Neuroactive compounds produced by bacteria from the marine sponge Halichondria panicea: activation of the neuronal NMDA receptor

Abstract Previous studies revealed that the marine sponge Halichondria panicea habors symbiotic- and commensalic bacteria ( Althoff et al., 1998 . Marine Biol. 130, 529–536). In the present study the hypothesis was tested whether some of those bacteria synthesize neuroactive compounds. For the first time the effect of bacterial bioactive compounds on the neuronal ionotropic glutamate receptors [iGluR], subtype N -methyl- d -aspartate (NMDA) receptor, was checked. In cortical neurons from rats as cell system the supernatant of two bacterial cultures isolated from H. panicea proved to agonize the NMDA receptor. The response of the NMDA receptor to the bioactive compounds was determined by mea…

Caspase-mediated apoptosis in sponges: cloning and function of the phylogenetic oldest apoptotic proteases from Metazoa

AbstractSponges (phylum Porifera) represent the phylogenetically oldest metazoan phylum. These animals have complex cell adhesion and powerful immune systems which allow the formation of a distinct body plan. Consequently, an apoptotic machinery has to be predicted that allows sponges to eliminate unwanted cells accumulating during development. With the marine sponge Geodia cydonium, it is shown that allografts of these animals undergo apoptosis as demonstrated by apoptotic DNA fragmentation. Extracts from allografts contain an enzymic activity characteristic for caspases; as substrate to determine the cleavage activity, Ac-DEVD-AMC was applied. cDNAs encoding predicted caspase-3-related pr…

Flupirtine protects both neuronal cells and lymphocytes against induced apoptosis in vitro: Implications for treatment of AIDS patients

In the present study we demonstrate that flupirtine, an already clinically used, centrally acting, non-opiate analgesic agent, protects rat cortical neurons against HIV-gp120 induced apoptotic cell death. The drug was active at concentrations between 1 and 10 microg/ml. Furthermore we show inhibition of in vitro induced apoptosis in human blood mononuclear cells, using flupirtine. Induced apoptosis in peripheral blood mononuclear cells from healthy individuals and HIV-1 infected patients was reduced to approximately 50% after in vitro preincubation with flupirtine at concentrations between 0.1 and 10 microg/ml. The anti-apoptotic effect of flupirtine was restricted to CD3+ lymphocytes and i…

Synthesis of the neurotoxin quinolinic acid in apoptotic tissue from Suberites domuncula: cell biological, molecular biological and chemical analyses

Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms. In the present study we document for the first time that in tissue from sponges which underwent apoptosis, a toxic compound is produced which very likely controls the elimination of the dying tissue. The marine sponge Suberites domuncula develops in the field occasionally apoptotic tissue areas which are rapidly eliminated. In the present study apoptosis was induced in S. domuncula by exposing the specimens in aquaria to 5 µg/ml Dip or by maintaining the sponges for 3 - 5 days under non-aeration conditions. After that treatment only one eukaryotic epibiont, the mollus…

Dinoflagellates from marine algal blooms produce neurotoxic compounds: effects on free calcium levels in neuronal cells and synaptosomes

In this report, evidence is presented that the marine unicellular eukaryotic dinoflagellates can cause neurotoxicity very likely by an increase in intracellular free calcium ions ([Ca(2+)](i)). Determinations of the effects of culture supernatants from different clones of the dinoflagellate Alexandrium sp. isolated from algal blooms on the viability of rat primary neuronal cells revealed that all clones tested were toxic for these cells. In addition, all Alexandrium clones tested, except for A. ostenfeldii BAH ME-141, were found to be toxic for rat pheochromocytoma PC12 cells. No toxicity was observed for culture supernatants from Gonyaulax and Coolia monotis. Calcium ions are important in …

Effect of flupirtine on Bcl-2 and glutathione level in neuronal cells treated in vitro with the prion protein fragment (PrP106-126).

Flupirtine, trade name Katadolon, is a centrally acting nonopioid analgesic that has recently been found to display cytoprotective activity in vitro and in vivo on neurons induced to undergo apoptosis. This report shows that the PrP106-126 fragment of the prion protein, which is the likely etiological agent for a series of encephalopathies, is toxic to cortical neurons in vitro. Simultaneously, PrP106-126 influences the molecular GSH content and the bcl-2 expression in neurons. Significant toxicity (32% reduction in cell viability) was observed at a concentration of 50 microM of the peptide after 9 days of incubation, while at higher concentrations toxicity increased to 70%. Neurotoxicity w…

Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation

SUMMARY Sponges (Porifera) represent the evolutionary oldest multicellular animals. They are provided with the basic molecules involved in cell–cell and cell–matrix interactions. We report here the isolation and characterization of a complementary DNA from the sponge Suberites domuncula coding for the sponge homeobox gene, SUBDOIRX-a. The deduced polypeptide with a predicted Mr of 44,375 possesses the highly conserved Iroquois-homeodomain. We applied in situ hybridization to localize Iroquois in the sponge. The expression of this gene is highest in cells adjacent to the canals of the sponge in the medulla region. To study the expression of Iroquois during development, the in vitro primmorph…

Protection of Flupirtine on β-Amyloid-Induced Apoptosis in Neuronal Cells In Vitro: Prevention of Amyloid-Induced Glutathione Depletion

Effective drugs are not available to protect against beta-amyloid peptide (A beta)-induced neurotoxicity. Cortical neurons from rat embryos were treated with the toxic fragment A beta25-35 at 1 microM in the presence or absence of flupirtine, a triaminopyridine, successfully applied clinically as a nonopiate analgesic drug. Five days later 1 microM A beta25-35 caused reduction of cell viability to 31.1%. Preincubation of cells with flupirtine (1 or 5 microg/ml) resulted in a significant increase of the percentage of viable cells (74.6 and 65.4%, respectively). During incubation with A beta25-35 the neurons undergo apoptosis as determined by appearance of the characteristic stepladder-like D…

Background Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase.

Exposure to gp120 of HIV-1 induces an increased release of arachidonic acid in rat primary neuronal cell culture followed by NMDA receptor-mediated neurotoxicity.

After incubation of highly enriched neurons from rat cerebral cortex with the HIV-1 coat protein gp120 for 18 h, cells showed fragmentation of DNA at internucleosomal linkers followed by NMDA receptor-mediated neurotoxicity. We report that in response to exposure to gp120 cells react with an increased release of arachidonic acid (AA) via activation of phospholipase A2. This process was not inhibited by NMDA receptor antagonists. To investigate the role of AA on the sensitivity of the NMDA receptor towards its agonist, low concentrations of NMDA were co-administered with AA. This condition enhanced the NMDA-mediated cytotoxicity. Administration of mepacrine reduced cytotoxicity caused by gp1…

Origin of neuronal-like receptors in Metazoa: cloning of a metabotropic glutamate/GABA-like receptor from the marine sponge Geodia cydonium.

To date, no conclusive evidence has been presented for the existence of neuronal-like elements in Porifera (sponges). In the present study, isolated cells from the marine sponge Geodia cydonium are shown to react to the excitatory amino acid glutamate with an increase in the concentration of intracellular calcium [Ca2+]i. This effect can also be observed when the compounds L-quisqualic acid (L-QA) or L-(+)-2-amino-4-phosphonobutyric acid (L-AP-4) are used. The effect of L-QA and L-AP-4, both agonists for metabotropic glutamate receptors (mGluRs), can be abolished by the antagonist of group I mGluRs, (RS)-alpha-methyl-4-carboxyphenylglycine. These data suggest that sponge cells contain an mG…

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both R…

Cultivation of primmorphs from the marine sponge Suberites domuncula: morphogenetic potential of silicon and iron.

Abstract Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula , the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and—with main emphasis—of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the f…

Interaction of prion protein mRNA with CBP35 and other cellular proteins: possible implications for prion replication and age-dependent changes.

Abstract A study of the intracellular distribution of prion protein (PrP) in N2a neuroblastoma cells which had been infected with prions (ScN2a cells) revealed that most PrP is present in the cytoplasm. However, a significant amount of PrP is also present in the nucleus (predominantly in the nucleoli) of these cells, as analyzed by confocal laser scanning microscopy. By contrast, no PrP could be detected in the nucleus of uninfected N2a cells. The steady-state level of PrP mRNA did not markedly differ between the two cell strains. Likewise, no changes were found in the rate of transcription and in the half-life of PrP mRNA. A number of cellular proteins, among them the nuclear lectin CBP35,…

Sarcophytolide: a new neuroprotective compound from the soft coral Sarcophyton glaucum

Abstract Bioactivity-guided fractionation of an alcohol extract of the soft coral Sarcophyton glaucum collected from the intertidal areas and the fringing coral reefs near Hurghada, Red Sea, Egypt resulted in the isolation of a new lactone cembrane diterpene, sarcophytolide. The structure of this compound was deduced from its spectroscopic data and by comparison of the spectral data with those of known closely related cembrane-type compounds. In antimicrobial assays, the isolated compound exhibited a good activity towards Staphylococcus aureus, Pseudomonas aeruginosa , and Saccharomyces cerevisiae. Sarcophytolide was found to display a strong cytoprotective effect against glutamate-induced …

Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s)

Porifera (sponges) constitute the lowest metazoan phylum, Experiments examined whether sponges can recognize self/nonself molecules. Cells from the marine sponge Suberites domuncula were incubated with membranes from either S. domuncula or another marine sponge, Geodia cydonium, as well as with recombinant alpha-integrin from G. cydonium. The cells responded immediately with a rise of intracellular Ca2+ ([Ca-i(2+)]) if they were treated with membranes from G. cydonium but not after treatment by those from S. domuncula. This change of [Ca-i(2+)] was also recorded with G. cydonium alpha-integrin. In parallel, the expression of two genes was strongly upregulated; one codes for a cytokine-relat…

Pharmacological intervention in age-associated brain disorders by Flupirtine: Alzheimer’s and Prion diseases

Alzheimer's disease, a major form of dementia in the elderly has become an increasingly important health problem in developed countries. In vitro studies on primary neurons demonstrate that Flupirtine (Katadolon) at a concentration of 1 microg/ml, significantly reduces the neurotoxic (apoptotic) effect displayed by A beta25-35, a segment of the amyloid beta-protein precursor the etiologic agent of Alzheimer's disease. Flupirtine, which has been in clinical use since 10 years ago, prevents the toxic effect of PrP, the presumed etiologic agent of the Creutzfeldt-Jakob disease as well as the excitatory amino acid glutamate on cortical neurons. Flupirtine displays a bimodal activity. Its strong…

Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells

Abstract Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca 2+ level [Ca 2+ ] i , and with an upregulation of the expression of (at least) two genes, a Ca 2+ /calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schroder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Muller, W.E.G. Muller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca 2+ ] i . Next, the promoter for the sponge SDSNZERR gene was isolated …

Changes of ICE protease activities caused by toxic supernatants of dinoflagellates (Prorocentrum species) from marine algal blooms

Summary Marine phytotoxins may become a major health problem for humans because of their ability to contaminate seafood and to cause shellfish poisoning. In this report, the cytotoxic effects and the effects on intracellular caspase activities of culture supernatants from different dinoflagellate Prorocentrum clones were determined. Among the clones tested, P. tepsium BAH ME-140 and P. lima BAH ME-130 K1 and K2 clones but not P. minimum and P. micans were found to be toxic on rat pheochromocytoma PC12 cells, mouse lymphoma L5178Y cells and rat primary neurons. A significant increase in the specific activities of caspase 1 (ICE), caspase 3 (CPP32) and caspase 6 (Mch2) to 149–167% was observe…

The triaminopyridine flupirtine prevents cell death in rat cortical cells induced by N-methyl-D-aspartate and gp120 of HIV-1.

Abstract Flupirtine, a triaminopyridine derivative, is a non-opiate centrally acting analgesic agent with muscle relaxant properties. Now we show that this drug displays a potent cytoprotective effect on neurons (rat cortical cells) treated with (i) the excitatory amino acid N-methyl- d -aspartate (NMDA) or (ii) with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120. In the absence of the drug the two agents cause a >90% reduction of cell viability after a 18 h incubation. During this period the DNA in the cells undergoes fragmentation and shows a pattern which is typical for cell death. If the neurons were preincubated with flupirtine for 2 h and subsequently exposed to th…

Neurotoxicity in Rat Cortical Cells Caused by N-Methyl-D-Aspartate (NMDA) and gp120 of HIV-1: Induction and Pharmacological Intervention

Incubation of highly enriched neurons from rat cerebral cortex with the human immunodeficiency virus type 1 (HIV-1) coat protein gpl20 for 18 h results in fragmentation of DNA at internucleosomal linkers, a feature of apoptosis. We report that neurons respond to exposure to gp120 with an increased release of arachidonic acid via activation of phospholipase A2. This process is not inhibited by antagonists of the N-methyl-D-aspartate (NMDA) receptor channels. To investigate the influence of arachidonic acid on the sensitivity of NMDA receptor towards its aganist, low concentrations of NMDA were coadministered with arachidonic acid. Under these conditions the NMDA-mediated cytotoxicity was enh…

Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis.

Sponges (phylum Porifera) live in an aqueous milieu that contains dissolved organic carbon. This is degraded photochemically by ultraviolet radiation to alkenes, particularly to ethylene. This study demonstrates that sponge cells (here the demosponge Suberites domuncula has been used), which have assembled to primmorphs, react to 5 microM ethylene with a significant up-regulation of intracellular Ca(2+) concentration and with a reduction of starvation-induced apoptosis. In primmorphs from S. domuncula the expression of two genes is up-regulated after exposure to ethylene. The cDNA of the first gene (SDERR) isolated from S. domuncula encodes a potential ethylene-responsive protein, termed ER…

Flupirtine increases the levels of glutathione and Bcl-2 in hNT (human ) neurons: mode of action of the drug-mediated anti-apoptotic effect

Flupirtine is a triaminopyridine analogue which has been successfully applied in clinics as a non-opiate analgesic drug. Previously we described that flupirtine acts like an N-methyl-D-aspartate (NMDA) receptor antagonist in neuronal cells both in vitro and in vivo. Here we show that flupirtine displays its anti-apoptotic effect also in hNT (human Ntera/D1) neurons. hNT neurons were induced to apoptosis applying glutamate (Glu; at concentrations > or = 1 mM) or NMDA (> or = 1 mM). During Glu/NMDA-mediated apoptosis the levels of the intracellular anti-apoptotic agents Bc1-2 and glutathione dropped by more than 50%. Flupirtine completely abolished this reduction of Bc1-2 and glutathione leve…

Oxygen as a morphogenic factor in sponges: expression of a tyrosinase gene in the sponge Suberites domuncula

Sponges live in a symbiotic relationship with microorganisms, especially bacteria. Here we show, using the demosponge Suberites domuncula as a model, that the sponge expresses the enzyme tyrosinase which synthesizes diphenols from monophenolic compounds. It is assumed that these products serve as carbon source for symbiotic bacteria to grow.