Werner E. G. Müller

Evolutionary relationships of Metazoa within the eukaryotes based on molecular data from Porifera

Recent molecular data provide strong support for the view that all metazoan phyla, including Porifera, are of monophyletic origin. The relationship of Metazoa, including the Porifera, to Plantae, Fungi and unicellular eukaryotes has only rarely been studied by using cDNAs coding for proteins. Sequence data from rDNA suggested a relationship of Porifera to unicellular eukaryotes (choanoflagellates). However, ultrastructural studies of choanocytes did not support these findings. In the present study, we compared amino acid sequences that are found in a variety of metazoans (including sponges) with those of Plantae, Fungi and unicellular eukaryotes, to obtain an answer to this question. We used the four sequences from 70 kDa heat–shock proteins, the serine–threonine kinase domain found in protein kinases, β–tubulin and calmodulin. The latter two sequences were deduced from cDNAs, isolated from the sponge Geodia cydonium for the phylogenetic analyses presented. These revealed that the sponge molecules were grouped into the same branch as the Metazoa, which is statistically (significantly) separated from those branches that comprise the sequences from Fungi, Plantae and unicellular eukaryotes. From our molecular data it seems evident that the unicellular eukaryotes existed at an earlier stage of evolution, and the Plantae and especially the Fungi and the Metazoa only appeared later.

Evolutionary relationships of the metazoan beta gamma-crystallins, including that from the marine sponge Geodia cydonium.

βγ‐crystallins are one major component of vertebrate lenses. Here the isolation and characterization of a cDNA, coding for the first βγ‐crystallin molecule from an invertebrate species, the marine sponge Geodia cydonium, is described. The size of the transcript as determined by Northern blotting was 0.7 kb in length. The deduced amino acid sequence consists of 163 aa residues and comprises four repeated motifs which compose the two domains of the βγ‐crystallin. Motif 3 contains the characteristic βγ‐crystallin ‘Greek key’ motif signature, while in each of the three other repeats, one aa residue is replaced by an aa with the same physico‐chemical property. The sponge peptide shows striking similarities to vertebrateβγ‐crystallins. Analysis by neighbour joining of the sponge motifs with the two motifs present in spherulin 3a of Physarum polycephalum shows that motif 4 of the sponge βγ‐crystallin was added as the last single sequence to the tree. The data support the view that the βγ‐crystallin superfamily, present in eukaryotes, evolved from a common ancestor including also the sponge βγ‐crystallin.

Cold stress defense in the freshwater sponge Lubomirskia baicalensis

The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Baikal in winter (samples from March have been studied) under complete ice cover at near 0°C, and in summer in open water at 17 °C (September). In March, specimens show high metabolic activity as reflected by the production of gametes. L. baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum. Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein‐bound state. In metazoans OA inhibits both protein phosphatase‐2A and protein phosphatase‐1 (PP1). Only cDNA corresponding to PP1 could be identified in L. baicalensis and subsequently isolated from a L. baicalensis cDNA library. The deduced polypeptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases. As determined by western blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer). PP1 is not inhibited by low OA concentrations (100 nm); concentrations above 300 nm are required for inhibition. A sponge cell culture system (primmorphs) was used to show that at low temperatures (4 °C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured at 17 °C. In the enriched extract, PP1 activity at 4 °C is close to that measured at 17 °C. Immunoabsorption experiments revealed that hsp70 contributes to the high protein phosphatase activity at 4 °C. From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge.

Biomedical Inorganic Polymers

Vanadate is widely used as an inhibitor of protein tyrosine phosphatases (PTPase) and is routinely applied in cell lysis buffers or immunoprecipitations of phosphotyrosyl proteins. Additionally, vanadate has been extensively studied for its antidiabetic and anticancer effects. In most studies, orthovanadate or metavanadate was used as the starting compound, whereas these “vanadate” solutions may contain more or less oligomerized species. Whether and how different species of vanadium compounds formed in the biological media exert specific biological effect is still a X.-G. Yang • K. Wang (*) State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China e-mail: wangkuipku@bjmu.edu.cn W.E.G. Müller et al. (eds.), Biomedical Inorganic Polymers, Progress in Molecular and Subcellular Biology 54, DOI 10.1007/978-3-642-41004-8_1,

Effect of Bacterial Infection on Stem Cell Pattern in Porifera

Multicellular organisms derived from one common ancestor, the Urmetazoa. The only living fossils, which can testify about the earliest evolutionary processes in Metazoa on the molecular level are sponges (phylum: Porifera). The present study outlines that stem cells may play essential roles in cellular specialization, embryogenesis and sponge Bauplan formation, using the demosponge Suberites domuncula as a model. Data indicate that the archaeocytes represent, besides the germ/embryonic cells, totipotent stem cells. First marker genes have been identified, which are expressed in totipotent stem cells and in cells from gemmules. Furthermore, genes have been described that are characteristic for the three main cell lineages in sponges; they all originate from archaeocytes and are involved in the differentiation of skeletal cells, epithelial cells and contractile cells. Finally it is shown that after exposure to the endotoxin LPS (lipopolysaccharide) a differential gene expression occurs, leading to an upregulation of the gene encoding perforin and a concomitant down-regulation of noggin, a stem cell marker. In parallel with this process an increased phosphorylation of the mitogen-activating protein kinase p38 occurs. This modification of the p38 kinase has been quantified with a novel ELISA assay. Our data suggest that in response to bacterial infection the number of stem cells in sponges decreases.