Recombinant protein expression in Leishmania tarentolae.

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.

Yeast-secreted recombinant extracellular domain of human CD105 antigen is able to bind TGF-beta type II receptor in vitro.

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.

Targeting of PED/PEA-15 molecular interaction with phospholipase D1 enhances insulin sensitivity in skeletal muscle cells.

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.

Design, synthesis, and in vitro activity of peptidomimetic inhibitors of myeloid differentiation factor 88.

We describe the design and synthesis of a peptidomimetic library derived from the heptapeptide Ac-RDVLPGT-NH 2, belonging to the Toll/IL-1 receptor (TIR) domain of the adaptor protein MyD88 and effective in inhibiting its homodimerization. The ability of the peptidomimetics to inhibit protein-protein interaction was assessed by yeast 2-hybrid assay and further validated in a mammalian cell system by evaluating the inhibition of NF-kappaB activation, a transcription factor downstream of MyD88 signaling pathway that allows production of essential effector molecules for immune and inflammatory responses.

Polyomavirus BK-specific immunity after kidney transplantation.

Failure to mount or maintain a protective immune response may influence the development of polyomavirus BK (BKV)-associated nephropathy (PVAN). However, limited data are so far available on BKV-specific immunity after kidney transplantation. BKV-specific cellular immune response was retrospectively analyzed in kidney recipients with or without BKV infection/reactivation by measuring the frequency of interferon (IFN)-gamma-secreting cells in peripheral blood. Patients with BKV-active infection and good renal function (n=6) had a mean BKV-specific lymphocyte frequency 2 log lower than healthy controls and in the same range as BKV-seropositive recipients without active infection (n=7). Patients with PVAN (n=5) revealed undetectable levels of BKV-specific cells. However, two patients from the latter cohort treated with immunosuppression reduction showed the emergence of specific immunity, with IFN-gamma production in the same range as healthy controls. Our preliminary data suggest that lack of protective immunity toward BKV may favor the occurrence of BKV active infection and influence the progression to PVAN.

Recurrence of focal segmental glomerulosclerosis after renal transplantation in patients with mutations of podocin.

BACKGROUND: Posttransplant recurrence of focal segmental glomerulosclerosis (FSGS) occurs in a relevant proportion of FSGS patients and represents an important clinical emergency. It is taken as a proof of the existence of circulating permeability plasma factor(s) that are also putative effectors of original proteinuria in these patients. Familial forms of FSGS do not recur, but the discovery of numerous patients with sporadic FSGS and mutations of podocin (NPHS2, that is actually an inherited disease) who received a renal graft require a re-evaluation of the problem. METHODS: To evaluate the incidence of posttransplant recurrence of FSGS in patients with NPHS2, the authors screened for podocin mutations in 53 patients with the clinical and pathologic stigmata of FSGS who had renal failure and who had undergone renal transplantation.Results. Twelve children were found to carry a homozygous (n9) or a heterozygous (n4) mutation of podocin and were classified, according to current criteria, as patients with inherited FSGS. In 5 patients of this group (38%), proteinuria recurred after renal graft and in 2, renal biopsy results showed recurrence of FSGS. Prerecurrence serum of 3 patients of this cohort was tested for antipodocin antibodies with indirect immuno-Western utilizing human podocyte extracts and were found negative. The rate of FSGS recurrence was comparable in non-NPHS2-FSGS children (12 of 27) and adults (3 of 13). Also clinical outcome of recurrence and response to plasmapheresis and immunosuppressors were comparable, suggesting a common mechanism. CONCLUSION: These data show a high rate of FSGS recurrence in patients with NPHS2 mutations that is comparable with idiopathic FSGS and describe the successful therapeutic approach. Recurrence of an apparently inherited disease should stimulate a critical review of the mechanisms of recurrence and of original proteinuria in these cases.