Deconvolution Analysis of the Non-Ionic Iomeprol, Iobitridol and Iodixanol Contrast Media-Treated Human Whole Blood Thermograms: A Comparative Study.

To study the effect of non-ionic contrast media on anticoagulated and non-anticoagulated human whole blood samples, calorimetric measurements were performed. The anticoagulated plasma showed the greatest fall in the total ΔH after Iodixanol treatment. The plasma-free erythrocytes revealed a pronounced shift in the Tmax and a decrease in the ΔH of hemoglobin and transferrin. The total ΔH of Iodixanol treatment showed the highest decline, while Iomeprol and Iobitridol had fewer adverse effects. Similarly, the non-anticoagulated samples revealed a decrease both in the Tmax and the ΔH of albumin and immunoglobulin-specific transitions. The total ΔH showed that Iodixanol had more influence on the serum. The serum-free erythrocyte samples resulted in a significant drop in the Tmax of erythrocyte and transferrin (~5-6 °C). The ΔH of deconvolved hemoglobin and transferrin decreased considerably; however, the ΔH of albumin increased. Surprisingly, compared to Iomeprol and Iobitridol treatments, the total ΔH of Iodixanol was less pronounced in the non-anticoagulated erythrocyte samples. In sum, each non-ionic contrast medium affected the thermal stability of anticoagulated and non-anticoagulated erythrocyte proteins. Interestingly, Iodixanol treatment caused more significant effects. These findings suggest that conformational changes in blood components can occur, which can potentially lead to the increased prevalence of cardiovascular dysfunctions and blood clotting.

A Possible Way to Relate the Effects of SARS-CoV-2-Induced Changes in Transferrin to Severe COVID-19-Associated Diseases.

SARS-CoV-2 infections are responsible for the COVID-19 pandemic. Transferrin has been found to explain the link between diseases associated with impaired iron transport and COVID-19 infection. The effect of SARS-CoV-2 on human whole blood was studied by differential scanning calorimetry. The analysis of the thermal transition curves showed that the melting temperature of the transferrin-related peak decreased in the presence of SARS-CoV-2. The ratio of the under-curve area of the two main peaks was greatly affected, while the total enthalpy of the heat denaturation remained nearly unchanged in the presence of the virus. These results indicate that SARS-CoV-2, through binding to transferrin, may influence its Fe3+ uptake by inducing thermodynamic changes. Therefore, transferrin may remain in an iron-free apo-conformational state, which depends on the SARS-CoV-2 concentration. SARS-CoV-2 can induce disturbance in erythropoiesis due to toxicity generated by free iron overload.

Effectiveness and safety of anti-ischemic trimetazidine in patients with stable angina pectoris and Type 2 diabetes.

Aim & methods: This 6-month prospective, observational, noninterventional, open-label clinical study assessed the effectiveness/safety of trimetazidine in 737 patients with stable angina pectoris and Type 2 diabetes mellitus (OGYI/51534-1/2014). RESULTS: Trimetazidine-based therapy was effective in stable coronary artery disease, with significant improvements from baseline (p < 0.05) in: number of angina attacks/week (2.9 ± 2.4 vs 1.1 ± 1.6), angina severity (Canadian Cardiovascular Society Classification 1.9 ± 0.8 vs 1.2 ± 0.8), exercise capacity (metabolic equivalents 6.1 ± 1.7 vs 6.5 ± 1.7), and exercise-induced myocardial ischemia (min 5.5 ± 2.5 vs 6.5 ± 2.6). DISCUSSION: Trimetazidine treatment significantly (p < 0.05) improved glucose metabolism, lowered HbA1c (7.1 ± 1.1% vs 6.6 ± 1.0%), glucose levels (7.7 ± 2.1 mmol/l vs 6.9 ± 1.6 mmol/l) and decreased arterial stiffness (pulse wave velocity 11.2 ± 2.1 m/s vs 10.4 ± 2.2 m/s). In most patients, the tolerability of trimetazidine was rated as excellent to good, with a low incidence of adverse events.

Spectroscopic characterization of the effect of mouse twinfilin-1 on actin filaments at different pH values.

The effect of mammalian twinfilin-1 on the structure and dynamics of actin filaments were studied with steady state fluorescence spectroscopy, total internal reflection fluorescence microscopy and differential scanning calorimetry techniques. It was proved before that the eukaryotic budding yeast twinfilin-1 can efficiently bind and severe actin filaments in vitro at low pH values. In the present work steady-state anisotropy measurements revealed that twinfilin can bind efficiently to F-actin. Dilution-induced depolymerization assay proved that mammalian twinfilin-1 has an actin filament severing activity. This severing activity was more pronounced at low pH values. Total internal reflection fluorescence microscopy measurements could support the severing activity of mouse twinfilin-1. The average rate of depolymerization was more apparent at low pH values. The differential scanning calorimetry measurements demonstrated that mammalian twinfilin-1 could reduce the stiffness within the actin filaments before the detachment of the actin protomers. The structural and dynamic reorganization of actin can support the twinfilin-1 induced separation of actin protomers. The measured data indicated that mammalian twinfilin-1 was able to accelerate the monomers dissociation and/or sever the filaments effectively at low pH values. It was concluded that twinfilin-1 can affect the F-actin in biological processes or under stress situations when the pH is markedly under the physiological level.

Safety and effectiveness of combined antihypertensive and cholesterol-lowering therapy in high-/very high-risk patients.

AIM: Controlling cardiovascular (CV) risk factors is paramount in reducing atherosclerotic events. This 6-month prospective noninterventional trial assessed the safety and effectiveness of fixed-combination lisinopril-amlodipine plus rosuvastatin. PATIENTS & METHODS: Patients with mild/moderate hypertension and hypercholesterolemia, at high-/very high-CV risk, received lisinopril-amlodipine (10/5, 20/5 or 20/10 mg/day) plus rosuvastatin (10 or 20 mg/day). Primary end points: systolic/diastolic blood pressure, low-density lipoprotein cholesterol. RESULTS: At 6 months, 91% of 2241 evaluable patients achieved blood pressure target (<140/90 mmHg); low-density lipoprotein cholesterol targets, <3, <2.5 and 1.8 mmol/l, were achieved by 67, 49 and 40% of patients, respectively. Adverse events (4.4%) were mostly mild. CONCLUSION: Lisinopril-amlodipine plus rosuvastatin was well tolerated and effective in patients with mild/moderate hypertension and hypercholesterolemia at high/very high CV risk.

The effect of mouse twinfilin-1 on the structure and dynamics of monomeric actin.

The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1.

The effect of toxofilin on the structure and dynamics of monomeric actin.

The effects of toxofilin (an actin binding protein of Toxoplasma gondii) on G-actin was studied with spectroscopy techniques. Fluorescence anisotropy measurements proved that G-actin and toxofilin interact with 2:1 stoichiometry. The affinity of toxofilin to actin was also determined with a fluorescence anisotropy assay. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of toxofilin. The results can be explained by the shift of the nucleotide binding cleft into a closed conformational state. Differential scanning calorimetry measurements revealed that actin monomers become thermodynamically more stable due to the binding of toxofilin.

The other side of the coin: functional and structural versatility of ADF/cofilins.

Several cellular processes rely on the fine tuning of actin cytoskeleton. A central component in the regulation of this cellular machinery is the ADF-H domain proteins. Despite sharing the same domain, ADF-H domain proteins produce a diverse functional landscape in the regulation of the actin cytoskeleton. Recent findings emphasize that the functional and structural features of these proteins can differ not only between ADF-H families but even within the same family. The structural and evolutional background of this functional diversity is poorly understood. This review focuses on the specific functional characteristics of ADF-H domain proteins and how these features can be linked to structural differences in the ADF-H domain and also to different conformational transitions in actin. In the light of recent discoveries we pay special attention to the ADF/cofilin proteins to find tendencies along which the functional and structural diversification is governed through the evolution.

The effect of ADF/cofilin and profilin on the dynamics of monomeric actin.

The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.

Myosin and tropomyosin stabilize the conformation of formin-nucleated actin filaments.

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.

The effect of toxins on inorganic phosphate release during actin polymerization.

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.

Effects of PACAP in UV-A radiation-induced retinal degeneration models in rats.

The retina is constantly exposed to ultraviolet (UV) light with different wavelengths, which may lead to chronic UV-induced retinal injury. In our previous studies, we have shown the protective effects of pituitary adenylate cyclase activating polypeptide (PACAP) in toxic and ischemic retinal injuries. The aim of the present study was to investigate the effects of PACAP in UV-A-induced retinal lesion. We used diffuse UV-A radiation (315-400 nm) to induce acute retinal damage over a short period of exposure. Using standard histological (morphological and morphometrical) analysis, we assessed the actions of intravitreal PACAP (100 pmol/5 µl) treatment on acute UV-A-induced retinal damage. We measured the thickness of nuclear and plexiform layers as well as the number of cells in the outer nuclear and inner nuclear layers and in the ganglion cell layer. Outer limiting membrane-inner limiting membrane distances in the cross-section of the retina were also examined. Our results show that UV-A light-induced retinal damage led to severe degeneration in the photoreceptor layer, and in the outer and inner nuclear layers. Alteration in the plexiform layers was also observed. We found that post-irradiation PACAP treatment significantly attenuated the UV-A-induced retinal damage. Our results provide the basis for future clinical application of PACAP treatment in retinal degeneration and may have clinical implications in several ophthalmic diseases.

Conformational dynamics of actin: effectors and implications for biological function.

Actin is a protein abundant in many cell types. Decades of investigations have provided evidence that it has many functions in living cells. The diverse morphology and dynamics of actin structures adapted to versatile cellular functions is established by a large repertoire of actin-binding proteins. The proper interactions with these proteins assume effective molecular adaptations from actin, in which its conformational transitions play essential role. This review attempts to summarise our current knowledge regarding the coupling between the conformational states of actin and its biological function.

The effects of detergents on the polymerization properties of actin.

Effects of some detergents-most frequently used in membrane raft studies-on the polymerization properties of actin were examined under in vitro and in vivo conditions, for protein and cellular investigations, respectively. Under in vitro conditions the polymerization rates were measured with pyrene-labeled actin. We found that polymerization rate depended on the detergent concentration by following either biphasic characteristics or only decreasing tendency. The strongest effects were observed at relatively low detergent concentrations. SDS-PAGE electrophoresis and dynamic light-scattering measurements provided further evidences for the size distribution of actin filaments formed under the influence of detergents. Comparing the polymerization rates measured in the presence of different detergents to those obtained with various magnesium and KCl concentrations showed that detergents may influence the actin polymerization at three levels by modifying: (i) the monomer-monomer interaction, (ii) the local ionic strength, and (iii) the affinity of actin for various cations. In vivo studies on NIH 3T3MDR1 cells using TRITC-phalloidin detected fast depolymerization of large extent around the critical micellar concentrations of the detergents. We concluded that microdomain insolubility observed in the presence of detergents is hardly to be the result of the stabilization of the submembrane actin cytoskeleton merely; rather inter-lipid and lipid-protein interactions are also involved within the detergent-resistant membranes.

The effects of formins on the conformation of subdomain 1 in actin filaments.

In this study we investigated the effects of formins on the conformation of actin filaments by using the method of fluorescence quenching. Actin was labelled with IAEDANS at Cys(374) and the quencher was acrylamide. The results showed that formin binding induced structural changes in the subdomain 1 of actin protomers which were reflected by greater quenching constants (K(SV)). Simultaneously the fraction of the fluorophore population accessible for the quencher (alpha) decreased. These observations suggest that the conformational distribution characteristic for the actin protomers became broader after the binding of formins, for which the structural framework was provided by a more flexible protein matrix in the microenvironment of the label. The effects of formins depended on the formin:actin molar ratio, and also on the ionic strength of the medium. These observations are in agreement with previous results and underline the importance of the intramolecular conformational changes induced by formins in the structure of actin filaments.

Time course and strain dependence of ADP release during contraction of permeabilized skeletal muscle fibers.

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P approximately NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12 degrees C of 10(5) M(-1) s(-1). This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P approximately NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa(2+) of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 +/- 0.6 microM and 4.7 +/- 1.5 microM at 12 and 20 degrees C, respectively. The rates of P approximately NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s(-1) at 12 and 20 degrees C, respectively. The time courses of isometric force and P approximately NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P(i) and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s(-1)) in the cross-bridge turnover during isometric contraction. At 12 degrees C, the A.M.ADP.P(i) and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20 degrees C, the force-bearing A.M.ADP.P(i) state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P approximately NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the P(i) transient during force recovery at 20 degrees C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.

The effects of ADF/cofilin and profilin on the conformation of the ATP-binding cleft of monomeric actin.

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer's ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound epsilon-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound epsilon-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound epsilon-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.

Effect of tropomyosin on formin-bound actin filaments.

Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods. The temperature dependence of the Förster-type resonance energy transfer showed that the formin-induced increase of flexibility of actin filaments was diminished by the binding of tropomyosin to actin. Fluorescence anisotropy decay measurements also revealed that the structure of flexible formin-bound actin filaments was stabilized by the binding of tropomyosin. The stabilizing effect reached its maximum when all binding sites on actin were occupied by tropomyosin. The effect of tropomyosin on actin filaments was independent of ionic strength, but became stronger as the magnesium concentration increased. Based on these observations, we propose that in cells there is a molecular mechanism in which tropomyosin binding to actin plays an important role in forming mechanically stable actin filaments, even in the case of formin-induced rapid filament assembly.

Noncooperative stabilization effect of phalloidin on ADP.BeFx- and ADP.AlF4-actin filaments.

Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actin-bound ATP is hydrolyzed to ADP and P i. We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4. The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x- or ADP.AlF 4-actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4-bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.

Nucleotide dependent differences between the alpha-skeletal and alpha-cardiac actin isoforms.

The thermodynamic properties of the actin filaments prepared from cardiomyocytes were investigated with differential scanning calorimetry. This method could distinguish between the alpha-cardiac and alpha-skeletal components of the actin filaments polymerised from ADP-actin monomers by their different melting temperatures (T(m)). Similar separation was not possible with filaments polymerised from ATP-actin monomers. Further analyses revealed that the activation energy (E(act)) was greater for filaments of alpha-skeletal actin than for alpha-cardiac actin monomers when the filaments were polymerised from ADP-actin monomers. These results showed that the alpha-cardiac actin filaments were thermodynamically less stable than the filaments of alpha-skeletal actin and their difference was nucleotide dependent. Based on these results and considering previous observations it was concluded that the existence of two actin isoforms and their nucleotide dependent conformational differences are part of the tuning regulatory mechanism by which the cardiac muscle cells can maintain their biological function under pathological conditions.