Glycans with Antiviral Activity from Marine Organisms.

There remains today a critical need for new antiviral agents, particularly in view of the alarming increase in drug resistance and associated issues. The marine environment has been a prolific contributor towards the identification of novel therapeutic agents in the recent few decades. Added to this, glycans (or carbohydrate- or sugar-based compounds) have in very recent decades made outstanding contributions to the development of novel therapeutics. This review brings together these significant facets of modern drug discovery by presenting the reported literature on glycans derived from marine organisms that possess antiviral activity.The glycans have been grouped together based on the marine organism they were isolated from, namely, (1) bacteria, (2) chromists, (3) plants and (4) animals. For chromists, glycans are further subsectioned into Ochrophyta (brown algae), Miozoa (according to www.algaebase.org ; also called Myzozoa according to WoRMS, www.marinespecies.org ) (dinoflagellates) and Bacillariophyta (diatoms). For plants, glycans are further subsectioned into Chlorophyta, Rhodophyta and Tracheophyta. Glycans isolated to date are reported as alginates, chitosan, extracellular polysaccharides, fucans (e.g. fucoidans), galactans (e.g. carrageenans), glycolipids, glycosaminoglycans, glycosides, glycosylated haemocyanin, laminarans, mannans, polysaccharides (not defined), rhamnans and xylomannans. Interestingly, many of the glycans displaying antiviral properties are sulfated.Reports indicate that marine-sourced glycans have exhibited antiviral activity against African swine fever virus, cytomegalovirus, dengue virus, Epstein-Barr virus, encephalomyocarditis virus, human immunodeficiency virus, hepatitis C virus, herpes simplex virus, human cytomegalovirus, human papilloma virus, human rhino virus, influenza virus, Japanese encephalitis virus, murine leukaemia virus, murine sarcoma virus, Newcastle disease virus, parainfluenza virus, respiratory syncytial virus, Semliki Forest virus, tobacco mosaic virus, vaccinia virus, varicella zoster virus, viral haemorrhagic septicaemia virus and vesicular stomatitis virus. Selected representative glycan structures are presented in Fig. 20.1.

Cnidarian Jellyfish: Ecological Aspects, Nematocyst Isolation, and Treatment Methods of Sting.

Cnidarians play an important role in ecosystem functioning, in the competition among species, and for possible utilization of several active compounds against cardiovascular, nervous, endocrine, immune, infective, and inflammatory disorders or having antitumoral properties, which have been extracted from these organisms. Nevertheless, notwithstanding these promising features, the main reason for which cnidarians are known is due to their venomousness as they have a serious impact on public health as well as in economy being able to affect some human activities. For this reason a preeminent subject of the research about cnidarians is the organization of proper systems and methods of care and treatment of stinging. This chapter aims to present the data about the morphological, ecological, toxicological, epidemiological, and therapeutic aspects regarding cnidarians with the purpose to summarize the existing knowledge and to stimulate future perspectives in the research on these organisms.

Cytotoxicity in vitro analysis of ceramic materials for ''metal free'' prosthetic substructures.

AIM: The aim of this study was to investigate the in vitro cytotoxicity of 5 ceramic materials for metal-free fixed prosthodontics: In-Ceram, Cergo, IPS Empress II, Cercon ZrO2, Finesse All Ceram compared each other and to commercially pure Titanium (CpTi). METHODS: The materials, prepared directly from manufacturers as 10 mm diameter and 3 mm thickness disks, have been tested following the ISO 10993-l guidelines, performing the in vitro cytotoxicity test with the use of mouse's cells, fibroblasts L-929, isolated by muscular tissue and cultured in an appropriate medium. The MTT test has been used to evaluate the cell viability through the succinate dehydrogenase enzyme activity. The originality of this investigation is that all the materials examined have been tested under the same conditions: the cytotoxicity test has been performed on these materials at the same time, in the same period, under the same conditions of temperature and humidity and by the same operator. RESULTS: Not all tested materials were free from cytotoxicity. Cercon, within the limits of this in vitro study, showed the lower cytotoxicity. CONCLUSIONS: This in vitro study suggested that ceramic materials for metal free prosthetic substructures are in competition with the CpTi which is very used in implant prosthodontics.

Video-based eye tracking: our experience with Advanced Stimuli Design for Eye Tracking software.

We present an independent, flexible, and easily programmable software program for generating a wide set of visual stimuli paradigms in eye-movement studies. The software, called ASTIDET (Advanced Stimuli Design for Eye Tracking), has been interfaced in real time with a high speed video-based eye tracking system in order to obtain a reliable measurement of saccades. Two saccadic paradigms have been tested (gap and memory guided tasks) in 10 normal subjects. The preliminary results confirm that ASTIDET is a user-friendly software and can be interfaced with a video-based eye-tracking device in order to obtain reliable measurement of saccades.

Cytotoxicity of the venom of Pelagia noctiluca forskål (Cnidaria: Scyphozoa).

The activity of Pelagia noctiluca venom was never assessed on cultured cells; therefore, we have evaluated on V79 cells the cytotoxicity, genotoxicity and ATP depletion induced after treatment. Venom did not cause alteration on cell DNA, but showed remarkable cytotoxic properties. With the highest nematocyst concentration (150,000 nematocyst/ml) 74 and 39% cells survived after 1 and 3 h, respectively, when evaluated by Trypan blue. Treated cells showed increased ATP levels during the same time. Preliminary HPLC analyses have showed the occurrence of a protein containing peak.

Variations of ATP content in V79 cells treated with crude toxins of--Aequorea aequorea (Cnidaria: Hydrozoa) and Rhizostoma pulmo (Cnidaria: Scyphozoa). A preliminary study.

The toxicity of Cnidaria exerts a noticeable influence on some human activities, such as fishery and bathing, and on public health. As toxins of Mediterranean Cnidaria are located in nematocysts and in tissues, in this study the influence of crude toxins (nematocyst and surrounding tissue venom) extracted from the jellyfish Aequorea aequorea and Rhizostoma pulmo on ATP content of cultured V79 cells was assessed. Using the crude toxin of A. aequorea an increase of ATP levels in treated cells was noted; highest values (41.2 10(-7) mM/ml after 180 min treatment) were reached using the highest dose. Otherwise, a generalized decrease of ATP levels was observed treating cells with crude toxin of R. pulmo; recorded values showed the complete depletion of cell ATP at 115 min treatment with the highest dose. A statistical significance was recorded between treatment times and between doses using crude toxin of R. pulmo, and only between treatment times for A. aequorea.

An ultrastructural study of the Antarctic calanoid Copepod Metridia gerlachei giesbrecht, 1902. Adult female.

Scanning electron microscopy (SEM) techniques can highly contribute to the knowledge of body structures in order to differentiate between different species or between varieties within the same species. This is particularly important in extreme environments, such as in Antarctic waters, where the evolution efforts have promoted the development of endemisms. In this work the external anatomy of Metridia gerlachei (Copepoda, Calanida) adult females, sampled during the Italian Oceanographic Campaign in Antarctica 1987-88, was described by SEM, particularly considering some swimming legs and the genital abdominal joint. The descriptions already reported have been verified and some morphological details have been better emphasized. As concerns the P2, the hook process of the first segment of endopod and a series of spines vaguely indicated, but not defined, in previous descriptions have been clearly evidenced. In the P5 the occurrence of three well separated free segments and the location of a marginal sets have been shown. The ultrastracture of the genital segment showed that a clear areola surrounds the genital field.

Application of the principal component analysis (PCA) to the ecological study of an artificial environment: the tunny-fishing net of Camogli (Ligurian Sea).

The management of the marine environment and resource exploitation depend on the knowledge of both water conditions and ecological relationships between organisms. In the framework of fishing problems, an adequate food availability is important in order to allow maintaining and growth of fish stocks. The tunny-fishing of Camogli, owing to the coco-fibre texture of its net, can improve the trophic resources allowing the settlement of organisms eaten by fish. The distribution and composition of settled organisms was studied during the campaign 1988 by microscopical methods. The results have been elaborated by using multivariate (PCA) methods. Foraminifers, ciliates, hydroids, nematodes and copepods were the mainly observed groups. Their variations with season and depth and the relationships with caught fish species are presented. The elaboration of data by PCA allowed an easy and complete interpretation of the obtained complex data set showing the existence of a "rope" and of a "depth" effect.

In vitro evaluation of the cytotoxic, hemolytic and clastogenic activities of Rhizostoma pulmo toxin(s).

Cytotoxic, hemolytic and clastogenic activities of Rhizostoma pulmo toxin(s) contained in the jelly tissue free of nematocysts were investigated in mammalian cells with in vitro procedures. At the concentration of 37.6 microg/ml the tissue protein produced the death of 50% V79 cells; a similar potency was observed in terms of hemolytic activity. The toxin(s) was not clastogenic for human lymphocytes in culture at the concentration of 5 microg/ml.

Toxicity of Actinia equina (Cnidaria: Anthozoa) crude venom on cultured cells.

The toxicity of Cnidaria is a subject of high concern owing to its influence on human activities and public health and to the implications in ecological relationships between organisms. In order to simulate as much as possible the natural conditions and the consequences of stinging against injured cells, as well considering that Cnidarian venoms are located not only in nematocysts but also in tissues, the cytotoxicity and genotoxicity of crude extracts from nematocyst and surrounding tissues of the sea-anemone Actinia equina (Cnidaria: Anthozoa) were assessed on cultured fibroblasts (V79). The cytotoxicity was remarkable and cell survival was highly reduced at highest tested concentration (150,000 nematocysts/ml); using the Trypan blue dye exclusion test, only 40% of treated cells survived after the first hour of treatment and viable cells were not counted after the second hour. Also the results obtained utilizing the Neutral Red assay showed serious effects of crude toxin (only 31.5% and 12.6% cell survival after one and three hours respectively). Genotoxic effects were not observed at tested doses.

Toxicity of jellyfish and sea-anemone venoms on cultured V79 cells.

Cnidarian toxins exert an influence on human activities and public health. The cytotoxicity of crude toxins (nematocyst and surrounding tissue venom) of Aequorea aequorea, Rhizostoma pulmo and Anemonia sulcata was assessed on V79 cells. Rhizostoma pulmo and Anemonia sulcata crude venoms showed remarkable cytotoxicity and killed all treated cells at highest tested concentration within 2 and 3 hr, respectively. Aequorea aequorea crude venom greatly affected growth rate during long-term experiments. No genotoxic effect was observed.

Mitochondrial alteration caused by cnidarian toxins: a preliminary study.

Cnidarians contain different toxins causing cytotoxic and cytolytic effects probably due to cell membrane alterations. Mitochondria were used here as an experimental model system to characterize the action of cnidarian toxins. Toxin from the sea anemone Anemonia sulcata affects the function of rat liver mitochondria. Effects are dose- and time-dependent and they disappear when Ca2+ is absent. This toxin could therefore have a phospholipase-like action, as reported for other cnidarians. Mitochondrial alterations by cnidarian toxins could therefore be a particular case of a general mechanism of toxicity directed towards biological membranes.

HPLC separation of toxic fraction components extracted from planktonic and benthic Cnidaria.

HPLC separation of crude extract components derived from nematocysts and extranematocystic tissues of macroplanktonic jellyfish Aequorea aequorea and Rhizostoma pulmo and benthic sea-anemones Actinia equina and Anemonia sulcata was carried out by different columns. A satisfactory peak separation was obtained analyzing the toxin of Rhizostoma pulmo by cationic and C18 columns. Low molecular weight fragments were separated by C18 column and U.V. monitored varying pH values and obtaining the displacement of significant peaks. Clear differences between chromatographic plots concerning planktonic and benthic species was evidenced by anionic column; this result can point out a clear ecological analogy between species living in the same environment and a similar toxin biosynthesis, due to selective actions related to both the environment and the phylogenetic relationships; these organisms could have developed similar mechanisms useful to tackle the environment.

Gamma-interferon and retinoic acid synergize in inhibiting the growth of human neuroblastoma cells in nude mice.

We have investigated the effects of retinoic acid (RA), human recombinant gamma interferon (gamma-IFN), and the association of both agents on the growth of human neuroblastoma (NB) cells in [CD1(nu/nu)] nude mice. Two human NB cell lines, namely LAN-5 and GI-LI-N, were previously adapted to grow in syngeneic animals for 7 consecutive passages. At the eighth passage, only animals which developed 10-mm diameter tumors within 40 days from xenograft were admitted to the study. RA and/or gamma-IFN were administered subcutaneously 3-5 days per week for 3 consecutive weeks. The number of days necessary for each tumor mass to grow up to 20 mm diameter (in vivo doubling time, ivDT) was then evaluated. Tumor growth was significantly inhibited in gamma-IFN (P less than 0.005) and RA (P less than 0.05) treated mice grafted with GI-LI-N. The combination of the two agents did not further enhance ivDT. The tumor growth inhibition was not statistically significant in LAN-5 bearing mice treated with RA or gamma-IFN alone, while a synergistic effect between the two drugs was observed (P less than 0.05). We conclude that parenteral combined administration of RA and gamma-IFN may prove to be useful in inhibiting the growth of tumors derived from human NB cells resistant to single inducers.

A new human highly tumorigenic neuroblastoma cell line with undetectable expression of N-myc.

A peculiar human cell line (GI-ME-N) derived from the metastatic bone marrow of a 2-yr-old patient with stage IV neuroblastoma (NB) was extensively characterized. Cell-type-specific markers, tumorigenicity in nude mice, morphology, cytogenetics, and amplification/expression of the N-myc gene were evaluated. All metaphases presented the typical 1p deletion. Surface markers specific for NB cells, vimentin, and neurofilament proteins were all clearly detectable with immunofluorescence and/or western blot procedures. Moreover, it was found that GI-ME-N cells did not express N-myc oncogene or HLA class 1 antigens, and were not classified as peripheral neuroectodermal tumor cells. However, extremely short latency and survival times, comparable to peripheral neuroectodermal tumor cells, were observed in nude mice grafted with GI-ME-N. In addition, no correlations were observed in tumorigenicity of N-myc amplified (IMR32) versus unamplified (SK-N-SH GI-ME-N) human NB cell lines in nude mice. We conclude that N-myc amplification/expression do not correlate with the aggressiveness of human NB in athymic animals, which is not always explained by the peripheral neuroectodermal tumor cell nature of the malignant cells, either.