Patch-clamp recordings in isolated sponge cells (Axinella polypoides)

Sponges are the most ancient known metazoans. Their cells are specialised but not organised into tissues or organs. Recordings of action potential-like propagating electrical impulses suggested that electrical signalling may occur between sponge cells, but the characterization of ionic channels in these cells is still at the beginning. Actually, sponge cell surfaces are covered by a complex glycocalyx and long-chain fatty acids are present in the lipid core of their membranes. In these experimental conditions, a low percentage of tight seals (3%) was obtained applying the patch-clamp technique to cells isolated from the Mediterranean Demospongia Axinella polypoides. This paper shows in detail how difficulties can be overcome making use of trivalent cations in the extracellular solution and how electrophysiological measurements can be performed on sponge cell membranes. A potassium selective conductance is shown as an example. We suggest that the presented methodology could also be applied to other cell types.     

Minor and trace sterols in marine invertebrates 47. A re-investigation of the 19-nor stanols isolated from the sponge Axinella polypoides

The aim of this research was to establish the true composition of the 19-nor stanols isolated from the sponge Axinella polypoides and to determine accurate stereochemistry for each 19-nor stanol isolated. The following new 19-nor stanols were collected from this sponge: (i) (22E,24S)-24-methyl-19,27-bisnor-5 alpha-cholest-22-en-3 beta-ol, (ii) (22R,23R)-22,23-methylene-19-nor-5 alpha-cholestan-3 beta-ol, (iii) (24 xi)-24-propyl-19-nor-5 alpha-cholestan-3 beta-ol and (iv) (23R,24R)-23,24-dimethyl-19-nor-5 alpha-cholestan-3 beta-ol. The general structure and stereochemistry of all fifteen 19-nor stanols were established by analysis of the MS and H-NMR (300 MHz, CDCl3) data measured for each compound. The relative percentage of 19-nor stanols having delta 22 double bonds should be sufficient to suggest that this sponge could be a potential source of starting material for the partial synthesis of certain oral contraceptives, which also have a 19-nor steroid nucleus.     

Studies on the relationship between lectins from Axinella polypoides agglutinating bacteria and human erythrocytes

A new lectin, called Axinella IV, with bacteria agglutinating activity was detected in the crude extract of the sponge Axinella polypoides. Immunofluorescence studies and immunoelectrophoresis revealed no cross-reactivities with the already known lectins Axinella I. II, and III. d-Galactose and oligosaccharides with d-Galactose in terminal nonreducing position are inactive in agglutination inhibition tests demonstrating also different specificity of Axinella IV lectin when compared with the three aforementioned lectins.     

The fine structure of pinacocytes in the marine sponge Microciona prolifera (Ellis and Solander)

Summary The primitive nature of the sponge epithelium was revealed primarily by the mode of intercellular attachment. The zonula adhaerens (terminal bar) and macula adhaerens (desmosome) which occur in most metazoa are totally lacking in the exo- and endopinacoderms of Micoricona prolifera. Although a careful search was made for examples of the zonula occludens (tight junction complex), none were seen. There is still the possibility that they occur, but if so, they occupy only a small portion of the junctional area.Both exo- and endopinacoderms are single-layered and composed of cells whose thickness is usually 0.5–2 . The similarity of architecture of the endopinacoderm to that of capillary endothelium suggests that the flattened shape of the endopinacocytes is a specialization for rapid diffusion between the water channels and the mesenchyme. Exopinacocytes are distinguished by the location of their nucleus in a club-shaped projection extending below the level of the exopinacoderm and by the mat of fibrous material which usually covers their flattened external surface. The presence of phagosomes in both the endo- and exopinacocytes suggests a nutritive role for these cells.Contrary to the reports of Wilson and Penney (1930) the pinacoderms are not syncytial. Reasons for their failure to see cell borders are discussed.This investigation was supported by N. I. H. Biomedical Support Grant 5 S05 FR0708802 to the University of Tennessee and by a Faculty Research Grant from the University of Tennessee.The author is indebted to Prof. Tracy L. Simpson for his suggestions and critical evaluation of this study.     

Phagocytic efficiency and cytotoxic responses of Indian freshwater sponge (Eunapius carteri) cells isolated by density gradient centrifugation and flow cytometry: a morphofunctional analysis

The freshwater sponge Eunapius carteri (Porifera: Demospongiae: Spongillidae), a resident of Indian freshwater ecosystems, has pharmaceutical and ecological potential, but there is inadequate information on its cellular spectrum and cell-mediated immune responses. Microscopical analysis revealed the existence of eight distinct cellular variants, i.e. blast-like cells, choanocytes, small amoebocytes, granular cells, pinacocytes, large amoebocytes, archaeocytes and sclerocytes. The cells were isolated by density gradient centrifugation and flow cytometry and used for a morphofunctional analysis. We investigated the phagocytic efficiency of E. carteri cells under the challenge of yeast particles in vitro and spectrophotometrically quantified the generation of cytotoxic molecules (superoxide anions and nitric oxide) in different isolated cellular fractions. The two cell separating technologies did not yield any significant differences in the major findings on morphology, phagocytic response and generation of superoxide anions and nitric oxide. Archaeocytes, granular cells and large amoebocytes were identified as chief phagocytes with a high phagocytic potential as recorded by light microscopy. Archaeocytes were the principal generators of superoxide anions, whereas nitric oxide was recorded in the fractions rich in archaeocytes and large amoebocytes. The present investigation thus provides useful information regarding cellular variation, cytotoxic status and innate phagocytic response of the cells of E. carteri, a common but less studied sponge of India.     

The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.     

Relative importance of bacteria, microalgae and yeast for growth of the sponge Halichondria melanadocia (De Laubenfels, 1936): A laboratory study

Bacteria, microalgae and yeast less than 10 μm in size are the primary food source of sponges but their relative contribution to somatic growth is poorly understood. In a laboratory study, the sponge Halichondria melanadocia was fed for 6 weeks a diet consisting solely of four bacterial strains, or a mixed diet consisting of bacteria, microalgae and yeast. Both diets were fed at three concentrations, based on the natural concentration (NC) of particles available to sponges: 1/5, 1 and 5NC. Mean final size of H. melanadocia was 40% greater on a mixed diet than on the bacteria diet, probably because of the greater supply of carbon and other essential nutrients in microalgae and yeast. Cell concentration also significantly affected the growth of H. melanadocia, with greatest growth for sponges fed at the highest cell concentration. The estimated carbon requirement for H. melanadocia to meet metabolic costs was 0.356 mg C l^− 1 or 103 μg C h^− 1 gDW^− 1. Many H. melanadocia appeared to be optimizing their surface area for food uptake.     

Isolation and characterization of a protease from the marine sponge Spheciospongia vesparia

A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC-anion exchange chromatographies. The purified protease showed a single band in SDS-PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10-phenanthroline, so it seemed to be a metalloprotease.     

Scanning electron microscopy of taxonomic diagnostic criteria of the freshwater sponge,Heteromeyenia tubisperma (Potts, 1881) (Porifera : Spongillidae)

Taxonomic diagnostic criteria of the spongillid freshwater sponge, Heteromeyenia tubisperma (Potts, 1881) were examined using scanning electron microscopy. The species is characterized by a gemmule which bears an unusually long, prominent porous tube. The application of SEM to the systematic studies of the freshwater sponges provides diagnostic capabilities not available with the light microscope. It is desirable that a key, coupled with a reference atlas of scanning electron micrographs illustrating taxonomic diagnostic criteria of freshwater sponge species, particularly utilizing type specimens, be developed.     

Spicule formation in the calcareous sponge Sycon ciliatum

Summary The spicule primordium is formed in an intercellular cavity within a group of sclerocytes. This cavity contains organic material which ensheaths the growing spicule but does not appear to determine the nature of the mineral morph (magnesian calcite) or the crystallographic orientation of the spicule. The tip of each growing spicule ray is seated in a dense cup in the cytoplasm of the sclerocyte concerned. Both ends of monaxons are initially inserted each into a dense cup. As rays elongate the sclerocyte membrane around the tip becomes invaginated and forms a system of converging spaces that possibly indicate high secretory activity in that region. Spicule growth involves the displacement and expansion of the organic sheath by the enlarging spicule. Fully formed spicules which are exposed to the mesohyl become surrounded by collagen fibrils. However, these fibrils are in no way concerned with the process of mineral deposition and are never found within the spicule calcite.     

Retagging identifies dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN) protein as a novel receptor for a major allergen from house dust mite

Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.     

The effects of lectins on the interaction between macrophages and bone in vitro

Summary Recent studies have demonstrated that the attachment of elicited rat macrophages to bone is mediated by specific saccharides located on the cell and/or bone surfaces. We have used a macrophage-bone culture system to study the effects of two lectins, concanavalin A (con A) and soybean agglutinin (SBA), on the morphology of macrophage attachment to a devitalized bone surface and subsequent functional activity. Macrophages were obtained from 3- to 4-week-old rats by peritoneal lavage and the adherent pool was used to prepare cell suspensions. Con A-treated, SBA-treated or control cell suspensions were aliquoted onto the endocranial surface of devitalized rat calvariae. The cells were allowed to attach for 1 h at 37° C, after which, the bone samples were removed from culture and prepared for scanning electron microscopy (SEM). The morphology of con A-treated macrophages attached to bone was markedly different from that of control or SBA-treated cells. Con A altered the attachment and subsequent spreading of macrophages on bone as visualized by SEM. Furthermore, the number of con A-treated cells that attached to bone and the average surface area of cell membrane apposed to the matrix was significantly different from that of control or SBA-treated cells. A ⁴⁵Ca bone-release assay was performed to evaluate the functional significance of the morphological findings. Lectin-treated or control cell suspensions were allowed to attach to the endocranial surface of ⁴⁵Ca pre-labeled calvariae for 1 h. Following attachment, the samples were cultured for 72 h. The con A-treated cultures demonstrated a significant decrease in the release of ⁴⁵Ca after 48 and 72 h in comparison to control cultures, while the ⁴⁵Ca released from SBA-treated cultures did not differ significantly from controls. These results suggest that certain sugar residues common to membrane-associated glycoconjugates and the organic component of the bone matrix regulate the attachment of macrophages to bone and their subsequent bone-resorbing activity.     

Latitudinal variation in spongivorous fishes and the effectiveness of sponge chemical defenses

It has been proposed that predation pressure declines with increasing latitude and a positive correlation exists between predation intensity and the investment into chemical defenses. However, little direct evidence supports the idea that tropical species are better defended chemically than their temperate counterparts. Temperate reefs of the South Atlantic Bight (SAB) off Georgia, USA, provide a unique opportunity to study tropical sponges in a temperate environment. We documented sponge species richness and abundance, sponge predator density, and examined the ability of eight sponge species to chemically deter predation by fishes on two reefs in the SAB. We used rarefaction analysis and ANOVA to compare our results for sponge species richness and density, respectively, with similar published studies conducted on reefs of the sub-tropical Atlantic (i.e., Florida Keys). These analyses were combined with similar statistical comparisons for spongivorous fish species richness and density. Results showed that sponge species richness was lower, but sponge density was higher, on the temperate SAB reefs than on the subtropical reefs. Both spongivorous fish diversity and density were lower on the SAB reefs. The greater abundance of sponges and lower density of predators on SAB reefs suggest a lower frequency of predation on sponges on SAB reefs. Of the eight sponge species assayed from the SAB reefs, five possessed chemical extracts that were significantly less deterrent to fish predators than their tropical/subtropical conspecifics. When the results were combined across all sponge species, the chemical deterrence of fish predators was significantly lower for extracts obtained from the temperate sponge community as compared to the tropical/subtropical assemblage. These results support the more general hypothesis that a lower density and diversity of sponge predators occurs at high as compared to low latitudes in the western Atlantic and may contribute to decreased investment in chemical defenses.     

Early Cambrian radiolarians and sponge spicules from the Niujiaohe Formation in South China

Most of the reported Cambrian radiolarians are from middle Cambrian (Miaolingian Series, Wuliuan Stage) and onward, the radiolarians from lower Cambrian (Terreneuvian and Cambrian Series 2), on the other hand, are poorly documented, thus their morphological characteristics have not been well understood. In this study, we extracted spherical radiolarians and sponge spicules from the chert of the Cambrian Series 2 Niujiaohe Formation in southern Jiangxi Province, China. The well-preserved radiolarian fossils, identified as Paraantygopora porosa, consist of perforate plate shells pierced by dense pores with elevated rims, and display similarities to those from Lower Ordovician. Seven forms of sponge spicules are recognized, including monaxons, diaxons, tetractines, hexactines, triaxon tetractines, triaxon pentactines and irregular tetraxon tetractines, which are similar to those from the Qiongzhusian Stage in South China. Our results, combined with previously reported early Cambrian radiolarians from South China, indicate that the early Cambrian radiolarians developed advanced spherical skeletons.     

The presence of an extracellular matrix between cells involved in the determination of the mesoderm bands in embryos ofPatella vulgata (Mollusca,gastropoda)

Summary In 32-cell stage embryos ofPatella vulgata one of the macromeres contacts the animal micromeres, and as a result is induced to differentiate into the stem cell of the mesodermal cell line. In this study we show the presence of an extracellular matrix (ECM) between these two interacting cell types. The ECM appears to be formed by the micromeres during the 32-cell stage. Staining experiments with alcian blue and tannic acid indicate that in contains glycoconjugates, possibly in the form of proteoglycans. The characteristics of the ECM were examined further by fluorescein isothiocyanate (FITC)-lectin labelling. Of 17 lectins tested, concanavalin A (ConA), succinyl-ConA, LCH-B (Lens culinaris) and PEA (Pisum sativum) showed a positive labelling of the ECM. These results are in accordance with the electron microscopic data. The appearance of the ECM at this specific stage and place suggests that it might play an important role in the induction of the mesodermal cell line.     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

A novel lectin from the sponge Haliclona cratera: isolation,characterization and biological activity

A lectin from the Adriatic sponge Haliclona cratera was purified by ion-exchange and gel chromatography. The molecular mass of the lectin is approximately 29 kDa. Purified lectin is rich in hydrophobic and basic amino acids and has an isoelectric point at pH 8.6. H. cratera lectin is relatively heat- and pH-stable. It agglutinates native and trypsinized, papainized and neuraminidase-treated human A, B, O, AB and sheep erythrocytes, and the hemagglutinating activity is independent of Ca(2+), Mn(2+) and Mg(2+) ions; D-galactose and N-acetyl-D-galactosamine are found to be moderate inhibitors of the activity. H. cratera lectin displays cytotoxic effect on HeLa and FemX cells and weak mitogenic effect on human T-lymphocytes pretreated with phytohemagglutinin (PHA).     

p-Aminophenol-induced cytotoxicity in Jurkat T cells: protective effect of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid

Acetaminophen (APAP) is hepatotoxic and can cause toxicity in Jurkat T cells. p-Aminophenol (PAP), an industrial chemical and APAP metabolite, is nephrotoxic and hepatotoxic. Its potential toxicity in Jurkat T cells was investigated. PAP (10-250 μM) caused toxicity (decreased survival and increased LDH activity in incubation medium) and GSH depletion. At a concentration of 100 μM but not 250 μM, PAP increased DNA fragmentation. It decreased p-Akt levels (Elisa) and at higher concentrations decreased p-Akt expression (Western blotting). It had no effect on FasL expression. The cysteine precursor 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (250 μM) attenuated the PAP (100 μM)-induced decrease in viability and prevented GSH depletion and increased DNA fragmentation. It attenuated the PAP-induced decrease in p-Akt levels and protected against the decrease in p-Akt expression. The results demonstrate PAP-induced toxicity and suggest that it is due at least in part to apoptosis and involves GSH depletion and p-Akt inactivation.