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.     

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and sequenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa–Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.     

Experimental Indication in Favor of the Introns-Late Theory: The Receptor Tyrosine Kinase Gene from the Sponge Geodia cydonium

We have analyzed the gene that encodes receptor tyrosine kinase (RTK) from the marine sponge Geodia cydonium, which belongs to the most ancient and simple metazoan groups, the Porifera. RTKs are enzymes found only in metazoa. The sponge gene contains two introns in the extracellular part of the protein. However, the rest of the protein (transmembrane and intracellular part), including the tyrosine kinase (TK)-domain, is encoded by a single exon. In contrast, all TK genes, so far known only from higher animals (vertebrates), contain several introns especially in the TK-domain. The TK-domain of G. cydonium shows similarity with numerous members of receptor as well as nonreceptor TKs. Phylogenetic analysis of the sponge TK-domain indicates that this enzyme branched off first from the common tree of metazoan TK proteins. Consequently, we assume that introns, found in the TK-domains of genes from higher animals, were inserted into these genes after splitting off the sponge taxa from other metazoan organisms (over 600 million years ago). Our results support the view that ancient genes were not ``in pieces.' Received: 8 August 1996 / Accepted: 4 November 1996     

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.     

Aggregative properties of different cell types of the fresh-water spongeEphydatia fluviatilis isolated on ficoll gradients

Summary Cell suspensions of the fresh-watersponge Ephydatia fluviatilis have been fractionated by means ofFicoll gradient centrifugation. Three fractions were isolated. The densest contains archeocyte-like cells only; the intermediate fraction is very rich in choanocytes, and the lightest is a mixture of cell types. Earch fraction shows specificaggregative properties and potentialities to reconstitute functional sponges.It appears that the sequence of reconstitution events can be selectively altered by certain disequilibria in the cell populationThese preliminary results constitute a first approach to the analysis ofcell type specificity in sponges.     

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.     

Investigations on the lectin-producing cells in the sponge Axinella polypoides (Schmidt)

Summary The localization of two carbohydrate binding proteins, so-called lectins, was studied in the sponge tissue of Axinella polypoides by light and immunofluorescence microscopy. They do not occur at the cellular surface of any cell type, but they are stored in vesicles of the spherulous cells. After short formaldehyde fixation spherulous cells can be isolated and they release the active lectins upon lysis in distilled water.Electron microscopical studies of spherulous cells show that they contain almost nothing else but a small nucleus and vesicles of different size and number. Small vesicles are full of an electron dense material, whereas the content of large vesicles has a fluffy and fibrillar structure. Spherulous cells are large and tightly packed in the outer layer of the ectosome and in the mesh work of the spongin fibres of the central axis. They are small and scattered in the inner layer of the ectosome, and they are found throughout the choanosome. The function of the lectins is not clearly defined, and different alternatives such as participation in glycoprotein synthesis, immunological defense, or carbohydrate transport are possible.This study was supported by a grant from the Deutsche ForschungsgemeinschaftWe are gratefully indebted to Dr. D. Keyser for his help in our electron microscopical studies     

Recent advances in marine drug research

Structures and properties of promising marine anti-cancer, anti-inflammation and anti-infectious (HIV, HSV, malaria, leishmania) compounds reported during 2008–2011 are discussed. Wherever possible, attempts have also been made to highlight their possible biogenesis or structure–activity relationships (SAR). Since the stress is on identifying and short-listing potential drug molecules, this review is restricted to only those compounds exhibiting promising in vitro activity, the arbitrary cut off being IC₅₀ < 15 μM, reported during the above period.     

The Drosophila DOCK family protein sponge is involved in differentiation of R7 photoreceptor cells

The Drosophila sponge (spg)/CG31048 gene belongs to the dedicator of cytokinesis (DOCK) family genes that are conserved in a wide variety of species. DOCK family members are known as DOCK1–DOCK11 in mammals. Although DOCK1 and DOCK2 involve neurite elongation and immunocyte differentiation, respectively, the functions of other DOCK family members are not fully understood. Spg is a Drosophila homolog of mammalian DOCK3 and DOCK4. Specific knockdown of spg by the GMR-GAL4 driver in eye imaginal discs induced abnormal eye morphology in adults. To mark the photoreceptor cells in eye imaginal discs, we used a set of enhancer trap strains that express lacZ in various sets of photoreceptor cells. Immunostaining with anti-Spg antibodies and anti-lacZ antibodies revealed that Spg is localized mainly in R7 photoreceptor cells. Knockdown of spg by the GMR-GAL4 driver reduced signals of R7 photoreceptor cells, suggesting involvement of Spg in R7 cell differentiation. Furthermore, immunostaining with anti-dpERK antibodies showed the level of activated ERK signal was reduced extensively by knockdown of spg in eye discs, and both the defects in eye morphology and dpERK signals were rescued by over-expression of the Drosophila raf gene, a component of the ERK signaling pathway. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rap1 in and around the plasma membrane of the eye disc cells. Together, these results indicate Spg positively regulates the ERK pathway that is required for R7 photoreceptor cell differentiation and the regulation is mediated by interaction with Rap1 during development of the compound eye.