Molecular basis of the allosteric mechanism of cAMP in the regulatory PKA subunit

The second messenger cyclic Adenosine MonoPosphate (cAMP) mediates many biological process by interacting with structurally conserved nucleotide binding domains (cNBD's). Here, we use molecular dynamics simulations on RIIbeta-PKA, one of the best characterized members of the cNBD family, in presence and absence of cAMP. The results of our calculations are fully consistent with the available experimental data and suggest that the key factor of the cAMP allosteric mechanism in cNBDS's is the increased flexibility of the protein upon ligand release along with a mechanical coupling between helical segments. In addition, our calculations provide a rationale for the experimentally observed cAMP selective binding to PKA.     

Molecular dynamics simulations on HIV-1 Tat

Molecular dynamics simulations are used to investigate dynamics and intramolecular interactions of the HIV-1 transactivator (Tat) in aqueous solution. The calculations are based on the AMBER force field with particle mesh Ewald treatment for long-range electrostatics. The Tat structure exhibits a large flexibility, consistent with its absence of secondary structure elements. From an analysis of the correlation matrix and of electrostatic interactions we suggest that segments expressed by the two exons (amino acids 1-72 and 73-86, respectively) exhibit rather separated dynamic and energetic properties. We also identify intramolecular interactions of importance for structure stabilization. In particular, significant electrostatic interactions are recognized between the N-terminus and the basic domain of the protein, consistent with site-directed mutagenesis performed in this work.     

Efficacy and safety of rituximab treatment in early primary Sj?gren's syndrome: a prospective,multi-center,follow-up study

Introduction

Primary Sjögren’s syndrome (pSS) is an autoimmune disorder affecting exocrine glands; however, a subgroup of pSS patients experience systemic extra-glandular involvement leading to a worsening of disease prognosis. Current therapeutic options are mainly empiric and often translated by other autoimmune diseases. In the last few years growing evidence suggests that B-cell depletion by rituximab (RTX) is effective also in pSS. Patients with early active disease appear to be those who could benefit the most from RTX. The aim of this study was to investigate the efficacy and safety of RTX in comparison to disease modifying anti-rheumatic drugs (DMARDs) in early active pSS patients.

Methods

Forty-one patients with early pSS and active disease (EULAR Sjogren’s syndrome disease activity index, ESSDAI ≥ 6) were enrolled in the study. Patients were treated with either RTX or DMARDs in two different Rheumatology centers and followed up for 120 weeks. Clinical assessment was performed by ESSDAI every 12 weeks up to week 120 and by self-reported global disease activity pain, sicca symptoms and fatigue on visual analogic scales, unstimulated saliva flow and Schirmer’s I test at week 12, 24, 48, 72, 96, and 120. Laboratory assessment was performed every 12 weeks to week 120. Two labial minor salivary gland (MSG) biopsies were obtained from all patients at the time of inclusion in the study and at week 120.

Results

Our study demonstrated that RTX treatment results in a faster and more pronounced decrease of ESSDAI and other clinical parameters compared to DMARDs treatment. No adverse events were reported in the two groups. We also observed that RTX is able to reduce glandular infiltrate, interfere with B/T compartmentalization and consequently with the formation of ectopic lymphoid structures and germinal center-like structures in pSS-MSGs.

Conclusions

To our knowledge, this is the first study performed in a large cohort of early active pSS patients for a period of 120 weeks. We showed that RTX is a safe and effective agent to be employed in pSS patients with systemic, extra-glandular involvement. Furthermore, our data on pSS-MSGs provide additional biological basis to employ RTX in this disease.     

The role of macrophages polarization in predicting prognosis of radically resected gastric cancer patients

Tumour‐associated Macrophages (TAM) present two different polarizations: classical (M1) characterized by immunostimulation activity and tumour suppression; alternative (M2) characterized by tumour promotion and immune suppression. In this retrospective study, we evaluated the correlation between the two forms of TAM with survival time in radically resected gastric cancer patients. A total of 52 chemo‐ and radio‐naive patients were included. Two slides were prepared for each patient and double‐stained for CD68/NOS2 (M1) or CD68/CD163 (M2) and five representative high‐power fields per slide were evaluated for TAM count. The median value of the two macrophage populations density and the median value of M1/M2 ratio were used as cut‐off. Twenty‐seven patients with M1 density above‐the‐median had a significantly higher survival compared to those below the median. Twenty‐six patients with M1/M2 ratio above the median showed median OS of 27.2 months compared to 15.5 months of the patients below the median. No association between M2 macrophage density and patient's outcome was found. In multivariate analysis, M1/M2 was a positive independent predictor of survival. The M1 macrophage density and M1/M2 ratio, as confirmed in multivariate analysis, are factors that can help in predicting patients survival time after radical surgery for gastric cancer.     

Genetic variability of glutathione S-transferase enzymes in human populations: Functional inter-ethnic differences in detoxification systems

Glutathione S-Transferase enzymes (GSTs) constitute the principal Phase II superfamily which plays a key role in cellular detoxification and in other biological processes. Studies of GSTs have revealed that genetic polymorphisms are present in these enzymes and that some of these are Loss-of-Function (LoF) variants, which affect enzymatic functions and are related to different aspects of human health.     

Adenoviral Gene Transfer of PLD1-D4 Enhances Insulin Sensitivity in Mice by Disrupting Phospholipase D1 Interaction with PED/PEA-15

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg _ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.     

Traceability in clinical enzymology

The primary goal of standardisation for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the assay is carried out. In order to pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has established reference systems for the most important clinical enzymes. These systems are based on three requirements: a) reference measurement procedures that are extensively evaluated and carefully described; b) certified reference materials; and c) a network of reference laboratories operating in a highly controlled manner. Using these reference systems and the manufacturer's standing procedures, industry can assign traceable values to commercial calibrators. Clinical laboratories, which use routine procedures with validated calibrators to measure human specimens, can finally obtain values which are traceable to higher-order reference procedures. These reference systems constitute the structure of the traceability chain to which the routine methods can be linked via an appropriate calibration process, provided that they have a comparable specificity (i.e. they are measuring the same quantity).     

Genetic characterization of p27kip1 and stathmin in controlling cell proliferation in vivo

The CDK inhibitor p27^kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27^kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27^kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27^kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27^kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27^kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27^kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.     

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.