The gene for human chromogranin A (CgA) is located on chromosome 14

Chromogranin A (CgA) is a protein that is present in most neuroendocrine tissues and is co-secreted with their resident hormones. We have assigned the CgA gene to human chromosome 14 by hybridization of a CgA cDNA probe cloned from a cDNA library of human medullary thyroid carcinoma cells to spots of individual human chromosomes flow-sorted onto nitrocellulose filters. Southern analysis of human genomic DNA with the same probe revealed only 1-3 restriction bands. These studies indicate that the CgA gene is probably single copy and not a member of a dispersed, multigene family. The CgA gene is not co-localized with the genes of any of the CgA-associated hormones.     

Proteolytic cleavage of chromogranin A (CgA) by plasmin. Selective liberation of a specific bioactive CgA fragment that regulates catecholamine release

Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.     

An Italian program of external quality control for chromogranin A (CgA) assay: state of the art of CgA measurement

Chromogranin A (CgA) is a secretory protein produced by many neuroendocrine cells. Circulating levels of CgA have been found to be elevated in a variety of neuroendocrine tumors and may facilitate the diagnosis and management of patients with functioning as well as non-functioning forms. However, up to now the analytical methods used for assaying intact CgA and CgA-derived peptides in the circulation of patients have not been monitored in Italy by an external quality control program. Within the framework of a Ministry of Health project an external quality control program was developed to investigate the state of the art of CgA determination in Italy and to monitor the performance of laboratories carrying out this assay. This paper deals primarily with the former of these aspects. Every laboratory received the study protocol together with a questionnaire to be returned before receipt of the samples to be assayed. Serum and plasma samples obtained from a pool of routine specimens were prepared at three different concentrations of CgA, aliquoted, frozen at -80 degrees C and mailed in dry ice to the participating laboratories. Of the 43 laboratories, 21 used IRMA, 21 used ELISA and one used RIA. There was a wide range in the time of kit utilization and the number of samples assayed per year, which indicated that the participating group was heterogeneous with regard to their experience in the determination of CgA. Most laboratories routinely used serum and plasma for IRMA and ELISA, respectively, and different data fitting approaches were employed. Further analyses will investigate the possible influence of these preanalytical factors on laboratory performance.     

Immunocytochemical localisation of pancreastatin and chromogranin A in porcine neuroendocrine tissues.

Pancreastatin is a 49 amino acid peptide with a C-terminal glycine amide originally isolated from porcine pancreas. In the present study the cellular localisation of pancreastatin in porcine neuroendocrine tissue was examined immunocytochemically using an antiserum raised against porcine pancreastatin (33-49) that does not cross-react with porcine chromogranin A. In order to study the possible precursor-product relationship between chromogranin A and pancreastatin the cellular localisation of both peptides was examined in peripheral tissues using simultaneous double immunostaining. The pancreastatin antiserum immunostained cells and nerve fibers throughout the neuroendocrine system. In most of the examined tissues we found colocalisation of pancreastatin and chromogranin A immunostaining. These results support the precursor-product concept for chromogranin A and pancreastatin. However, in the gastrointestinal tract and the adenohypophysis a minor population of the endocrine cells exhibited immunostaining with only one of the two antibodies. This discrepancy between immunostaining with pancreastatin antiserum and monoclonal chromogranin A antibody could be due to absence of, or extensive, processing of chromogranin A in certain cell populations.     

Significance of plasma chromogranin A determination in neuroendocrine tumour (NET) diagnosis

The secretory nature of NETs implies the determination of the CgA concentration as a standard marker. The concentration of CgA in plasma correlates with the degree of histopathological differentiation, tumor stage, and is an essential prerequisite for therapy. A retrospective analysis of the results of the plasma CgA concentrations in relation to histopathological and clinical findings (type of NET according to the WHO classification, severity of disease based on the presence of metastases and clinical symptoms) as well as somatostatin receptor scintigraphy was performed in 41 patients with NET. The patients were treated in The Regional Oncology of Lublin from February 2005 to May 2008. Data from the literature and results of this study suggest the use of CgA in the diagnosis and prognosis of NET. Plasma CgA concentration analysed together with histopathological assessment of tumor and the clinical picture is a useful marker in the diagnosis of neuroendocrine tumours. High plasma CgA concentrations may indicate the presence of highly-differentiated NET (WDNEC), and also may indicate the presence of tumor metastasis. The highest CgA concentrations were observed in patients with neuroendocrine tumors associated with carcinoid symptoms and the presence of metastases to the liver.     

Sequence complexity of cDNA transcribed from a diverse mRNA population

Mouse liver poly(A)+mRNA was reverse transcribed using oligo-p(dT) or random oligonucleotides as primers to yield cDNA about equal to the mass of the template RNA. The size profile of the oligo-p(dT)-primedd cDNA was similar to that of the template RNA. RNA or cDNA driven saturation annealing of labeled single copy genomic DNA (scDNA) showed that 2% of the scDNA was complementary in either case indicating the sequence complexity of cDNA was equivalent to that of the template mRNA. These results establish for the first time that cDNA represents essentially all of the sequence complexity of a diverse template RNA population in which individual mRNA species are present in vastly different concentrations. RNA driven hydridization of the cDNA showed that about 40% of the cDNA mass represents most of the sequence complexity of the template RNA. Also, kinetics of this hybridization indicate a complexity of 58,000 kb for the template RNA, a value similar to that obtained by scDNA hybridization. We conclude that appropriately characterized cDNA probes can be used to make valid qualitative and quantitative comparisons of the complex, infrequent class mRNAs of different cells and tissues.     

A cloned cDNA encoding MAP1 detects a single copy gene in mouse and a brain-abundant RNA whose level decreases during development

Screening of a bacteriophage lambda gt11 cDNA expression library with a polyclonal anti-microtubule associated protein (MAP) antiserum resulted in the isolation of two non-cross-hybridizing sets of cDNA clones. One set was shown to encode MAP2 (Lewis, S. A., A. Villasante, P. Sherline, and N. J. Cowan, 1986, J. Cell Biol., 102:2098-2105). To determine the specificity of the second set, three non-overlapping fragments cloned from the same mRNA molecule via a series of "walking" experiments were separately subcloned into inducible plasmid expression vectors in the appropriate orientation and reading frame. Upon induction and analysis by immunoblotting, two of the fusion proteins synthesized were shown to be immunoreactive with an anti-MAP1-specific antibody, but not with an anti-MAP2-specific antibody. Since these MAP1-specific epitopes are encoded in non-overlapping cDNAs cloned from a single contiguous mRNA, these clones cannot encode polypeptides that contain adventitiously cross-reactive epitopes. Furthermore, these cDNA clones detected an abundant mRNA species of greater than 10 kb in mouse brain, consistent with the coding requirement of a 350,000-D polypeptide and the known abundance of MAP1 in that tissue. The MAP1-specific cDNA probes were used in blot transfer experiments with RNA prepared from brain, liver, kidney, stomach, spleen, and thymus. While detectable quantities of MAP1-specific mRNA were observed in these tissues, the level of MAP1 expression was approximately 500-fold lower than in brain. The levels of both MAP1-specific and MAP2-specific mRNAs decline in the postnatal developing brain; the level of MAP1-specific mRNA also increases slightly in rat PC12 cells upon exposure to nerve growth factor. These surprising results contrast sharply with reported dramatic developmental increases in the amount of MAP1 in brain and in nerve growth factor-induced PC12 cells. The cDNA clones encoding MAP1 detect a single copy sequence in mouse DNA, even under conditions of low stringency that would allow the detection of related but mismatched sequences. The cDNAs cross-hybridize with genomic sequences in rat, human, and chicken DNA, but not with DNA from frog, Drosophila, or sea urchin. These data are discussed in terms of the evolution and possible biological role of MAP1.     

Chromogranin A (CgA) in the gastro-entero-pancreatic (GEP) endocrine system. II. CgA in mammalian entero-endocrine cells

Chromogranin A (CgA) and related acidic proteins are widely distributed in the organism. They are also present in entero-endocrine cells and in other members of the paraneuron family. Therefore, CgA has been claimed as an universal marker of this cellular community. To yield precise data about the distribution of CgA in entero-endocrine cells, all segments of the gastro-intestinal tract of five mammalian species (man, cattle, pig, cat, guinea-pig) were investigated immunohistochemically for CgA. In serial semithin plastic sections, all CgA-immunoreactive endocrine cells were identified for resident amines or peptides. CgA could be found in ten hormonally identified endocrine cell types and in two or three other endocrine cell types. Entero-endocrine cells containing amines (histamine, serotonin) regularly exhibited CgA-immunoreactivities. In contrast, peptide-containing endocrine cells were largely heterogeneous: Their CgA-immunoreactivities varies among the species, among the gastro-intestinal segments, and even among the members of the same cell population. Hence, seen histochemically, CgA is no universal marker for entero-endocrine cells. Seen biochemically, the observed heterogeneities of CgA-immunoreactivities theoretically can be attributed to various factors (species-specificities of CgA, subclasses of chromogranins, processing of CgA or its pro-protein). Most probably, these heterogeneities are caused by species- or cell-specific differences in the extent of processing of CgA. In addition, some findings point to certain interrelations between the processing or storage of CgA and resident peptides in the secretion granules of enteroendocrine cells.     

Intestinal glucagon mRNA identified by hybridization to a cloned islet cDNA encoding a precursor

Poly(A) RNA was prepared from the intestine of anglerfish and was translated in a wheat germ cell-free system supplemented with ³⁵S-methionine. SDS polyacrylamide gel electrophoresis of the labeled translation products revealed that the intestinal poly(A) RNA directs the synthesis of many proteins. Immunoprecipitations of the intestinal cell-free translation products with an antiserum to glucagon known to recognize anglerfish islet pre-proglucagon failed to identify an intestinal glucagon precursor. However, sensitive techniques of hybridization with a ³²P-labelled cDNA containing the coding sequence for pancreatic glucagon identified a complementary RNA in the intestine. The mRNA of 620 bases is similar in size to the pre-proglucagon RNA in the islets (620–650 bases). These observations indicate that a gene encoding glucagon is expressed in the intestine, and that the mRNA encoding the intestinal glucagon precursor is of similar size to the pre-proglucagon mRNAs identified in the islets.     

Analyses of ornithine decarboxylase antizyme mRNA with a cDNA cloned from rat liver

Ornithine decarboxylase antizyme is a unique inhibitory protein induced by polyamines and involved in the regulation of ornithine decarboxylase. A cDNA was isolated from a rat liver cDNA library by the screening with monoclonal antibodies to rat liver antizyme as probes. The expression products of the cDNA in bacterial systems inhibited rat ornithine decarboxylase activity in a manner characteristic of antizyme and rabbit antisera raised against its direct expression product reacted to rat liver antizyme, confirming the authenticity of the cDNA. On RNA blot analysis with the cDNA probe, an antizyme mRNA band of 1.3 kb was detected in rat tissues. Antizyme mRNA did not increase upon administration of putrescine, an inducer of antizyme, and its half-life after actinomycin D treatment was as long as 12 h in rat liver, suggesting that antizyme mRNA is constitutively expressed and antizyme synthesis is regulated at the translational level. Similar-sized mRNAs hybridizable to the cDNA were also found in various mammalian and non-mammalian vertebrate tissues under physiological conditions. In addition, chicken and frog antizymes showed immunocrossreactivity with rat antizyme. The ubiquitous presence and the evolutionally conserved structure of antizyme in vertebrate tissues suggest that it has an important function.     

Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA

Chromogranin A (CGA), also referred to as secretory protein I, is an acidic protein that has been detected in all neuroendocrine cell types examined and is often present in large amounts relative to other secreted proteins. For example, CGA comprises at least 40% of the soluble protein of the adrenal chromaffin granule, and it appears to be the major secretory protein in the parathyroid secretory granules. CGA complementary DNAs (cDNAs) from bovine adrenal and pituitary have recently been cloned and sequenced and found to be nearly identical. A region of bovine CGA has a high degree of amino acid sequence identity to pancreastatin, a recently isolated porcine peptide that inhibits glucose-induced insulin secretion. This suggests that CGA may be a prohormone. We have cloned and sequenced a human cDNA encoding CGA. This human CGA cDNA has an overall 86% nucleic acid identity to the bovine cDNA. Like the bovine CGA cDNA, the human cDNA has little homology to pancreastatin at the 5' region of this peptide but significant amino acid homology to the carboxyl-terminal portion of pancreastatin where the biologic activity resides. There is an area within the pancreastatin region of human CGA and porcine pancreastatin with a 70% amino acid identity to the calcium-binding moiety of the E-F hand proteins such as parvalbumin and oncomodulin. These data suggest that CGA and pancreastatin may both be members of a larger family of calcium-binding proteins.     

Chromogranin A (CgA) in the gastro-entero-pancreatic (GEP) endocrine system

Summary Chromogranin A (CgA) and related acidic proteins are widely distributed in the organism. They are also present in entero-endocrine cells and in other members of the paraneuron family. Therefore, CgA has been claimed as an universal marker of this cellular community. To yield precise data about the distribution of CgA in entero-endocrine cells, all segments of the gastro-intestinal tract of five mammalian species (man, cattle, pig, cat, guinea-pig) were investigated immunohistochemically for CgA. In serial semithin plastic sections, all CgA-immunoreactive endocrine cells were identified for resident amines or peptides. CgA could be found in ten hormonally identified endocrine cell types and in two or three other endocrine cell types. Entero-endocrine cells containing amines (histamine, serotonin) regularly exhibited CgA-immunoreactivities. In contrast, peptide-containing endocrine cells were largely heterogeneous: Their CgA-immunoreactivities varied among the species, among the gastro-intestinal segments, and even among the members of the same cell population. Hence, seen histochemically, CgA is no universal marker for entero-endocrine cells. Seen biochemically, the observed heterogeneities of CgA-immunoreactivities theoretically can be attributed to various factors (species-specificities of CgA, subclasses of chromogranins, processing of CgA or its proprotein). Most probably, these heterogeneities are caused by species- or cell-specific differences in the extent of processing of CgA. In addition, some findings point to certain interrelations between the processing or storage of CgA and resisdent peptides in the secretion granules of entero-endocrine cells.The results were partly presented at the 7th Workshop of the Anatomische Gesellschaft, Würzburg (FRG), 1988 (see Cetin and Grube 1989)     

The effect of a chromogranin A-derived peptide (CgA4-16) in the writhing nociceptive response induced by acetic acid in rats

The nociceptive effects of i.p administration of a synthetic peptide (CgA4-16) derived from chromogranin A (CgA) were studied on a model of inflammatory (somato-visceral) pain. Inflammatory mediators participate in controlling the activity of enterochromaffin cells that store and release chromogranins. Adult male Wistar rats were injected i.p with diluted acetic acid (AA) to induce abdominal writhes. Pharmacological agents were injected prior to CgA4-16 and/or AA together. While i.p CgA4-16 alone did not produce any effect, the peptide increased the number of abdominal constrictions induced by i.p AA administration in a dose-related manner. To determine the possible mechanisms involved in CgA4-16 produced pronociceptive effect, i.p diltiazem or indomethacin were tested. The pronociceptive effect induced by CgA4-16 was blocked by pretreatment of either substance. I.p administration of CGRP, substance P (SP) or capsaicin evoked dose-related abdominal writhing. CgA4-16, 20 min prior to CGRP or capsaicin, potentiated the nociceptive effects induced by CGRP or capsaicin, but not those induced by SP. Taken together, these data suggest for the first time that a CgA-derived peptide may modulate inflammatory pain.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.     

Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.     

Developmental expression and localization of IA-2 mRNA in mouse neuroendocrine tissues

Islet antigen (IA)-2 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM), and belongs to a new class within the receptor-type protein tyrosine phosphatase (PTP) family characterized by lack of PTP enzymatic activity with conventional substrates. Its expression is restricted primarily to the pancreas, pituitary, and brain with the highest level in the brain. IA-2 mRNA expressions in the brain, pituitary and pancreas of 1-, 4-, and 8-week-old mice were examined. In situ hybridization of the brain revealed that IA-2 mRNA was expressed in the cerebral cortex, hippocampus, thalamus, choroid plexus, hypothalamus, Purkinje cells, and granular layer of the cerebellum. In the pituitary, IA-2 mRNA was located in the anterior and posterior pituitary by in situ hybridization. The pattern of IA-2 mRNA expression in normal male mouse brain at 1, 4, and 8 weeks of age by the Northern blot analysis was similar to that in the pituitary by RT-PCR analysis. The expression level was higher at 4 weeks and lower at 1 week of age. In the pancreas, IA-2 mRNA expressions detected by RT-PCR were highest at 8 weeks of age. These results indicated that the amount of mRNA expression increased in accordance to development in brain, pituitary, and pancreas.