Regulated Secretion of Acid Sphingomyelinase: IMPLICATIONS FOR SELECTIVITY OF CERAMIDE FORMATION*

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase^WT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C₁₆-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase^WT exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase^S508A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C₁₆-ceramide. Taken together, these data support a crucial role for Ser⁵⁰⁸ in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.     

LCA mainstreaming conditions in Latin America—based on learnings from 2005 to 2014

Purpose

Based on the 2005–2014 developments in the Latin American and the Caribbean region (LAC), this paper aims to understand the conditions’ levels for mainstreaming life cycle assessment/life cycle management (LCA/LCM) and map key next actions.

Methods

Along the paper, four mainstreaming conditions are analyzed: expansion of LCM/LCA training activities, availability of LCA studies, national LCA database operating, and existence and activity of national life cycle network(s). Assuming that countries with better conditions are in a better position to develop national LCA based regulations, policies are also researched to complement this study.

Results and discussion

With nine life cycle (LC) networks in 2014, the LAC region has positively developed its networking capacities since 2005 but not the databases area (only one LCA database, Mexicaniuh, is fully operational). It was found that countries with no networks, lack all LCA trainings, studies, and databases.Local capacities are limited which in best case, Chile, does not exceed 18 practitioners per 10 million inhabitants. Based on the total score on mainstreaming conditions, Mexico and Brazil are the most advanced countries, but their markets for LCA professionals are still small (Valdivia et al. 2015), which suggests that tailored made strategies are needed for stronger uptake of LCA by industrial sectors.Argentina, Peru, Chile, and Colombia are in the second tier but still lack a critical mass of business cases and the political will to improve their mainstreaming conditions.

Conclusions

LCA development in the LAC region since 2005 is overall positive but still insufficient to serve the growth of prosperous LCA markets. Well-functioning LC networks are essential to leapfrog LCA. In 2014, about 27 % of LAC countries counted on a LC network. A common language in the region (except for Portuguese in Brazil) has been instrumental for expanding LCA through regional cooperation. LCA-based policies are boosted when local capacities and databases are available following the cases of Mexico, Chile, and Brazil. More data and research are needed to understand the women role in advancing LCA and the causalities and motivations of LAC companies to decide for LCA implementation. The application of the methodology was possible thanks to good quality data available and delivered key findings to develop national road maps for advancing LCA. No indicator used is specific for the LAC region and similar exercises are encouraged in other regions such as Africa and Asia.

     

Differential intestinal mucosal protein expression in hypercholesterolemic mice fed a phytosterol-enriched diet

The molecular mechanisms involved in the phytosterol-induced decrease in intestinal cholesterol absorption remain unclear. Further, other biological properties such as immunomodulatory activity and protection against cancer have also been ascribed to these plant compounds. To gain insight into the mechanisms underlying phytosterol actions, we conducted a proteomic study in the intestinal mucosa of phytosterol-fed apolipoprotein E-deficient hypercholesterolemic (apoE-/-) mice. With respect to control-fed apoE-/- mice, nine differentially expressed proteins were identified in whole-enterocyte homogenates using 2-D DIGE and MALDI-TOF MS. These proteins are involved in plasma membrane stabilization, cytoskeleton assembly network, and cholesterol metabolism. Four of these proteins were selected for further study since they showed the highest abundance change or had a potential functional relationship with known effects of phytosterols. Annexin A2 (ANXA2) and beta-actin decrease and annexin A4 (ANXA4) and annexin A5 (ANXA5) increase were confirmed by Western blot analysis. Intestinal gene expression of ANXA2 and A5 and beta-actin was reduced, whereas that of ANXA4 was unchanged. The main results were retested in normocholesterolemic C57BL/6J mice. ANXA4 and ANXA5 protein upregulation and ANXA2 and beta-actin downregulation were reproduced in these animals. However, no changes in gene expression were found in C57BL/6J mice in either of the four proteins selected. ANXA2, A4, and A5 and beta-actin are proteins of special interest given their pleiotropic functions that include cholesterol-ester transport from caveolae, apoptosis, and anti-inflammatory properties. Therefore, the protein expression changes identified in this study might be involved in the biological effects of phytosterols.     

Crucial role of TrkB ligands in the survival and phenotypic differentiation of developing locus coeruleus noradrenergic neurons

The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1) the lack of alterations in double Gdnf/Nt3 null mutant mice; and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this, treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells, our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.     

Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively.     

Further research at the Oldowan site of Ain Hanech,North-eastern Algeria

Further investigations were carried out at Ain Hanech, Algeria in 1998 and 1999 to explore its potential for investigating early hominid behavioral patterns and adaptation. Research concentrated on the stratigraphy and dating, identifying new archaeological deposits, and excavating the Ain Hanech and El-Kherba localities. To enhance the chronological control within a biostratigraphic framework, the Ain Boucherit fossil-bearing stratum, yielding a Plio-Pleistocene fauna, is correlated with the regional stratigraphy. In the stratigraphic sequence, the Ain Boucherit stratum, located 13m below the Ain Hanech Oldowan occurrences, is found in Unit Q of the Ain Hanech Formation. Unit Q shows a paleomagnetically reversed polarity, which may be correlated with an age earlier than the Olduvai normal subchron (1.95-1.77Ma). Based on test trenches and stratigraphic analyses, additional Oldowan deposits A, B, and C are identified at Ain Hanech. All three deposits and the El-Kherba site contain Mode I technology artefacts associated with an Early Pleistocene fauna. El-Kherba is stratigraphically equivalent to Ain Hanech. These two archaeological sites are estimated to be dated to about 1.8Ma.     

Abundance of Mepraia spinolai in a Periurban zone of Chile

Mepraia spinolai is a silvatic species of Triatominae which prefers microhabitats near to or in rock piles. It is also able to maintain similar or higher size populations near houses. The density of bugs in quarries near Santiago, Chile, differed within microhabitats and varied significantly within sites according to season. M. spinolai was not found in sites characterized by human perturbation of quarries. Our results confirm M. spinolai as a silvatic triatomine whose importance as a vector of Chagas disease will depend on contact with humans. This could occur if the habitats where populations of this species are found become exploited for the building of urban areas.     

Assessing the Impact of Disease Vectors on Animal Populations

Many studies have attempted to assess the relative effects of different vectors of a disease on animal populations. To this end, three measures have been proposed: Vectorial efficiency, Vectorial capacity and recently Vectorial effectiveness (or Vectorial impact). In this study we relate these measures to derive some of their properties emphasising in the vectorial impact for its importance in both, population performance of parasites and the proportion of the prevalence of one parasite due to a given vector. We applied the quantitative expressions advanced in this study to a simple Chilean example with one parasite (Trypanosoma cruzi), two vectors (Triatoma infestans and Mepraia spinolai) and one animal population (humans: Chagas's disease).     

Pathogenic Aeromonas hydrophila Serogroup O:14 and O:81 Strains with an S Layer

Five autoagglutinating Aeromonas hydrophila isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme. They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10^4.61 to 10^7.11), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy. A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of A. hydrophila TF7 (O:11 reference strain). Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein. The S-layer proteins of the O:14 and O:81 A. hydrophila strains seemed to be primarily different from those previously purified from strains A. hydrophila TF7 and Aeromonas salmonicida A450 on the basis of colony hybridizations with both the structural genes vapA and ahsA. This is the first report of the presence of an S layer in mesophilic Aeromonas strains not belonging to serogroup O:11.     

Target identification of the novel antiobesity agent tungstate in adipose tissue from obese rats

Adipose tissue plays an active role in the development of obesity, and thus characterization of the molecular changes related to obesity in this tissue is a priority. Recently, we identified tungstate as a potent body weight reducing agent in obese animals, adipose tissue being one of the targets of its action. In this study a proteomics approach combining 2-DE and MS was used to identify proteins associated with obesity and targets of tungstate in white adipose tissue. Twenty-nine proteins were found differentially expressed between lean and diet-induced obese rats. Expression changes in transferrin, vimentin, vinculin, peroxiredoxins, Rho-GTP dissociation inhibitor, grifin, guanine deaminase and 3-phosphoglycerate dehydrogenase were associated here for the first time with obesity. Furthermore, tungstate treatment of obese rats reverted expression changes of 70% of the proteins modulated by obesity and another ten proteins were regulated by tungstate independently of the body weight reduction. The results suggest that the tungstate antiobesity effect can be mediated by the modulation of cellular structure, metabolism, redox state and signalling processes in adipose tissue. These findings open new avenues for the study of the aetiology of obesity and its treatment.