Characterisation of ACP5 missense mutations encoding tartrate-resistant acid phosphatase associated with spondyloenchondrodysplasia.

Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism

Plant products with antifungal activity. From field to biotechnology strategies

In this chapter, informations on the recent advances regarding antifungal activity of natural products obtained from plants collected directly from their natural habitat or from plant cell and organ, cultures have been reported. The biotechnological approaches could increase uniformity and predictability of the extracts and overcome problems associated with geographical, seasonal, and environmental variations. Human fungal pathogens are the cause of severe diseases associated with high morbidity and mortality. The major human fungal pathogens are Candida species, dermatophytes, Aspergillus species, and Cryptococcus neoformans. Side effects and resistance are frequently attributed to the current antifungal agents. Moreover, the treatments often require long-term therapy and are not resolving. Plants represent a source of antifungal agents, but up to date, the number of new phytochemicals reaching the market is very low. This review attempts to summarize the current status of botanical screening efforts, as well as in vitro and in vivo studies on antifungal activity of plant products. Despite the currently non-uniform regulatory framework in all the states, the plant-derived products are increasingly in demand for their effectiveness. The basic conclusion from these studies is that rigorous, well-designed clinical trials are needed to validate the effectiveness and safety of plant extracts for their use as antifungals