amine precursor uptake decarboxylation) concept, the diffuse neuroendocrine system (DNES) or the paraneuron concept have been put forth to classify neuroendocrine cells as a cohesive group. Neuroendocrine characteristics have been used as evidence of a common embryological origin for normal and neoplastic cells. However, it is now recognized that neuroendocrine characteristics can be observed in various cell types, such as immunocytes, that are not of a common embryological origin with either neurons or endocrine cells. We propose to redefine previous "neuroendocrine" concepts to include the notion that activation of specific genetic switches can lead to the expression of a partial or full neuroendocrine phenotype in a variety of cell types, including immune cells.     

Endocrine markers of cellular immunity: defining the endocrine phenotype

The term neuroendocrine has been used to define cells that secrete their products in a regulated manner, in response to a specific stimulus. The neuroendocrine system includes neurons and endocrine cells sharing a common phenotypic program characterized by the expression of markers such as neuropeptides, chromogranins, neuropeptide processing enzymes SPC2 and SPC3 (subtilase-like pro-protein convertases) or dense core secretory granules. Various theories such as the APUD (amine precursor uptake decarboxylation) concept, the diffuse neuroendocrine system (DNES) or the paraneuron concept have been put forth to classify neuroendocrine cells as a cohesive group. Neuroendocrine characteristics have been used as evidence of a common embryological origin for normal and neoplastic cells. However, it is now recognized that neuroendocrine characteristics can be observed in various cell types, such as immunocytes, that do not share a common embryological origin with either neurons or endocrine cells. We propose to redefine previous "neuroendocrine" concepts to include the notion that activation of specific genetic switches can lead to the expression of a partial or full neuroendocrine phenotype in a variety of cell types, including immune cells.     

The dynamics of morphological changes in the pyloric endocrine cells of rats with uremia

Disturbances in renal homeostatic function lead to changes in endocrine cell secretory activity. The aim of this study was the histomorphological estimation of dependence of gastric APUD system cell morphology and function on the time after subtotal nephrectomy in Wistar rats. Fragments of gastric pylorus were collected 1. 2, 4, and 6 weeks after nephrectomy. Paraffin sections were stained with H+E and by silver impregnation. Immunohistochemical reactions with the use of specific antibodies against calcitonin gene-related peptide (CGRP), synaptophysin (SPh). somatostatin (ST), and neuron-specific enolase (NSE) were also performed. Immunoreactivity of the examined substances in the pyloric mucosa in the first week after nephrectomy was lower than in the control group. However, in the following time intervals, endocrine cells showed stronger immunostaining in comparison with the control rats. The results suggest that chronic renal failure can modulate secretory activity of APUD system cells.     

Immunoreactivity of neuroendocrine cells in the respiratory tract in rats with experimental uremia after thyroparathyroidectomy

Animals with experimental uremia, which underwent thyroparathyroidectomy, reveal numerous metabolic disorders that can influence morphology and activity of endocrine cells of the scattered neuroendocrine system. The aim of the study was the evaluation of the influence of thyroparathyroidectomy in rats with chronic renal failure on APUD system cells localized in the respiratory tract. The examination was conducted on the group of 20 rats. Thyroparathyroidectomy was performed 30 days after nephrectomy. Fragments of the lungs and trachea were collected 14 days after the operation. Routinely prepared paraffin sections were stained with H+E and with silver method. The immunohistochemical reactions were conducted with the use of antibodies against calcitonin (CT), synaptophysin (SPh), somatostatine (ST), and neuron-specific enolase (NSE) The results were estimated in light microscope on the basis of stain reaction of endocrine cells. Our examination showed that chronic renal failure affects the functioning of endocrine cells. We also observed the increase in APUD system cell number in the trachea and the lungs after thyroparathyroidectomy in uremic rats.     

胎儿胰腺发育中CgA和NSE的表达及其意义

目的检测人胎儿胰腺中CgA和NSE的表达特征,初步探讨胎儿胰腺发育过程弥散神经内分泌系统的形成及其生物学作用.方法用免疫组织化学SP法,检测嗜铬素A(chromograin A,CgA)和神经元特异性烯醇化酶(neuron specific enolase,NSE)在不同胎龄胎儿胰腺组织中的表达.结果CgA在16-38周的胰腺中均有表达,随胎儿发育,CgA阳性细胞分布状态和反应程度均有差异变化;NSE在22-38周胎儿胰腺中表达,集中定位于胰腺的内分泌部细胞.结论CgA和NSE在人胎儿胰腺中的阳性表达,反映出弥散神经内分泌系统在胎儿胰腺中的形成过程;表明胎儿胰岛的形成及其DNES细胞的分泌,有调节胰腺外分泌部发育分化的作用,也提示胰岛DNES细胞通过调节血糖可能对胎儿个体发育具有重要影响.     

Fine structure and serotonin immunohistochemistry of the neuroendocrine cells in the lungs of the bichirs Polypterus delhezi and P. ornatipinnis

Neuroendocrine cells (NE) occurring in the pulmonary epithelium of the fishes Polypterus delhezi and P. ornatipinnis are studied by electron microscopy and by immunostaining for serotonin which is often present in such cells in the mammalian lung. With the electron microscopy NE are found to occur single, resting upon the basement membrane and forming a narrow cytoplasmic extension towards the air lumen. They contain dense-cored vesicles of 80-165 nm which form exocytotic profiles at the level of the basal membrane. An immunoreactivity for serotonin is demonstrated for the first time in the NE of these species. The role of this mediator may involve a paracrine or endocrine function as postulated for the respiratory neuroendocrine mammalian cells. NE of the species studied are considered similar to those found within the wall of lung airways in mammals and submammalian vertebrates. Although much immunocytochemical investigations remain to be executed, they may also be included in the APUD (or DNES) cell system.     

Neuron specific enolase: a marker for the early development of nerves and endocrine cells in the human lung

Neuron specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase, has been established by immunocytochemical means as a marker of morphological and functional maturation in central neurons and appears late in development. However, little is known about the presence of NSE in developing peripheral neurons and endocrine cells and its relationship to the development of classical neurotransmitters and peptides. We therefore investigated the appearance of NSE immunoreactivity in nerves and mucosal endocrine cells of the human respiratory tract in foetal, neonatal and adult life. NSE was found to be present in neuroblasts, nerve fibres and endocrine cells from the earliest period of gestation examined (8 weeks), before the appearance of acetylcholinesterase activity (10-12 weeks), dopamine-beta-hydroxylase (20 weeks), vasoactive intestinal polypeptide (20 weeks) or calcitonin (20 weeks). Bombesin-like immunoreactivity was found in a small proportion of mucosal endocrine cells as early as eight weeks in the foetal respiratory tract. These findings indicate that unlike central neurons and their processes, peripheral neurons of the lung contain NSE immunoreactivity well before full maturation and establishment of synaptic contact with end organs.     

NSE/αNAE positivity in B-lineage acute lymphoblastic leukemia: revisiting a potential cytochemical diagnostic pitfall

Cytochemical staining for leukemia typing is declining in hematology laboratories, but the use of flow cytometry may not be possible in some settings. Aberrant cytochemical nonspecific esterase/α-naphthyl acetate esterase (NSE/αNAE) positive B-lymphoblasts can cause confusion with monoblasts, a potentially dangerous pitfall. This unusual cytochemical NSE/αNAE positivity had been associated with relatively poorer outcome of acute lymphoblastic leukemia (ALL) in the era prior to the advent of routine multicolor flow cytometric immunophenotyping. We reviewed morphological, cytochemical and flow-cytometric data from five cases of B-lineage ALL that showed NSE/αNAE positivity and were diagnosed definitively using multi-parametric flow cytometric immunophenotypic analysis. Diffuse or dot-like (localized) strong cytochemical NSE/αNAE activity was detected in all cases and all showed one or more features of high risk disease. The number of NSE/αNAE positive blasts in the marrow varied from 10 to 75%. The morphological differential diagnoses included T-lymphoid lineage ALL and acute monoblastic leukemia (AML-M5). Flow cytometric data revealed B-lineage antigens and the absence of monocytic or other myeloid markers resolved the diagnosis. These cases underscore the importance of immunophenotyping in all cases of suspected ALL regardless of the cytochemical findings. Although the numbers are small, the association with high risk disease observed in all five of our cases may corroborate the previously reported poor prognostic value of such aberrant cytochemical staining.     

Localization and possible function of polyamines in protein and peptide secreting cells

Three different cytochemical methods, the formaldehyde-fluorescamine and the ortho-phtalaldehyde fluorescence cytochemical methods as well as immunocytochemistry have been developed for localising the polyamines spermidine and spermine in tissue. All three methods produce identical results and show polyamines to occur inter alia in high concentrations in certain protein- and peptide-secreting cells. Many of these cells also show the capacity to metabolise monoamines and belong to the amine content or amine precursor uptake and decarboxylation (APUD) series. In cell types where fluorescence microscopical resolution allows, most polyamines appear to be localised to secretory granules. Moreover, studies on isolated pancreatic islets reveal active and glucose-dependent polyamine biosynthesis to occur in insulin cells. Possible function of polyamines in secretory granules are discussed in the light of the above findings.     

Cytochemistry of membrane proteases

Summary Membrane proteases that are detectable by cytochemical means are the classified exopeptidases, aminopeptidases A and M (or N), -glutamyl transpeptidase (which also acts as transferase), dipeptidyl peptidase IV and the endopeptidase, enteropeptidase (also known as enterokinase). Not yet classified are the possible expeptidase, tripeptidyl peptidase and endopeptidases I (Ala-endopeptidase) and II (Arg-endopeptidase). All these membrane proteases can be investigated with either chromogenic or fluorogenic procedures using synthetic peptide substrates. The most useful substrates are 4-methoxy-2-naphthylamine amino acids and peptides for cytochemical localizations at the light and electron microscope levels, for cytophotometric quantification and the study of membrane protease isoenzymes after analytical isoelectric focusing. Amino acid or peptide derivatives of naphthylamine AS can be recommended for light microscopical localization and cytofluorometric quantification, and 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin amino acids and peptides for the development of enzyme bands after isoelectric focusing. Cytochemistry reveals the heterogeneity in the distribution and species differences of membrane proteases in adult cells, tissues and organs and during development. It also reveals some common localizations, such as in small intestinal enterocytes and proximal tubule cells. The species and organ differences are substantiated and extended considerably by isoelectric focusing in combination with methods for the cytochemical detection of proteases. In addition, continuous cytophotometry or cytofluorometry (section and cultured cell biochemistry) allows the kinetic characteristics, initial reaction rates and maximum activities of all membrane proteases to be determined.The physiological functions of the endopeptidases and exopeptidases are still a matter of debate. However, from cytochemical inhibition studies with natural peptide substrates, e.g. peptide hormones, there is increasing evidence that the proteases detected with synthetic peptides play a decisive role in many physiological circumstances, e.g. in endocrine regulation mechanisms or the regulation of blood pressure. In this respect, capillary endothelium-linked surface membrane proteases may be especially important.     

The efficient isolation of murine splenic dendritic cells and their cytochemical features

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.     

Biomarkers and molecular imaging in gastroenteropancreatic neuroendocrine tumors

Neuroendocrine gastrointestinal and pancreatic tumors (GEP-NETs) are a heterogenous group of cancers with various clinical expressions. All tumors produce and secret various amines and peptides, which can be used as tissue and circulating markers. Chromogranin A (CgA) is a general tumor marker stored in secretory granules within the tumor cell and released upon stimulation. CgA is the best general tumor marker at the moment, expressed in 80-90% in all patients with GEP-NETs. CgA and NSE are used as tissue markers for the delineation of the neuroendocrine features of the tumors, but recently also the proliferation marker Ki-67 has been included in the standard procedure for evaluation of the proliferation. GEP-NETs are classified into well differentiated neuroendocrine tumors (Ki-67<2%), well-differentiated neuroendocrine carcinoma (Ki-67 2-20%), poorly differentiated neuroendocrine carcinoma (Ki-67>20%). The molecular imaging of NETs is based on the ability of these tumor cells to express somatostatin receptors as well as the APUD features. Octreoscan has been applied for imaging and staging of the disease for more than 2 decades and will nowadays be replaced by 68Ga-DOTA-Octreotate, with higher specificity and sensitivity. 18Fluoro-DOPA and 11C-5HTP are specific tracers for NETs with high specificity and selectivity. A new potential biomarker is auto-antibodies to paraneoplastic antigen MA2, which might indicate early recurrence of carcinoids after surgery with a curative intent. Circulating tumor cells (CTC) have been applied in GEP-NETs quite recently. There is still an unmet need for new markers.     

A cytochemical and immunofluorescence study of endocrine cells in the gut of the ascidian Styela clava

Summary Strong secretin-like immunofluorescence has been demonstrated in endocrine-like cells from the gastric epithelium of Styela. These cells also stain with lead haematoxylin and exhibit a brilliant formaldehyde-induced fluorescence, but do not show any other cytochemical features characteristic of the mammalian APUD series. Tests with antisera to glucagon, gastrin and somatostatin all proved negative. In the oesophagus tests with all four antisera proved negative. The significance of these results is discussed in relation to the phylogeny of vertebrate gastro-intestinal hormones.     

Uptake of L-dopa and L-5-hydroxytryptophan by endocrine-like cells in the rat larynx

Synopsis In the larynx of the rat, and to a lesser extentin the trachea and main bronchi, there are numerous cells which possess the amine-handling characteristics, but not the enzymatic ones, of the APUD series of endocrine polypeptide cells. The cytochemical properties of their contained granules, which are ultrastructurally atypical in reaching sizes up to 2000 nm on their longest axis, are not those of the APUD series since their protein is predominantly basic and arginine-rich, rather than acidic.A weak formaldehyde-induced autofluorescence is present in the cells, due to a substance with an excitation maximum at 400 nm and an emission maximum at 490 nm. This may be dopamine.The true nature of these endocrine-like cells is not apparent. They are regarded, for the present, as being related to cutaneous melanocytes.     

Demonstration of the neural crest origin of type I (APUD) cells in the avian carotid body,using a cytochemical marker system

Summary The biogenic amines present in the carotid body Type 1 cells of two avian species (Japanese quail and chicken) were identified, by microspectrofluorometry of formaldehyde-induced fluorescence, as dopamine and 5-hydroxytryptamine respectively. These and other cytochemical properties establish the cells as members of the APUD series.Grafts of the neural rhombencephalic primordium from 6 to 10-somite quail embryos were implanted in the appropriate region of chick embryos of the same age. After up to 11 days incubation the carotid bodies of the host were freeze-dried and treated with hot formaldehyde vapour. The carotid body Type 1 cells in the chick host were identified, by the presence of dopamine and the absence of 5-HT, as cells from the quail neural crest.The dopamine phenotype in cells of quail origin thus provides a cytochemical marker which may be used for other allograft experiments. The present work confirms earlier findings, using a biological (nuclear chromatin) marker, which showed the avian carotid body to be of neural crest origin.     

APUD cells the human pulmonary neuroendocrine system

Literature data are presented on the anatomy of the diffuse neuroendocrine system (DNES) of the human lungs. The following subjects are discussed: physiological effects of the biologically active substances synthesized, stored, and secreted by the pulmonary neuroendocrine cells; their immunomodulating function; and the role of the DNES in the pathogenesis of bronchial asthma and other bronchopulmonary diseases.     

Localization of immunoreactive beta-endorphin and adrenocorticotropic hormone and pro-opiomelanocortin mRNA to rat testicular interstitial tissue macrophages.

Pro-opiomelanocortin (POMC) gene expression and POMC peptides have been demonstrated in the Leydig cells of the testis, although selective removal of the Leydig cells with the cytotoxic drug ethane dimethane sulfonate did not significantly reduce levels of testicular POMC mRNA or peptides in adult rats. Since macrophages in the rat spleen synthesize POMC peptides, we investigated whether isolated macrophages from the adult rat testis may be an additional source of POMC-derived peptides. Testicular macrophages were isolated by collagenase treatment of adult rat testes and adherence to siliconized glass coverslips; the biological, cytochemical and immunological characteristics of the attached cells were compared with those of Leydig cells purified by Percoll gradient centrifugation. Macrophages in the cell preparations were identified by positive esterase cytochemical staining, latex bead ingestion, and immunocytochemical staining with ED2 (a macrophage-specific monoclonal antibody), and an absence of 3 beta-hydroxysteroid dehydrogenase cytochemical staining. Leydig cells in the purified preparations were positive for 3 beta-hydroxysteroid dehydrogenase and esterase staining but negative with ED2, and were not phagocytic. Based on these criteria, the purities of the macrophage and Leydig cell preparations employed in this study were estimated to be 87 +/- 4% and 91 +/- 3%, respectively. Cytoplasmic beta-endorphin (beta EP) immunoreactivity (ir) was present in 62 +/- 9% of cells in the purified Leydig cell preparations--confirming these cells as a source of POMC-derived peptides. In addition, ir-beta EP and ir-ACTH were localized to the cytoplasm of a similar proportion of cells (beta EP, 62.5 +/- 5%; ACTH, 64 +/- 5%) in macrophage preparations.(ABSTRACT TRUNCATED AT 250 WORDS)