Amalia Diez

Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchus labrax)

Abstract A growth trial was conducted with groups of European seabass having an initial weight of 6 g to study the lipogenic action of dietary protein and non-protein energy supplies. Six experimental diets were formulated to contain one of two crude protein levels (43 and 52%) with digestible protein (DP) to digestible energy (DE) ratios ranging from 19 to 26 mg/kJ. At the end of the growth trial (12 weeks), the activities of liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), malic enzyme (ME, EC 1.1.1.40), ATP citrate lyase (ACL, EC 4.1.3.8), acetyl-CoA carboxylase (ACoAC, EC 6.4.1.2) and fatty acid synthetase (FAS, EC 2.3.1.38) were measured. Digestibility of main dietary components was also determined over a three-week period. At each protein level, an increase in dietary DE led to improved growth performance, protein efficiency, daily N deposition and to a reduction of N loss. Best results were achieved at 40% DP and a DP/DE ratio of 19–20 mg/kJ. G6PD, ME and ACoAC were found to be the key regulatory enzymes in the lipogenic pathway, with G6PD being the main NADPH-generating enzyme. Activities of G6PD, ME, ACL and FAS were reduced with increasing fat intake. Activities of G6PD, ME and ACL were increased with increasing starch intake. ACoAC activity was negatively correlated with starch intake and positively with fat intake.

Short-term modulation of lipogenesis by macronutrients in rainbow trout ( Oncorhynchus mykiss ) hepatocytes

Rainbow trout (Oncorhynchus mykiss) hepatocytes were cultured under simulated conditions of varying nutritional status to explore the short-term modulation by dietary substrates of the main lipogenic enzymes: glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACoAC) and fatty acid synthetase (FAS). Primary cultures were individually exposed to varying amounts of glucose, hydrolysed casein and long-chain polyunsaturated fatty acids (PUFA) for 12 h. A second set of experiments was designed to evaluate the effects of mixing different relative amounts of these macronutrients in the culture medium. Glucose concentrations of up to 20-25 mm showed a stimulatory effect on G6PD, ME, ACL and ACoAC activity while an earlier inhibitory effect on FAS was observed at 10-20 mm glucose The use of hydrolysed casein as a nutritional source of amino acids inhibited the activity of FAS and ME and stimulated G6PD, ACoAC and ACL activity Low levels of linolenic acid exerted a stimulatory effect on all the lipogenic enzymes assayed with the exception of FAS, and increased amounts showed some inhibition of lipogenic activities Eicosapentaenoic acid and docosahexaenoic acid showed a similar effect, although the former strongly inhibited FAS activity while the latter showed greater potential to inhibit ACoAC and G6PD. A complete change in the relative levels of glucose, hydrolysed casein and PUFA in turn led to changes in the enzyme activity patterns observed. The present study shows the feasibility of exploring the direct regulation of lipogenesis in isolated fish cells by varying the relative amounts of main macronutrients, mimicking in vivo dietary conditions. It is felt that such an approach may serve to investigate the macronutrient regulation of other metabolic pathways.

Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily

Nnal has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nnal has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nnal homologues in bacteria, Protista, and Anima‐lia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nnal‐like sequences demonstrates a major divergence between Nnal‐like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational mod‐eling of representative Nnal‐like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nnal‐like peptidases from Caenσrhabditis elegans and demonstrated that this protein is a fully functional metaUocarboxypeptidase that cleaves a range of C‐terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt‐inactivated, and is preferentially inhibited by Z‐Glu‐Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal (1). Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nnal‐like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.—Rodriguez de la Vega, M., Sevilla, R. G., Hermoso, A., Lorenzo, J., Tanco, S., Diez, A., Fricker, L. D., Bautista, J. M., Avilés, F. X. Nna1‐like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily. FASEB J. 20, 851–865 (2007)

Synchronous culture of Plasmodium falciparum at high parasitemia levels

This protocol describes a method for preparing cultures of Plasmodium falciparum synchronized at any intraerythrocytic stage. Using this method, around 60% parasitized cells may be obtained. On the basis of Trager and Jensens original continuous culture method, our approach relies on the use of fresh human blood not older than 2 weeks, a low hematocrit between 0.8 and 1.5%, a starting frozen inoculum of 10% ring-stage parasitemia, human serum replaced with AlbuMAX I and alternating sorbitol and Percoll synchronization methods to shorten the cycle window to 4–6 h and reduce sorbitol toxicity. From our synchronized high parasite density cultures, 3–5 ml of infected red blood cells can be obtained in 1 week, corresponding to 1.2 mg of total parasite protein per ml of harvested culture. On the basis of the variables parasitemia and packed cell volume, we provide an equation to accurately calculate the amount of complete medium required every 24 h corrected for the cycle stage and capacity of the culture flask. Ten days suffice to complete the protocol from a frozen stock of parasites.

Dietary fish oil and digestible protein modify susceptibility to lipid peroxidation in the muscle of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax).

The effects of dietary fish oil and digestible protein (DP) levels on muscle fatty acid composition and susceptibility to lipid peroxidation were studied in two representative fish species for human nutrition, from fresh and seawater, rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax). In rainbow trout, higher concentrations of dietary fat and DP led to higher weight gain (g/d) (P = 0.001 and P = 0.043 respectively). Additionally, an interaction effect was observed in this species, since the effect of DP was only evident when the dietary fat concentration was low (P = 0.043). A similar tendency was also observed in European sea bass, although with less marked differences among nutritional treatments. Trout fed on diets with a higher concentration of dietary fat had higher concentrations of intramuscular total and neutral lipids in the dorsal muscle (P = 0.005). Increased levels of dietary DP led to significantly lower concentrations of polar lipids in the dorsal muscle of both rainbow trout (P = 0.005) and European sea bass (P = 0.006). In the neutral fraction of intramuscular lipids of dorsal muscle the concentration of n-3 fatty acids was positively affected by the dietary fat concentration in both rainbow trout (P = 0.04) and sea bass (P = 0.001). Muscle homogenates from trout and sea bass fed on diets rich in fish oil showed a significantly higher susceptibility to oxidation than muscle homogenates from fish fed on low-fat diets (P = 0.001). The higher DP concentration also increased susceptibility to oxidation. Moreover, in rainbow trout an interaction effect was observed where the pro-oxidant effect was of higher magnitude when the dietary concentration of both nutrients, fat and protein, was high (P = 0.004).

Molecular characterization of three peroxisome proliferator-activated receptors from the sea bass (Dicentrarchus labrax)

Peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that control the expression of genes involved in lipid homeostasis in mammals. We searched for PPAR in sea bass, a marine fish of particular interest to aquaculture, after hypothesizing that the physiological and molecular processes that regulate lipid metabolism in fish are similar to those in mammals. Here, we report the identification of complementary DNA and corresponding genomic sequences that encode three distinct PPAR from sea bass. The sea bass PPAR are the structural homologs of the mammalian PPARα, β/δ and γ isotypes. As revealed by RNase protection, the tissue expression profile of the fish PPAR appears to be very similar to that of the mammalian PPAR homologs. Thus, PPARα is mainly expressed in the liver, PPARγ in adipose tissue, and PPARβ in all tissues tested, with its highest levels in the liver, where it is also the dominant isotype expressed. Like mammalian PPAR, the sea bass isotypes recognize and bind to PPAR response elements of both mammalian and piscine origin, as heterodimers with the 9-cis retinoic acid receptor. Through the coactivator-dependent receptor ligand assay, we also demonstrated that natural FA and synthetic hypolipidemic compounds can act as ligands of the sea bass PPARα and β isotypes. This suggests that the sea bass PPAR act through similar mechanisms and perform the same critical lipid metabolism functions as mammalian PPAR.

Chloroquine mediates specific proteome oxidative damage across the erythrocytic cycle of resistant Plasmodium falciparum.

Resistance of Plasmodium falciparum to chloroquine hinders malaria control in endemic areas. Current hypotheses on the action mechanism of chloroquine evoke its ultimate interference with the parasites oxidative defence systems. Through carbonyl derivatization by 2,4-dinitrophenylhydrazine and proteomics, we compared oxidatively modified proteins across the parasites intraerythrocytic stages in untreated and transiently IC(50) chloroquine-treated cultures of the chloroquine-resistant P. falciparum strain Dd2. Functional plasmodial protein groups found to be most oxidatively damaged were among those central to the parasites physiological processes, including protein folding, proteolysis, energy metabolism, signal transduction, and pathogenesis. While an almost constant number of oxidized proteins was detected across the P. falciparum life cycle, chloroquine treatment led to increases in both the extent of protein oxidation and the number of proteins oxidized as the intraerythrocytic cycle progressed to mature stages. Our data provide new insights into early molecular effects produced by chloroquine in the parasite, as well as into the normal protein-oxidation modifications along the parasite cycle. Oxidized proteins involved in the particular parasite drug-response suggest that chloroquine causes specific oxidative stress, sharing common features with eukaryotic cells. Targeting these processes might provide ways of combating chloroquine-resistance and developing new antimalarial drugs.

Multi-targeted activity of maslinic acid as an antimalarial natural compound

Most drugs against malaria that are available or under development target a single process of the parasite infective cycle, favouring the appearance of resistant mutants which are easily spread in areas under chemotherapeutic treatments. Maslinic acid (MA) is a low toxic natural pentacyclic triterpene for which a wide variety of biological and therapeutic activities have been reported. Previous work revealed that Plasmodium falciparum erythrocytic cultures were inhibited by MA, which was able to hinder the maturation from ring to schizont stage and, as a consequence, prevent the release of merozoites and the subsequent invasion. We show here that MA effectively inhibits the proteolytic processing of the merozoite surface protein complex, probably by inhibition of PfSUB1. In addition, MA was also found to inhibit metalloproteases of the M16 family by a non‐chelating mechanism, suggesting the possible hindrance of plasmodial metalloproteases belonging to that family, such as falcilysin and apicoplast peptide‐processing proteases. Finally, in silico target screening was used to search for other potential binding targets that may have remained undetected. Among the targets identified, the method recovered two for which experimental activity could be confirmed, and suggested several putative new targets to which MA could have affinity. One of these unreported targets, phospholipase A2, was shown to be partially inhibited by MA. These results suggest that MA may behave as a multi‐targeted drug against the intra‐erythrocytic cycle of Plasmodium, providing a new tool to investigate the synergistic effect of inhibiting several unrelated processes with a single compound, a new concept in antimalarial research.

Transient silencing of Plasmodium falciparum bifunctional glucose‐6‐phosphate dehydrogenase− 6‐phosphogluconolactonase

The bifunctional enzyme glucose‐6‐phosphate dehydrogenase‐6‐phosphogluconolactonase (G6PD‐6PGL) found in Plasmodium falciparum has unique structural and functional characteristics restricted to this genus. This study was designed to examine the effects of RNA‐mediated PfG6PD‐6PGL gene silencing in cultures of P. falciparum on the expression of parasite antioxidant defense genes at the transcription level. The highest degree of G6PD‐6PGL silencing achieved was 86% at the mRNA level, with a recovery to almost normal levels within 24 h, indicating only transient diminished expression of the PfG6PD‐6PGL gene. PfG6PD‐6PGL silencing caused arrest of the trophozoite stage and enhanced gametocyte formation. In addition, an immediate transcriptional response was shown by thioredoxin reductase suggesting that P. falciparum G6PD‐6PGL plays a physiological role in the specific response of the parasite to intracellullar oxidative stress. P. falciparum transfection with an empty DNA vector also promoted intracellular stress, as determined by mRNA up‐regulation of antioxidant genes. Collectively, our findings point to an important role for this enzyme in the parasites infection cycle. The different characteristics of G6PD‐6PGL with respect to its homologue in the host make it an ideal target for therapeutic strategies.

Insights into the preclinical treatment of blood‐stage malaria by the antibiotic borrelidin

Blood‐stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl‐tRNA synthetases have been validated as antimalarial drug targets. We explored long‐term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria.