C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind l-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N^G-nitro-l-arginine, but not its inactive d-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.     

Genetic diversity and differentiation of three Brazilian populations of Scarlet ibis (Eudocimus ruber)

The possible origin of the Scarlet ibis population of Cubat?o in southern Brazil, and its levels of genetic diversity and differentiation in relation to populations from the country’s northern coast were investigated through the sequences of 980 base pairs of β-fibrinogen intron 7 from a sample of 37 specimens. A total of 19 haplotypes were recorded in the three populations. Despite observed discrepancies in the levels of genetic diversity (π = 0.0017–0.0033; h = 0.60–0.95), AMOVA, K*st and Fst values all indicated that genetic differentiation among the populations was relatively low. This suggests that the Cubat?o population was isolated recently from the panmictic population that was once distributed all along the Brazilian coast, although it does not totally refute its possible derivation from a specific population on the north coast. Given our results, genetic management should focus on the minimization of inbreeding, especially in the smaller populations, such as Cubat?o. However, a more definitive study, including markers with higher evolutionary rates (e.g. microsatellites) and a much larger sample, would be required before any such actions can be taken.     

One-year clinical follow-up of a registry evaluating a percutaneous revascularisation strategy combining a pre-specified simple selection process with the use of a new thin-strut bare cobalt-chromium stent

Objectives. To evaluate clinical events in a specifically selected cohort of patients with obstructive coronary artery disease (CAD), using a new generation thin-strut bare cobalt-chromium coronary stent. Methods. Patients with single- or multi-vessel, stable or unstable CAD eligible for percutaneous implantation of at least one bare cobalt-chromium stent were evaluated in a single-centre registry. Prospective pre-specified criteria for bare cobalt-chromium stent implantation in our centre were: any acute ST-elevation myocardial infarction (MI), otherwise 1) de novo coronary lesion, and 2) lesion length <20 mm, and 3) reference vessel diameter >2.6 mm, and 4) no diabetes, unless reference vessel diameter >3.5 mm. Endpoints, retrospectively collected, were death, MI and clinically driven target-lesion revascularisation (TLR) and target-vessel revascularisation (TVR) after 12 months. Results. Between September 2005 and June 2007, 712 patients (48.7% one-vessel, 29.9% two-vessel, 20% three-vessel and 1.4% left main disease; 7.9% diabetics) were treated with 800 bare cobalt-chromium stents, for stable angina (40.9%), unstable angina (20.9%) or acute ST-elevation MI (38.2%). The procedural success rate was 99.3%. Peri-procedural MI rate was 2.2% in the semi-elective group. At 12 months there were 17 deaths (2.4%), of which nine non-cardiac, 20 (2.8%) MI, 19 (2.7%) TLR and 29 (4.1%) TVR. Early and late definite stent thrombosis occurred in four (0.6%) and three (0.4%) patients, respectively. Conclusion. A strategy aimed at minimising drug-eluting stent use and combining a pre-specified simple selection process with the use of a new thin-strut bare cobalt-chromium stent is safe and effective at one-year clinical follow-up. (Neth Heart J 2010;18:486-92.)     

Association between AKT1 but not AKTIP genetic variants and increased risk for suicidal behavior in bipolar patients

We tested the hypothesis that the presence of AKT1 and AKTIP polymorphisms, target genes that encode key proteins in the signaling of dopaminergic and serotonergic systems, is associated with suicidal behavior in bipolar patients. The subjects were 273 patients diagnosed with bipolar disorder I or II (age = 41.4 ± 12.9). TaqMan single‐nucleotide polymorphism genotyping assays (AKT1: rs2494731, rs3803304, rs3730358, rs10149779, rs2494746, rs1130214 and rs249878; AKTIP: rs9302648 and rs7189819) were used. We found that the AKT1 marker showed an association with suicide attempts (rs1130214, P < 0.05) and attempted violent attacks (rs2494746, P < 0.05). One out of the seven tested markers of AKT1 attained significant genotype association with violent attempt (rs2494731; P < 0.05). A significant association was detected in the AKT1 haplotype test. We did not observe an association between suicidal behavior and AKTIP variants and also did not find an interaction between AKTIP and AKT1 polymorphisms. In addition, we found that demographic and clinical data are associated with lifetime history of suicide attempts. Our data suggest that demographic and clinical characteristics and AKT1 single markers and haplotypes, but not AKTIP polymorphisms or interactions between AKT1 and AKTIP, are associated with increased risk for suicidal behavior in bipolar patients.     

Cost (Non)-Recovery by Platform Technology Facilities in the Bio21 Cluster

Platform technologies (PT) are techniques or tools that enable a range of scientific investigations and are critical to today''s advanced technology research environment. Once installed, they require specialized staff for their operations, who in turn, provide expertise to researchers in designing appropriate experiments. Through this pipeline, research outputs are raised to the benefit of the researcher and the host institution.¹ Platform facilities provide access to instrumentation and expertise for a wide range of users beyond the host institution, including other academic and industry users. To maximize the return on these substantial public investments, this wider access needs to be supported. The question of support and the mechanisms through which this occurs need to be established based on a greater understanding of how PT facilities operate. This investigation was aimed at understanding if and how platform facilities across the Bio21 Cluster meet operating costs. Our investigation found: 74% of platforms surveyed do not recover 100% of direct operating costs and are heavily subsidized by their home institution, which has a vested interest in maintaining the technology platform; platform managers play a major role in establishing the costs and pricing of the facility, normally in a collaborative process with a management committee or institutional accountant; and most facilities have a three-tier pricing structure recognizing internal academic, external academic, and commercial clients.     

Role of Sonic hedgehog signaling and the expression of its components in human embryonic stem cells

Human embryonic stem cells (hESC) are characterized by their ability to self-renew and differentiate into all cell types of the body, making them a valuable resource for regenerative medicine. Yet, the molecular mechanisms by which hESC retain their capacity for self-renewal and differentiation remain unclear. The Hedgehog signaling pathway plays a pivotal role in organogenesis and differentiation during development, and is also involved in the proliferation and cell-fate specification of neural stem cells and neural crest stem cells. As there has been no detailed study of the Sonic hedgehog (SHH) signaling pathway in hESC, this study examines the expression and functional role of SHH during hESC self-renewal and differentiation. Here, we show the gene and protein expression of key components of the SHH signaling pathway in hESC and differentiated embryoid bodies. Despite the presence of functioning pathway components, SHH plays a minimal role in maintaining pluripotency and regulating proliferation of undifferentiated hESC. However, during differentiation with retinoic acid, a GLI-responsive luciferase assay and target genes PTCH1 and GLI1 expression reveal that the SHH signaling pathway is highly activated. Besides, addition of exogenous SHH to hESC differentiated as embryoid bodies increases the expression of neuroectodermal markers Nestin, SOX1, MAP2, MSI1, and MSX1, suggesting that SHH signaling is important during hESC differentiation toward the neuroectodermal lineage. Our findings provide a new insight in understanding the SHH signaling in hESC and the further development of hESC differentiation for regenerative medicine.     

The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

Background

Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB₁ and CB₂ receptors mediate this therapeutic effect is unclear.

Principal Findings

We generated astrocytoma subclones that express set levels of CB₁ and CB₂, and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB₁, CB₂ and AKT, but still through a mechanism involving ERK1/2.

Significance

The high expression level of CB₁ and CB₂ receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB₁ and CB₂ receptors, yet still activate ERK1/2.     

Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages

Background

Sand fly saliva contains molecules that modify the host''s hemostasis and immune responses. Nevertheless, the role played by this saliva in the induction of key elements of inflammatory responses, such as lipid bodies (LB, also known as lipid droplets) and eicosanoids, has been poorly investigated. LBs are cytoplasmic organelles involved in arachidonic acid metabolism that form eicosanoids in response to inflammatory stimuli. In this study, we assessed the role of salivary gland sonicate (SGS) from Lutzomyia (L.) longipalpis, a Leishmania infantum chagasi vector, in the induction of LBs and eicosanoid production by macrophages in vitro and ex vivo.

Methodology/Principal Findings

Different doses of L. longipalpis SGS were injected into peritoneal cavities of C57BL/6 mice. SGS induced increased macrophage and neutrophil recruitment into the peritoneal cavity at different time points. Sand fly saliva enhanced PGE₂ and LTB₄ production by harvested peritoneal leukocytes after ex vivo stimulation with a calcium ionophore. At three and six hours post-injection, L. longipalpis SGS induced more intense LB staining in macrophages, but not in neutrophils, compared with mice injected with saline. Moreover, macrophages harvested by peritoneal lavage and stimulated with SGS in vitro presented a dose- and time-dependent increase in LB numbers, which was correlated with increased PGE₂ production. Furthermore, COX-2 and PGE-synthase co-localized within the LBs induced by L. longipalpis saliva. PGE₂ production by macrophages induced by SGS was abrogated by treatment with NS-398, a COX-2 inhibitor. Strikingly, SGS triggered ERK-1/2 and PKC-α phosphorylation, and blockage of the ERK-1/2 and PKC-α pathways inhibited the SGS effect on PGE₂ production by macrophages.

Conclusion

In sum, our results show that L. longipalpis saliva induces lipid body formation and PGE₂ production by macrophages ex vivo and in vitro via the ERK-1/2 and PKC-α signaling pathways. This study provides new insights regarding the pharmacological mechanisms whereby L. longipalpis saliva influences the early steps of the host''s inflammatory response.