Effects of Blood Transfusion on Exercise Capacity in Thalassemia Major Patients

Anemia has an important role in exercise performance. However, the direct link between rapid changes of hemoglobin and exercise performance is still unknown.To find out more on this topic, we studied 18 beta-thalassemia major patients free of relevant cardiac dysfunction (age 33.5±7.2 years,males = 10). Patients performed a maximal cardiopulmolmonary exercise test (cycloergometer, personalized ramp protocol, breath-by-breath measurements of expired gases) before and the day after blood transfusion (500 cc of red cell concentrates). After blood transfusion, hemoglobin increased from 10.5±0.8 g/dL to 12.1±1.2 (p<0.001), peak VO₂ from 1408 to 1546mL/min (p<0.05), and VO₂ at anaerobic threshold from 965 to 1024mL/min (p<0.05). No major changes were observed as regards heart and respiratory rates either at peak exercise or at anaerobic threshold. Similarly, no relevant changes were observed in ventilation efficiency, as evaluated by the ventilation vs. carbon dioxide production relationship, or in O₂ delivery to the periphery as analyzed by the VO₂ vs. workload relationship. The relationship between hemoglobin and VO₂ changes showed, for each g/dL of hemoglobin increase, a VO₂ increase = 82.5 mL/min and 35 mL/min, at peak exercise and at anaerobic threshold, respectively. In beta-thalassemia major patients, an acute albeit partial anemia correction by blood transfusion determinates a relevant increase of exercise performance, observed both at peak exercise and at anaerobic threshold.     

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.     

Biochemical characterisation of the D60A mutant of the elongation factor 1α from the archaeon Sulfolobus solfataricus

The D60A mutant of the elongation factor (EF) 1α from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1α; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.     

Multimeric Scaffolds Displaying the HIV-1 Envelope MPER Induce MPER-Specific Antibodies and Cross-Neutralizing Antibodies when Co-Immunized with gp160 DNA

Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.     

Alum and Squalene-Oil-in-Water Emulsion Enhance the Titer and Avidity of Anti-Aβ Antibodies Induced by Multimeric Protein Antigen (1–11)E2, Preserving the Igg1-Skewed Isotype Distribution

The development of active immunotherapy for Alzheimer''s disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward Aβ, while minimizing the risks of adverse reactions. Multimeric protein (1–11)E2 induces a robust and persistent antibody response to Aβ in mice, when formulated in Freund''s adjuvant. The goal of this translational study was to evaluate the immunogenicity of (1–11)E2 formulated in alum (Alhydrogel 2%), or in a squalene oil-in-water emulsion (AddaVax), or without adjuvant. A IgG1-skewed isotype distribution was observed for the anti-Aβ antibodies generated in mice immunized with either the non-adjuvanted or the adjuvanted vaccine, indicating that (1–11)E2 induces a Th2-like response in all tested conditions. Both Alhydrogel 2% and AddaVax enhanced the titer and avidity of the anti-Aβ response elicited by (1–11)E2. We conclude that (1–11)E2 is a promising candidate for anti-Aβ immunization protocols that include alum or squalene-oil-in-water emulsion, or no adjuvant.     

NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease

NGF has been implicated in forebrain neuroprotection from amyloidogenesis and Alzheimer's disease (AD). However, the underlying molecular mechanisms are still poorly understood. Here, we investigated the role of NGF signalling in the metabolism of amyloid precursor protein (APP) in forebrain neurons using primary cultures of septal neurons and acute septo‐hippocampal brain slices. In this study, we show that NGF controls the basal level of APP phosphorylation at Thr668 (T668) by downregulating the activity of the Ser/Thr kinase JNK(p54) through the Tyr kinase signalling adaptor SH2‐containing sequence C (ShcC). We also found that the specific NGF receptor, Tyr kinase A (TrkA), which is known to bind to APP, fails to interact with the fraction of APP molecules phosphorylated at T668 (APP^pT668). Accordingly, the amount of TrkA bound to APP is significantly reduced in the hippocampus of ShcC KO mice and of patients with AD in which elevated APP^pT668 levels are detected. NGF promotes TrkA binding to APP and APP trafficking to the Golgi, where APP–BACE interaction is hindered, finally resulting in reduced generation of sAPPβ, CTFβ and amyloid‐beta (1‐42). These results demonstrate that NGF signalling directly controls basal APP phosphorylation, subcellular localization and BACE cleavage, and pave the way for novel approaches specifically targeting ShcC signalling and/or the APP–TrkA interaction in AD therapy.     

The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with magnesium and GDP

Recent studies have shown that elongation factors extracted from archaea/eukarya and from eubacteria exhibit different structural and functional properties. Along this line, it has been demonstrated that, in contrast to EF-Tu, Sulfolobus solfataricus EF-1alpha in complex with GDP (SsEF-1alpha.GDP) does not bind Mg(2+), when the ion is present in the crystallization medium at moderate concentration (5 mM). To further investigate the role that magnesium plays in the exchange process of EF-1alpha and to check the ability of SsEF-1alpha.GDP to bind the ion, we have determined the crystal structure of SsEF-1alpha.GDP in the presence of a nonphysiological concentration (100 mM) of Mg(2+). The analysis of the coordination of Mg(2+) unveils the structural bases for the marginal role played by the ion in the nucleotide exchange process. Furthermore, nucleotide exchange experiments carried out on a truncated form of SsEF-1alpha, consisting only of the nucleotide binding domain, demonstrate that the low affinity of SsEF-1alpha.GDP for Mg(2+) is due to the local architecture of the active site and does not depend on the presence of the other two domains. Finally, considering the available structures of EF-1alpha, a detailed mechanism for the nucleotide exchange process has been traced. Notably, this mechanism involves residues such as His14, Arg95, Gln131, and Glu134, which are strictly conserved in all archaea and eukarya EF-1alpha sequences hitherto reported.     

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.     

Co-immunization with multimeric scaffolds and DNA rapidly induces potent autologous HIV-1 neutralizing antibodies and CD8+ T cells

To obtain proof of concept for HIV vaccines, we generated recombinant multimeric particles displaying the HIV-1 Envelope (Env) third hypervariable region (V3) as an N-terminal fusion protein on the E2 subunit of the pyruvate dehydrogenase complex of Geobacillus stearothermophilus. The E2 scaffold self-assembles into a 60-mer core that is 24 nm in diameter, with a molecular weight of 1.5 MDa, similar to a virus like particle with up to 60 copies of a heterologous protein accessible on the surface. Env(V3)-E2 multimers were tested alone and in combination with Env(gp160) DNA in mice and rabbits. Following two or more co-immunizations with Env(V3)-E2 and Env gp160 DNA, all 18 rabbits developed potent autologous neutralizing antibodies specific for V3 in six weeks. These neutralizing antibodies were sustained for 16 weeks without boosting, and comparable responses were obtained when lipopolysaccharide, a contaminant from expression in E. coli, was removed. Co-immunizations of Env(V3)-E2 and DNA expressing gp160 elicited moderate CD8-specific responses and Env-specific antibodies in mice. Co-immunization with DNA and E2 was superior to individual or sequential vaccination with these components in eliciting both neutralizing antibodies in rabbits and CD8(+) T cell responses in mice. Co-immunization with DNA and multimeric E2 scaffolds appears to offer a highly effective means of eliciting rapid, specific, and sustained immune responses that may be a useful approach for other vaccine targets.