Renate Steffen

Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells

Sponges (Porifera) represent the lowest metazoan phylum, characterized by a pronounced plasticity in the determination of cell lineages. In a first approach to elucidate the molecular mechanisms controlling the switch from the cell lineage with a putative indefinite growth capacity to senescent, somatic cells, the activity of the telomerase as an indicator for immortality has been determined. The studies were performed with the marine demosponges Suberites domuncula and Geodia cydonium. It was found that the activity for the telomerase in the tissue of both sponges is high; a quantitative analysis revealed that the extract from S. domuncula contained 10.3 TPG units per 5000 cell equivalents and the one from G. cydonium 8.3 TPG units; hence the activity reached approximately 30-20% of the activity seen in telomerase-positive reference cells. In contrast, dissociated spherulous cells from G. cydonium, after an incubation period of 24 h, contained no detectable telomerase activity. From earlier studies it is known that isolated sponge cells do not proliferate. Based on these findings it is assumed that the separation of the senescent sponge cell lineage from the immortal germ/somatic cell lineage is triggered by the loss of contact with cell adhesion factors. First evidence is included which suggests that the final progress of the senescent, telomerase-negative cells to cell death is caused by apoptosis.

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

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 formation of larger primmorphs (size of 10 mm) as well as of a canal system in primmorphs; canals are probably required for an improved oxygen and food supply. We conclude that the primmorph system will facilitate a sustainable use of sponges in the production of bioactive compounds; it may furthermore allow new and hitherto not feasible insights into basic questions on the origin of Metazoa.

Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula.

Dissociated cells from marine demosponges retain their proliferation capacity if they are allowed to form special aggregates, the primmorphs. On the basis of incorporation studies and septin gene expression, we show that Fe3+ ions are required for the proliferation of cells in primmorphs from Suberites domuncula. In parallel, Fe3+ induced the expression of ferritin and strongly stimulated the synthesis of spicules. This result is supported by the finding that the enzymatic activity of silicatein, converting organosilicon to silicic acid, depends on Fe3+. Moreover, the expression of a scavenger receptor molecule, possibly involved in the morphology of spicules, depends on the presence of Fe3+. We conclude that iron is an essential factor in proliferative and morphogenetic processes in primmorphs.

Molecular Mechanism of Spicule Formation in the Demosponge Suberites domuncula: Silicatein-Collagen-Myotrophin

In living organisms four major groups of biominerals exist: (1) iron compounds, which are restricted primarily to Prokaryota; (2) calcium phosphates, found in Metazoa; (3) calcium carbonates, used by Prokaryota, Protozoa, Plantae, Fungi and Metazoa and (4) silica (opal) present in sponges and diatoms (reviewed in: Bengtson 1994; Baeuerlein 2000). It is surprising that the occurrence of silica as a major skeletal element is restricted to some Protozoa and to sponges (Porifera). The element silicon (Si) contributes to 28% of the earth crust and is - after oxygen - the second most abundant element on earth (Windholz 1983).

Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium.

Abstract A cDNA encoding a receptor tyrosine kinase (RTK) was previously cloned and expressed from the marine sponge (Porifera) Geodia cydonium. In addition to the two intracellular regions characteristic for RTKs, two immunoglobulin (Ig)-like domains are found in the extracellular part of the sponge RTK. In the present study it is shown that no further Ig-like domain is present in the upstream region of the cDNA as well as of the gene hitherto known from the sponge RTK. Two different full-length cDNAs have been isolated and characterized in the present study, which possess two Ig-like domains, one transmembrane segment, and only a short intracellular part, without a TK domain. The two deduced polypeptides were preliminarily termed sponge adhesion molecules (SAM). The longer form of the SAM, GCSAML, encodes a deduced aa sequence, GCSAML, which comprises in the open reading frame 505 amino acids (aa) and has a calculated Mr of 53911. The short form, GCSAMS, has 313 aa residues and an Mr of 33987. The two Ig-like domains in GCSAML and GCSAMS are highly similar to the corresponding Ig-like domains in the RTKs from G. cydonium; the substitutions on both the aa and nt level are restricted to a few sites. Phylogenetic analyses revealed that the Ig-like domain 1 is similar to the human Ig lambda chain variable region, while the Ig-like domain 2 is related more closely to the human Ig heavy chain variable region. Transplantation experiments (autografting) were performed to demonstrate that the level of expression of the two new genes, GCSAML and GCSAMS, is upregulated during the self/self fusion process. Immunohistochemical analyses using antibodies raised against the two Ig-like domains demonstrate a strong expression in the fusion zone between graft and host. This finding has been supported by northern blotting experiments that revealed that especially GCSAML is strongly upregulated after autografting (up to 12-fold); the expression of GCSAMS reaches a value of 5-fold if compared with the controls. The results presented here demonstrate that the expression of the new molecules described, comprising two Ig-like domains, is upregulated during the process of autograft fusion.

FORMATION OF SPICULES BY SCLEROCYTES FROM THE FRESHWATER SPONGE EPHYDATIA MUELLERI IN SHORT-TERM CULTURES IN VITRO

SummaryCells from the freshwater spongeEphydatia muelleri were isolated by dissociating hatching gemmules. During the first 24 h the cells reaggregated, but the aggregates progressively disintegrated again to single cells, among which the spicule-forming sclerocytes were recognized. Such cultures were used to study spicule (megascleres) formation in vitro. The isolated sclerocytes formed the organic central axial filament onto which they deposited inorganic silicon. The size of the spicules (200 to 350µm in length) as well as the rate of spicule formation (1 to 10µm/h) under in vitro conditions were similar to the values measured in vivo. Immediately after completion of spicule formation, or even before, the sclerocyte could start formation of a new spicule; 5% of the cells were in the process of forming two spicules simultaneously. Cultivation of sclerocytes in the absence of silicon resulted in the formation of the axial filament only. We succeeded in maintaining the sclerocytes in a proliferating and spicule-forming state for up to 3 mo. These results demonstrate that the establishment of short-term cell cultures fromE. muelleri is possible; however, future studies must be undertaken to identify the growth factors required for a permanent culture of sponge cells.

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

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 neurotoxicity in primary cortical cells from rat embryos. Preincubation of the neurons with 1 or 10 microg/ml of sarcophytolide resulted in a significant increase of the percentage of viable cells from 33 +/- 4% (treatment of the cells with glutamate only) to 44 +/- 4 and 92 +/- 6%, respectively. Administration of sarcophytolide during the post-incubation period following glutamate treatment did not prevent neuronal cell death. Pretreatment of the cells with sarcophytolide for 30 min significantly suppressed the glutamate-caused increase in the intracellular Ca2+ level ([Ca2+]i). Evidence is presented that the neuroprotective effect of sarcophytolide against glutamate may be partially due to an increased expression of the proto-oncogene bcl-2. The coral secondary metabolite, sarcophytolide, might be of interest as a potential drug for treatment of neurodegenerative disorders.

DIFFERENTIAL IN VITRO ANTI-HIV ACTIVITY OF NATURAL LIGNANS

Abstract Two naturally occurring lignanolides, isolated from the tropical climbing shrub Ipomoea cairica, (-)-arctigen in and (-)-trachelogen in , were found to inhibit strongly replication of human immunodeficiency virus type 1 (HIV-1; strain HTLV-III B) in vitro. At a concentration of 0.5 (μм , (-)-arctigenin and (-)-trachelogenin inhibited the expression of HIV-1 proteins p 17 and p24 by 80 -90 % and 60 -70 % , respectively. The reverse transcriptase activity in the culture fluids was reduced by 80 -90 % when the cells (HTLV-III B/H 9) were cultivated in the presence of 0.5 μм (-)-arctigen in or 1 μм (-)-trachelogenin . At the same concentrations, the formation of syncytia in the HTLV-III B/H 9-Jurkat cell system was inhibited by the compounds by more than 80%. A series of other lignan type compounds displayed no anti-HIV activity. Studying the molecular mechanism of action of (-)-arctigenin and (-)-trachelogenin we found that both compounds are efficient inhibitors of the nuclear matrix-associated DNA topoisomerase II activity, particularly of the enzyme from HIV -1-infected cells. Our results suggest that both compounds prevent the increase of topoisomerase II activity, involved in virus replication, after infection of cells with HIV -1.

Histocompatibility reaction in tissue and cells of the marine sponge Suberites domuncula in vitro and in vivo: central role of the allograft inflammatory factor 1

Abstract. Sponges (Porifera) are the phylogenetically oldest still extant metazoan phylum. Recently elements of their immune system have been cloned and analyzed, primarily from the demosponges Suberites domuncula and Geodia cydonium. By differential display, two genes were identified in S. domuncula, whose translation products are involved in graft rejection/fusion: the allograft inflammatory factor (AIF-1) and the Tcf-like transcription factor (TCF). Since the AIF-1 and TCF genes are upregulated in vivo after tissue transplantation, especially in allografts, we investigated whether this reaction can be monitored in vitro. Therefore, the autogeneic and the allogeneic mixed sponge cell reaction (MSCR) system was applied for the first time to identify distinct factors in sponges in vitro. The results confirm that the two AIF-1 and TCF genes are induced during allogeneic MSCR. Furthermore, the recombinant sponge AIF-1 causes an upregulation of the expression of the TCF. We conclude that the AIF-1 and TCF genes are upregulated in sponges during histoincompatibility reactions; the data support the view that sponges have immune systems composed of highly complex elements related to those found in mammalian systems.

The multixenobiotic resistance mechanism in the marine sponge Suberites domuncula: its potential applicability for the evaluation of environmental pollution by toxic compounds

Experiments were carried out with the marine sponge Suberites domuncula to determine whether sponges may express-like mammalian tumor cells-a multidrug-like transporter system. The results demonstrate that sponge cells possess such a protective system termed multixenobiotic resistance (MXR) pump or P-glycoprotein-like pump. The protein was identified by antisera for the mammalian P170 multidrug resistance protein as a 130 kDa molecule. Binding studies were performed with 3H-vincristine (3H-VCR) and membrane vesicles; this process is ATP-dependent and inhibited by verapamil, which is known to reverse the multidrug-resistance phenotype in mammalian systems. Accumulation experiments were performed to demonstrate that the uptake of 3H-VCR is time-dependent, and increases at elevated extracellular levels of 3H-VCR. Application of the dyeing technique with calcein-AM, a suitable functional assay for multidrug transporter systems in mammal cells, also revealed the existence of the MXR pump in S. domuncula plasma membranes. These data demonstrate that S. domuncula is provided with a multidrug-like transporter, the MXR pump, which might function as a protection system for sponges in polluted environments.