Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes.

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630 Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1-40, but the remaining sequence, positions 41-73 or 41-72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH(41-76) sequence in the 3'-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH(1-40)/SG-CHH(1-40) sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3'-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.

Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts.

The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.

Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1.

In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.

Delta and kappa opioid receptors in eyestalk ganglia of a crustacean.

Crustacean eyestalk ganglia are part of the protocerebrum and have been demonstrated to produce numerous neurohormones. 3H(2-D-Pen, 5-D-Pen)-enkephalin, 3H-(-)-ethylketocyclazocine and 3H(D-Ala2-NMePhe-Glyol5)-enkephalin were used as ligands for opioid receptors on neuronal membrane preparations of eyestalk ganglia under consideration of their stereospecific binding properties. In context with saturation binding isotherms, association and dissociation plots, we demonstrate here two opioid receptors; a delta-type receptor with high affinity (Bmax 68.5 fmol/mg protein, Kd = 4.0 nM) and low affinity (Bmax 493 fmol/mg, Kd = 83.6 nM) and a second receptor of kappa-type with Bmax of 3.1 pmol/mg protein and Kd = 68.6 nM.

Isolation and identification of multiple neuropeptides of the allatostatin superfamily in the shore crab Carcinus maenas.

20 neuropeptides belonging to the allatostatin superfamily were isolated from extracts of cerebral and thoracic ganglia of the shore crab Carcinus maenas. They were purified by HPLC, monitored by radioimmunoassay and identified by mass spectrometry and amino acid sequencing. The allatostatins are characterised by a common C-terminal pentapeptide sequence -YXFGL-NH2. Previously such peptides have only been reported from insects. In insects the variable post-tyrosyl residue is restricted to Ala, Asn, Asp, Gly or Ser. In C. maenas, however, there are only two types; thirteen of the peptides having a post-tyrosyl Ala and the other seven, a post-tyrosyl Ser. The crab peptides include the shortest allatostatins so far identified (YAFGL-NH2 and YSFGL-NH2) as well as the longest, a 27-residue peptide. The total of 20 peptides exceeds the highest number of allatostatins found in any of the insects investigated so far (14 in Periplaneta americana). It is of interest that, despite their clear homology, none of the peptides of C. maenas is identical to any of the more than 50 known insect allatostatins. The crab allatostatins show evidence of gene duplication and mutation that has resulted in several sub-groups with close structural similarities. For example, there are four heptapeptides with the common C-terminus -PYAFGL-NH2 that differ only at the N-terminal residue, which is either Glu, Asp, Asn or Ser. Other motifs, variously extended at the N-terminus, include -GPY(A/S)FGL-NH2 (three peptides), -DMY(A/S)FGL-NH2 (three peptides), and -GQY(A/S)FGL-NH2 (two peptides). Unique among the allatostatin superfamily, one of the crab peptides has a Tyr for Phe substitution at position three from the C-terminus (GGPYSYGL-NH2). Immunocytochemistry has provided clues to the functions of the allatostatins in crustaceans by showing their widespread presence in the central and stomatogastric nervous systems.

Opioid receptor types for endogenous enkephalin in the thoracic ganglion of the crab, Carcinus maenas.

In crustaceans, the endogenous opioid peptides, enkephalins, are known to be concentrated in the thoracic ganglion, although they have been demonstrated in all parts of the nervous system. Bmax and Kd measurements have been obtained for the binding of ligands used to characterize delta- and kappa-type opioid receptors in vertebrates. High- and low affinity binding of [3H] [2-D-Pen5-D Pen] enkephalin ([3H]DPDPE) has been measured with a Kd = 9.2 +/- 2.4 nM, Bmax = 153 fmol/mg, and Kd = 243 +/- 27 nM, Bmax = 1.785 pmol/mg, respectively. In addition a kappa-type receptor with Kd 85.5 +/- 12.6 nM and Bmax = 21.138 pmol/mg protein has been recorded. Binding characteristics of several ligands were monitored. Electrophoretic studies of affinity chromatographically purified receptor fractions revealed a molecular mass of 60 kDa. Isoelectric focusing showed a specific binding of [3H]DPDPE to thoracic ganglion membranes at a pl of 5.5.

Physiological studies of visual cortex reorganization following cortical deafferentation in neonatal cats.

Whether restoration takes place in the visual cortex of neonates was physiologically studied in cortical cells of cats following their deafferentation. Deafferentation was performed by a parasagittal incision made in the visual cortex, separating the medial part of it from the thalamocortical and other visual fibers. Responsiveness (percentage of responsive cells) in the middle zone (the middle sector along the cortical incision) of the deafferented region was 82.5%, compared with 91.7% in the afferented (lateral to the incision) region (p = 0.5). In comparison, the responsiveness level was 32.3 and 81.3% (p < 0.05) in the respective zones of the similarly deafferented adult controls. The ocular dominance distribution and binocularity were almost normal in the deafferented region of the neonatally operated cats, whereas binocularity was remarkably diminished in the adult controls. Recovery was also found in the specificity of the cells to orientation and direction in the neonatally operated cats, but not in the adult-operated cats. Thus, functional reorganization of the columnar organizations takes place in the neonatally deafferented but not in the adult-operated cats.

Physiological studies in deafferented visual cortex cells of cats following transplantation of fetal xenografts from the rat's cortex.

We have studied the physiological properties of cells (N = 822) in visual cortex area 17 of seven adult cats transplanted with visual cortex xenografts from fetal (E15-E17) rats. The transplants were assumed to induce recovery of adjacent neurons partially deafferented from visual input. The control group (eight cats, 564 cells) had just analogous sectioning in the cortex. The level of activity found, following visual stimulation, in the deafferented cortical region (medially to the graft) was 48.6% compared to the result (34.5%) obtained in the control cats, indicating the preservation of visual responsiveness. Furthermore, no disturbance could be found in the ocular dominance distribution or binocularity (64.4%) of the cells in the grafted region compared to those in the control cats, indicating preservation of the columnar organization. The deafferented cells in the grafted cortex thus demonstrated the absence of adverse immunological reaction there due to the presence of the xenogeneic tissue, indicating that the visual cortex is immunologically privileged.

Autoradiographic localization of opioid binding sites combined with immunogold detection of Leu-enkephalin, crustacean hyperglycaemic hormone and moult inhibiting hormone at the electron microscopic level in the sinus gland of the shore crab, Carcinus maenas.

Double labelling experiments were performed on the same tissue section at the electron microscopic level, in order to show the involvement of the opioid leucine-enkephalin (Leu-enk) in the modulation of crustacean hyperglycaemic hormone (CHH) mobilization. Both neuropeptides were stored in distinct axon terminals of the sinus gland of Carcinus maenas. A post-embedding immunogold cytochemical technique for Leu-enk, CHH and the CHH neurohormone related moult inhibiting hormone (MIH) was combined with a scintillator intensified autoradiographic method to demonstrate binding of the opioid antagonist [3H] naloxone. Ultrathin sections were successively incubated with antisera against Leu-enk, CHH or MIH, and the corresponding colloidal gold labelled antisera, followed by autoradiographic processing. At the ultrastructural level [3H] naloxone binding sites were easily recognized by their silver tracks after development. Opioid binding sites for [3H] naloxone were visualized only at membranes of CHH-containing axon terminals. These results provide morphological evidence for direct enkephalinergic control of CHH containing neurons in the sinus gland of C. maenas and are furthermore the first autoradiographic demonstration of opioid binding sites in the nervous system of invertebrates.

Isolation and purification of calmodulin from the shrimp, Crangon crangon.

Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins.

Isolation, sequence analysis, and physiological properties of enkephalins in the nervous tissue of the shore crab Carcinus maenas L.

[Leu]- and [Met]enkephalin from thoracic ganglia of the shore crab Carcinus maenas have been purified to homogeneity by a reversed-phase HPLC procedure. Automated gas-phase sequencing revealed a primary structure identical with that of enkephalins from vertebrates. The immunoreactive material in different parts of the nervous system of C. maenas was measured by RIA. Isolated crustacean [Leu]enkephalin shows physiological properties identical to synthetic [Leu]enkephalin in our bioassay. The enkephalinergic effect has been monitored by two bioassays: (i) inhibition of release of crustacean hyperglycemic hormone from isolated eyestalks of C. maenas, and (ii) decreased effect on blood glucose level in the fiddler crab Uca pugilator. The decrease in blood sugar level induced by [Leu]enkephalin was antagonized by naloxone, indicating a receptor-mediated process. In sequence analysis, the thoracic ganglion yielded 3 pmol of [Leu]enkephalin and approximately 1 pmol of [Met]enkephalin. The presence of enkephalins in crustaceans shows an earlier phylogenetic origin of opioids than presumed.

Biological properties and partial purification of a growth factor from porcine spleen.

3T3-Fibroblast growth is enhanced in a dose-dependent way by a factor isolated from porcine spleen after limited proteolysis. The factor is not dialyzable and is stable for 5 min at 100 degrees C and at pH 2 at room temperature. Trypsin or collagenase treatment does not affect its biological activity. The stimulation of cell growth is independent of serum concentration in the culture medium but is accelerated by insulin supplement. The biochemical properties of this factor indicate a novel growth promotor different from other growth factors.

The hyperglycemic neuropeptide of the terrestrial isopod, Porcellio dilatatus. II. Immunocytochemical demonstration in neurosecretory structures of the nervous system.

By use of an rabbit antiserum raised against the crustacean hyperglycemic hormone (CHH) of the shore crab, Carcinus maenas, CHH-producing perikarya were detected in the brain of the isopod, Porcellio dilatatus, by the double-antibody immunofluorescence technique. The course of the secretory axons to the neurohemal organ, the sinus gland, could be traced. The sinus gland also exhibited strong fluorescence. The reaction was very specific, no other structures gave appreciable immunofluorescence. There are two CHH cells in each hemisphere of the brain, located in the medio-rostral part of the protocerebrum. This area appears to correspond to the medulla terminalis of decapods. The cells are aldehyde fuchsin-positive and have been previously described as beta 1 cells. The immunofluorescence could be inhibited by preabsorption of the antiserum with pure Carcinus-CHH.

The hyperglycemic neuropeptide of the terrestrial isopod, Porcellio dilatatus. I. Isolation and characterization.

From isolated sinus glands of Porcellio dilatatus, a hyperglycemic neuropeptide (CHH) was purified by means of a single, two-step purification procedure which consisted of gel chromatography on Sephadex G-50, followed by high-performance liquid chromatography. The 5800- to 6100-Da peptide contains 50-52 amino acids residues. The amino acid composition is (Pro, Ile)1; His1-2; (Thr, Ser, Gly, Met, Tyr)2; (Val, Phe, Lys)3; (Ala, 1/2Cys, Leu, Arg)4; Glx5; Asx7; Trp, n.d. The amino acid composition differs from that of two decapod CHHs analyzed thus far. The N-terminus is blocked. The neuropeptide exhibits little or no interspecific hyperglycemic effect in the brachyuran, Uca pugilator, and its cross-reactivity and potency in the RIA for Carcinus-CHH is very low.

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect, Carausius morosus.

Histological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.

Immunocytochemical demonstration of the neurosecretory X-organ complex in the eyestalk of the crab Carcinus maenas.

An antiserum was obtained by immunizing rabbits with sinus gland extracts from Carcinus maenas. The antiserum is almost exclusively directed against neurosecretory material in the medulla terminalis X-organ (MTGXO), as demonstrated by the peroxidase--antiperoxidase (PAP) staining method in light and electron microscopic studies. Radioimmunological binding studies indicate the presence of antibodies against the crustacean hyperglycemic hormone (CHH) or the black pigment dispersing hormone (BPDH) in the antiserum. The results suggest that the neurosecretory perikarya of the MTGXO are the sites of production of CHH and/or BPDH.

Radioimmunoassay of an invertebrate peptide hormone - the crustacean neurosecretory hyperglycemic hormone.

Antibodies against hyperglycemic hormone (CHH) of Carcinus were raised in rabbits by injection of extract from sinus glands which contain high concentrations of CHH. The antiserum neutralizes the biological activity of CHH and binds 125-J-CHH. A RIA for CHH was established and was used to measure the hormone content of sinus glands.

Immunocytochemical identification of hyperglycemic hormone-producing cells in the eyestalk of Carcinus maenas.

Antiserum raised in rabbits against extracts of sinus glands from Carcinus annd shown by several criteria to contain antibodies directed against the neurosecretory hyperglycemic hormone was used to locate the hormone-producing perikarya in the optic ganglia. By means of the double antibody fluorescence technique, selective staining of the large neurosecretory perikarya of the medulla terminalis ganglionic X-organ (MTGXO) and their axons is obtained. The axon endings of the sinus gland are also stained. None of the other groups of neurosecretory cells in the eyestalk shows fluorescence. Preabsorption of the antiserum with pure hyperglycemic hormone abolishes the fluorescence.

Tracing of neurosecretory neurons in crayfish optic ganglia by cobalt iontophoresis.

The axonal connections between the medulla terminalis ganglionic X-organ (MTGXO) and the sinus gland are traced by iontophoretic application of cobalt dye to the neurosecretory system in the eyestalks of the crayfish, Orconectes limosus. The MTGXO consists of about 15 large perikarya, forming a distinct subgroup of neurosecretory cells in the medulla terminalis and giving rise to a prominent fibre bundle. Additional axons reaching the sinus gland from the medulla interna, the medulla externa and the optic nerve are less conspicuous.