Purification and chemical characterization of peptide G1, an invertebrate neuropeptide that stimulates cyclic GMP metabolism.

Bioassay analysis of extracts of the major neurosecretory structures of the American lobster have revealed several different agents with stimulatory effects on the cyclic GMP metabolism of various lobster tissues. The most potent of these is a peptide extracted from the sinus gland, a neurohemal organ found in the animal's eyestalk. This molecule, called peptide G1 (for its effects on cyclic GMP metabolism), can increase the cyclic GMP content of every lobster tissue tested, sometimes by as much as 200-fold. In this article, we describe the purification and some of the chemical properties of peptide G1. Purification was accomplished by sequential anion exchange and reverse-phase HPLC. The purified peptide is a large, extremely hydrophobic molecule. Its apparent molecular mass on a reducing sodium dodecyl sulfate-containing gel is 6.4 kDa, and its calculated molecular mass (based on an amino acid analysis of the purified material) is 8.2 kDa. Amino acid analysis reveals a high proportion of leucine and valine residues. The amino terminus of the molecule is not susceptible to Edman degradation, but sequencing studies were successfully carried out on tryptic fragments. Based on the estimated size of the molecule, these studies provide approximately 60% of the total sequence. No homologies with any previously sequenced peptide were observed, but biochemical similarities to as yet unsequenced peptides found in extracts of sinus glands from other crustaceans (hyperglycemic hormone and moult-inhibiting hormone) are described.

A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines.

Synaptophysin is a Mr 38,000 integral membrane glycoprotein expressed by a variety of normal and neoplastic neuroendocrine cells. We studied synaptophysin as an immunocytochemical marker for neuroendocrine differentiation in lung cancer and compared it to the immunocytochemical expression of chromogranin A, a marker for dense core (endocrine) granules, and the biochemical activity of L-dopa decarboxylase (DDC), the key amine-handling enzyme. Of the 250 cell lines available to us, we selected examples representative of the following cell types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell lung cancers (n = 18) whose neuroendocrine status had been previously determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of synaptophysin than chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (b) 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines expressed synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non-small cell lung cancer cell lines previously shown to express multiple neuroendocrine markers were positive for synaptophysin, chromogranin A, and DDC activity; (d) none of the other 11 non-small cell lung cancer cell lines expressed synaptophysin or chromogranin A; and (e) formalin fixation and paraffin embedding reduced synaptophysin immunoreactivity in 11 of 14 (79%) of the cell lines, as compared to freshly prepared specimens fixed in 95% ethanol. Western blot analysis using the synaptophysin antibody (SY38) demonstrated immunoreactive proteins ranging from Mr 43,000 to 45,000 in five representative cell lines. The concordance of expression of all three neuroendocrine markers was statistically significant when values for all cell lines were totalled. Synaptophysin was a more commonly expressed marker for variant SCLC cell lines, which rarely showed DDC activity. We conclude that synaptophysin may be a more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines which express only part of the neuroendocrine program.

Isolation and structure of a novel charged member of the red-pigment-concentrating hormone-adipokinetic hormone family of peptides isolated from the corpora cardiaca of the blowfly Phormia terraenovae (Diptera).

A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species.

Atypical endocrine granules in atypical endocrine tumor (AET) of the lung. An immunoelectron microscopic study.

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecretory-type granules: moderately dense granules (mean size 373.7 nm) and "target" granules with a central dense core (425.1 nm), both apparently larger than the highly dense granules (223.3 nm). Immunoelectron microscopical studies demonstrated that all three types of granule were positive for gastrin-releasing peptide (GRP), human chorionic gonadotropin alpha-subunit (hCG alpha), calcitonin or serotonin. Although the size profiles of positive granules were similar for calcitonin and hCG alpha, they were different from those of GRP or serotonin granules. The presence of atypical granules and the different size profiles of hormonal products in AET indicate that caution is required in ultrastructural evaluation of granules in lung carcinomas.

Primary structures of hypertrehalosaemic neuropeptides isolated from the corpora cardiaca of the cockroaches Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica and Blatta orientalis and of the stick insect Extatosoma tiaratum assigned by tandem fast atom bombardment mass spectrometry.

Hypertrehalosaemic peptides were isolated by reversed-phase high-performance liquid chromatography from corpora cardiaca of four species of cockroaches (Leucophaea maderae, Gromphadorhina portentosa, Blattella germanica, and Blatta orientalis) and one stick insect species (Extatosoma tiaratum), and their primary sequences were assigned by collision-induced decomposition tandem fast atom bombardment mass spectrometry (FABMS/CID/MS). The members of the cockroach families Blaberidae (L. maderae and G. portentosa) and Blattellidae (B. germanica) contained an identical decapeptide (Glu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-ThrNH2), whereas the member of the cockroach family Blattidae (B. orientalis) had two octapeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-TrpNH2 and Glu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH2). The structure of the stick insect hypertrehalosaemic compound was assigned as a decapeptide (Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2). The respective synthetic peptides elevated blood carbohydrates in their respective acceptor species. The results are discussed in the light of family-specificity of members of the adipokinetic hormone/red pigment-concentrating hormone family.

Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycoproteins. Demonstration by combined use of lectins and specific glycosidases, and by the administration of Tunicamycin.

Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, beta-galactosidase, alpha-mannosidase, alpha-glucosidase and beta-N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunica-mycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera).

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.

Pulmonary bombesin and calcitonin in hamsters during exposure to hyperoxia and diethylnitrosamine.

Combined exposure of hamsters to 60% hyperoxia and the carcinogen diethylnitrosamine for 6 wk resulted in the development of lung tumors. This was associated with progressive loss of body weight as well as increases in the pulmonary-associated peptides, mammalian bombesin (MB) and immunoreactive calcitonin (iCT). After 3 wk of exposure, multiple bronchial epithelial hyperplastic foci were noted, along with increased lung levels of MB and iCT as well as increased serum levels of MB. At this time, immunocytochemistry revealed the presence of MB and iCT within hyperplastic pulmonary neuroendocrine (PNE) cells. In addition, the localization of MB to alveolar type II cells was noted, along with the presence of lamellar bodies and secretion granules in these cells on electron microscopy. After 6 wk of exposure, distinctive microscopic pulmonary tumorlets were seen. These tumorlets were associated with a marked increase in lung and serum MB, and to a lesser extent lung and serum iCT. At this time, MB and iCT were localized exclusively to these abnormal PNE cell sites. These results, which may have relevance in humans, suggest that endogenous peptides may be important components in the process of development of neuroendocrine cancer.

The structurally similar neuropeptides adipokinetic hormone I and II are derived from similar, very small mRNAs.

The grasshopper neuropeptides adipokinetic hormone (AKH) I and II were among the first of an extensive family of structurally similar arthropod hormones and neuroregulators to be isolated and sequenced. This paper reports the cloning of cDNAs derived from the unusually small mRNAs (550 bases) which code for the precursors of AKH I and II from Schistocerca nitans. Sequence analysis of the cDNAs indicates that AKH I and II are derived from small precursor proteins (63 and 61 amino acids) which are 55% identical in amino acid sequence. Each contains a 22-amino acid hydrophobic leader sequence followed by the AKH I or II sequence and an additional 28-amino acid carboxyl-terminal peptide of unknown function. Significant homology at the nucleic acid level (64% identity) is confined to the coding region of the mRNA sequences. Preliminary DNA blot analyses suggest that a single gene codes for each, and that the genes for AKH I and II may be linked. Genomic blots from various tissues fail to suggest that the high level of expression of AKH in the corpora cardiaca is due to tissue specific gene amplification.

The hypertrehalosaemic peptides of cockroaches: a phylogenetic study.

Hypertrehalosaemic peptides from the corpora cardiaca of 14 different species were compared with respect to phylogenetic relationships within the insect suborder Blattaria (cockroaches). Gland extracts from members of the family Blattidae (Periplaneta americana, P. brunnea, P. australasiae, P. fuliginosa, and Blatta orientalis) contain two hypertrehalosaemic octapeptides with identical properties to the recently sequenced peptides M I and M II from the American cockroach, whereas corpora cardiaca from members of the families Blaberidae and Blattellidae (Nauphoeta cinerea, Leucophaea maderae, Blaberus discoidalis, B. trapezoideus, Diploptera punctata, and Gromphadorhina portentosa) possess one hypertrehalosaemic decapeptide with identical properties as the peptide recently sequenced from B. discoidalis and N. cinerea. A member of the family of Polyphagidae (Polyphaga aegyptiaca), placed at the origin of the phyletic tree of Blattaria, has two hypertrehalosaemic factors in its corpus cardiacum which are each different from M I, M II, and HTH.

Expression of the alpha subunit of human chorionic gonadotropin in normal and neoplastic neuroendocrine cells. An immunohistochemical study.

The alpha subunit of human chorionic gonadotropin (HCG) was localized Immunohistochemically in paraffin sections of normal human tissues and neuroendocrine tumors. A small subset of dispersed neuroendocrine cells was positive in normal adult tissues, including gastric antrum, urachal remnant, anal glands and prostate. Positive cells were consistently present in perinatal lung but rare in adult lung. In contrast, the beta subunit was absent from these cells. Seventy-two of 151 extrapituitary neuroendocrine tumors (48%) were alpha subunit-positive. Thirty-three of 37 bronchial carcinoids (92%) were immunoreactive, with a high percentage of the tumors (54%) containing moderate to large numbers of positive cells. The alpha subunit was further demonstrated in 9 of 45 small cell lung carcinomas (20%), 19 of 35 extrapulmonary carcinoids (54%), 3 of 11 islet cell tumors (27%) and 8 of 13 medullary thyroid carcinomas (62%). Two of three malignant islet cell tumors were positive. Positive cells were usually few in number, except for two small cell lung carcinomas, two rectal carcinoids, one thymic carcinoid and one malignant islet cell tumor. Pheochromocytomas (n = 10) were negative. Eleven of 19 pulmonary tumorlets (58%) were alpha subunit-immunoreactive. A few beta subunit-positive cells were detected in only 6 lung lesions. The physiological significance of the imbalance of expression of HCG subunits by certain neuroendocrine cells and their tumors remains unknown.

An immunohistochemical study of neuroendocrine cells in gynecologic tumors.

Various gynecologic tumors with argyrophilia were studied immunohistochemically for chromogranin using two antibodies, antichromogranin and antineuroendocrine. Of seven small cell carcinomas of the cervix, four were immunoreactive with antichromogranin and seven with antineuroendocrine. Argyrophil cells of six cervical adenocarcinomas were all immunoractive with both antibodies. Type I argyrophil cells of 20 endometrial carcinomas were likewise stained positively. However, of the 30 endometrial carcinomas with type II argyrophil cells, 19 showed positive immunoreactivity for chromogranin and 22 for neuroendocrine. Of the ovarian tumors tested, argyrophil cells of 11 mucinous tumors, three carcinoid tumors, and the pancreatic tissue of a malignant mixed germ cell tumor were all chromogranin- and neuroendocrine-immunoreactive. Type I argyrophil cells of five endometrioid carcinomas of the ovary were also immunoreactive with both antibodies. Of the 13 endometrioid carcinomas with type II argyrophil cells, only four showed positive immunoreactivity for chromogranin and only five for neuroendocrine. In conclusion, both antichromogranin and antineuroendocrine detect the specific neuroendocrine markers in close association with argyrophilia in gynecologic tumors, the latter being more sensitive for small cell carcinoma of the cervix, and for type II argyrophil cells in adenocarcinoma of the endometrium and endometrioid carcinoma of the ovary.

Pancreastatin-like immunoreactivity in epidermal Merkel cells of pig and man.

By use of light microscopic immunohistochemistry it was shown that Merkel cells of pig and man stained for pancreastatin. In pig, evaluation of paired consecutive sections revealed a coexistence of pancreastatin and chromogranin A (CGA) in individual Merkel cells from all localisations. However, in man pancreastatin expression was variable and depended from the localization and the developmental stage. The present findings suggest that pancreastatin is not specific for the pancreas and the intestine. However, it remains to be elucidated whether pancreastatin is a widely distributed in peptide-containing neuroendocrine granules of cells of the diffuse neuroendocrine system (DNES) as has been established for CGA.

Amino acid sequence of locust neuroparsins.

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two-step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N-terminal sequence of both native neuroparsins was determined: the N-terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N-terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu-C, comprises 78 residues.

Partial characterization of a thyroid-stimulating hormone-like peptide in neuroendocrine cells of the human prostate gland.

Immunohistochemical identification of the most prevalent type of neuroendocrine (NE) cells in the human prostate gland can be made with polyclonal antisera against human thyroid-stimulating hormone (TSH). A TSH-like peptide was characterized by analysis of prostatic tissue homogenates with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis followed by immunoblotting. A single protein band, with an apparent mass of about 32 kDa after reduction, was identified both with polyclonal antisera against human TSH and with a polyclonal antiserum raised against a synthetic peptide corresponding to the carboxyterminal part of the beta-subunit of human TSH. The TSH-like prostatic peptide identified here is, on the basis of its molecular mass and absence of immunoreactivity with an antiserum raised against a synthetic peptide representing the mid-portion of the beta-subunit of TSH, not identical with the pituitary beta-subunit of TSH. On the other hand, this 32 kDa prostatic peptide may have certain structural elements in common with the pituitary beta-subunit of TSH, since it is recognized both with polyclonal antisera against TSH and with an antiserum against the carboxyterminal part of the beta-subunit of TSH.

Immunohistochemical investigation of locust neuroparsin-like substances in several insects, in some other invertebrates, and vertebrates.

Anti-neuroparsin serum was immunohistochemically tested on brain and/or neurohemal organ from 40 insect species belonging to 13 orders, and from 8 non-insect invertebrate and 5 vertebrate representatives using the peroxidase-antiperoxidase procedure. In insects, immunostaining of only the A1 type of the protocerebral median neurosecretory cells was revealed in all species tested of Odonata, Dictyoptera, Isoptera and Orthoptera and in 2 species from the 9 other orders out of 13 orders tested. No immunostaining was detected in vertebrate and non-insect invertebrate species except in 2 annelid species out of 4 tested. The distribution of neuroparsin-like products in Coelomata appears to be restricted mainly to 4 phylogenetically close insect orders.