Lecithin cholesterol acyltransferase (LCAT) activity in the presence of Apo-AI-derived peptides exposed to disorder-order conformational transitions.

Although the association of Apo AI with HDLs has been proposed to activate LCAT activity, the detailed molecular mechanisms involved in the process are not known. Therefore, in this study we have investigated how conformational changes in several exposed regions of Apo-AI might cause LCAT activation and for this purpose, designed a strategy to investigate three Apo AI-derived peptides. Since these peptides present the ability to adopt several secondary structure conformations, they were used to determine whether LCAT activity could be modulated in the presence of a particular conformation. Circular dichroism experiments showed that Apo AI-derived peptides in PBS displayed a disordered arrangement, with a strong tendency to adopt ß-sheet and random conformational structures as a function of concentration. However, in the presence of Lyso-C12PC, maximal percentages of α-helical structures were observed. Performed in human plasma, time-course experiments of LCAT activity under control conditions reached the highest level of (3)H-cholesteryl esters after 2.5 h incubation. In the presence of Apo AI-derived peptides, a significant increase in the production of (3)H-cholesteryl esters was observed. The present study provides an important insight into the potential interactions between LCAT and lipoproteins and also suggests that peptides, initially present in a disordered conformation, are able to sense the lipid environment provided by lipoproteins of plasma and following a disorder-to-order transition, change their conformation to an ordered α-helix.

Pioglitazone improves the cardiovascular profile in patients with uncomplicated systemic lupus erythematosus: a double-blind randomized clinical trial.

OBJECTIVE: We studied the effect of pioglitazone on insulin levels, inflammation markers, high-density lipoprotein (HDL) composition and subclasses distribution, in young women with uncomplicated systemic lupus erythematosus (SLE). METHODS: This double-blind trial included 30 premenopausal women (30 ±8 years old) with SLE, who were randomized to pioglitazone (30 mg/day) or placebo treatment for 3 months. Plasma and HDL lipids were determined by colorimetric enzymatic assays, insulin by radioimmunometric assay, inflammation by immunonephelometry and HDL size and subclasses distribution by a native 4-30% polyacrylamide gradient gel electrophoresis. RESULTS: Compared with placebo, pioglitazone significantly increased HDL-cholesterol plasma levels (14.2%), reduced fasting insulin plasma levels (23.6%) and the homeostasis model assessment-insulin resistance (31.7%). C-reactive protein (70.9%) and serum amyloid A (34.9%) were also significantly reduced with the pioglitazone use, whereas the HDL particle size was increased (8.80 nm vs. 8.95 nm; p = 0.044) by changes in the distribution of HDL(2b), HDL(3b), and HDL(3c) subclasses. The change in HDL size correlated with a rise in free and cholesterol-ester content in the HDL particles. CONCLUSION: Pioglitazone significantly enhanced insulin sensitivity, reduced inflammation, and modified HDL characteristics, suggesting a potential beneficial effect of this drug in patients with SLE with a risk to develop cardiovascular disease. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov Protocol Registration System, with the number NCT01322308.

Rheology and DWS microrheology of concentrated suspensions of the semiflexible filamentous fd virus.

Microrheology measurements were performed on suspensions of bacteriophage fd with diffusive wave spectroscopy in the concentrated regime, at different values of ionic strength. Viscosity vs. shear rate was also measured, and the effect of bacteriophage concentration and salt addition on shear thinning was determined, as well as on the peaks in the viscosity vs. shear curves corresponding to a transition from tumbling to wagging flow. The influence of concentration and salt addition on the mean square displacement of microspheres embedded in the suspensions was determined, as well as on their viscoelastic moduli up to high angular frequencies. Our results were compared with another microrheology technique previously reported where the power spectral density of thermal fluctuations of embedded micron-sized particles was evaluated. Although both results in general agree, the diffusive wave spectroscopy results are much less noisy and can reach larger frequencies. A comparison was made between measured and calculated shear modulus. Calculations were made employing the theory for highly entangled isotropic solutions of semiflexible polymers using a tube model, where various ways of calculating the needed parameters were used. Although some features are captured by the model, it is far from the experimental results mainly at high frequencies.

Proapoptotic role of novel gene-expression factors.

The mechanisms that control cellular proliferation, as well as those related with programmed cell death or apoptosis, require precise regulation systems to prevent diseases such as cancer. Events related to cellular proliferation as well as those associated with apoptosis involve the regulation of gene expression carried out by three basic genetic expression regulation mechanisms: transcription, splicing of the primary transcript for mature mRNA formation, and RNA translation, a ribosomal machinery-dependent process for protein synthesis. While development of each one of these processes requires energy for recognition and assembly of a number of molecular complexes, it has been reported that an increased expression of several members of these protein complexes promotes apoptosis in distinct cell types. The question of how these factors interact with other proteins in order to incorporate themselves into the different transduction cascades and stimulate the development of programmed cell death, although nowadays actively studied, is still waiting for a clear-cut answer. This review focuses on the interactions established between different families of transcription, elongation, translation and splicing factors associated to the progression of apoptosis.

Synthesis of ATP catalyzed by the (Ca2+ + Mg2+)-ATPase from erythrocyte ghosts. Energy conservation in plasma membranes.

The (Ca2+ + Mg2+)-ATPase from erythrocyte ghosts catalyzed the hydrolysis of ATP together with the synthesis of ATP or ATP in equilibrium 'Pi exchange. The modulation of the ATPase reaction cycle was controlled by high- and low-affinity calcium-binding sites asymmetrically located on the enzyme. Calmodulin accelerated the reaction cycle in both directions, stimulating the overall turnover of the enzyme. Calcium transport was achieved utilizing optimal conditions for the expression of the ATP in equilibrium Pi exchange system.

Cholesterol effect on enzyme activity of the sarcolemmal (Ca2+ + Mg2+)-ATPase from cardiac muscle.

The effect of cholesterol incorporation and depletion of the cardiac sarcolemmal sacs on (Ca2+ + Mg2+)-ATPase activity was examined. Cholesterol incorporation to the sarcolemmal sacs was achieved utilizing an in vivo and an in vitro procedure. Cholesterol depleted membranes were obtained in vitro after incubation of the sarcolemmal sacs with inactivated plasma. Arrhenius plots of the (Ca2+ + Mg2+)-ATPase activity showed a triphasic curve when the assays were carried out using a temperature range between 0 and 40 degrees C. The sarcolemmal (Ca2+ + Mg2+)-ATPase activity was shown to be inversely proportional to the cholesterol concentration of the membranes, showing a low ATPase activity with a high cholesterol content and a high ATPase activity when the cholesterol concentration was low. Although the (Ca2+ + Mg2+)-ATPase activity was found to be inhibited in the cholesterol incorporated sarcolemmal sacs, the withdrawal of small amounts of cholesterol from the membranes produced an important stimulatory effect. Changes in (Ca2+ + Mg2+)-ATPase activity due to variation in the membrane cholesterol concentration were shown to be reversible. Our results indicate the possibility of a slow exchange of cholesterol between the tightly bound lipid surrounding the (Ca2+ + Mg2+)-ATPase and the bulk lipid of the sarcolemma.

Cholesterol increases the thermal stability of the Ca2+/Mg(2+)-ATPase of cardiac microsomes.

The effect of membrane cholesterol on the thermal inactivation of Ca2+/Mg(2+)-ATPase activity of bovine cardiac microsome was measured and compared to the thermal denaturation profiles of the microsomes as measured by differential scanning calorimetry (DSC). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Both thermal inactivation of Ca2+/Mg(2+)-ATPase activity and thermal denaturation were shifted to higher temperatures in microsomes enriched with cholesterol (37 +/- 5 micrograms cholesterol/mg protein, cholesterol/phospholipid molar ratio 0.31) compared to control microsomes (15 +/- 3 micrograms cholesterol/mg protein, molar ratio 0.12). Thermal inactivation was measured by two methods: first, measuring activity at room temperature as a function of heating to elevated temperatures at 1 K/min, where inactivation temperatures (T1, temperature of half activity) were 58.9 +/- 0.3 degrees C for control membranes and 59.9 +/- 0.1 degrees C for cholesterol-enriched membranes, respectively. Second, measuring ATPase activity as a function of time at constant temperature, where T1 values of 57.6 +/- 0.5 degrees C and 59.2 +/- 0.5 degrees C were determined for control and cholesterol-enriched membranes, respectively. DSC profiles of microsomal membranes consisting of a number of overlapping peaks were obtained. A well resolved component (transition C) was observed with a transition temperature (T 1/2) of 58.2 degrees C. This T 1/2, which is a measure of conformational stability, correlates with the T1 for Ca2+/Mg(2+)-ATPase activity and is 1.9 +/- 0.6 K higher in cholesterol-enriched membranes. Thus, the increased resistance to inactivation appears to be due to increased conformational stability of the protein induced by cholesterol, demonstrating that a change in lipid composition can influence the stability of an integral membrane protein in a natural membrane. The increased stability is of sufficient magnitude to account for the previously observed correlation between cholesterol content and resistance to heat shock in several cell lines.

Stability of the C-terminal peptide of CETP mediated through an (i, i + 4) array.

Based on circular dichroism (CD), we have found an essential (i, i + 4) alpha-helix stabilizing array in the C-terminus region for the cholesteryl ester transfer protein (CETP) between histidine 466 and aspartic acid 470. This region apparently corresponds to an amphipathic alpha-helix. The behavior of this peptide in solution in comparison with a mutant peptide (D470N) was also analyzed by dynamic light scattering (DLS). The results showed that alpha-helix stabilization is not due to peptide aggregation. The thermodynamic estimation of stability supports the idea that the phenomenon is carried out through an (i, i + 4) array. The representation of the C-terminal region as an amphipathic alpha-helical peptide shows that lipid-binding activity might be in part due to both the asymmetric polar/non-polar residue distribution and to the presence of an (i, i + 4) array important for helix stability.

Regulation of the (Ca2+ + Mg2+)-ATPase activity and calcium transport in reconstituted cardiac sarcolemmal vesicles. Effect of sodium and potassium.

A reconstitution procedure for a cardiac sarcolemmal enriched fraction is described. In the reconstituted cardiac sarcolemmal inside-out vesicles, a difference in calcium transport and (Ca2+ + Mg2+)-ATPase activity was found depending on the side of the membrane at which sodium and potassium were placed. Having inhibited the (Na+ + K+)- ATPase activity with ouabain, the active transport of calcium was increased when potassium was located outside and sodium inside the reconstituted vesicles. Nevertheless, this activity was maximal having potassium present on both sides. During calcium transport it was also shown that 86Rb moves opposite to calcium. When the experiment was carried out having 22Na located at the inside, there was no movement of this cation despite the low calcium transport observed. The present study supports the possibility of potassium having a stimulatory effect upon the sarcolemmal (Ca2+ + Mg2+)-ATPase activity and suggests the existence of a Ca2+, K+ co-transport carried out by this enzyme.

The effect of verapamil on the Ca2+-transporting and Ca2+-ATPase activity of isolated cardiac sarcolemmal preparations.

1 The effect of (+/-)-, (+)- and (-)-verapamil on the Ca2+-binding, Ca2+-transporting activity, and Ca2+-dependent adenosine triphosphatase (ATPase) activity of isolated cardiac sarcolemmal preparations was studied. Enzymatic treatment was used to establish the nature of the sites facilitating [14C]-(+/-)-verapamil binding. 2 (+/-)-Verapamil 1 microM inhibited the passive binding of 45Ca2+. The (+/-)- and (-)-isomers were equiactive. 3 (+/-)-Verapamil 1 microM inhibited the ATP-dependent transport of 45Ca2+ and the associated activation of the Ca2+-sensitive ATPase. The activity resided in the (-)-isomer. 4 Lineweaver-Burk plots for the initial rates of ATP-dependent transport showed that the inhibition induced by the (-)-isomer was accompanied by a reduced Km and Vmax. 5 Enzymatic removal of N-acetyl neuraminic acid and galactose residues increased [14C]-(+/-)-verapamil binding; removal of N-acetylglucosamine and treatment with phospholipase C and trypsin decreased the binding. 6 These results have been interpreted to mean that (-)-verapamil interferes with the ATP-dependent Ca2+-transporting properties of the sarcolemma, and that this effect is accompanied by an altered activity of the intrinsic Ca2+-sensitive ATPase. N-acetylneuramic acid and galactose residues do not provide binding sites for verapamil at the cell surface.

Beta 1-adrenoceptors in rat hepatoma. Desensitization by isoproterenol and phorbol-myristate-acetate.

The beta-adrenergic responsiveness of hepatocytes obtained from hypothyroid rats and of a transplantable hepatoma cell line (AS-30D) were studied by measuring the accumulation of cyclic AMP. The potency order for agonists in hepatocytes was: isoproterenol greater than epinephrine much greater than norepinephrine whereas in the hepatoma cells the potency order was: isoproterenol greater than norepinephrine greater than or equal to epinephrine. The effect of isoproterenol was antagonized in hepatocytes by low concentrations of ICI 118551 and only partially by concentrations of atenolol as high as 100 microM. In hepatoma cells the effect of isoproterenol was inhibited by both antagonists with the potency order atenolol greater than ICI 118551. These data indicate that in hepatocytes the effect is mediated by beta 2-adrenoceptors whereas in hepatoma cells it is through beta 1-adrenoceptors. Preincubation of hepatoma cells with isoproterenol or phorbol-myristate-acetate diminished the subsequent beta-adrenergic responsiveness of the cells. Interestingly, when both isoproterenol and phorbol-myristate-acetate were present during the preincubation the beta-adrenergic desensitization observed was bigger than that induced by any of these agents alone.

Apoptosis and cell death channels in prostate cancer.

Apoptosis, a type of programmed cell death, is a decisive mechanism in cell processes such as homeostasis, development, and many diseases including cancer. In mammals, the mechanisms that trigger and control the process of apoptosis are complex, because it has been observed that many molecules might be involved, acting in distinct ways and depending on the cellular type. The process of apoptosis is characterized by specific biochemical and morphologic changes. However, important specific messengers such as Ca(2)+ act in active proliferation as well as in apoptosis. At present, there is convincing evidence that a sustained increase in intracellular Ca(2)+ can activate cytotoxic mechanisms in various cells and tissues. Several ionic channels located in the cytoplasmic membrane might participate in the entry of calcium into the cytosol during apoptosis. Among these ionic channels, the purinoreceptors P2X and the channels of capacitative entry of calcium have been described. Pro- and anti-apoptotic molecules such as bax and bcl-2, respectively, have also been shown to participate in the process. We have recently found the activation of a Ca(2)+-permeable, nonselective cation channel of 23 pS conductance in prostatic cancer (LNCaP) exclusively in cells previously induced to apoptosis. Our findings are discussed taking into account the different ion channels that might participate in programmed cell death in prostate cancer.

Forces between hydrophilic surfaces adsorbed with apolipoprotein AII alpha helices.

To provide better understanding of how a protein secondary structure affects protein-protein and protein-surface interactions, forces between amphiphilic alpha-helical proteins (human apolipoprotein AII) adsorbed on a hydrophilic surface (mica) were measured using an interferometric surface force apparatus (SFA). Forces between surfaces with adsorbed layers of this protein are mainly composed of electrostatic double layer forces at large surface distances and of steric repulsive forces at small distances. We suggest that the amphiphilicity of the alpha-helix structure facilitates the formation of protein multilayers next to the mica surfaces. We found that protein-surface interaction is stronger than protein-protein interaction, probably due to the high negative charge density of the mica surface and the high positive charge of the protein at our experimental conditions. Ellipsometry was used to follow the adsorption kinetics of this protein on hydrophilic silica, and we observed that the adsorption rate is not only controlled by diffusion, but rather by the protein-surface interaction. Our results for dimeric apolipoprotein AII are similar to those we have reported for the monomeric apolipoprotein CI, which has a similar secondary structure but a different peptide sequence and net charge. Therefore, the observed force curves seem to be a consequence of the particular features of the amphiphilic alpha-helices.

ARP2 a novel protein involved in apoptosis of LNCaP cells shares a high degree homology with splicing factor Prp8.

The mechanism of apoptosis has been recognized as an important event in processes such as cellular development and homeostasis, as well as degenerative conditions like cancer. Prostate cancer during its advanced stages develops androgen independent cells that ultimately overgrow and promote metastatic events. Our group employing androgen independent LNCaP cells have previously proposed, based on electrophysiological findings, that apoptosis induced cells overexpress a cell death calcium channel-like molecule. Here we report the cloning and expression in Xenopus laevis oocytes of apoptosis regulated protein 2 (ARP2), a protein overexpressed in apoptosis induced LNCaP cells capable to induce calcium inward currents and apoptosis typical morphology changes in oocytes injected with arp2 mRNA. Our results also indicate that clone arp2 cDNA (1.3Kb) shares a 99% homology with a small fragment that corresponds to 18% of the complete sequence of Prp8 cDNA (7.0 Kb), a molecule that codifies for an important protein in the assembly of the spliceosome. We propose that protein ARP2 as a fragment of protein Prp8, corresponds to a molecule with a new function in apoptosis related phenomena.

Effect of calmodulin on sarcoplasmic reticulum Ca(2+)-ATPase isolated from cardiac muscle.

A Ca(2+)-dependent ATPase, purified from cardiac microsomal membranes by solubilization and chromatography, is identified as cardiac sarcoplasmic reticulum ATPase on the basis of its electrophoretic mobility and its trypsin digestion pattern. The ATPase (both in membranous and purified form) is stimulated by calmodulin, while the skeletal muscle ATPase is not. Rapid kinetic experiments demonstrate that the calmodulin stimulation is already present within the first enzyme cycle following the addition of ATP, and consists of an increased turnover of the phosphorylated enzyme intermediate. The calmodulin effect does not involve the phosphorylation of any protein other than the ATPase. Following the incubation of ATPase with [gamma-32P]ATP, even in conditions of calmodulin stimulation, radioactive phosphorus is found only on the ATPase electrophoretic band, corresponding to the phosphorylated enzyme intermediate. These observations, together with the results obtained for [125I]calmodulin binding to the ATPase, suggest that the stimulation in turnover produced by calmodulin on the ATPase is due to a direct effect on the enzyme. This may provide an independent regulation of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, in addition to the known regulation mediated by other accessory proteins.

Effect of lead on the erythrocyte (Ca2+,Mg2+)-ATPase activity. Calmodulin involvement.

I have investigated the effect of lead on the erythrocyte ghosts (Ca2+,Mg2+)-ATPase, with special attention to the role of calmodulin in this phenomena. Under regular incubation conditions, lead inhibits the enzyme with an IC50 of 6.0 microM. The presence of exogenously added calmodulin apparently does not change this inhibitory value. DTT added during the incubation period does not affect the inhibitory action of lead. However, when the membranes are preincubated with DTT, an important IC50 displacement is observed, either with or without added calmodulin. Since [125I]calmodulin binding to the membranes is enhanced when lead is used, the possibility of a lead/calmodulin complex that optimally stimulates the enzyme using lead concentrations between 1.0 and 10.0 microM, is suggested. Based on the experimental data, I propose two well defined actions of lead; first, an inhibitory action upon the ATPase above 1.0 microM lead, most probably related to essential sulphydryl groups in the enzyme; and second, a direct action of lead upon calmodulin at lead concentrations below 1.0 microM.

Cholesterol effect on thermostability of the (Ca2+, Mg2+)-ATPase from cardiac muscle sarcolemma.

When the cholesterol concentration in the sarcolemmal system is raised, the (Ca2+,Mg2+)-ATPase activity acquires an important degree of thermostability; phenomena that is completely lost if the experiment is carried out with cholesterol depleted sarcolemma. In this system, a gradual depletion of sarcolemmal cholesterol, renders the ATPase remarkably sensitive to temperature. At different concentrations of ATP, it is found that cholesterol affects the Vmax of the (Ca2+,Mg2+)-ATPase but not its Km. These results support our earlier suggestion of a direct effect of cholesterol upon the enzyme, and opens a possible mode of action of cholesterol on the enzyme. It is suggested that the inverse relationship between catalysis and thermostability is due to differences in the flexibility of the enzyme directly related to hydrophobicity changes caused by cholesterol.

A novel cholesterol transfer protein in cardiac sarcolemma. Purification and initial characterization.

In contrast to several sterol carrier proteins isolated from soluble cytosolic fractions, a cholesterol transfer protein (CHTP) with an apparent molecular weight of 73,000 was isolated from a cardiac sarcolemmal fraction by detergent solubilization, column chromatography, and preparative electrophoresis using nondissociating polyacrylamide gels. This protein must be reconstituted into an artificial membrane in order to mediate cholesterol transfer activity. For the expression of its full activity, CHTP must also be present in the membrane in a multimeric form, since the monomer was shown not to be active. We believe this novel protein might represent an important molecule in the regulation of the homeostasis of cholesterol in cardiac sarcolemma.