Circulating exosomes deliver free fatty acids from the bloodstream to cardiac cells: Possible role of CD36.

Regulation of circulating free fatty acid (FFA) levels and delivery is crucial to maintain tissue homeostasis. Exosomes are nanomembranous vesicles that are released from diverse cell types and mediate intercellular communication by delivering bioactive molecules. Here, we sought to investigate the uptake of FFAs by circulating exosomes, the delivery of FFA-loaded exosomes to cardiac cells and the possible role of the FFA transporter CD36 in these processes. Circulating exosomes were purified from the serum of healthy donors after an overnight fast (F) or 20 minutes after a high caloric breakfast (postprandial, PP). Western blotting, Immunogold Electron Microscopy and FACS analysis of circulating exosomes showed that CD36 was expressed under both states, but was higher in postprandial-derived exosomes. Flow cytometry analysis showed that circulating exosomes were able to take-up FFA directly from serum. Importantly, preincubation of exosomes with a blocking CD36 antibody significantly impeded uptake of the FFA analogue BODIPY, pointing to the role of CD36 in FFA exosomal uptake. Finally, we found that circulating exosomes could delivery FFA analogue BODIPY into cardiac cells ex vivo and in vivo in a mice model. Overall, our results suggest a novel mechanism in which circulating exosomes can delivery FFAs from the bloodstream to cardiac tissue. Further studies will be necessary to understand this mechanism and, in particular, its potential involvement in metabolic pathologies such as obesity, diabetes and atherosclerosis.

Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes.

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.

Electrogenerated chemiluminescence reactions between the [Ru(bpy)3](2+) complex and PAMAM GX.0 dendrimers in an aqueous medium.

Electrogenerated chemiluminescence, ECL, reactions between tris(2,2'-bipyridine)ruthenium(II), [Ru(bpy)3](2+), and PAMAM GX.0 (X=1 and 2) dendrimers in an aqueous medium were carried out at pH10 (fully deprotonated dendrimer surface). ECL was detected in the presence of GX.0 dendrimers without addition of any known coreactant. Atomic force microscopy, AFM, measurements for GX.0 dendrimers in the presence of the [Ru(bpy)3](2+) complex were also done. AFM images showed the existence of aggregates (pillars) of globular shape, as well as interdendrimer networks forming fibers in the x-y direction for dendrimer aqueous solutions. ECL and AFM results in cooperation suggest that the coreactant effect of the end amine groups is improved by both the dendritic branched shells and the globular z-type aggregates. The ECL efficiency trends as a function of [GX.0] (whole range) can be interpreted taking into account the coreactant effect modulated by the presence of the z and x-y type aggregates. Importantly, ECL efficiency values can be taken as a measure of the change induced on the dendrimer aggregation in aqueous solutions when their concentrations rise. Redox potentials of the [Ru(bpy)3](3+/2+) couple in the presence of the G1.0 and G2.0 dendrimers were also determined.

Free energy of binding of cationic metal complexes to AuNPs through electron-transfer processes.

AuNP effects on the oxidation reaction of [Ru(NH3)5pz](2+) with S2O8(2-) were studied. Experimental results show the effects produced by gold nanoparticles of different sizes, which are then discussed by using an approach based on the two-state model. Changes in the observed reactivity are explained by a change in the degree of association of the reactants to the nanoclusters, which depends on the negative surface potential of the gold nanoparticles. TEM and zeta potential measurements show that this potential determines the binding strength of one of the reactants ([Ru(NH3)5pz](2+)) to the citrate gold surface. The number of binding sites on a citrate nanoparticle receptor has also been determined. The experimental results confirm that an electron transfer reaction can be used as a probe for the determination of the free energy of binding of cationic metal complexes to gold nanoparticles.

Noncovalent interactions of tiopronin-protected gold nanoparticles with DNA: two methods to quantify free energy of binding.

The binding of gold nanoparticles capped with N-(2-mercaptopropionyl)glycine (Au@tiopronin) with double-stranded DNA has been investigated and quantified in terms of free energies by using two different approaches. The first approach follows the DNA conformational changes induced by gold nanoparticles using the CD technique. The second methodology consists in the use of pyrene-1-carboxaldehyde as a fluorescent probe. This second procedure implies the determination of the "true" free energy of binding of the probe with DNA, after corrections through solubility measurements. Working at different salt concentrations, the nonelectrostatic and electrostatic components of the binding free energy have been separated. The results obtained revealed that the binding is of nonelectrostatic character, fundamentally. The procedure used in this work could be extended to quantify the binding affinity of other AuNPs/DNA systems.

Electrochemiluminescence of the [Ru(bpy)3]2+ complex: the coreactant effect of PAMAM dendrimers in an aqueous medium.

Electrogenerated chemiluminescence (ECL) from aqueous solutions of tris(2,2'-bipyridine)ruthenium(II), [Ru(bpy)(3)](2+), in the presence of PAMAM G1.5 and G4.5 dendrimers, was observed without the addition of coreactants. The ECL efficiency, Φ(ECL), was enhanced with the addition of increasing amounts of G1.5 dendrimer. Indeed, the ECL efficiency for the [Ru(bpy)(3)](2+)/G1.5 dendrimer became about 10 times higher than that for the [Ru(bpy)(3)](2+)/ oxalate anion system. However, the ECL efficiency in the presence of the G4.5 dendrimer was smaller than that for the G1.5 dendrimer at concentrations similar to those for G1.5 with identical medium conditions. Besides, the addition of NaCl at a given concentration of G1.5 dendrimer decreased the ECL efficiency. The results of Φ(ECL) were interpreted by taking into account the coreactant effect and the electrostatic (long-range and short-range) interactions between the ruthenium(II) complex and the electric field of the dendrimer surface. Standard formal potentials of the [Ru(bpy)(3)](3+/2+) couple in the presence of G1.5 and G4.5 dendrimers were also determined.

Compaction and decompaction of DNA induced by the cationic surfactant CTAB.

A multifaceted study on the interaction of the cationic surfactant CTAB with calf thymus DNA was carried out by using different techniques. The measurements were done at different molar ratios X = [CTAB]/[DNA]. Results show the conformational change that DNA suffers due to the interaction with surfactant molecules at low molar ratios: the condensation of the polynucleotide, from an extended coil state to a globular state. The effect observed at the higher molar ratios is worth noting: the decondensation of DNA, that is, the transition from a compact state to a more extended conformation. Experimental data obtained confirm that this latter state is not exactly the same as that found in the absence of the surfactant. Attractive interactions between different parts of the molecule by ion correlation effects are the driving force to produce both the compaction and decompaction events. Results also show the importance of choosing both a proper system for the study and the most seeming measuring technique to use. The study demonstrates that, in some cases, the use of several techniques is desirable in obtaining reliable and accurate results.

Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties.

The binding of [Ru(PDTA-H(2))(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.

Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration.

The peritoneal mesothelium exhibits a high regenerative ability. Peritoneal regeneration is concomitant with the appearance, in the coelomic cavity, of a free-floating population of cells whose origin and functions are still under discussion. We have isolated and characterized this cell population and we have studied the process of mesothelial regeneration through flow cytometry and confocal microscopy in a murine model lethally irradiated and reconstituted with GFP-expressing bone marrow cells. In unoperated control mice, most free cells positive for mesothelin, a mesothelial marker, are green fluorescent protein (GFP). However, 24 hrs after peritoneal damage, free mesothelin(+)/GFP(+) cells appear in peritoneal lavages. Cultured lavage peritoneal cells show colocalization of GFP with mesothelial (mesothelin, cytokeratin) and fibroblastic markers. Immunohistochemical staining of the peritoneal wall also revealed colocalization of GFP with mesothelial markers and with procollagen-1 and smooth muscle α-actin. This was observed in the injured area as well as in the surrounding not-injured peritoneal surfaces. These cells, which we herein call peritoneal repairing cells (PRC), are very abundant 1 week after surgery covering both the damaged peritoneal wall and the surrounding uninjured area. However, they become very scarce 1 month later, when the mesothelium has completely healed. We suggest that PRC constitute a type of monocyte-derived cells, closely related with the tissue-repairing cells known as 'fibrocytes' and specifically involved in peritoneal reparation. Thus, our results constitute a synthesis of the different scenarios hitherto proposed about peritoneal regeneration, particularly recruitment of circulating progenitor cells and adhesion of free-floating coelomic cells.

Kinetic study of the cetyltrimethylammonium/DNA interaction.

A kinetic study of the interaction of the surfactant cetyltrimethylammonium (CTA(+)) with DNA was carried out in water and in salt (NaCl) solutions. The results can be explained in terms of a reaction mechanism involving two consecutive reversible steps. The first step corresponds to the union/separation of the surfactant with/from the DNA. The second step corresponds to a conformational change of the surfactant/DNA complex. The equilibrium constant, calculated from the forward and reverse rate constants of these steps, agrees with the results of a previous thermodynamic study.

Kinetic approach for the study of noncovalent interaction between [Ru(NH3)5pz]2+ and gold nanoparticles.

Gold nanoparticles (AuNPs) capped with N-(2-mercaptopropionyl)glycine have been used to study the strength and character of the binding of a cationic metal complex, [Ru(NH3)5pz]2+ (pz = pyrazine), at pH = 8, to these nanoparticles. The strength of the binding has been studied using a kinetic approach consisting of the study of the kinetics of the oxidation of this ruthenium complex by S2O82- at different NaCl concentrations. When the ionic strength increases, the strength of the binding decreases, as a consequence of the partial neutralization of the charge on the AuNPs which, at pH = 8, has the tiopronin residue negatively charged. The increase of the ionic strength also produces a change in the character of the binding, which changes from anticooperative to noncooperative when the ionic strength increases. The nonelectrostatic and electrostatic components of the free energy of binding are determined. From the latter, we have obtained the values of the electrostatic potential differences at the AuNPs/solutions interface.

Clomethiazole Acute Stroke Study in ischemic stroke (CLASS-I): final results.

BACKGROUND AND PURPOSE: A previous trial (the Clomethiazole Acute Stroke Study) generated the hypothesis that clomethiazole is effective in patients with a major ischemic stroke (total anterior circulation syndrome), and this was tested in the present study. METHODS: A total of 1198 patients with major ischemic stroke and a combination of limb weakness, higher cortical dysfunction, and visual field deficits were randomly assigned to clomethiazole (68 mg/kg IV over 24 hours) or placebo. The study drug was initiated within 12 hours of symptom onset. Functional outcome and neurological recovery were assessed at days 7, 30, and 90, with the proportion of patients with a Barthel Index > or =60 at last follow-up as the primary outcome measure. RESULTS: The patients were randomly assigned equally, and the two treatment groups were well matched for baseline characteristics, including stroke severity (mean National Institutes of Health Stroke Scale score 16.9+/-5.2). Ninety-six percent were classified as total anterior circulation syndrome. The proportion of patients reaching a Barthel Index score of > or =60 was 42% in the clomethiazole-treated group and 46% in the placebo-treated group (odds ratio, 0.81; 95% CI, 0.62 to 1.05; P=0.11). There was no evidence of efficacy on any secondary outcome variables (modified Rankin Score, National Institutes of Health Stroke Scale, Scandinavian Stroke Scale, and 30-day CT infarct volumes) compared with placebo. Subgroup analysis showed a similar lack of treatment effect in patients treated early (<6 hours) and in those treated later (6 to 12 hours). Somnolence was an expected pharmacological effect of clomethiazole, and this occurred during treatment as an adverse event in half of the patients randomly assigned to study drug. CONCLUSIONS: The target population was selected, and sufficient drug was given to produce the expected pharmacological effect in the brain. Clomethiazole does not improve outcome in patients with major ischemic stroke.

The anorectic effect of oestradiol does not involve changes in plasma and cerebrospinal fluid leptin concentrations in the rat.

Oestradiol is a potent anorectic agent that reduces both food intake and body weight. Since leptin is known to reduce food intake, we first analysed if the anorectic effect of oestradiol is driven by an increased leptin concentration in either cerebrospinal fluid or plasma. Oestradiol also reduces body weight and fat mass. Accordingly, a decrease in plasma leptin concentration can also be expected after an oestradiol-driven reduction in fat mass. To test this hypothesis was the second aim of this study. Female Wistar rats received oestradiol chronically during 14 days. During the first week of treatment there was a reduction in food intake, body weight and fat mass that returned to initial values during the second week, but no changes in ob mRNA levels were found in white adipose tissue depots. There was no effect of treatment or time on plasma and cerebrospinal fluid leptin concentrations. Therefore, the anorectic effect of oestradiol is not driven by an increase in leptin concentration either in plasma or in cerebrospinal fluid, and the reduction in fat mass that oestradiol produces is not followed by a reduction leptin concentration.

Plasma and cerebrospinal fluid leptin levels are maintained despite enhanced food intake in progesterone-treated rats.

OBJECTIVES: For adipostatic control, increases in food intake are followed by increased leptin levels that in turn reduce food intake. However, progesterone administration increases both food intake and body weight. The aim of this study was to analyze changes in the white adipose tissue-leptin system in rats with enhanced plasma levels of progesterone. METHODS: Female Wistar rats received progesterone chronically by means of subcutaneous implants over 30 days. RESULTS: They showed an increased food intake followed by increased body weight and heavier fat depots. An enhanced ob-mRNA level was detected in inguinal white adipose tissue depot on day 2 of treatment but the increase was transient, disappearing on day 6 of treatment. No changes in ob-mRNA levels were found in parametrial and retroperitoneal white adipose tissue depots. Plasma and cerebrospinal fluid leptin levels were unchanged either during the treatment or between corresponding treated and control rats. Leptin concentrations in cerebrospinal fluid were ten times lower than in plasma (0.2--0.3 ng/ml versus 2--3 ng/ml respectively). CONCLUSIONS: These results indicated that progesterone favours a positive energy balance not only by enhancing food intake but also by inhibiting the concurrent enhancement in plasma and cerebrospinal fluid leptin levels expected from the increased fat mass.

Characterization of an expanded glutamine repeat androgen receptor in a neuronal cell culture system.

Spinal and bulbar muscular atrophy (SBMA) is an inherited form of lower motor neuron degeneration caused by expansion of a CAG repeat in the androgen receptor (AR) gene. To study the mechanism by which this mutation causes neuronal pathology, we stably transfected a motor neuron hybrid cell line with human AR cDNAs containing either 24 or 65 repeats (AR24 and AR65, respectively). Both forms of receptor were able to bind ligand and activate transcription of a reporter construct equally well. Likewise, the subcellular localizations of AR24 and AR65 were similar, in both the presence and the absence of ligand. AR24- and AR65-expressing clones were phenotypically indistinguishable. They survived equally well after differentiation and were equally susceptible to damage by oxidative stress. Our studies thus demonstrate that, in a neuronal system, the expanded repeat AR functions like the normal repeat AR in several important ways. Because levels of AR65 expression were consistently lower than levels of AR24 expression, we propose that the loss of function of AR seen in SBMA may be due to decreased levels of receptor expression rather than to a difference in intrinsic properties. The postulated gain of function responsible for neuronal degeneration remains to be determined.

Developmental shift of myosin heavy chain mRNA expression due to neural factor(s) and muscle activity.

The adult ventricular isoform of chicken myosin heavy chain (MHC-V) is transiently expressed in all skeletal muscle primordia analyzed and is completely repressed around embryonic days 10-12, when functional innervation is established. By ribonuclease protection assay, we demonstrated that denervation of the adult anterior latissimus dorsi muscle resulted in reexpression of MHC-V mRNA. In contrast, treatment of primary cultures of fetal breast or leg muscles with embryonic brain extract or conditioned media from glial or neuroblastoma cell lines, but not from a myogenic cell line or primary muscle cell cultures, led to inhibition of MHC-V expression. This inhibitory activity was abolished by heating and increased with protein concentration. The acquisition of both brain inhibitory activity and the competence of myogenic cells to downregulate MHC-V mRNA expression were age dependent. Furthermore, either paralysis of muscle in ovo by curare or contraction arrest of cultured myotubes resulted in persistent expression of MHC-V mRNA. Thus a putative soluble factor(s) of nerve origin as well as muscle activity are involved in the developmental downregulation of MHC-V expression in muscle primordia.

Human T-cell lymphotropic virus type-I infection in the severe combined immunodeficiency mouse.

Human T-cell lymphotropic virus type-I (HTLV-I) is the etiologic agent of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). HAM/TSP and ATL occur infrequently among HTLV-I-infected individuals, and rarely develop in the same individual. To study host and viral factors involved in the induction, tissue tropism, as well as pathogenesis of HAM/TSP, peripheral blood lymphocytes (PBL) from 14 patients with HAM/TSP and from 9 controls were introduced into severe combined immunodeficiency (SCID) mice by intraperitoneal injection. Mice were followed for up to 26 weeks. Human IgG was produced from 2 to 14 weeks after reconstitution in all animals. Thirty-two of 44 mice (72%) showed circulating human antibody against the major viral protein products of HTLV-I. Analysis of viral sequences by polymerase chain reaction (PCR) demonstrated HTLV-I sequences in 21/38 (55%) brains and in 7/17 (41%) spinal cords from HTLV-I-hu SCID mice. No animal had clinical evidence of neurological impairment or pathological findings similar to those seen in HAM/TSP. Seven mice who received PBL from Epstein Barr virus (EBV)-seropositive patients developed an intraperitoneal lymphoma. In 2 mice an infiltration of brain by a lymphoblastic tumor of B/T cell type was observed. By PCR, all the tumors were EBV-positive; HTLV-I sequences were detected in 5 of them. Our study suggests that the HTLV-I-hu-SCID mouse provides a potentially valuable system for studying the production, kinetics, and pathogenicity of anti-HTLV-I antibody, and may help clarify the interaction of EBV and retroviruses in the development of disease.

Functional receptor for GDNF encoded by the c-ret proto-oncogene.

Glial-cell-line-derived neutrophic factor (GDNF) promotes the survival and phenotype of central dopaminergic noradrenergic and motor neurons, as well as various subpopulations of peripheral sensory and sympathetic neurons. GDNF is structurally related to members of the transforming growth factor (TGF)-beta superfamily, several members of which have well-characterized receptor systems; however, GDNF receptors still remain undefined. Here we show that GDNF binds to, and induces tyrosine phosphorylation of, the product of the c-ret proto-oncogene, an orphan receptor tyrosine kinase, in a GDNF-responsive motor-neuron cell line. Ret protein could also bind GDNF and mediate survival and growth responses to GDNF upon transfection into naive fibroblasts. Moreover, high levels of c-ret mRNA expression were found in dopaminergic neurons of the adult substantia nigra, where exogenous GDNF protected Ret-positive neurons from 6-hydroxydopamine-induced cell death. Thus the product of the c-ret proto-oncogene encodes a functional receptor for GDNF that may mediate its neurotrophic effects on motor and dopaminergic neurons.

Amyotrophic lateral sclerosis and viruses.

Amyotrophic lateral sclerosis (ALS) is a disease of unknown etiology. A number of theories have been pursued to explain the cause of ALS, including viral infection. This review examines the evidence implicating viruses in the pathogenesis of ALS, as well as current studies of naturally occurring and experimental models of virus-induced motor neuron disease (MND). The association of viruses and ALS remains to be established. The study of animal models of virus-induced MND may shed light on processes relevant to the etiology of ALS.