Approaches for a sustainable use of the bioactive potential in sponges: analysis of gene clusters, differential display of mRNA and DNA chips.

In recent years, analyses of the genome organization of marine sponges have begun that have led to the elucidation of selected genes and gene arrangements that exist in gene clusters (e.g. the receptor tyrosine kinase cluster and the allograft inflammatory factor cluster). Most of these studies were performed with the demosponge Suberites domuncula; but Geodia cydonium (Demospongiae), Aphrocallistes vastus (Hexactinellida) and Sycon raphanus (Calcarea) were also investigated. Both S. domuncula and G. cydonium possess a surprisingly large genome of approximately 1.7 pg DNA per haploid set. Taking the high gene density in these sponges into account and considering that predominantly single-copy DNA exists, the gene number of S. domuncula and G. cydonium was estimated to be approximately 300,000. Presumably, the large gene number in the sponge genome is due to regional gene duplication; so far evidence for a transposition in sponges has been presented. Data indicate that only 0.25 % of the total sponge genome comprises CA/TG microsatellites, and until now also no SINEs/transposable elements have been identified. Due to the rapid progress in the field of molecular biology of sponges the application of sponge genes for expression studies by DNA-array techniques (microarray) has become possible. These achievements will be further supported by the systematic analysis of the expressed genome of sponges; the results will be (partially) released (http://spongebase.uni-mainz.de/cgi-bin/blast/blastserver.cgi). In our efforts employing the results from the analysis of the genome to molecular biotechnology, we applied the technique of differential display of mRNA. One example, the effect of silicate on gene expression in S. domuncula, is outlined here. Future results will allow the identification of the genes involved in the synthesis of bioactive compounds from sponges [Porifera]. This progress will contribute considerably to a fruitful and fast development in the field of molecular marine biotechnology.

Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa).

Recently the term Urmetazoa, as the hypothetical metazoan ancestor, was introduced to highlight the finding that all metazoan phyla including the Porifera (sponges) are derived from one common ancestor. Sponges as the evolutionarily oldest, still extant phylum, are provided with a complex network of structural and functional molecules. Analyses of sponge genomes from Demospongiae (Suberites domuncula and Geodia cydonium), Calcarea (Sycon raphanus) and Hexactinellida (Aphrocallistes vastus) have contributed also to the reconstruction of the evolutionary position of Metazoa with respect to Fungi. Furthermore, these analyses have provided evidence that the characteristic evolutionary novelties of Metazoa, such as the extracellular matrix molecules, the cell surface receptors, the nervous signal transduction molecules as well as the immune molecule existing in Porifera, share high sequence and in some aspects also functional similarities to related polypeptides found in other metazoan phyla. During the transition to Metazoa new domains occurred; as one example, the formation of the death domain from the ankyrin is outlined. In parallel, domanial proteins have been formed, such as the receptor tyrosine kinases. The metazoan essentials have been defined by analyzing and comparing the sponge sequences with the related sequences from the metazoans Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the fungus Saccharomyces cerevisiae and the plant Arabidopsis thaliana. The data revealed that those sponge molecules grouped to cell adhesion cell recognition proteins are predominantly found in Protostomia and Deuterostomia while they are missing in Fungi and Viridiplantae. Moreover, evidence is presented allowing the conclusion that the sponge molecules are more closely related to the corresponding molecules from H. sapiens than to those of C. elegans or D. melanogaster. Especially surprising was the finding that the Demospongiae are provided with elements of adaptive immunity.

Molecular evolution of the metazoan extracellular matrix: cloning and expression of structural proteins from the demosponges Suberites domuncula and Geodia cydonium.

One crucial event during evolution to multicellularity was the development of either direct cell-cell contact or indirect interaction via extracellular matrix (ECM) molecules. The identification of those polypeptides provides conclusive data on the phylogenetic relationship of metazoan phyla and helps us to understand the position of the Metazoa among the other kingdoms. Recently it became evident that the ECM of sponges is amazingly complex; it is composed of fibrous molecules, e.g., collagen, and their corresponding receptors, which are highly similar to those existing in other metazoan phyla. While these data already support the view of monophyly of Metazoa, additional studies are required to understand whether these molecules, which are similar in their primary sequence, also have the same function throughout the metazoan kingdom. In the present study we identified the ligand for one of the autopomorphic characters of Metazoa, the single-transmembrane receptor protein with the receptor tyrosine kinase (RTK) from G. cydonium, as an example: the putative mucus-like protein from G. cydonium. This protein was upregulated during autograft fusion in the homologous system with kinetics similar to those of the RTK. Additionally, a cDNA was isolated from S. domuncula whose deduced polypeptide displays a high sequence similarity to dermatopontin, an ECM molecule found exclusively in Metazoa. Furthermore, it is documented that expression of the fibrous ECM molecule collagen is regulated by the characteristic metazoan morphogens myotrophin and endothelial monocyte-activating polypeptide. These data indicate that the ECM of sponges is not an unstructured ground substance but provides the basis for integrated cell communication.

Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula.

The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophin-like polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval-shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up-regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G-x-y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.

[Immobilized forms of daunorubicin in patients with acute leukemia]. / Primenenie immobilizirovannykh form daunorubitsina u bol'nykh ostrymi leikozami.

AIM: To study pharmacokinetics of liposomal daunorubicine DaunoXome and daunorubicine (rubomycin) associated with red cells: to assess their effectiveness and toxicity in patients with acute leukemia. MATERIALS AND METHODS: 7 patients with resistant or recurrent acute leukemia entered the trial. Of them 2 patients had acute myeloid leukemia. They received DaunoXome in dose 100 mg in days 1, 2 and 3 of 7 + 3 program. 1 patient had pretreated acute promyelocytic leukemia. This patient received 5-day course of DaunoXome in a dose 100 mg in the presence of ATRA therapy. 4 patients were given single dose daunorubicin associated with autoerythrocytes in the courses RACOP and 7 + 3 in a dose 45 mg/m2. Concentrations of free, bound and liposomal daunorubicin were determined spectrofluorimetrically in chlorophorm extracts of plasm, blood, liquor and bone marrow specimens. RESULTS: Immobilization of daunorubicin on the red cells and liposomes changes pharmacokinetics of the drug: peak concentrations change and the area under the concentration curve increases. Tolerance of DaunoXome and daunorubicine associated with red cells was satisfactory in all the cases: clinical and echo-CG signs of cordiotoxicity were absent, myelotoxicity was similar to that of free daunorubicine. DaunoXome was effective in 2 of 3 patients with acute myeloblastic leukemia. CONCLUSION: The findings are of practical interest for physicians designing new programs of therapy of acute leukemia.

Gene structure and function of tyrosine kinases in the marine sponge Geodia cydonium: autapomorphic characters in Metazoa.

Porifera (sponges) represent the most ancient, extant metazoan phylum. They existed already prior to the 'Cambrian Explosion'. Based on the analysis of aa sequences of informative proteins, it is highly likely that all metazoan phyla evolved from only one common ancestor (monophyletic origin). As 'autapomorphic' proteins which are restricted to Metazoa only, integrin receptors, receptors with scavenger receptor cysteine-rich repeats, neuronal-like receptors and protein-tyrosine kinases (PTKs) have been identified in Porifera. From the marine sponge Geodia cydonium, a receptor tyrosine kinase (RTK) has been cloned that comprises the characteristic structural topology known from other metazoan RTKs; an extracellular domain, the transmembrane region, the juxtamembrane region and the TK domain. Only two introns, within the coding region of the RTK gene, could be found, which separate the two highly polymorphic immunoglobulin-like domains, found in the extracellular region of the enzyme. The functional role of this sponge RTK could be demonstrated both in situ (grafting experiments) and in vitro (increase of intracellular Ca2+ level). Upstream of this RTK gene, two further genes coding for tyrosine kinases (TK) have been identified. Both are intron-free. The deduced aa sequence of the first gene shows no transmembrane segment; from the second gene--so far--only half of its catalytic domain is known. A phylogenetic analysis with the TK domains from these sequences and a fourth, from a novel scavenger RTK (all domains comprise the signature for the TK class II receptors), showed that they are distantly related to the insulin and insulin-like receptors. The presented findings support the 'introns-late' hypothesis for such genes that encode 'metazoan' proteins. It is proposed that the TKs evolved from protein-serine/threonine kinases through modularization and subsequent exon shuffling. After formation of the ancestral TKs, the modules lost the framing introns to protect the evolutionary novelty. Since cell culture systems of sponges are now available, it can be expected that soon also those mechanisms that control the developmental programs will be unravelled.

Origin of insulin receptor-like tyrosine kinases in marine sponges.

One autapomorphic character restricted to all Metazoa including Porifera [sponges] is the existence of transmembrane receptor tyrosine kinases (RTKs). In this study we screened for molecules from one subfamily within the superfamily of the insulin receptors. The subfamily includes the insulin receptors (InsR), the insulin-like growth factor I receptors, and the InsR-related receptors--all found in vertebrates--as well as the InsR-homolog from Drosophila melanogaster. cDNAs encoding putative InsRs were isolated from the hexactinellid sponge Aphrocallistes vastus, the demosponge Suberites domuncula, and the calcareous sponge Sycon raphanus. Phylogenetic analyses of the catalytic domains of the putative RTKs showed that the sponge polypeptides must be grouped with the InsRs. The relationships revealed that all sponge sequences fall into one branch of this group, whereas related sequences from mammals (human, mouse, and rat), insects and molluscs, and polypeptides from one cephalochordate, fall together into a second branch. We have concluded that (i) the InsR-like molecules evolved in sponges prior to the "Cambrian Explosion" and contributed to the rapid appearance of the higher metazoan phyla; (ii) the sponges constitute a monophyletic taxon, and (iii) epidermal growth factor (EGF)-like domains are present in sponges, which allows the insertion of this domain into potential receptor and matrix molecules.

[Detrimental effects of Daunorubicin and Doxorubicin on human erythrocytes in vitro]. / Povrezhdaiushchee vozdeistvie Rubomitsina i Doksorubitsina na ritrotsity cheloveka in vitro.

In isotonic medium, daunorubicin in the concentration range of 0.5-5.0 mg/ml of cells and doxorubicin in the concentration range of 0.2-1.0 mg/ml of cells caused hemoglobin (Hb) and K+ to efflux from red blood cells (RBC), to increase RBC size and to lower their deformability. Hb and K+ efflux rates were proportional to the antibiotic concentrations and remained stable for a few hours. Hb efflux did not change significantly in the 4-21 degrees C range (exempt at an experimental concentration of daunorubicin of 5.0 mg/ml of cells) but soared sharply at 37 degrees C. At the daunorubicin and doxorubicin concentration of 0.2 mg/ml of cells, Hb and K+ efflux virtually did not differ from control values. At the antibiotic concentration of 1.0 mg/ml of cells, 37 degrees C, Hb efflux rate was 0.34-5.6%, while that of K+(-) 1.0-8.2%, per hour, of possible maximum value. For the daunorubicin level of 5.0 mg/ml of cells, the respective values were 10 and 17%. At the daunorubicin concentration of 5.0 mg/ml, at 4 degrees C, Hb efflux from RBC was significantly higher than at 21 degrees C. RBC malleability, which was determined as their ability to pass through membrane filters having 3 mm dia pores, did not differ significantly from control values for a few hours antibiotic concentrations not exceeding 0.3 mg/ml of cells. At the antibiotic concentration of 1.0 mg/ml and higher RBC deformability dropped to zero within 10 min. ATP level in RBC practically remained identical to control values during incubation with the antibiotics for several hours.

Receptor tyrosine kinase, an autapomorphic character of metazoa: identification in marine sponges.

In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulin-like receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, mouse and rat, including also invertebrate sequences, together form a second branch. It is concluded that the RTK molecules have evolved in sponges prior to the "Cambrian Explosion" and have contributed to the rapid appearance of the higher metazoan phyla and that sponges are, as a taxon, also monophyletic. Due to the fact that protein tyrosine kinases in general and RTKs in particular have only been identified in Metazoa, they are, as a group qualified, to be considered as an autapomorphic character of all metazoan phyla.

Multiple Ig-like featuring genes divergent within and among individuals of the marine sponge Geodia cydonium.

The receptor tyrosine kinase of the marine sponge Geodia cydonium features two extracellular Ig-like domains in which we recently documented RT-PCR polymorphism among individuals. Genomic-PCR analysis presented here revealed 14 unique sequences from four sponges, differing predominantly in the sequence of an intron which splits the Ig-like domains. Nevertheless, analysis of putative coding regions in 19 distinct clones (156-159 aa) from seven sponges revealed 69 positions of nucleotide substitutions, 67.6% of them non-synonymous, translating into 43 positions of divergent residues. Excluding aa deletions, these 19 sequences share pairwise aa identities of 89-99%. In three sponges, four or five unique Ig-like coding regions were scored. PCR amplification across this intron revealed multiple bands, polymorphic among five of six sponges. Further substantiated by Southern and Northern blots, it is evident that the genome of G. cydonium harbors multicopies of moderately divergent Ig-like domains. Presently, this only appears paralleled by the human KIR multigene family of NK cells MHC class I-specific receptors, which consist of two or three moderately divergent extracellular Ig-like domains.

Immunochemical localization of the phylogenetically oldest receptor tyrosine kinase: existence in the marine sponge Geodia cydonium.

Until now molecular data, elucidating the basis of invertebrate immunity are lacking. Previously both the gene and different cDNAs, coding for the ancestor of metazoan receptor tyrosine kinases (RTK), have been isolated from the marine sponge Geodia cydonium. The sponge RTK shows high polymorphism in the coding as well as in the non-coding parts of the gene. To further elucidate if the sponge RTK might be a molecule involved in self/non-self recognition the intracellular portion of the sponge RTK was expressed in Escherichia coli. The 59 kDa recombinant protein was used to raise monoclonal antibodies (McAb). The McAb recognized three polypeptides of sizes 135, 68 and 26 kDa by Western blotting. The McAb recognized only the plasma membranes of sponge cells as analyzed by immunohisto- and cytochemical studies. Northern blotting analysis revealed that the expression of the RTK gene depends on environmental conditions and on seasonal variations. Based on the high degree of polymorphism and on the immunochemical data we suggest that the RTK in G. cydonium might be involved in sponge immunity.

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."

Isolation of a second nonallelic insulin-like growth factor I gene from the salmon genome.

We have characterized a second nonallelic insulin-like growth factor-I (IGF-I) gene in the chum salmon (Oncorhynchus keta) genome. This gene, IGF-I.2, differs from the previously described chum salmon IGF-I gene, IGF-I.1, in the E peptide-coding portion of exon 3; specifically, the IGF-I.2 gene lacks one codon present in the IGF-I gene and contains two potential splice donor sites at the 3' end of exon 3 rather than the single, more distal site present in the IGF-I.1 gene. The expression of these two IGF-I genes could give rise to as many as six IGF-I mRNA species, each of which would encode a unique E-peptide moiety of the IGF-I prohormone. Thus, the presence of multiple, distinct IGF genes adds an additional level of complexity to IGF-I gene expression and IGF-I biosynthesis in salmon.