Enhanced cytotoxic activity of a bifunctional chimeric protein containing a type 1 ribosome-inactivating protein and a serine protease inhibitor

Both ribosome-inactivating proteins (RIPs) and plant proteinase inhibitors, belong to protein families known to regulate cellular homeostasis and likely involved in plant defense. Nevertheless the interest in these protein classes is due to their potential use for the treatment of several important human diseases such as cancer. Thus, in the present study, type 1 ribosome-inactivating protein and wheat subtilisin/chymotrypsin inhibitor, were engineered into a chimeric protein with cytotoxic action selective for murine tumor cells, while lacking any appreciable toxicity on murine normal cells. This chimeric protein selectively sensitizes to apoptotic death cells derived from Simian-virus-40-transformed mouse fibroblasts (SVT2 cells). The cytotoxicity of this new recombinant product has been detected also on three different human malignant cells. Therefore action on tumor cells of this protein could represent a potentially very attractive novel tool for anticancer drug design.     

Corticoadrenal activity regulates in rat betaine-homocysteine <Emphasis Type="Italic">S</Emphasis>-methyltransferase expression with opposites effects in liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly corticosteroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys.     

Complex impact of an invasive crayfish on freshwater food webs

Invasive alien species can have complex effects on native ecosystems, and interact with multiple components of food webs, making it difficult a comprehensive quantification of their direct and indirect effects. We evaluated the relationships between the invasive crayfish, Procambarus clarkii, amphibian larvae and predatory insects, to quantify crayfish impacts on multiple levels of food webs, and to evaluate whether crayfish predation of aquatic insects has indirect consequences for their preys. We used pipe sampling to assess the abundance of crayfish, amphibian larvae and their major predators (Ditiscidae, Notonectidae and larvae of Anisoptera) in invaded and uninvaded ponds within a human dominated landscape. We disentangled the multivariate effects of P. clarkii on different components of food web through a series of constrained redundancy analyses. The crayfish had a negative, direct impact on both amphibian communities and their predators. Amphibian abundance was negatively related to both predators. However, the negative, direct effects of crayfish on amphibians were much stronger than predation by native insects. Our results suggest that this crayfish impacts multiple levels of food webs, disrupting natural prey-predator relationships.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

Toll-like receptor 3 activation induces antiviral immune responses in mouse sertoli cells

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.     

The carboxyl-terminal domain of closely related endotoxin-binding proteins determines the target of protein-lipopolysaccharide complexes.

The bactericidal/permeability increasing (BPI) and lipopolysaccharide (LPS)-binding (LBP) proteins are closely related two-domain proteins in which LPS binding is mediated by the NH(2)-terminal domain. To further define the role of the COOH-terminal domain of these proteins in delivery of LPS to specific host acceptors, we have compared interactions of LBP, BPI, LBP(N)-BPI(C) (NH(2)-terminal domain of LBP, COOH-terminal domain of BPI), and BPI(N)-LBP(C) with purified (3)H-LPS and, subsequently, with purified leukocytes and soluble (s)CD14. The COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both polymorphonuclear leukocytes and monocytes resulting in cell activation. In the presence of Ca(2+) and Mg(2+), LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent M(r) > 20 x 10(6) Da), but only LPS associated with LBP and BPI(N)-LBP(C) is disaggregated in the presence of CD14. BPI and LBP(N)-BPI(C) promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these closely related endotoxin-binding proteins dictates the route and host responses to complexes they form with endotoxin.     

A Proposito della Vaccinazione Nelle Piante Superiori

Riassunto

L'A. discute le varie interpretazioni date alle sue esperienze intorno ai fenomeni detti di vaccinazione nelle piante superiori e alla ipotesi emessa, a questo riguardo.     

Identification of an evx1-Dependent Joint-Formation Pathway during FIN Regeneration

Joints are essential for skeletal flexibly and form, yet the process underlying joint morphogenesis is poorly understood. Zebrafish caudal fins are comprised of numerous segmented bony fin rays, where growth occurs by the sequential addition of new segments and new joints. Here, we evaluate joint gene expression during fin regeneration. First, we identify three genes that influence joint formation, evx1, dlx5a, and mmp9. We place these genes in a common molecular pathway by evaluating both their expression patterns along the distal-proximal axis (i.e. where the youngest tissue is always the most distal), and by evaluating changes in gene expression following gene knockdown. Prior studies from our lab indicate that the gap junction protein Cx43 suppresses joint formation. Remarkably, changes in Cx43 activity alter the expression of joint markers. For example, the reduced levels of Cx43 in the sof ^b123 mutant causes short fin ray segments/premature joints. We also find that the expression of evx1-dlx5a-mmp9 is shifted distally in sof ^b123, consistent with premature expression of these genes. In contrast, increased Cx43 in the alf ^dty86 mutant leads to stochastic joint failure and stochastic loss of evx1 expression. Indeed, reducing the level of Cx43 in alf ^dty86 rescues both the evx1 expression and joint formation. These results suggest that Cx43 influences the pattern of joint formation by influencing the timing of evx1 expression.