PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax–caspase9–caspase3 pathway

This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24?h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24?h, cells were treated with ox-LDL for 24?h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.     

Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy

Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA _{P.fluorescens} indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K _i for glyphosate compared to those of the AroA _E.coli , while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA _{P.fluorescens} gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.     

The dynamics of melittin-induced membrane permeability

The transport of co-encapsulated solutes through the melittin-induced pores in the membrane of giant phospholipid vesicles was studied, and the characteristics of the pore formation process were modeled. Molecules of two different sizes (dextran and the smaller, fluorescent marker Alexa Fluor) were encapsulated inside the vesicles. The chosen individual vesicles were then transferred by micromanipulation from the stock suspension to the environment with the melittin (MLT). The vesicles were observed optically with a phase-contrast microscope and by monitoring the fluorescence signal. Such an experimental setup enabled an analysis of a single vesicle’s response to the MLT on the basis of simultaneous, separate measurements of the outflow of both types of encapsulated molecules through the MLT-induced pores in the membrane. The mechanisms of the MLT’s action were suggested in a model for MLT pore formation, with oligomeric pores continuously assembling and dissociating in the membrane. Based on the model, the results of the experiments were explained as a consequence of the membrane’s permeability dynamics, with a continuously changing distribution of pores in the membrane with regard to their size and number. The relatively stable “average MLT pore” characteristics can be deduced from the proposed model.     

Regulation of neuronal differentiation at the neurogenic wavefront

Signaling mediated by the Delta/Notch system controls the process of lateral inhibition, known to regulate neurogenesis in metazoans. Lateral inhibition takes place in equivalence groups formed by cells having equal capacity to differentiate, and it results in the singling out of precursors, which subsequently become neurons. During normal development, areas of active neurogenesis spread through non-neurogenic regions in response to specific morphogens, giving rise to neurogenic wavefronts. Close contact of these wavefronts with non-neurogenic cells is expected to affect lateral inhibition. Therefore, a mechanism should exist in these regions to prevent disturbances of the lateral inhibitory process. Focusing on the developing chick retina, we show that Dll1 is widely expressed by non-neurogenic precursors located at the periphery of this tissue, a region lacking Notch1, lFng, and differentiation-related gene expression. We investigated the role of this Dll1 expression through mathematical modeling. Our analysis predicts that the absence of Dll1 ahead of the neurogenic wavefront results in reduced robustness of the lateral inhibition process, often linked to enhanced neurogenesis and the presence of morphological alterations of the wavefront itself. These predictions are consistent with previous observations in the retina of mice in which Dll1 is conditionally mutated. The predictive capacity of our mathematical model was confirmed further by mimicking published results on the perturbation of morphogenetic furrow progression in the eye imaginal disc of Drosophila. Altogether, we propose that Notch-independent Delta expression ahead of the neurogenic wavefront is required to avoid perturbations in lateral inhibition and wavefront progression, thus optimizing the neurogenic process.     

Synthesis and evaluation of substituted hexahydronaphthalenes as novel inhibitors of the Mcl-1/BimBH3 interaction

Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in a broad range of human cancers and plays a critical role in conferring resistance to chemotherapy. In the course of screening a natural product-like library of sesquiterpenoid analogs, we identified substituted hexahydronaphthalenes that showed activity against the Mcl-1/BimBH3 interaction in vitro. Here, we describe the synthesis of a small library of analogs and their biological evaluation. The most potent inhibitor in the series (19) exhibits an IC(50) of 8.3μM by ELISA and disrupts the interaction between endogenously expressed Mcl-1 and Bim in cultured MDA-MB-468 breast cancer cells.     

Paxillus involutus Strains MAJ and NAU mediate K(+)/Na(+) homeostasis in ectomycorrhizal Populus x canescens under sodium chloride stress

Salt-induced fluxes of H(+), Na(+), K(+), and Ca(2+) were investigated in ectomycorrhizal (EM) associations formed by Paxillus involutus (strains MAJ and NAU) with the salt-sensitive poplar hybrid Populus × canescens. A scanning ion-selective electrode technique was used to measure flux profiles in non-EM roots and axenically grown EM cultures of the two P. involutus isolates to identify whether the major alterations detected in EM roots were promoted by the fungal partner. EM plants exhibited a more pronounced ability to maintain K(+)/Na(+) homeostasis under salt stress. The influx of Na(+) was reduced after short-term (50 mm NaCl, 24 h) and long-term (50 mm NaCl, 7 d) exposure to salt stress in mycorrhizal roots, especially in NAU associations. Flux data for P. involutus and susceptibility to Na(+)-transport inhibitors indicated that fungal colonization contributed to active Na(+) extrusion and H(+) uptake in the salinized roots of P. × canescens. Moreover, EM plants retained the ability to reduce the salt-induced K(+) efflux, especially under long-term salinity. Our study suggests that P. involutus assists in maintaining K(+) homeostasis by delivering this nutrient to host plants and slowing the loss of K(+) under salt stress. EM P. × canescens plants exhibited an enhanced Ca(2+) uptake ability, whereas short-term and long-term treatments caused a marked Ca(2+) efflux from mycorrhizal roots, especially from NAU-colonized roots. We suggest that the release of additional Ca(2+) mediated K(+)/Na(+) homeostasis in EM plants under salt stress.     

Fossil human remains from Bolomor Cave (Valencia, Spain)

Systematic excavations carried out since 1989 at Bolomor Cave have led to the recovery of four Pleistocene human fossil remains, consisting of a fibular fragment, two isolated teeth, and a nearly complete adult parietal bone. All of these specimens date to the late Middle and early Late Pleistocene (MIS 7-5e). The fibular fragment shows thick cortical bone, an archaic feature found in non-modern (i.e. non-Homo sapiens) members of the genus Homo. Among the dental remains, the lack of a midtrigonid crest in the M₁ represents a departure from the morphology reported for the majority of Neandertal specimens, while the large dimensions and pronounced shoveling of the marginal ridges in the C¹ are similar to other European Middle and late Pleistocene fossils. The parietal bone is very thick, with dimensions that generally fall above Neandertal fossils and resemble more closely the Middle Pleistocene Atapuerca (SH) adult specimens. Based on the presence of archaic features, all the fossils from Bolomor are attributed to the Neandertal evolutionary lineage.