Peter Ruth

The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (Mr = ∼3.5 million), assembled from 10 identical copies of an ∼350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of ∼50 kDa each (FUs a–g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5′ UTR of 58 bp, a 3′ UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (Mr = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin “OdH” from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 ± 24 million years ago, in close agreement with fossil records from the early Devonian.

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda).

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.

CYTOBIOLOGICAL STUDIES ON HEMOCYANIN METABOLISM IN THE BRANCHIAL HEART COMPLEX OF THE COMMON CUTTLEFISH SEPIA OFFICINALIS (CEPHALOPODA, DIBRANCHIATA)

Abstract The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with 125I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.

Distribution and function of biogenic amines in the heart of Nautilus pompilius L. (Cephalopoda, Tetrabranchiata)

SummaryBiogenic amines (serotonin and catecholamines), play an important role in the control of the blood flow not only in vertebrates, but also in invertebrates such as cephalopods. In contrast to the well investigated hearts of the ȁ8modern,ȁ9 coleoid cephalopods, the innervation of the heart of the archaic Nautilus pompilius L. has not been studied in detail. In this study the distribution and effects of biogenic amines in the Nautilus heart were investigated. Serotonin and catecholamines were visualised by the glyxoylic acid induced fluorescence. High performance liquid chromatotography analysis was performed to discriminate between the catecholamines, which showed a high content of noradrenaline in the 4 auricles, the aorta and the ventricle, whereas the ventricle showed a high dopamine content. Adrenaline was found at a very low concentration in the ventricle. Serotonin and dopamine were also immunohistochemically localised to larger nerves and throughout the heart, respectively. In organ bath experiments, the auricles showed little spontaneous activity. After adding serotonin, they displayed rhythmical contractions, which were accelerated dose-dependently by noradrenaline. In summary, these data suggest an important role for biogenic amines in the control of the heart of Nautilus pompilius L., with serotonin possibly stimulating excitatory nerve fibres, whereas noradrenaline is likely to influence the muscle contraction itself.

Hemocyanin and the branchial heart complex of Sepia officinalis: are the hemocytes involved in hemocyanin metabolism of coleoid cephalopods?

Abstract. Cytobiological experiments using isotopic- and cytochemical-labeled Sepia hemocyanin as well as immunocytochemical localization of the respiratory pigment were carried out to investigate the function of the hemocytes in hemocyanin metabolism of the common cuttlefish Sepia officinalis. For comparison, the rhogocytes (ovoid cells) of the branchial heart complex were included in this study. Hemocyanin molecules were immunocytochemically detected in the lysosomal compartment of the rhogocytes and, at lower levels, in adhesive and circulating hemocytes. 125I-labeled Sepia hemocyanin was taken up by the rhogocytes only, whereas gold- and/or fluorescein-labeled Sepia hemocyanin was solely taken up by the adhesive and the circulating hemocytes, even though the level of uptake is different. There are also differences in the uptake of pure gold particles and/or fluorescein between rhogocytes and hemocytes. These findings give evidence that circulating and adhesive hemocytes of the branchial heart complex are not involved in hemocyanin turnover, but are a component of the cellular defense and detoxification system of adult coleoid cephalopods.

Immunohistochemical Localization of Cardio-Active Neuropeptides in the Heart of a Living Fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata)

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neuro-secretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

Tracer studies of food absorption in the digestive tract of Nautilus pompilius (Cephalopoda, Tetrabranchiata)

Abstract. In Nautilus pompilius, tracer experiments with 14C-labelled food show that the midgut gland, caecum and crop are involved in absorption of nutrients. According to liquid scintillation and light- and electron-microscopic autoradiography, the midgut gland exhibits the highest activity, followed by the caecum and crop. The density of silver precipitates is highest in the terminal alveoli of the midgut gland. Precipitates are also seen in the main cells of the caecal epithelium. Few precipitates are found in the lamina epithelialis mucosae of the crop, indicating that, in addition to food storage, digestive processes begin in this organ. These results have been confirmed by injection of the protein ferritin into the buccal cavity. The largest amount of ferritin is seen in the dense bodies of the main cells of the midgut gland, whereas those of the main cells of the caecum and crop contain less ferritin.

Hemocyanin re-uptake in the renal and branchial heart appendages of the coleoid cephalopod Sepia officinalis.

Abstract. The renal and branchial heart appendages of Sepia officinalis L. were investigated in order to elucidate a possible involvement of their excretory epithelia in hemocyanin metabolism. Immunocytochemical findings and tracer experiments indicate that after passing the barrier of ultrafiltration the hemocyanin molecules are taken up by the epithelial cells of the renal and branchial heart appendages and are subsequently carried back to the circulatory system, suggesting a mechanism of hemocyanin recycling. Apart from a function in maintaining constant hemocyanin levels, the present study indicates that the renal and branchial heart appendages are also sites of temporary hemocyanin storage.

Comparative Immunohistochemical and Immunocytochemical Investigations on the Location of Haemocyanin Synthesis in Dibranchiate and Tetrabranchiate Cephalopods (Sepia and Nautilus)

Our comparative immunohistochemical and immunocytochemical investigations on dibranchiate and tetrabranchiate cephalopods showed that haemocyanin synthesis is localized in a special type of cell which is cytomorphologically similar in both genera. In Sepia these haemocyanin-producing cells are located in the branchial gland (polygonal cells) and also in the midgut gland (triangular replacement cells), especially in newly hatched animals. In Nautilus these cells can be identified in the terminal alveoli of the midgut gland, where they are described as basal cells. Some haemocyanin synthesizing cells are scattered in the tissue of the ligamentum branchiale in the gill complex.