Regulation of various proteolytic pathways by insulin and amino acids in human fibroblasts

Intracellular protein degradation is a regulated process with several proteolytic pathways. Although regulation of macroautophagy has been investigated in some detail in hepatocytes and in few other cells, less is known on this regulation in other cells and proteolytic pathways. We show that in human fibroblasts insulin and amino acids reduce protein degradation by different signalling pathways and that this inhibition proceeds in part via the mammalian target of rapamycin, especially with amino acids, which probably increase lysosomal pH. Moreover, the regulatory amino acids (Phe, Arg, Met, Tyr, Trp and Cys) are partially different from other cells. Finally, and in addition to macroautophagy, insulin and amino acids modify, to different extents and sometimes in opposite directions, the activities of other proteolytic pathways.     

Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1

Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan-binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1.     

Production of bioalcohols and antioxidant compounds by acid hydrolysis of lignocellulosic wastes and fermentation of hydrolysates with Hansenula polymorpha

The effect of the H₂SO₄ concentration in the hydrolysis of sunflower‐stalk waste, at 95ºC and using a liquid/solid relation of 20, was studied. In a later stage, the hydrolysates were fermented at different temperatures with the aim of ethanol and xylitol production. A total conversion of the hemicellulose at the acid concentration of 0.5 mol/L was achieved; whereas an acid concentration of 2.5 mol/L was needed to reach the maximum value in the conversion of the cellulose fraction. The analysis of the hydrolysis kinetics has enabled to determine the apparent reaction order, which was 1.3. The hydrolysates from hydrolysis process with H₂SO₄ 0.5 mol/L, once detoxified, were fermented at pH 5.5, temperatures 30, 40, and 50ºC with the yeast Hansenula polymorpha (ATCC 34438), resulting in a sequential uptake of sugars. In relation to ethanol and xylitol yields, the best results were observed at 50°C ( = 0.11 g/g;  = 0.12 g/g). Instantaneous xylitol yields were higher than in ethanol, at the three temperatures essayed. Different phenolic compounds were analyzed in the hydrolysates; hydroxytyrosol was the most abundant (3.79 mg/L). The recovery of these compounds entails the elimination of inhibitors in the fermentation process and the production of high value‐added antioxidant products.     

Feasibility of anaerobic co-digestion as a treatment option of meat industry wastes

The anaerobic biodegradability of meat industry wastes was investigated in mesophilic batch reactors and combined with a mathematical model for describing their biodegradable fractions. The characteristics and methane yield achieved when digesting waste sludge, suggested the use of this as co-substrate for enhancing the biodegradability of the other wastes. The co-digestion experiments showed that it would be feasible to co-digest cow manure or ruminal waste with waste sludge, but biodegradability of pig/cow slurries was not improved, being strongly influenced by the ammonium concentration of co-digestion mixture. By applying the mathematical model, it was observed that when increasing the amount of waste sludge in the co-digestion mixtures, the amount of inert and slowly biodegradable fractions decreased leading to an increase in readily biodegradable fractions, volatile solid removal efficiencies and methane yields. These results suggest that using readily biodegradable wastes as co-substrate, the anaerobic biodegradability of complex organic wastes can be improved.     

Oleoylethanolamide,a natural ligand for PPAR-alpha,inhibits insulin receptor signalling in HTC rat hepatoma cells

Oleoylethanolamide (OEA) is a lipid mediator belonging to the fatty acid ethanolamides family. It is produced by intestine and adipose tissue. It inhibits food intake and body weight gain, and has hypolipemiant action in vivo, as well as a lipolytic effect in vitro. OEA is a PPAR-alpha agonist, and recently it has been found that OEA is an endogenous ligand of an orphan receptor. Previously, we have shown that OEA inhibits insulin-stimulated glucose uptake in isolated adipocytes, and produces glucose intolerance in rats. In the present work, we have studied another insulin target cell, the hepatocyte using a rat hepatoma cell line (HTC), and we have studied the cross-talk of OEA signalling with metabolic and mitotic signal transduction of insulin receptor. OEA dose-dependently activates JNK and p38 MAPK, and inhibits insulin receptor phosphorylation. OEA inhibits insulin receptor activation, blunting insulin signalling in the downstream PI3K pathway, decreasing phosphorylation of PKB and its target GSK-3. OEA also inhibits insulin-dependent MAPK pathway, as assessed by immunoblot of phosphorylated MEK and MAPK. These effects were reversed by blocking JNK or p38 MAPK using pharmacological inhibitors (SP 600125, and SB 203580). Since OEA is an endogenous PPAR-alpha agonist, we investigated whether a pharmacologic agonist (WY 14643) may mimic the OEA effect on insulin receptor signalling. Activation of PPAR-alpha by the pharmacological agonist WY14643 in HTC hepatoma cells is sufficient to inhibit insulin signalling and this effect is also dependent on p38 MAPK but not JNK kinase. In summary, OEA inhibits insulin metabolic and mitogenic signalling by activation of JNK and p38 MAPK via PPAR-alpha.     

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5′-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.     

Eight polymorphic microsatellite markers in the Andalusian belladonna (Atropa baetica, Solanaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for the critically endangered Atropa baetica. These are the first microsatellite loci reported for Atropa species. The total number of alleles found was 18, the expected heterozygosity ranged from 0.198 to 0.505. These markers will be useful to establish the real census of individuals and the genetic diversity both within and among the different populations of A. baetica.     

Kinetic Analysis of Ex Vivo Human Blood Infection by Leishmania

The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0–5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k₊₁) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills ∼95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k₊₁ for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which ∼50% and ∼25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209⁺ dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.     

Antagonistic interactions among halobacteria due to halocin production

Abstract By using 3 different methods for the detection of antagonism, complex patterns of this type of interaction were evident within a group comprising 79 halobacterial strains, which included the most divergent types of culture collection strains available, as well as freshly isolated ones. The experiments carried out showed that such interactions are not due to phages, but to the effects of bacteriocin-like substances, known as halocins. With the exception of only 2 strains, all the remainder caused inhibition to a greater or lesser degree, showing a wide variety of activity spectra. Comparative numerical analysis of these spectra revealed the existence of at least 15 different groups of halocins.