Demonstration and characterization of hippocampal cholinergic neurostimulating peptide (HCNP) processing enzyme activity in rat hippocampus

Hippocampal cholinergic neurostimulating peptide (HCNP) stimulates cholinergic activity of cultured medial septal nuclei explants. It consists of eleven amino acids that are located at the N-terminal region of its precursor protein. This report concerns the demonstration and characterization of an HCNP processing enzyme that cleaves the bioactive undecapeptide from the precursor. The enzyme was purified from the hippocampus of young Wistar rats. A synthetic deacetylated peptide (peptide_1–26) consisting of the N-terminal 26 amino acids of the HCNP precursor protein served as substrate. The product of the enzyme reaction was identified and quantitated by HPLC using deacetylated HCNP as standard. The amount of undecapeptide generated was directly proportional to the time of incubation of the enzyme reaction mixture. From molecular sieving chromatography it was estimated that the molecular mass of the enzyme is close to 68 kDa. The HCNP processing enzyme has a pH optimum of 6.0 and a K_m of 0.50 mM for peptide_1–26. Preincubation at 56°C causes rapid inactivation of the HCNP processing activity. Enzyme activity is enhanced by EDTA and 1,4-dithiothreitol, and inhibited by antipain, chymostatin and E-64. These findings suggest that the enzyme probably has a thiol group in its active site. This novel enzyme of the hippocampus may represent a valuable tool for further studies on the general protein metabolism in the central nervous system, as well as for elucidating the neurochemical aspects of neurodegenerative disorders.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

T-cadherin as a receptor regulating angiogenesis and blood vessel remodeling

The search for a membrane receptor responsible for hormone-like effects of low density lipoproteins (LDL) has revealed two proteins (Mol. wt. 105 and 130 kDa) in the membrane fraction of human aortic smooth muscle cells. These proteins were identified as mature T-cadherin and its unprocessed precursor. T-cadherin was originally cloned from chick embryo brain, where it was implicated in axon guidance in the developing nervous system. Our study on the T-cadherin distribution in human organs and tissues has indicated that T-cadherin is specifically expressed in nervous and cardiovascular system. However, physiological significance of T-cadherin expression in the vasculature, as well as intracellular signaling pathways mediating its effects remain obscure. This review summarizes our current knowledge about intracellular signaling utilized by T-cadherin and discusses possible functions of T-cadherin in the vasculature.     

The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.     

Immunohistochemical localization of insulin-like growth factor I in the adult rat

Rabbit antisera against native human insulin-like growth factor I (IGF-I; somatomedin C) or a synthetic tetradecapeptide, representing the carboxyterminal amino acids 57-70 of human IGF-I, were used to map immunohistochemically the distribution of IGF-I immunoreactive material in adult rats. Both antisera were specific for IGF-I, as characterized by immunoabsorption, immunoblotting and radioimmunoassay. There was no cross-reactivity to IGF-II, relaxin or pro-insulin; substances having a high degree of structural homology with IGF-I. High IGF-I immunoreactivity was observed in spermatocytes of the testis; in oocytes, granulosa and theca interna cells of the ovary during early stages of follicle development; in some lymphocytes and in reticular cells of lymphoid and hematopoietic organs; in salivary gland duct cells; in the adrenal medulla, the parathyroid gland and the Langerhans' islets. Chondrocytes in the epiphyseal and rib growth plates and at articular surfaces showed strong IGF-I immunoreactivity. Brown but not white fat cells were stained. Nerve cells in the peripheral and autonomic nervous system showed faint to intense IGF-I immunoreactivity. In contrast, neurons and neuroglial cells in the central nervous system were generally negative; motor neurons being an exception. Erythropoietic, thrombocytopoietic and myeloic cells in the bone marrow showed IGF-I immunoreactivity, but only at defined developmental stages. Hepatocytes showed faint IGF-I immunoreactivity, but became more intensely stained after pretreatment with colchicine. The present results suggest that IGF-I is synthetized by cells in several tissues and organs in the adult rat. There was an apparent association between the localization of IGF-I and cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of insulin-like growth factor I in the adult rat

Summary Rabbit antisera against native human insulin-like growth factor I (IGF-I; somatomedin C) or a synthetic tetradeca peptide, representing the carboxyterminal amino acids 57–70 of human IGF-I, were used to map immunohistochemically the distribution of IGF-I immunoreactive material in adult rats. Both antisera were specific for IGF-I, as characterized by immunoabsorption, immunoblotting and radioimmunoassay. There was no cross-reactivity to IGF-II, relaxin or pro-insulin; substances having a high degree of structural homology with IGF-I.High IGF-I immunoreactivity was observed in spermatocytes of the testis; in oocytes, granulosa and theca interna cells of the ovary during early stages of follicle development; in some lymphocytes and in reticular cells of lymphoid and hematopoetic organs; in salivary gland duct cells; in the adrenal medulla, the parathyroid gland and the Langerhans' islets. Chondrocytes in the epiphyseal and rib growth plates and at articular surfaces showed strong IGF-I immunoreactivity. Brown but not white fat cells were stained. Nerve cells in the peripheral and autonomic nervous system showed faint to intense IGF-I immunoreactivity. In contrast, neurons and neuroglial cells in the central nervous system were generally negative; motor neurons being an exception. Erythropoeitic, trombocytopoeitic and myeloic cells in the bone marrow showed IGF-I immunoreactivity, but only at defined developmental stages. Hepatocytes showed faint IGF-I immunoreactivity, but became more intensely stained after pretreatment with colchicine.The present results suggest that IGF-I is synthetized by cells in several tissues and organs in the adult rat. There was an apparent association between the localization of IGF-I and cell differentiation. Certain cells involved in secretory processes also displayed high IGF-I immunoreactivity. The wide distribution of IGF-I indicates that the circulating pool of IGF-I has multiple origins.     

The Expression of GHS-R in Primary Neurons Is Dependent upon Maturation Stage and Regional Localization

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer’s disease.     

Selective fixation of methylselenobenzoic acid by the pineal gland]

Investigation of the localization in the central nervous system of the rat of an epileptogenic agent, methylseleno-2-benzoic acid, did not lead to its selective distribution in the cerebral cortex or in the brainstem in relation with its biological activity. But a systematic study of the distribution of this compound labelled with 75Se at a high specific activity has revealed a rate of fixation by the pineal gland 4 to 5 times higher than that of other tissues of the central nervous system. After a survival time of 4 hours, the radioactivity of the pineal gland exceeds that of the blood. A parallel study of the distribution of the 75SeO3- ion on the one hand of the 35S homolog of the 75Se compound on the other hand has demonstrated that the fixation by the pineal gland is bound to the molecular structure of the selenium compound.     

Localization of epidermal growth factor precursor in tooth and lung during embryonic mouse development

The murine epidermal growth factor (EGF) precursor is a 1217 amino acid protein which contains mature EGF (amino acid residues 977-1029) as well as eight EGF-like repeats. Although the highest levels of EGF are found in the adult male mouse submandibular gland, the results of in situ hybridization studies and mRNA analyses suggest that EGF precursor mRNA is synthesized in several adult mouse tissues including the lung and the incisor. To determine if EGF precursor gene expression is intrinsic to the developmental program for either embryonic tooth or lung organogenesis, sense and antisense oligodeoxyribonucleotide probes corresponding to amino acids 1070-1081 of the precursor were used to localize cellular sites of synthesis of EGF precursor mRNA by in situ hybridization. Antibodies directed against amino acid residues 348-691 of the precursor were used in immunodetection techniques to identify either EGF precursor protein or processed derivatives. In contrast to earlier reports indicating that embryonic mouse tissues do not synthesize EGF precursor mRNA, we found that EGF precursor mRNA is present in clusters of ectoderm-, mesoderm-, and ectomesenchyme-derived cells associated with embryonic teeth and lung organs. Moreover, epitopes common to the EGF precursor were immunolocalized in both the epithelial and mesenchymal tissues of embryonic mouse tooth and lung organs. These results suggest that the EGF precursor and/or motifs contained within the precursor molecule, including mature EGF, may play an instructive or permissive role in epithelial-mesenchymal interactions pursuant to organogenesis.     

Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails

The distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, were examined in the nervous system of pulmonate snails. We chemically identified the authentic NdWFamide from a land snail (Euhadra congenita) and a freshwater snail (Lymnaea stagnalis). NdWFamide potentiated the heartbeat of those snails. Immunohistochemistry using anti-NdWFamide antibody demonstrated the distribution of NdWFamide-containing neurons and fibers in the central nervous system, as well as peripheral tissues, such as the cardiovascular region and accessory sex organs. These results suggest that NdWFamide is a neuropeptide mediating the neural regulation of the activity of the cardiovascular and reproductive systems of snails.     

First case of feline spongiform encephalopathy in a captive cheetah born in France: PrP<Superscript>sc</Superscript> analysis in various tissues revealed unexpected targeting of kidney and adrenal gland

Feline spongiform encephalopathy (FSE), affecting domestic and captive feline species, is a prion disease considered to be related to bovine spongiform encephalopathy. Here we report an immunohistological analysis of the first FSE-affected cheetah born in France. The duration of clinical signs, of which ataxia was the main one, was about 8 weeks. The distribution of abnormal prion protein (PrP(sc)) was studied by immunohistochemistry within 27 different tissues. Different antibodies were used to visualise abnormal PrP deposits in situ. PrP(sc )accumulation was detected in the central nervous system (cerebral cortex, cerebellum, brain stem, spinal cord, retina), in peripheral nerves and in lymphoid organs. PrP(sc) deposits were not observed within the enteric nervous system nor in several other organs, such as pancreas, ovary, liver and muscle. More interestingly, unusual PrP(sc )deposits were observed within the zona fasciculata/reticularis of the adrenal gland and within some glomeruli of the kidney raising the question of possible PrP(sc) excretion. The sympathetic innervation of these two organs was visualised and compared to the distribution of PrP(sc) deposits. Our results suggest the possibility that the infectious agent is spread by both haematogenous and nervous pathways.     

Distribution of Peripheral PrPSc in Sheep with Naturally Acquired Scrapie

Accumulation of prion protein (PrPSc) in the central nervous system is the hallmark of transmissible spongiform encephalopathies. However, in some of these diseases such as scrapie or chronic wasting disease, the PrPSc can also accumulate in other tissues, particularly in the lymphoreticular system. In recent years, PrPSc in organs other than nervous and lymphoid have been described, suggesting that distribution of this protein in affected individuals may be much larger than previously thought. In the present study, 11 non-nervous/non-lymphoid organs from 16 naturally scrapie infected sheep in advanced stages of the disease were examined for the presence of PrPSc. Fourteen infected sheep were of the ARQ/ARQ PRNP genotype and 2 of the VRQ/VRQ, where the letters A, R, Q, and V represent the codes for amino-acids alanine, arginine, glutamine and valine, respectively. Adrenal gland, pancreas, heart, skin, urinary bladder and mammary gland were positive for PrPSc by immunohistochemistry and IDEXX HerdChek scrapie/BSE Antigen EIA Test in at least one animal. Lung, liver, kidney and skeletal muscle exhibited PrPSc deposits by immunohistochemistry only. To our knowledge, this is the first report regarding the presence of PrPSc in the heart, pancreas and urinary bladder in naturally acquired scrapie infections. In some other organs examined, in which PrPSc had been previously detected, PrPSc immunolabeling was observed to be associated with new structures within those organs. The results of the present study illustrate a wide dissemination of PrPSc in both ARQ/ARQ and VRQ/VRQ infected sheep, even when the involvement of the lymphoreticular system is scarce or absent, thus highlighting the role of the peripheral nervous system in the spread of PrPSc.     

Immunohistochemical localization of carbonyl reductase in human tissues.

Carbonyl reductase, an NADPH-dependent oxidoreductase of broad specificity, is present in many human tissues. Its precise localization, however, has remained unclear, as well as its physiological and possible pathophysiological significance. The present study reports the immunohistochemical localization of the enzyme in normal human tissues. Immunostaining was detectable in all organs investigated. The highest concentrations were found in the parenchymal cells of the liver, the epithelial cells of the stomach and small intestine, the epidermis, the proximal tubules of the kidney, neuronal and glial cells of the central nervous system, and certain cells of the anterior lobe of the pituitary gland. Consistently pronounced staining was also observed in smooth muscle fibers and the endothelium of blood vessels. The results are in agreement with a housekeeping function of carbonyl reductase in the elimination of reactive carbonyl compounds.     

A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands

In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture system is a useful tool to analyze humoral communication between different tissues or cell populations.     

Distribution and characterization of immunoreactive prolactin-releasing peptide (PrRP) in rat tissue and plasma

We established a sensitive and specific two-site enzyme immunoassay (EIA) for prolactin-releasing peptide (PrRP) using two region-specific monoclonal antibodies. We investigated the tissue distribution and the plasma concentration of immunoreactive (ir-) PrRP in rats using this assay. Ir-PrRP was widely distributed in the central nervous system and pituitary gland. The highest concentration of ir-PrRP was found in the hypothalamus. In peripheral tissues, appreciable levels of ir-PrRP were found only in the adrenal gland. The mean plasma concentration of ir-PrRP was 0.13 +/- 0.01 fmol/ml (mean +/- SEM). In reverse-phase and gel-filtration high performance liquid chromatography, hypothalamic ir-PrRP eluted at a position identical to that of PrRP31 and PrRP20. On the other hand, ir-PrRP from the adrenal gland and plasma eluted only at the position of synthetic PrRP31, indicating that molecular forms of ir-PrRP in vivo differed among tissues.     

Organ repair and regeneration: An overview

A number of organs have the intrinsic ability to regenerate, a distinctive feature that varies among organisms. Organ regeneration is a process not fully yet understood. However, when its underlying mechanisms are unraveled, it holds tremendous therapeutic potential for humans. In this review, we chose to summarize the repair and regenerative potential of the following organs and organ systems: thymus, adrenal gland, thyroid gland, intestine, lungs, heart, liver, blood vessels, germ cells, nervous system, eye tissues, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone, and cartilage. For each organ, a review of the following is presented: (a) factors, pathways, and cells that are involved in the organ's intrinsic regenerative ability, (b) contribution of exogenous cells – such as progenitor cells, embryonic stem cells, induced pluripotent stem cells, and bone marrow‐, adipose‐ and umbilical cord blood‐derived stem cells – in repairing and regenerating organs in the absence of an innate intrinsic regenerative capability, (c) and the progress made in engineering bio‐artificial scaffolds, tissues, and organs. Organ regeneration is a promising therapy that can alleviate humans from diseases that have not been yet cured. It is also superior to already existing treatments that utilize exogenous sources to substitute for the organ's lost structure and/or function(s). (Part C) 96:1–29, 2012. © 2012 Wiley Periodicals, Inc.     

Cardiovascular actions of intrahypothalamic injections of atrial natriuretic peptide

Accumulating evidence implicates atrial natriuretic polypeptide (ANP) as a neurotransmitter in brain. The presence and distribution of ANP, its high affinity binding sites, and the messenger RNA of its precursor have been described in the central nervous system. However, the function(s) of ANP in specific brain areas is largely unknown. We have now determined the cardiovascular effects elicited by microinjection of atriopeptin-III (ANP-III) in hypothalamic and preoptic areas in rats. ANP-III (40 pmol) increased heart rate when injected into the anteromedial preoptic nucleus (AMPO), the medial preoptic area (MPA), the periventricular area, and in two regions of the dorsal hypothalamus. Other nuclei within the hypothalamus were unresponsive. The tachycardic effects elicited by AMPO-MPA injection of ANP-III were abolished by adrenalectomy. These data indicate that ANP-III acts at discrete sites to elicit tachycardia and the mechanism of action for at least one brain site appears to be through central pathways which selectively activate the adrenal gland.