A Proton ENDOR Study of Azurin

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cβ atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein

Structure and Backbone Dynamics of Apo- and Holo-cellular Retinol-binding Protein in Solution

Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo- and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most relevant difference between the NMR structure ensembles of apo- and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligand-binding site. These comprise alpha-helix II, the subsequent linker to beta-strand B, the hairpin turn between beta-strands C and D, and the betaE-betaF turn. The internal backbone dynamics, obtained from 15N relaxation data (T1, T2, and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the betaC-betaD and betaE-betaF turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment

Determinants of frontline tyrosine kinase inhibitor choice for patients with chronic-phase chronic myeloid leukemia: A study from the Registro Italiano LMC and Campus CML

Background: Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors (TKIs) approved in Italy for frontline treatment of chronic-phase chronic myeloid leukemia (CP-CML). The choice of TKI is based on a combined evaluation of the patient's and the disease characteristics. The aim of this study was to analyze the use of frontline TKI therapy in an unselected cohort of Italian patients with CP-CML to correlate the choice with the patient's features. Methods: A total of 1967 patients with CP-CML diagnosed between 2012 and 2019 at 36 centers throughout Italy were retrospectively evaluated; 1089 patients (55.4%) received imatinib and 878 patients (44.6%) received a second-generation (2G) TKI. Results: Second-generation TKIs were chosen for most patients aged <45 years (69.2%), whereas imatinib was used in 76.7% of patients aged >65 years (p < .001). There was a predominant use of imatinib in intermediate/high European long-term survival risk patients (60.0%/66.0% vs. 49.7% in low-risk patients) and a limited use of 2G-TKIs in patients with comorbidities such as hypertension, diabetes, chronic obstructive pulmonary disease, previous neoplasms, ischemic heart disease, or stroke and in those with >3 concomitant drugs. We observed a greater use of imatinib (61.1%) in patients diagnosed in 2018-2019 compared to 2012-2017 (53.2%; p = .002). In multivariable analysis, factors correlated with imatinib use were age > 65 years, spleen size, the presence of comorbidities, and ≥3 concomitant medications. Conclusions: This observational study of almost 2000 cases of CML shows that imatinib is the frontline drug of choice in 55% of Italian patients with CP-CML, with 2G-TKIs prevalently used in younger patients and in those with no concomitant clinical conditions. Introduction of the generic formulation in 2018 seems to have fostered imatinib use

Analysis of Early Events during the First Year of Tyrosine Kinase Inhibitor Therapy in Patients with Chronic Phase - Chronic Myeloid Leukemia: A "Campus CML" Study

Tyrosine kinase inhibitors (TKIs) revolutionized treatment of chronic myeloid leukemia (CML). However, the rst months of therapy are crucial, as optimal response is dened as the achievement of molecular milestones at 3, 6 and 12 months (mo.) and as many toxicities, also causing a TKI switch, are more frequent in the 1st yea

Managing chronic myeloid leukemia for treatment-free remission: a proposal from the GIMEMA CML WP

Several papers authored by international experts have proposed recommendations on the management of BCR-ABL1+ chronic myeloid leukemia (CML). Following these recommendations, survival of CML patients has become very close to normal. The next, ambitious, step is to bring as many patients as possible into a condition of treatment-free remission (TFR). The Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA; Italian Group for Hematologic Diseases of the Adult) CML Working Party (WP) has developed a project aimed at selecting the treatment policies that may increase the probability of TFR, taking into account 4 variables: the need for TFR, the tyrosine kinase inhibitors (TKIs), the characteristics of leukemia, and the patient. A Delphi-like method was used to reach a consensus among the representatives of 50 centers of the CML WP. A consensus was reached on the assessment of disease risk (EUTOS Long Term Survival [ELTS] score), on the definition of the most appropriate age boundaries for the choice of first-line treatment, on the choice of the TKI for first-line treatment, and on the definition of the responses that do not require a change of the TKI (BCR-ABL1 6410% at 3 months, 641% at 6 months, 640.1% at 12 months, 640.01% at 24 months), and of the responses that require a change of the TKI, when the goal is TFR (BCR-ABL1 >10% at 3 and 6 months, >1% at 12 months, and >0.1% at 24 months). These suggestions may help optimize the treatment strategy for TFR

Farmacocinetica della vitamina A: caratterizzazione della reazione metabolica responsabile della biosintesi di acido retinoico

Dottorato di ricerca in biofarmaceutica-farmacocinetica. 8. cicloConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Interactions of cytoplasmic retinol-binding proteins with phospholipid vesicles: insights into the physiological functions

Vitamin A plays a key role in vision, cell growth and differentiation. In the cell, retinol has several fates: (a) it can be stored as retinyl ester of fatty acids through the action of lecithin-retinol acyl transferase (LRAT) [1]; (b) most non-esterified retinol is bound to cellular carriers (CRBP); (c) it can enter the oxidative pathway for the synthesis of retinoic acid, through the action of retinol dehydrogenases (RDH) [2]. CRBP-I is ubiquitous, whereas the homologous CRBP-II is expressed solely in the enterocytes. Our current understanding of these processes remains largely incomplete, but there is evidence that the membrane-bound LRAT and RDH are inactive towards the protein/ligand complex, suggesting that the membrane microenvironment may trigger retinol transfer from the holo protein to the enzymes. To address this hypothesis we have performed a suite of NMR experiments with CRBP-I and CRBP-II in the presence of model membranes composed of either anionic or zwitterionic phospholipids, at varying protein:lipid molar ratios and ionic strength. The results will be discussed, in comparison with our previous data collected in buffer [3, 4]. All these studies may help to understand certain aspects of the physiological functions of CRBPs. [1] J. Amengual et al. J. Biol. Chem. 287, (2012) 24216-24227. [2] S. Portè et al. Chemico-Biological Interactions 202, (2013) 186-194. [3] T. Mittag, L. Franzoni et al. J. Am. Chem. Soc. 128, (2006) 9844-9848. [4] L. Franzoni et al. J. Lipid Res. 51, (2010) 1332-1343