Serum factors inhibitory for in vitro development of Plasmodium falciparum blood-stage parasites

Sera from 29 individuals residing in a malaria-endemic region of Colombia were evaluated by an inhibition assay for their capacity to retard the growth of Plasmodium falciparum in vitro. The inhibitory activity was found to be independent of antibody activity. Furthermore, the degree of inhibition of parasite development was variable, depending on the parasite isolate used for the assay and the season of malaria transmission. We selected sera with high inhibitory activity and carried out partial analytical characterization by anion exchange fast protein liquid chromatography (FPLC) to identify the chemical nature of the inhibitory factor(s). The results suggested that the in vitro inhibitory activity might result from the additive effect of different molecules. It appears that these molecules could be non-specifically induced by stimulation of the immune system, they seem to play a role in the immunity to malaria.

Oligodendroglia metabolically support axons and contribute to neurodegeneration.

Oligodendroglia support axon survival and function through mechanisms independent of myelination, and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been proposed. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and use. Here we show that the most abundant lactate transporter in the central nervous system, monocarboxylate transporter 1 (MCT1, also known as SLC16A1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and in mouse models of, amyotrophic lateral sclerosis, suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.

Propietats protectores de la lipoproteïna de alta densitat (HDL) sobre l'acció inflamatòria de la lipoproteïna de baixa densitat electronegativa (LDL(-)) en monòcits humans

Electronegative low-density lipoprotein (LDL(-)) is a modified fraction of LDL present in peripheral blood whose proportion is elevated in subjects with increased cardiovascular risk. LDL(-) has been shown to have an inflammatory effect on human endothelial cells and mononuclear blood cells. On the other hand, high-density lipoprotein (HDL) is known to have a protective effect against cardiovascular disease, partly mediated by its anti-inflammatory properties. The objective of the current work is to study the putative protective properties of HDL towards the inflammatory effect of LDL(-) in human monocytes, in order to elucidate the mechanisms behind their interaction. Total LDL and HDL were isolated by ultracentrifugation and LDL(-) was obtained from total LDL by anion exchange chromatography. HDL and LDL(-) were incubated together and then re-isolated, and their characteristics were compared to those of untreated lipoproteins. The inflammatory activity of the lipoproteins was determined by incubating monocytes with lipoproteins and measuring cytokine release from the cultured monocytes. The biochemical composition and electrophoretic mobility of the lipoproteins were also determined before and after their interaction. Incubation of HDL with LDL(-) reduced the inflammatory effect of LDL(-) and, in turn, HDL gained inflammatory properties. This indicates a transfer of inflammatory potential taking place during the interaction of LDL(-) and HDL. Additionally, LDL(-) lost non-esterified fatty acids (NEFAs) while HDL gained the same. We conclude that a transfer of NEFAs takes place between LDL(-) and HDL. These observations suggest that NEFAs play a role in the inflammatory effect mediated by LDL(-).

Glioprotective impact of cell-permeable peptides in preclinical models of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by progressive degeneration of upper and lower motor neurons. It is mostly sporadic, but about 2% of cases are associated with mutations in the gene encoding the enzyme superoxide dismutase 1 (SOD1). A major constraint to the comprehension of the pathogenesis of ALS has been long represented by the conviction that this disorder selectively affects motor neurons in a cell-autonomous manner. However, the failure to identify the events underlying the neurodegenerative process and the increased knowledge of the complex cellular interactions necessary for the correct functioning of the CNS has recently focused the attention on the contribution to neurodegeneration of glial cells, including astrocytes. Astrocytes can hurt motor neurons directly by secreting neurotoxic factors, but they can also play a deleterious role indirectly by losing functions that are supportive for neurons. Recently, we reported that a subpopulation of astrocytes degenerates in the spinal cord of hSOD1G93A transgenic mouse model of ALS. Mechanistic studies in cultured astrocytes revealed that such effect is mediated by the excitatory amino acid glutamate.On the bsis of these observations, we next used the established cell culture model as a tool to screen the glioprotective effect of innovative drugs, namely cell-permeable therapeutics. These consist of peptidic effector moieties coupled to the selective intracellular peptide transporter TAT protein. We initially validated the usefulness of these molecules demonstrating that a control fluorescent peptide enters astrocytes in culture and is retained within the cells up to 24-48 h, according to the timing of our cytotoxicity experiments. We then tested the impact of specific intracellular peptides with antiapoptotic properties on glutamate-treated hSOD1G93A- expressing astrocytes and we identified one molecule that protects the cells from death. Chronic treatment of ALS mice with this peptide had a positive impact on the outcome of the disease.

Perinatal nitric oxide therapy prevents adverse effects of perinatal hypoxia on the adult pulmonary circulation.

Adverse events in utero are associated with the occurrence of chronic diseases in adulthood. We previously demonstrated in mice that perinatal hypoxia resulted in altered pulmonary circulation in adulthood, with a decreased endothelium-dependent relaxation of pulmonary arteries, associated with long-term alterations in the nitric oxide (NO)/cyclic GMP pathway. The present study investigated whether inhaled NO (iNO) administered simultaneously to perinatal hypoxia could have potential beneficial effects on the adult pulmonary circulation. Indeed, iNO is the therapy of choice in humans presenting neonatal pulmonary hypertension. Long-term effects of neonatal iNO therapy on adult pulmonary circulation have not yet been investigated. Pregnant mice were placed in hypoxia (13% O2) with simultaneous administration of iNO 5 days before delivery until 5 days after birth. Pups were then raised in normoxia until adulthood. Perinatal iNO administration completely restored acetylcholine-induced relaxation, as well as endothelial nitric oxide synthase protein content, in isolated pulmonary arteries of adult mice born in hypoxia. Right ventricular hypertrophy observed in old mice born in hypoxia compared to controls was also prevented by perinatal iNO treatment. Therefore, simultaneous administration of iNO during perinatal hypoxic exposure seems able to prevent adverse effects of perinatal hypoxia on the adult pulmonary circulation.

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Non-coding small RNAs (sRNAs) have important regulatory functions in bacteria. In Pseudomonas spp., about 40 sRNAs have been reported until the end of 2008, a number that almost certainly is an underestimate. We provide a summary of the coding regions for these sRNAs is Pseudomonas aeruginosa. The functions of some Pseudomonas sRNAs can be deduced from those of homologous well-characterized sRNAs of Escherichia coli, e.g. 6S RNA (a stationary phase regulator of RNA polymerase) and tmRNA (a component of a machinery serving to eliminate truncated polypeptides). Two sRNAs of P. aeruginosa, PrrF1 and PrrF2, whose expression is repressed by the Fur repressor in the presence of iron, inhibit translation initiation of mRNAs specifying superoxide dismutase (sodB), succinate dehydrogenase (sdhABCD) and anthranilate degradation (antABC), via a base-paring mechanism. As a consequence, these mRNAs are poorly expressed under conditions of iron limitation. Two further sRNAs of P. aeruginosa, RsmY and RsmZ, whose expression is positively controlled by the GacS/GacA two-component system in response to unknown signals, act as scavengers of the RNA-binding protein RsmA. In this way, translational repression exerted by RsmA on target mRNAs can be relieved. The Gac/Rsm signal transduction pathway globally regulates motility and the formation of extracellular products in pseudomonas spp.

Reactive Nitrogen and Oxygen Intermediates in Patients with Cutaneous Leishmaniasis

The metabolisms of reactive nitrogen and oxygen intermediates (RNI and ROI) in patients with cutaneous leishmaniasis (CL) were investigated and compared with those of healthy subjects. To determine RNI metabolism, nitrite plus nitrate concentrations were measured spectrophotometrically. Nitrite concentration in plasma was determined directly by the Griess method. Nitrate levels in plasma were measured after reduction into nitrite by using copper-cadmium-zinc. ROI metabolism was evaluated by measuring erythrocyte superoxide dismutase, catalase and glutathione peroxidase activities. Plasma nitrite plus nitrate levels and erythrocyte superoxide dismutase activity were higher in the patient group than healthy subjects (p<0.01). In contrast, erythrocyte catalase and glutathione peroxidase activities were lower (p<0.05, p<0.01, respectively). ROI metabolism was altered in relation to hydrogen peroxide elevation in patients with CL. These alterations in ROI enable nitric oxide (NO) to amplify its leishmanicidal effect. The determination of ROI and RNI in patients with CL may be a useful tool to evaluate effector mechanisms of NO and clinical manifestations.

Hypoxic contraction of small pulmonary arteries from normal and endotoxemic rats: fundamental role of NO.

The present study was aimed at examining the role of nitric oxide (NO) in the hypoxic contraction of isolated small pulmonary arteries (SPA) in the rat. Animals were treated with either saline (sham experiments) or Escherichia coli lipolysaccharide [LPS, to obtain expression of the inducible NO synthase (iNOS) in the lung] and killed 4 h later. SPA (300- to 600-micrometer outer diameter) were mounted as rings in organ chambers for the recording of isometric tension, precontracted with PGF2alpha, and exposed to either severe (bath PO2 8 +/- 3 mmHg) or milder (21 +/- 3 mmHg) hypoxia. In SPA from sham-treated rats, contractions elicited by severe hypoxia were completely suppressed by either endothelium removal or preincubation with an NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME), 10(-3) M]. In SPA from LPS-treated rats, contractions elicited by severe hypoxia occurred irrespective of the presence or absence of endothelium and were largely suppressed by L-NAME. The milder hypoxia elicited no increase in vascular tone. These results indicate an essential role of NO in the hypoxic contractions of precontracted rat SPA. The endothelium independence of HPV in arteries from LPS-treated animals appears related to the extraendothelial expression of iNOS. The severe degree of hypoxia required to elicit any contraction is consistent with a mechanism of reduced NO production caused by a limited availability of O2 as a substrate for NOS.

Biochemical characterization of a trypanosomatid isolated from the plant Amaranthus retroflexus

A protozoan flagelate has recently been isolated from Amaranthus retroflexus. This plant grows near economically important crops in southeastern Spain, which are known to be parasitized by Phytomonas spp. The present study focuses on the characterization of the energy metabolism of this new isolate. These flagellates utilize glucose efficiently as their primary energy source, although they are unable to completely degrade it. They excrete ethanol, acetate, glycine, and succinate in lower amount, as well as ammonium. The presence of glycosomes was indicated by the early enzymes of the glycolytic pathway, one enzyme of the glycerol pathway (glycerol kinase), and malate dehydrogenase. No evidence of a fully functional citric-acid cycle was found. In the absence of catalase activity, these flagellates showed significant superoxide dismutase activity located in the glycosomal and cytosolic fractions. These trypanosomes, despite being morphologically and metabolically similar to other Phytomonas isolated from the same area, showed significant differences, suggesting that they are phylogenetically different species.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

CYP3A5 and ABCB1 genes influence blood pressure and response to treatment, and their effect is modified by salt.

The permeability-glycoprotein efflux-transporter encoded by the multidrug resistance 1 (ABCB1) gene and the cytochromes P450 3A4/5 encoded by the CYP3A4/5 genes are known to interact in the transport and metabolism of many drugs. Recent data have shown that the CYP3A5 genotypes influence blood pressure and that permeability-glycoprotein activity might influence the activity of the renin-angiotensin system. Hence, these 2 genes may contribute to blood pressure regulation in humans. We analyzed the association of variants of the ABCB1 and CYP3A5 genes with ambulatory blood pressure, plasma renin activity, plasma aldosterone, endogenous lithium clearance, and blood pressure response to treatment in 72 families (373 individuals; 55% women; mean age: 46 years) of East African descent. The ABCB1 and CYP3A5 genes interact with urinary sodium excretion in their effect on ambulatory blood pressure (daytime systolic: P=0.05; nighttime systolic and diastolic: P<0.01), suggesting a gene-gene-environment interaction. The combined action of these genes is also associated with postproximal tubular sodium reabsorption, plasma renin activity, plasma aldosterone, and with an altered blood pressure response to the angiotensin-converting enzyme inhibitor lisinopril (P<0.05). This is the first reported association of the ABCB1 gene with blood pressure in humans and demonstration that genes encoding for proteins metabolizing and transporting drugs and endogenous substrates contribute to blood pressure regulation.

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [214-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.

Determinants of basal fat oxidation in healthy Caucasians.

In a retrospective study, we examined several determinants of basal fat oxidation in 720 healthy Caucasian volunteers. Adult men (n = 427) and women (n = 293) were characterized for resting energy expenditure and substrate oxidation by indirect calorimetry (after a 12-h overnight fast), peak O2 consumption by a treadmill test to exhaustion, body composition by hydrodensitometry, food intake from a 3-day food diary, and hormonal status by fasting hormone concentrations. Fat oxidation was negatively correlated with fat mass in men (r = -0.11; P < 0.05), but no statistical relationship was found in women. In a stepwise multiple regression analysis, fat oxidation was best predicted by peak O2 consumption and fat-free mass in men (model R2 = 0.142) and by free thyroxine, fat-free mass, and fasting insulin in women (model R2 = 0.153). Relative rates of fat oxidation (fat oxidation adjusted for differences in resting energy expenditure) were not correlated with fat mass in either gender. Women showed a lower rate of basal fat oxidation (both absolute and adjusted) than did men. Our results show that fat oxidation is not greater in individuals with a greater fat mass. Furthermore, our results support a sexual dimorphism in basal rates of fat oxidation.

Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing.

BACKGROUND: In contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata). RESULTS: Transmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse. CONCLUSIONS: Altogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.

Oxygen uptake during early cardiogenesis of the chick.

Oxidative metabolism of the isolated embryonic heart of the chick has been determined using a spectrophotometric technique allowing global as well as localized micromeasurements of the O2 uptake. Entire hearts, excised from embryos of 10 somites (primordia fused, stage 10 HH) and 40 somites (S shaped, stage 20 HH) were placed in a special chamber under controlled metabolic conditions where they continued to beat spontaneously and regularly. During the 32 h of development, the O2 consumption of the whole heart increased from 0.9 +/- 0.1 to 5.3 +/- 0.8 nmol O2/h. These values corrected for protein content were, however, comparable (0.45 nmol O2.h-1.micrograms-1). At stage 10-12, the O2 uptake varied along the cardiac tube (from 0.74 to 1.0 nmol O2.h-1.mm-2). From stage 10 to 20, the O2 uptake per unit area of ventricle wall increased from 0.7 +/- 0.2 to 1.8 +/- 0.2 nmol O2.h-1.mm-2, and the O2 uptake per myocardial volume during one cardiac cycle varied from 7 to 2.5 nmol O2/cm3. These results indicate that, despite an intense morphogenesis, the cardiac tissue has a rather low and stable oxidative metabolism, although the O2 requirement of the whole heart increases significantly. Moreover, the normalized suprabasal aerobic energy expenditure decreases throughout early cardiogenesis. The functional integrity of the isolated embryonic heart combined with the experimental possibilities of the microtechnique make the preparation appropriate for studying the changes in cardiac metabolism during development.