A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals, and plants. Recently Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has been suggested as candidate for heterologous genes expression. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein; the oligosaccharide structures are similar to those of mammals with N-linked galactose, and fucose residues. To date several heterologous proteins have been expressed in L. tarentolae including both cytoplasmic enzymes and membrane receptors. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of those tools coupled with a better understanding of the biology of Leishmania species will lead to value and power in commercial and research labs alike.

1 We investigated the role of mast cells in granuloma-associated angiogenesis in rat by using: (i) a mast cell membrane stabilizer, ketotifen; (ii) a mast cell depleting agent, compound 48/80. Moreover, we focused on the role of chymases, which exhibit proinflammatory and proangiogenic properties by using: (i) chymostatin, an inhibitor of chymase activity; (ii) a specific antisense oligonucleotide (AS-ODN) designed against rat mast cell protease-5 (rMCP-5), the most abundantly expressed chymase in the skin. 2 The formation of granuloma was evaluated, as wet weight, 96 h after the subcutaneous implant of two l-carrageenin (1%)-soaked sponges on the back of male Wistar rats. Angiogenesis was evaluated as haemoglobin content in the granulomatous tissue and as level of tumour necrosis factor-a (TNF-a) in the exudates. 3 A single injection of ketotifen (1-5-25 mg kg À1 i.p.) significantly reduced granuloma formation by 31.6, 44.6 and 71.9%, and haemoglobin content by 17.0, 35.0 and 66.2%, suggesting that the release of mediator(s) from mast cells modulates the process. Chymostatin (5-10 nmol À1 site À1 day À1 ) reduced granuloma-associated angiogenesis by 57.3 and 70.0%. 4 RT-PCR analysis showed that rMCP-5 mRNA amounts were significantly reduced by rMCP-5 AS-ODN (1.25-2.5-5.0 nmol site À1 ) by 69.5, 72.5 and 81.8%. In parallel experiments, rMCP-5 AS-ODN (1.25, 2.5, 5.0 nmol site À1 ) strongly reduced granuloma weight (26.1, 45.0 and 56.3%) and haemoglobin content (22.2, 50.4, 62.03%), suggesting that the observed effect is mediated through an antisense mechanism. 5 In conclusion, these data suggest that: (i) inhibition of mast cell mediators release may represent a novel strategy to modulate angiogenesis; (ii) among the chymase family, rMCP-5 is a key promoter of angiogenesis in the rat.

Human CD105 antigen, a type I integral membrane glycoprotein, is expressed as homodimer and oligomer by human endothelial cells, and forms a heteromeric association with TGF-beta signaling receptors I and II. Several mutations of CD105 antigen gene are involved in a vascular disorder known as hereditary hemorrhagic telangiectasia type 1. The proposed mechanism by which CD105 is involved in said disorder is haploinsufficiency. We report expression and characterization of human CD105 antigen extracellular domain in yeast Saccharomyces cerevisiae. Different strategies to influence the release of heterologous proteins in the medium, such as alteration of cell wall integrity or coexpression of protein disulfide isomerase, were addressed. Purified extracellular domain of human CD105 antigen retains capacity to bind human TGF-beta receptor II in vitro.

In this work we describe the production of site-specific biotinylated human myeloid differentiation factor 88 (MyD88). A vector containing a coding sequence for a peptide derived from the carboxyl terminus of the Klebsiella pneumoniae oxalacetate decarboxylase alpha subunit was used to allow expression and biotinylation of MyD88 in Drosophila melanogaster Schneider 2 cell cytoplasm. As estimated by a comparison of Schneider 2 lysate with standard protein, the maximum expression level was 1.3 mug 107 cells-1. About 4 mg of biotinylated protein was purified by affinity chromatography on monomeric avidin from a 1-L culture. Exogenous biotin added to the culture medium increased the biotinylation efficiency of the expressed protein. Biotinylated MyD88 produced in Drosophila cells was able to precipitate recombinant MyD88 expressed in human embryonic kidney cells. The stable expression of MyD88 in Drosophila Schneider 2 cells offers a convenient and attractive method for large-scale production, which may be required to clarify the role of MyD88 in the inflammatory response. Moreover, site-specific biotinylation of MyD88 provides a useful tag for interaction assays where high sensitivity is required.