A patient-centered multidisciplinary cardiac rehabilitation program improves glycemic control and functional outcome in coronary artery disease after percutaneous and surgical revascularization.

BACKGROUND: Cardiac rehabilitation (CR) is strongly associated with all-cause mortality reduction in patients with coronary artery disease (CAD). The impact of CR on pathological risk factors, such as impaired glucose tolerance (IGT) and functional recovery remains under debate. The aim of the present study is to determine whether CR had a positive effect beside physical exercise improvement on pathological risk factors in IGT and diabetic patients with CAD. METHODS: One hundred and seventy-one consecutive patients participating in a 3-month CR from January 2014 to June 2015 were enrolled. The primary endpoint was defined as an improvement of peak workload and VO2-peak; glycated hemoglobin (HbA1c) reduction was considered as secondary endpoint. RESULTS: Euglycemic patients presented a significant improvement in peak workload compared to diabetic patients (from 5.75 ± 1.45 to 6.65 ± 1.84 METs vs. 4.8 ± 0.8 to 4.9 ± 1.4 METs , p = 0.018). VO2-peak improved in euglycemic patients (VO2-peak from 19.3 ± 5.3 to 22.5 ± 5.9 mL/min/kg, p = 0.003), while diabetic patients presented only a statistically significant trend (VO2-peak from 16.9 ± 4.4 to 18.0 ± 3.8 mL/min/kg, p < 0.056). Diabetic patients have benefited more in terms of blood glucose control compared to IGT patients (HbA1c from 7.7 ± 1.0 to 7.4 ± 1.1 compared to 5.6 ± 0.4 to 5.9 ± 0.5, p = 0.02, respectively). CONCLUSIONS: A multidisciplinary CR program improves physical functional capacity in CAD setting, particularly in euglycemic patients. IGT patients as well as diabetic patients may benefit from a CR program, but long-term outcome needs to be clarified in larger studies.

Structural basis of the lactate-dependent allosteric regulation of oxygen binding in arthropod hemocyanin.

Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H(+) and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k(obs) obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.

Reversibility and "pH-T phase diagrams" of Rapana venosa hemocyanin and its structural subunits.

We have studied the stability and reassociation behaviour of native molecules of Rapana venosa hemocyanin and its two subunits, termed RvH1 and RvH2. In the presence of different concentrations of Ca(2+) and Mg(2+) ions and pH values, the subunits differ not only in their reassociation behaviour, but also in their formation of helical tubules and multidecamers. RvH1 revealed a greater stability at higher pH values compared to RvH2. Overall, the stability of reassociated RvH and its structural subunits was found to be pH-dependent. The increasing stability of native Hc and its subunits, shown by pH-induced CD transitions (acid and alkaline denaturation), can be explained with the formation of quaternary structure. The absence of a Cotton effect at temperatures 20-40 degrees C in the pH-transition curves of RvH2 indicates that this subunit is stabilized by additional "factors", e.g.: non-ionic/hydrophobic stabilization and interactions of carbohydrate moieties. A similar behaviour was observed for the T-transition curves in a wide pH interval for RvH and its structural subunits. At higher temperatures, many of the secondary structural elements are preserved especially at neutral pH, even at extreme high temperatures above 90 degrees C the protein structures resemble a "globule state".

Respiration rate and oxy-regulatory capacity in cold stenothermal chironomids.

The effects of temperature and oxygen saturation on the respiration rate of two cold stenothermal chironomids, Diamesa insignipes and Pseudodiamesa branickii were investigated. Fourth instar larvae were collected in winter in a glacio-rhithral stream (1300 m a.s.l., Alps, NE-Italy) and their respiration rate was measured with a Clark's electrode in the range 0-14 degrees C. The respiration rate was significantly higher in D. insignipes than in P. branickii at low temperatures (

Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii. I. Apo form.

The effects of guanidinium chloride (GuHCl) on the stability of the apo form of the 5S non-reassociating subunit of hemocyanin from the crab Carcinus aestuarii (apo-CaeSS2) were investigated, using a variety of optical spectroscopy techniques (light scattering (LS), fluorescence (IF and EF) and circular dichroism (CD)). The fluorescence of 8-anilino-1-naphtalene sulphonate (ANS) was strongly enhanced in the presence of apo-CaeSS2, in contrast to holo-CaeSS2, suggesting the formation of a molten globule (MG)-like state, consequent to the removal of the two copper ions from the holo subunit. Other evidences, favouring the presence of this state in apo-CaeSS2, derive from an enhanced quenching of intrinsic fluorescence (IF) by acrylamide, a higher sensibility towards aggregation and a higher IF with respect to deoxy holo-CaeSS2. Aggregation of apo-CaeSS2 below 1.2 M GuHCl was detected by LS, suggesting the formation of an aggregation-prone intermediate, called I1. Due to this effect, fluorescence and CD data could only be collected above that denaturant concentration. Both IF (protein) and EF (ANS) fluorescence data were best fitted by a two-state cooperative transition, occurring between the intermediate I1 and the unfolded state U, with C(1/2) 1.6-1.7 M. A similar two-state transition, with a slightly higher C(1/2) value (1.9 M), was also inferred from far-UV CD data, suggesting the possible formation of another intermediate. Partial refolding of apo-CaeSS2 by dilution was found to occur above 1.2 M GuHCl, i.e. up to the level of I1, since at lower denaturant concentration protein aggregation took place, as also observed in unfolding. All thermodynamic parameters, derived from data above 1.2 M GuHCl, are therefore referred to transitions between intermediate and unfolded states only. Unfolding kinetics, followed by fluorescence stopped-flow, was biphasic in the whole GuHCl range investigated (3-5 M), suggesting the formation of a transient intermediate, possibly related to that observed under equilibrium conditions.

Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii. II. Holo form.

The effects of guanidinium hydrochloride (GuHCl) on the functional and structural properties of a 75-kDa, functionally active hemocyanin (Hc) subunit isolated from the crab Carcinus aestuarii (holo-CaeSS2) were investigated. The holo form of the protein contains two copper ions in the active site and is capable of reversibly binding dioxygen. The present results are compared with those previously described for the corresponding functionally inactive subunit (apo-CaeSS2), devoid of the two active site copper ions (accompanying paper [R. Favilla, M. Goldoni, A. Mazzini, M. Beltramini, P. Di Muro, B. Salvato, paper published in this issue]). As with apo-CaeSS2, both equilibrium and kinetic unfolding measurements were carried out using light scattering (LS), circular dichroism, intrinsic and extrinsic fluorescence (IF and EF, respectively). In addition here, absorbance spectroscopy was exploited to evaluate oxygen binding by holo-CaeSS2. These data were combined with those relative to the protein copper content to obtain information on the stability of the active site as a function of denaturant concentration. The different techniques used revealed several unfolding transitions. At GuHCl<1 M, an appreciable increase of LS intensity was observed, about an order of magnitude lower than with apo-CaeSS2, suggesting some reversible protein aggregation. A first cooperative transition as a function of GuHCl was detected as an increase of intensity of the protein IF (C(1/2)=1 M), followed by a second transition, characterised by a small intensity decrease and a red shift of the emission maximum (C(1/2)=1.4 M). Cooperative transitions with C(1/2) values near 1.4 M GuHCl were also detected by following the decrement of: (a) EF intensity of anilino-1-naphtalenesulphonate (ANS) bound to the protein; (b) absorbance at 340 nm, typical of oxy holo-CaeSS2; (c) copper-to-protein stoichiometry. A transition at higher GuHCl (C(1/2)=1.8 M) was also observed by far UV circular dichroism (far UV CD) and related to global unfolding. Unfolding kinetics was also studied using the fluorescence stopped-flow technique. All traces were best fitted by a sum of three or four exponential terms, depending on GuHCl concentration. A comprehensive unfolding model is proposed, which accounts for most of the complex behaviour of this protein subunit, including oxy and deoxy native and aggregation-prone intermediates, a highly fluorescent intermediate, molten globule-like apo and unfolded species.

Quaternary structure and functional properties of Penaeus monodon hemocyanin.

The hemocyanin of the tiger shrimp, Penaeus monodon, was investigated with respect to stability and oxygen binding. While hexamers occur as a major component, dodecamers and traces of higher aggregates are also found. Both the hexamers and dodecamers were found to be extremely stable against dissociation at high pH, independently of the presence of calcium ions, in contrast to the known crustacean hemocyanins. This could be caused by only a few additional noncovalent interactions between amino acids located at the subunit-subunit interfaces. Based on X-ray structures and sequence alignments of related hemocyanins, the particular amino acids are identified. At all pH values, the p50 and Bohr coefficients of the hexamers are twice as high as those of dodecamers. While the oxygen binding of hexamers from crustaceans can normally be described by a simple two-state model, an additional conformational state is needed to describe the oxygen-binding behaviour of Penaeus monodon hemocyanin within the pH range of 7.0 to 8.5. The dodecamers bind oxygen according to the nested Monod-Whyman-Changeaux (MWC) model, as observed for the same aggregation states of other hemocyanins. The oxygen-binding properties of both the hexameric and dodecameric hemocyanins guarantee an efficient supply of the animal with oxygen, with respect to the ratio between their concentrations. It seems that under normoxic conditions, hexamers play the major role. Under hypoxic conditions, the hexamers are expected not to be completely loaded with oxygen. Here, the dodecamers are supposed to be responsible for the oxygen supply.

Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii.

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.

Fluorescence spectroscopy of the tryptophan microenvironment in Carcinus aestuarii hemocyanin.

The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues.

Synergistic and antagonistic effects of thermal shock, air exposure, and fishing capture on the physiological stress of Squilla mantis (Stomatopoda).

This study is aimed at assessing the effects of multiple stressors (thermal shock, fishing capture, and exposure to air) on the benthic stomatopod Squilla mantis, a burrowing crustacean quite widespread in the Mediterranean Sea. Laboratory analyses were carried out to explore the physiological impairment onset over time, based on emersion and thermal shocks, on farmed individuals. Parallel field-based studies were carried out to also investigate the role of fishing (i.e., otter trawling) in inducing physiological imbalance in different seasonal conditions. The dynamics of physiological recovery from physiological disruption were also studied. Physiological stress was assessed by analysing hemolymph metabolites (L-Lactate, D-glucose, ammonia, and H+), as well as glycogen concentration in muscle tissues. The experiments were carried out according to a factorial scheme considering the three factors (thermal shock, fishing capture, and exposure to air) at two fixed levels in order to explore possible synergistic, additive, or antagonistic effects among factors. Additive effects on physiological parameters were mainly detected when the three factors interacted together while synergistic effects were found as effect of the combination of two factors. This finding highlights that the physiological adaptive and maladaptive processes induced by the stressors result in a dynamic response that may encounter physiological limits when high stress levels are sustained. Thus, a further increase in the physiological parameters due to synergies cannot be reached. Moreover, when critical limits are encountered, mortality occurs and physiological parameters reflect the response of the last survivors. In the light of our mortality studies, thermal shock and exposure to air have the main effect on the survival of S. mantis only on trawled individuals, while lab-farmed individuals did not show any mortality during exposure to air until after 2 hours.

Molecular basis of the Bohr effect in arthropod hemocyanin.

Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, k(on), rather than changes in k(off). In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349-1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s --> 4p(z) transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs.

Oxidized derivatives of Octopus vulgaris and Carcinus aestuarii hemocyanins at pH 7.5 and related models by x-ray absorption spectroscopy.

The binuclear copper sites of the met and met-azido derivatives of Octopus vulgaris and Carcinus aestuarii hemocyanins at pH 7.5 were characterized by high-resolution x-ray absorption spectroscopy in the low energy region (XANES) and in the higher region (EXAFS). The accuracy of the analysis of the data was tested with two mononuclear and six binuclear copper(II) complexes of the poly(benzimidazole) ligand systems 2-BB, L-5,5 and L-6,6 (Casella et al., 1993, Inorg. Chem. 32:2056-2067; 1996, Inorg. Chem. 35:1101-1113). Their structural and reactivity properties are related to those of the protein's derivatives. The results obtained for those models with resolved x-ray structure (the 2-BB-aquo and azido mononuclear complexes, and the binuclear L-5,5 Cu(II)-bis(hydroxo) (Casella et al., unpublished)), extends the validity of our approach to the other poly(benzimidazole)-containing complexes and to the hemocyanin derivatives. Comparison between the protein's and the complexes' data, support a description of the met-derivatives as a five-coordinated O-bridged binuclear copper(II) center and favors, for both species, a bis(hydroxo) structure with a 3-A Cu-Cu distance. For O. vulgaris met-azido derivative a mu-1,3 bridging mode for the ligand appears the most likely. The structural situation of C. aestuarii met-azido-derivative is less clear: a mu-1,1 mode is favored, but a terminal mode cannot be excluded.

Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution.

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.

A key structural role for active site type 3 copper ions in human ceruloplasmin.

Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.