Traditional and Modern Biomedical Prospecting: Part I—the History: Sustainable Exploitation of Biodiversity (Sponges and Invertebrates) in the Adriatic Sea in Rovinj (Croatia)

Nature, especially the marine environment, provides the most effective drugs used in human therapy. Among the metazoans, the marine sponges (phylum Porifera), which are sessile filter feeders, produce the most potent and highly selective bioactive secondary metabolites. These animals (or their associated symbiotic microorganisms) synthesize secondary metabolites whose activity and selectivity has developed during their long evolutionary history (evochemistry). The exploitation of these resources has become possible due to the progress in molecular and cell biology. BIOTECmarin, the German Center of Excellence follows this rationale. In the past, these animals have been successfully and extensively utilized to isolate bioactive compounds and biomaterials for human benefit. Pharmaceuticals prepared from marine animals, primarily sponges, have been applied since ancient times (Hippocrates, Aristotle and later Plinius). It has been reported that extracts and/or components from sponges can be used for the treatment of specific diseases. For a systematic and applied-oriented exploitation, the successful development of effective compounds largely depends on quality of the institutional infrastructure of marine stations and more so on the biodiversity. The Center for Marine Research in Rovinj (Croatia) fulfils these prerequisites. Founded in 1891, this institute has to its credit major discoveries related to exploitation of secondary metabolites/biomaterials from sponges for therapeutical application and to obtain biomaterials for general wellbeing. This is the first part of a review focusing on biomedical prospecting. Here, we have mainly described the historic background. The details of techniques, substances, approaches and outlooks will be discussed in the second part

Comparison between the Comet Assay and Fast Micromethod® for Measuring DNA Damage in HeLa Cells

The sensitivity and precision of the single cell gel electrophoresis (Comet) assay and Fast Micromethod® for DNA damage determinations in human HeLa cell line were compared. The first assay allows analysis of DNA breaks in individual cells while the second is a rapid and convenient procedure for DNA breaks determination in cell suspensions on single microplates. Both assays detect DNA strand breaks, alkali-labile sites and transient breaks occurring at sites of ongoing repair and might be applied for the assessment of surface water genotoxic potential as well as for clinical use. DNA damage in HeLa cells was induced by different doses of γ-rays generated by Cs137 (8 to 500 cGy), UV-C light (10 to 1000 J m-2) and by different concentrations of 4-nitroquinoline-V-oxide (0.026-2.6 μmol dm-3). Gamma rays induced a dose-depended response with the average Comet tail moment values from 7 mm for the negative control to 291 mm for 200 cGy, from 6.1 to 192 mm for 500 J m-2 of UV-C light and from 7.1 to 238 mm for 1.0 μmol dm-3 of 4-nitro-quinoline-N-oxide. The Fast Micromethod® strand scission factor varied from 0.010 for negative control to 0.701 for 500 cGy, from 0.019 to 1.196 for 1000 J m-2 and from 0.003 to 0.810 for 0.5 μmol dm-3 of 4-nitroquinoline-IV-oxide. Sensitivity was the same for both methods and in the case of 4-nitroquinoline-IV-oxide even better precision (lower variation coefficient) was achieved with the Fast Micromethod®. Since the time required for multiple analysis by the Fast Micromethod® is short (2 hours or less), its use in measuring DNA breakage in cells can be recommended for environmental genotoxicity monitoring

Traditional and Modern Biomedical Prospecting: Part II—the Benefits: Approaches for a Sustainable Exploitation of Biodiversity (Secondary Metabolites and Biomaterials from Sponges)

The progress in molecular and cell biology has enabled a rational exploitation of the natural resources of the secondary metabolites and biomaterials from sponges (phylum Porifera). It could be established that these natural substances are superior for biomedical application to those obtained by the traditional combinatorial chemical approach. It is now established that the basic structural and functional elements are highly conserved from sponges to the crown taxa within the Protostomia (Drosophila melanogaster and Caenorhabditis elegans) and Deuterostomia (human); therefore, it is obvious that the molecular etiology of diseases within the metazoan animals have a common basis. Hence, the major challenge for scientists studying natural product chemistry is to elucidate the target(s) of a given secondary metabolite, which is per se highly active and selective. After this step, the potential clinical application can be approached. The potential value of some selected secondary metabolites, all obtained from sponges and their associated microorganisms, is highlighted. Examples of compounds that are already in medical use (inhibition of tumor/virus growth [arabinofuranosyl cytosine and arabinofuranosyl adenine]), or are being considered as lead structures (acting as cytostatic and anti-inflammatory secondary metabolites [avarol/avarone], causing induction of apoptosis [sorbicillactone]) or as prototypes for the interference with metabolic pathways common in organisms ranging from sponges to humans (modulation of pathways activated by fungal components [aeroplysinin], inhibition of angiogenesis [2-methylthio-1,4-napthoquinone], immune modulating activity [FK506]) are discussed in this study. In addition, bioactive proteins from sponges are listed (antibacterial activity [pore-forming protein and tachylectin]). Finally, it is outlined that the skeletal elements—the spicules—serve as blueprints for new biomaterials, especially those based on biosilica, which might be applied in biomedicine. These compounds and biomaterials have been isolated/studied by members of the German Center of Excellence BIOTECmarin. The goal for the future is to successfully introduce some of these compounds in the treatment of human diseases in order to raise the public awareness on the richness and diversity of natural products, which should be sustainably exploited for human benefit

Porifera Lectins: diversity, physiological roles and biotechnological potential

An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell interaction, biomineralization and spiculogenesis, as well as host defense mechanisms and potentially in the association between the sponge and its microorganisms. In addition, these lectins exhibited a broad range of bioactivities, including modulation of inflammatory response, antimicrobial and cytotoxic activities, as well as anticancer and neuromodulatory activity. In view of their potential pharmacological applications, sponge lectins constitute promising molecules of biotechnological interest

Structural and Functional Characterization of Ribosomal Protein Gene Introns in Sponges

Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with “higher” metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales

Comparison between the Comet Assay and Fast Micromethod® for Measuring DNA Damage in HeLa Cells

The sensitivity and precision of the single cell gel electrophoresis (Comet) assay and Fast Micromethod® for DNA damage determinations in human HeLa cell line were compared. The first assay allows analysis of DNA breaks in individual cells while the second is a rapid and convenient procedure for DNA breaks determination in cell suspensions on single microplates. Both assays detect DNA strand breaks, alkali-labile sites and transient breaks occurring at sites of ongoing repair and might be applied for the assessment of surface water genotoxic potential as well as for clinical use. DNA damage in HeLa cells was induced by different doses of γ-rays generated by Cs137 (8 to 500 cGy), UV-C light (10 to 1000 J m-2) and by different concentrations of 4-nitroquinoline-V-oxide (0.026-2.6 μmol dm-3). Gamma rays induced a dose-depended response with the average Comet tail moment values from 7 mm for the negative control to 291 mm for 200 cGy, from 6.1 to 192 mm for 500 J m-2 of UV-C light and from 7.1 to 238 mm for 1.0 μmol dm-3 of 4-nitro-quinoline-N-oxide. The Fast Micromethod® strand scission factor varied from 0.010 for negative control to 0.701 for 500 cGy, from 0.019 to 1.196 for 1000 J m-2 and from 0.003 to 0.810 for 0.5 μmol dm-3 of 4-nitroquinoline-IV-oxide. Sensitivity was the same for both methods and in the case of 4-nitroquinoline-IV-oxide even better precision (lower variation coefficient) was achieved with the Fast Micromethod®. Since the time required for multiple analysis by the Fast Micromethod® is short (2 hours or less), its use in measuring DNA breakage in cells can be recommended for environmental genotoxicity monitoring