Salamanders on the bench - A biocompatibility study of salamander skin secretions in cell cultures.

Salamanders have evolved a wide variety of antipredator mechanisms and behavior patterns, including toxins and noxious or adhesive skin secretions. The high bonding strength of the natural bioadhesives makes these substances interesting for biomimetic research and applications in industrial and medical sectors. Secretions of toxic species may help to understand the direct effect of harmful substances on the cellular level. In the present study, the biocompatibility of adhesive secretions from four salamander species (Plethodon shermani, Plethodon glutinosus, Ambystoma maculatum, Ambystoma opacum) were analyzed using the MTT assay in cell culture and evaluated against toxic secretions of Pleurodeles waltl, Triturus carnifex, Pseudotriton ruber, Tylototriton verrucosus, and Salamandra salamandra. Their effect on cells was tested in direct contact (direct culture) or under the influence of the extract (indirect exposure) in accordance with the protocol of the international standard norm ISO 10993-5. Human dermal fibroblasts (NHDF), umbilical vein endothelial cells (HUVEC), and articular chondrocytes (HAC), as well as the cell lines C2C12 and L929 were used in both culture types. While the adhesive secretions from Plethodon shermani are cytocompatible and those of Ambystoma opacum are even advantageous, those of Plethodon glutinosus and Ambystoma maculatum appear to be cytotoxic to NDHF and HUVEC. Toxic secretions from Salamandra salamandra exhibited harmful effects on all cell types. Pseudotriton ruber and Triturus carnifex secretions affected certain cell types marginally; those from Pleurodeles waltl and Tylototriton verrucosus were generally well tolerated. The study shows for the first time the effect of salamander secretions on the viability of different cell types in culture. Two adhesive secretions appeared to be cell compatible and are therefore promising candidates for future investigations in the field of medical bioadhesives. Among the toxic secretions tested, only two of the five had a harmful effect on cells, indicating different cell toxicity mechanisms.

Immune suppressive effect of cinnamaldehyde due to inhibition of proliferation and induction of apoptosis in immune cells: implications in cancer.

BACKGROUND: Besides its anti-inflammatory effects, cinnamaldehyde has been reported to have anti-carcinogenic activity. Here, we investigated its impact on immune cells. METHODS: Activation of nuclear factor-κB by cinnamaldehyde (0-10 µg/ml) alone or in combination with lipopolysaccharide was assessed in THP1XBlue human monocytic cell line and in human peripheral blood mononuclear cells (PBMCs). Proliferation and secretion of cytokines (IL10 and TNFα) was determined in primary immune cells and the human cell lines (THP1, Jurkat E6-1 and Raji cell lines) stimulated with cinnamaldehyde alone or in conjunction with lipopolysaccharide. Nitric oxide was determined in mouse RAW264.7 cells. Moreover, different treated PBMCs were stained for CD3, CD20 and AnnexinV. RESULTS: Low concentrations (up to 1 µg/ml) of cinnamaldehyde resulted in a slight increase in nuclar factor-kB activation, whereas higher concentrations led to a dose-dependent decrease of nuclear factor-kB activation (up to 50%) in lipopolysachharide-stimulated THP1 cells and PBMCs. Accordingly, nitric oxide, interleukin 10 secretion as well as cell proliferation were reduced in lipopolysachharide-stimulated RAW264.7 cells, PBMCs and THP1, Raji and Jurkat-E6 immune cells in the presence of cinnamaldehyde in a concentration-dependent manner. Flow cytometric analysis of PBMCs revealed that CD3+ were more affected than CD20+ cells to apopotosis by cinnamaldehyde. CONCLUSION: We attribute the anti-inflammatory properties of cinnamaldehyde to its ability to block nuclear factor-κB activation in immune cells. Treatment with cinnamaldehyde led to inhibition of cell viability, proliferation and induced apoptosis in a dose-dependent manner in primary and immortalized immune cells. Therefore, despite its described anti-carcinogenic property, treatment with cinnamaldehyde in cancer patients might be contraindicated due to its ability to inhibit immune cell activation.

Crystallization and preliminary structure determination of the plant food allergen Pru av 2.

Thaumatin-like proteins (TLPs) have mostly been investigated in the context of their function as pathogenesis-related proteins and only in recent years have some of them been classified as allergens. Here, the purification and crystallization of the first allergenic TLP, Pru av 2, a 23.3 kDa protein isolated from ripe cherries, is reported. The crystals diffracted to 2.1 A resolution at a rotating-anode generator and were found to belong to space group P2(1), with unit-cell parameters a = 44.48, b = 41.04, c = 59.16 A, beta = 106.61 degrees and one molecule per asymmetric unit. In order to obtain high-resolution data, an annealing protocol was applied that improved the resolution limit from 1.6 to 1.3 A at a synchrotron.

Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana.

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.