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Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) by trapping the αβ dimers of Hb in the bloodstream. In turn, the Hp:Hb complexes display Hb-like reactivity. Here, the kinetics of NO dissociation from ferrous nitrosylated Hp:Hb complexes (i.e., Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively) are reported at pH 7.0 and 20.0 °C. NO dissociation from Hp:Hb(II)-NO complexes has been followed by replacing NO with CO. Denitrosylation kinetics of Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO are biphasic, the relative amplitude of the fast and slow phase being 0.495 ± 0.015 and 0.485 ± 0.025, respectively. Values of k off(NO)1 and k off(NO)2 (i.e., (6.4 ± 0.8) × 10 -5  s -1 and (3.6 ± 0.6) × 10 -5  s -1 for Hp1-1:Hb(II)-NO and (5.8 ± 0.8) × 10 -5  s -1 and (3.1 ± 0.6) × 10 -5  s -1 for Hp2-2:Hb(II)-NO) are unaffected by allosteric effectors and correspond to those reported for the α and β subunits of tetrameric Hb(II)-NO and isolated α(II)-NO and β(II)-NO chains, respectively. This highlights the view that the conformation of the Hb α 1 β 1 and α 2 β 2 dimers matches that of the Hb high affinity conformation. Moreover, the observed functional heterogeneity reflects the variation of energy barriers for the ligand detachment and exit pathway(s) associated to the different structural arrangement of the two subunits in the nitrosylated R-state. Noteworthy, the extent of the inequivalence of α and β chains is closely similar for the O 2 , NO and CO dissociation in the R-state, suggesting that it is solely determined by the structural difference between the two subunits., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The severity of heart disease varies widely among patients with transthyretin-related cardiac amyloidosis (ATTR-CA) at presentation, and availability of tools able to predict prognosis is essential for clinical and research purposes. Currently, two biomarker-based staging systems are available. The aim of this study was to compare their predictive performance., Methods: A total of 175 patients diagnosed with ATTR-CA (133 wild-type and 42 hereditary) were stratified into different stages based on 2 systems: the first system included N-terminal pro-B-type natriuretic peptide (NT-proBNP) and estimated glomerular filtration rate (eGFR), and the second one included NT-proBNP and troponin I (TnI). Survival estimates and age-adjusted survival for all-cause mortality were analysed over a median follow-up of 27 months (interquartile range 16-43 months)., Results: Predictive performance was more accurate when NT-proBNP and eGFR were used, resulting in effective survival stratification: 64.4 months for stage 1, 44.6 months for stage 2, and 20.5 months for stage 3 (P < 0.01 for stages 1 vs 2; P < 0.0001 for stages 1 vs 3; P < 0.0001 stages 2 vs 3). The combination of NT-proBNP and TnI was unable to effectively differentiate survival: 64.5 months for stage 1, 50.9 months for stage 2, and 27.3 months for stage 3 (P = 0.223 for stages 1 vs 2; P < 0.0001 for stages 1 vs 3; P < 0.0001 for stages 2 vs 3). The same results were seen after age adjustment., Conclusions: A staging system using NT-proBNP and eGFR had better prognostic accuracy for ATTR-CA patients compared with one using NTproBNP and TnI., (Copyright © 2019 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Human serum heme-albumin (HSA-heme-Fe) displays heme-based ligand binding and (pseudo-)enzymatic properties. Here, the effect of the prototypical drug warfarin on kinetics and thermodynamics of NO binding to ferric and ferrous HSA-heme-Fe (HSA-heme-Fe(III) and HSA-heme-Fe(II), respectively) and on the NO-mediated reductive nitrosylation of the heme-Fe atom is reported; data were obtained between pH5.5 and 9.5 at 20.0°C. Since warfarin is a common drug, its effect on the reactivity of HSA-heme-Fe represents a relevant issue in the pharmacological therapy management. The inhibition of NO binding to HSA-heme-Fe(III) and HSA-heme-Fe(II) as well as of the NO-mediated reductive nitrosylation of the heme-Fe(III) atom by warfarin has been ascribed to drug binding to the fatty acid binding site 2 (FA2), shifting allosterically the penta-to-six coordination equilibrium of the heme-Fe atom toward the low reactive species showing the six-coordinated metal center by His146 and Tyr161 residues. These data: (i) support the role of HSA-heme-Fe in trapping NO, (ii) highlight the modulation of the heme-Fe-based reactivity by drugs, and (iii) could be relevant for the modulation of HSA functions by drugs in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Chronic fluid overload is frequent in hemodialysis patients (P) and it associates with hypertension, left ventricular hypertrophy (LVH) and higher mortality. Moreover, echocardiographic data assessing fluid overload is limited. Our aim was to evaluate the relationship between fluid overload measured by bioimpedance spectroscopy (BIS) and different echocardiographic parameters., Methods: Cross-sectional observational study including 76 stable patients. Dry weight was clinically assessed. BIS and echocardiography were performed. Weekly time-averaged fluid overload (TAFO) and relative fluid overload (FO/ECW) were calculated using BIS measurements., Results: Based on TAFO three groups were defined: A- dehydrated, TAFO <-0.25 L 32 P (42%); B- normohydrated, TAFO between -0.25 and 1.5 l: 26 (34%); C- overhydrated, TAFO>1.5 l: 18 (24%). We found significant correlation between TAFO and left atrial volume index (LAVI) (r: 0.29; p=0.013) but not with FO/ECW (r 0.06; p=0.61). TAFO, but not FO/ECW kept a significant relationship with LAVI (p=0.03) using One-Way ANOVA test and linear regression methods. LVH was present in 73.7% (concentric 63.2%, eccentric in 10.5%). No differences between groups in the presence of LVH or left ventricular mass index were found., Conclusions: We found that left atrial volume index determined by echocardiographic Area-length method, but not left ventricle hypertrophy or dimensions of cavities, are related on hydration status based on bioimpedance measured time-averaged fluid overload (TAFO), and not with FO/ECW., (Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

Dystroglycan (DG) is a member of the glycoprotein complex associated to dystrophin and composed by two subunits, the β-DG, a transmembrane protein, and the α-DG, an extensively glycosylated extracellular protein. The β-DG ectodomain degradation by the matrix metallo-proteinases (i.e., MMP-2 and MMP-9) in both, pathological and physiological conditions, has been characterized in detail in previous publications. Since the amounts of α-DG and β-DG at the cell surface decrease when gelatinases are up-regulated, we investigated the degradation of α-DG subunit by MMP-2. Present data show, for the first time, that the proteolysis of α-DG indeed occurs on a native glycosylated molecule enriched from rabbit skeletal muscle. In order to characterize the α-DG portion, which is more prone to cleavage by MMP-2, we performed different degradations on tailored recombinant domains of α-DG spanning the whole subunit. The overall bulk of results casts light on a relevant susceptibility of the α-DG to MMP-2 degradation with particular reference to its C-terminal domain, thus opening a new scenario on the role of gelatinases (in particular of MMP-2) in the degradation of this glycoprotein complex, taking place in the course of pathological processes., (Copyright © 2014. Published by Elsevier B.V.)

We report a case of a Perforated Sinus of Valsalva Aneurysm (PSOV) closure using an Amplatzer muscular ventricular septal defect occluder (mVSD) device and describe a novel and potentially safer way for defect sizing. A literature review of the endovascular treatment of this disease is presented., (© 2014.)

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where [HSA]≥ 5×[NphOAc] and [NphOAc]≥ 5×[HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k(app) and k(obs) on [HSA] and [NphOAc], respectively, values of K(s), k(+2), and k(+2)/K(s) were determined. Values of K(s), k(+2), and k(+2)/K(s) obtained at [HSA]≥ 5×[NphOAc] and [NphOAc]≥ 5×[HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a) shift of the Tyr411 catalytic residue from 9.0 ± 0.1 in the substrate-free HSA to 8.1 ± 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To specify the incidence and risk factors for secondary autoimmune diseases (ADs) after HSCT for a primary AD, we retrospectively analyzed AD patients treated by HSCT reported to EBMT from 1995 to 2009 with at least 1 secondary AD (cases) and those without (controls). After autologous HSCT, 29 of 347 patients developed at least 1 secondary AD within 21.9 (0.6-49) months and after allogeneic HSCT, 3 of 16 patients. The observed secondary ADs included: autoimmune hemolytic anemia (n = 3), acquired hemophilia (n = 3), autoimmune thrombocytopenia (n = 3), antiphospholipid syndrome (n = 2), thyroiditis (n = 12), blocking thyroid-stimulating hormone receptor antibody (n = 1), Graves disease (n = 2), myasthenia gravis (n = 1), rheumatoid arthritis (n = 2), sarcoidosis (n = 2), vasculitis (n = 1), psoriasis (n = 1), and psoriatic arthritis (n = 1). After autologous HSCT for primary AD, the cumulative incidence of secondary AD was 9.8% ± 2% at 5 years. Lupus erythematosus as primary AD, and antithymocyte globulin use plus CD34(+) graft selection were important risk factors for secondary AD by multivariate analysis. With a median follow-up of 6.2 (0.54-11) years after autologous HSCT, 26 of 29 patients with secondary AD were alive, 2 died during their secondary AD (antiphospholipid syndrome, hemophilia), and 1 death was HSCT-related. This European multicenter study underlines the need for careful management and follow-up for secondary AD after HSCT.

Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O(2)-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O(2)-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k=9.8 × 10(-5) and 8.3 × 10(-4) s(-1), and h=1.3 × 10(-4) and 8.5 × 10(-4) s(-1), in the absence and presence of rifampicin, respectively, at pH=7.0 and T=20.0 °C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O(2)-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O(2) does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O(2)-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Type IV collagen remodeling plays a critical role in inflammatory responses, angiogenesis and metastasis. Its remodeling is executed by a family of matrix metalloproteinases (MMPs), of which the constitutive gelatinase A (MMP2) and the inducible gelatinase B (MMP9) are key examples. Thus, in many pathological conditions, both gelatinases act together. Kinetic data are reported for the enzymatic processing at 37 degrees C of type IV collagen from human placenta by MMP9 and its modulation by the fibronectin-like collagen binding domain (CBD) of MMP2. The alpha1 and alpha2 chain components of type IV collagen were cleaved by gelatinases and identified by mass spectrometry as well as Edman sequencing. Surface plasmon resonance interaction assays showed that CBD bound type IV collagen at two topologically distinct sites. On the basis of linked-function analysis, we demonstrated that CBD of MMP2 tuned the cleavage of collagen IV by MMP9, presumably by inducing a ligand-linked structural change on the type IV collagen. At low concentrations, the CBD bound the first site and thereby allosterically modulated the binding of MMP9 to collagen IV, thus enhancing the collagenolytic activity of MMP9. At high concentrations, CBD binding to the second site interfered with MMP9 binding to collagen IV, acting as a competitive inhibitor. Interestingly, modulation of collagen IV degradation by inactive forms of MMP2 also occurred in a cell-based system, revealing that this interrelationship affected neutrophil migration across a collagen IV membrane. The regulation of the proteolytic processing by a catalytically inactive domain (i.e., CBD) suggests that the two gelatinases might cooperate in degrading substrates even when either one is inactive. This observation reinforces the idea of exosite targets for MMP inhibitors, which should include all macromolecular substrate recognition sites.

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes beta-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward beta-amyloid. In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k(cat) (s(-1))=0.38 (+/-0.05); K(m) (M)=7.5 (+/-0.9) x 10(-6)] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic beta-amyloid through a decrease of the K(m) toward this substrate, which corresponds to the 10-25 amino acid sequence of the Abeta(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn(2+) ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic beta-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.

The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which neutrophil collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37 degrees C was investigated to identify functional differences in the processing of the two alpha-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of alpha-1 and/or alpha-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the alpha-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the alpha-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.

The use of 2,7-dimethyl-3,8-dinitrodipyrazolo[1,5-a:1',5'-d]pyrazine-4,9-dione as pre-column reagent for LC analysis of amino acids is proposed. The compound reacts (30 min at 68 degrees C in presence of 0.04 M triethylamine in a dimethylsulfoxide-water mixture) with primary and secondary amino function and the stable resulting adducts can be chromatographed under reversed-phase conditions and detected at lambda=280 nm. The derivatization conditions were optimized by a series of experiments. The effect of temperature, triethylamine concentration and reagent on the reaction was investigated. The yield of the glycine derivative was found to be quantitative at a reagent amino acid molar ratio of about 6 by comparison with an authentic specimen of synthesized glycine adduct. Application of the method to quality control of commercially available oral polyaminoacid formulations is described.

We characterized the overall early effect of chronic ochratoxin A (OTA) treatment on rat liver, analyzing different aspects related to: (i) fibrosis, by measuring collagen content and turnover, and alpha-smooth muscle actin (alphaSMA); (ii) oxidative stress and stress response, by analyzing protein carbonylation, superoxide dismutase (SOD) and heat shock protein (HSP70) gene expression; (iii) the possible tumor promoter effect, evaluating cadherin and connexin (CX) mRNA levels. Light microscopy analysis showed no histological differences in OTA-treated and control (CT) rats. Collagen content, determined by computer analysis of Sirius red-stained liver sections, was similar in both groups. In liver homogenates COL-I, COL-III, TIMP-1 and TGF-beta1 mRNA levels and alphaSMA were unaffected by OTA. Matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 protein levels were also similar in the two groups. Protein carbonylation, a marker of severe oxidative stress, was not evident in the homogenates of OTA-treated livers; superoxide dismutase (SOD) mRNA tended to be lower and HSP70 was strongly down-regulated. OTA reduced E-cadherin and DSC-2 transcription, and down-regulated liver CX26, CX32 and CX43. In conclusion, these in vivo results show that OTA-induced liver injury involves a reduction in the ability to counterbalance oxidative stress, maybe leading to altered gap junction intercellular communication and loss of cell adhesion and polarity. This suggests that mild oxidative damage might be a key factor, in combination with other cytotoxic effects, in triggering the promotion of liver tumors after exposure to OTA.

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).

Simple and rapid reversed-phase gradient column liquid chromatography (LC) with fluorescence detection at different wavelengths was developed for the simultaneous analysis of all-trans, 13-cis, 9-cis retinoic acids, vitamin A palmitate and beta-carotene in galenicals. The assay results agreed with those obtained by an LC method with diode-array UV detection. A post-column on-line photochemical reactor (irradiation at 254 and 366 nm) was inserted between the LC column and the fluorescence detector to enhance the performance of the method. Two fluorescence spectra (photoreactor on and off) were obtained for each analyte which proved useful for the unambiguous identification of the various analytes.