F. Van Herp

Circadian Rhythmicity of the Crustacean Hyperglycemic Hormone (CHH) in the Hemolymph of the Crayfish

The crustacean hyperglycemic hormone (CHH) is involved in the regulation of endogenous blood glucose metabolism. In this paper we describe the daily rhythmicity in the blood glucose and the blood CHH content of the crayfish Orconectes limosus. Both blood CHH and blood glucose levels increase during the first hours after the beginning of darkness. The bioactivity of released CHH is far higher than that of CHH stored in the sinus gland. Moreover, the released hyperglycemic material shows an affinity for high molecular weight proteins in the hemolymph. Preliminary results suggest that subunits of hemocyanin may act as potential carrier-proteins for bioactive CHH.

Species or group specificity in biological and immunological studies of crustacean hyperglycemic hormone.

Abstract Molecular differences of the hyperglycemic hormone have been reported previously for several decapod crustaceans. The relationships of these neuropeptides have been tested now, comparing their biological response by cross-injection experiments and their immunological properties by immunodiffusion. The data revealed cross-reactivity between several species within the appropriate group.

Demonstration of a cell-specific isomerization of invertebrate neuropeptides

Neurohemal organs of the lobster Homarus americanus contain isoforms of the crustacean hyperglycemic hormone, which differ by the third amino acid (phenylalanyl) residue that is either in the L- or in the D-configuration. Polyclonal antisera have been raised in rabbit against synthetic octapeptides with the sequence corresponding to the N-terminal part of the L- or D-phenylalanine-containing isoforms. Their specificity was shown by immunoassays, indicating that they discriminate the isoforms of the lobster hyperglycemic neuropeptides. It was demonstrated that the two major forms of the crayfish Orconectes limosus hyperglycemic hormone also correspond to peptide isomers containing the L- or D-phenylalanyl residue. The cellular distribution of the isoforms among the neurosecreting cells of the major neuroendocrine complex in lobster and crayfish has been studied by immunohistochemistry. Every hyperglycemic hormone-containing cell was labelled with the anti-L antisera while only some of them were visualized with the anti-D antisera. These results constitute the first observation of peptide isomerization at the cellular level and suggest that the isomerization process occurs in specialized neuroendocrine cells.

Localization of messenger RNAs encoding crustacean hyperglycemic hormone and gonad inhibiting hormone in the X-organ sinus gland complex of the lobster Homarus americanus

The localization of messenger RNAs encoding the crustacean hyperglycemic hormone, involved in regulation of carbohydrate metabolism and the gonad inhibiting hormone, which inhibits vitellogenesis, was studied in the eyestalk of the lobster Homarus americanus using complementary RNA probes for in situ hybridization. For the detection of gonad inhibiting hormone messenger RNA, we cloned and sequenced a partial complementary DNA encoding lobster gonad inhibiting hormone and for crustacean hyperglycemic hormone messenger RNA detection an available complementary DNA was used. This approach reveals that there is a frequent but inconsistent cellular co-localization of the two neurohormones. Furthermore, our data show that male lobsters contain an equal number of neuroendocrine gonad inhibiting hormone cells as female lobsters. An additional study, involving the use of in situ hybridization in combination with immunocytochemistry, shows that the synthetic activity of the crustacean hyperglycemic hormone- and gonad inhibiting hormone-producing cells can be followed at the messenger RNA as well as the protein level. This reveals that when strong immunostaining is present, the messenger RNA staining is usually weak or absent and vice versa. In conclusion, the presence of cells, containing only gonad inhibiting hormone messenger RNA or only crustacean hyperglycemic hormone messenger RNA, indicates that lobster crustacean hyperglycemic hormone and gonad inhibiting hormone originate from two different precursors. Co-localization of the two neurohormone messenger RNAs confirms the co-localization at the peptidergic level found by immunocytochemistry and thus these findings were not due to cross-reactions between the two antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.

Automated nanoflow liquid chromatography–tandem mass spectrometry for a differential display proteomic study on Xenopus laevis neuroendocrine cells

Many proteomic projects based on a comparison of protein profiles displayed on two-dimensional polyacrylamide gel electrophoresis rely on the identification of these proteins using peptide mass fingerprinting on a matrix-assisted laser desorption/ionization mass spectrometer after tryptic digestion. However, this approach is limited to an organism of which genomic information is largely available, i.e. when the total genome sequence is known. For other organisms, mass spectrometric sequence analysis is necessary for protein identification. We established a nano-LC-MS-MS system based on a quadrupole time-of-flight mass spectrometer, which allows automated sequence analysis of tryptic digestion mixtures from single gel spots. This system is applied in a differential-display proteomic study to identify differentially expressed proteins in the neuroendocrine cells of the neurointermediate pituitary of black- and white-background adapted Xenopus laevis.

Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell.

Members of the p24 family of putative cargo receptors (subdivided into p24-alpha, -beta, -gamma and -delta) are localized in the intermediate-and cis-Golgi compartments of the early secretory pathway, and are thought to play an important role in protein transport. In the present study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells, which in black- and white-adapted animals are biosynthetically highly active and virtually inactive respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24-alpha, -beta, -gamma and -delta proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black-adapted animals, the steady-state distribution of all four p24 members changed towards the intermediate compartment and subdomains of the endoplasmic reticulum (ER), most probably the ER exit sites. In the active melanotropes, the major cargo protein pro-opiomelanocortin was mostly localized to ER subdomains and partially co-localized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell.

Biochemical analyses of the crustacean hyperglycemic hormone of the crayfish Astacus leptodactylus

A biochemical analysis was made of the crustacean hyperglycemic hormone (CHH) of the crayfish Astacus leptodactylus, as present in the CHH-producing perikarya, in the axonal tract and in the sinus gland, respectively. Hyperglycemic material was analyzed by polyacrylamide gel electrophoresis (PAGE and SDS-PAGE) and high-pressure liquid chromatography (HPLC) in combination with a dotting immunobinding assay (DIA) and a bioassay for hyperglycemic activity. After electrophoretic analyses, the predominant biologically as well as immunologically detectable product present in all parts of the cell has an apparent molecular radius of approximately 7000 Da. In the perikarya extract, a second factor with lower electrophoretic mobility was found, which may represent the prohormone or precursor of CHH. The analyses by means of HPLC showed two predominant immunopositive peaks with an elution time of 28-29 and 52-54 min, respectively. For both HPLC peaks, electrophoretic analyses indicate a molecular weight of 7000 Da.

Characterization and Expression of Lobster Preprohormones Involved in Metabolism, Moulting and Reproduction

A combined molecular biological, (bio)chemical and immunocytochemical study was carried out on the role of neuropeptides in the control of the biannual reproductive cycle in the female lobster.