Refolding of hexameric porcine leucine aminopeptidase using a cationic detergent and dextrin-10 as artificial chaperones

The application of artificial chaperones in biotechnology has been inspired by the mechanism of molecular chaperones like GroEL/GroES. It involves addition of a capturing detergent during dilution of the chaotropic reagent, that prevents protein aggregation, and finally, addition of a oligosaccharide that removes the detergent allowing the protein to refold. Here, guanidinium hydrochloride-denatured hexameric leucine aminopeptidase is shown to be efficiently refolded by using the cationic detergent cetyltrimethylammonium bromide and the linear polysaccharide dextrin-10 as artificial chaperones. The effect of these additives and the time dependence on the recovery of total enzymatic activity, kinetic parameters (K_M, k_cat), intrinsic steady-state tryptophan fluorescence and oligomeric structure is presented. The method described is very promising since 92% of fully active and correct folded LAP could be produced. Moreover, we showed that the stripping process is relatively slow, it allows the protein to refold almost entirely to its native state.     

Endothelial transcytosis in health and disease

The visionaries predicted the existence of transcytosis in endothelial cells; the cell biologists deciphered its mechanisms and (in part) the molecules involved in the process; the cell pathologists unravelled the presence of defective transcytosis in some diseases. The optimistic perspective is that transcytosis, in general, and receptor-mediated transcytosis, in particular, will be greatly exploited in order to target drugs and genes to exclusive sites in and on endothelial cells (EC) or underlying cells. The current recognition that plasmalemmal vesicles (caveolae) are the vehicles involved in EC transcytosis has moved through various phases from intial considerations of caveolae as unmovable sessile non-functional plasmalemma invaginations to the present identification of a multitude of molecules and a crowd of functions associated with these ubiquitous structures of endothelial and epithelial cells. Further understanding of the molecular machinery that precisely guides caveolae through the cells so as to reach the target membrane (fission, docking, and fusion), to avoid lysosomes, or on the contrary, to reach the lysosomes, and discharge the cargo molecules will assist in the design of pathways that, by manipulating the physiological route of caveolae, will carry molecules of choice (drugs, genes) at controlled concentrations to precise destinations.     

A new Late Cretaceous (Maastrichtian) serial planktic foraminifer (Family Heterohelicidae) with early planispiral coil and revision of Spiroplecta Ehrenberg, 1844

A new planktic foraminifer, Hartella harti nov. gen., nov. sp. is described from the Maastrichtian sediments of the Atlantic Ocean. H. harti likely evolved from Fleisherites glabrans (Cushman). Spiroplecta Ehrenberg is reviewed and considered monospecific. The only species assigned to this genus is Spiroplecta americana Ehrenberg, which evolved from Heterohelix globulosa (Ehrenberg). It is demonstrated that the early planispiral coil developed in at least three separate lineages of serial planktic foraminifera in the Late Cretaceous (late Campanian-early Maastrichtian).     

Molecular identification of diazotroph microbial consortia in mountain soil

Nitrogen fixing microbial consortia from soil samples taken from five altitudinal vegetation zones (alpine, subalpine, coniferous, beech, Maleia flood plain) of Parang Massif, Romania, were isolated and identified. Molecular characterisation of nitrogen fixing consortia was carried out by PCR and nested PCR with 7 primer sets specific to nifH genes. All nifH genes are specific to nitrogen fixation and are found within phylogenetically related organisms which have the nitrogenase enzyme complex. These molecular studies allowed the assessment of nifH gene diversity within these nitrogen fixing microbial consortia from different type of soils. At high altitude, a consortium of nitrogen fixing bacteria dominated by Azotobacter chroococcum and Azospirillum brasilense was found. Clostridium, Rhizobiales, Herbaspirillum, Frankia species were also found in different rations depending on the altitudinal vegetation zone.     

PTEN tumor suppressor associates with NHERF proteins to attenuate PDGF receptor signaling

PTEN, a tumor suppressor frequently inactivated in many human cancers, directly antagonizes the activity of phosphatidylinositol-3-OH kinase (PI3K) by dephosphorylating phosphoinositides. We show here that PTEN interacts directly with the NHERF1 and NHERF2 (Na+/H+ exchanger regulatory factor) homologous adaptor proteins through the PDZ motif of PTEN and the PDZ1 domain of NHERF1 or both PDZ domains of NHERF2. NHERFs were shown to interact directly with platelet-derived growth factor receptor (PDGFR), and we demonstrate the assembly of a ternary complex between PTEN, NHERFs and PDGFR. The activation of the PI3K pathway after PDGFR stimulation was prolonged in NHERF1(-/-) mouse embryonic fibroblasts as compared to wild-type cells, consistent with defective PTEN recruitment to PDGFR in the absence of NHERF1. Depletion of NHERF2 by small interfering RNA similarly increased PI3K signaling. Phenotypically, the loss of NHERF1 enhanced the PDGF-induced cytoskeletal rearrangements and chemotactic migration of the cells. These data indicate that, in normal cells, NHERF proteins recruit PTEN to PDGFR to restrict the activation of the PI3K.     

RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells

AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE’s proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.