An Initial Proteomic Analysis of Biogas-Related Metabolism of Euryarchaeota Consortia in Sediments from the Santiago River, México.

In this paper, sediments from the Santiago River were characterized to look for an alternative source of inoculum for biogas production. A proteomic analysis of methane-processing archaea present in these sediments was carried out. The Euryarchaeota superkingdom of archaea is responsible for methane production and methane assimilation in the environment. The Santiago River is a major river in México with great pollution and exceeded recovery capacity. Its sediments could contain nutrients and the anaerobic conditions for optimal growth of Euryarchaeota consortia. Batch bioreactor experiments were performed, and a proteomic analysis was conducted with current database information. The maximum biogas production was 266 NmL·L-1·g VS-1, with 33.34% of methane, and for proteomics, 3206 proteins were detected from 303 species of 69 genera. Most of them are metabolically versatile members of the genera Methanosarcina and Methanosarcinales, both with 934 and 260 proteins, respectively. These results showed a diverse euryarcheotic species with high potential to methane production. Although related proteins were found and could be feeding this metabolism through the methanol and acetyl-CoA pathways, the quality obtained from the biogas suggests that this metabolism is not the main one in carbon use, possibly the sum of several conditions including growth conditions and the pollution present in these sediments.

Effects of Ornamental Plant Density and Mineral/Plastic Media on the Removal of Domestic Wastewater Pollutants by Home Wetlands Technology.

Wastewater treatment (WWT) is a priority around the world; conventional treatments are not widely used in rural areas owing to the high operating and maintenance costs. In Mexico, for instance, only 40% of wastewater is treated. One sustainable option for WWT is through the use of constructed wetlands (CWs) technology, which may remove pollutants using cells filled with porous material and vegetation that works as a natural filter. Knowing the optimal material and density of plants used per square meter in CWs would allow improving their WWT effect. In this study, the effect of material media (plastic/mineral) and plant density on the removal of organic/inorganic pollutants was evaluated. Low (three plants), medium (six plants) and high (nine plants) densities were compared in a surface area of 0.3 m2 of ornamental plants (Alpinia purpurata, Canna hybrids and Hedychium coronarium) used in polycultures at the mesocosm level of household wetlands, planted on the two different substrates. Regarding the removal of contaminants, no significant differences were found between substrates (p ≥ 0.05), indicating the use of plastic residues (reusable) is an economical option compared to typical mineral materials. However, differences (p = 0.001) in removal of pollutants were found between different plant densities. For both substrates, the high density planted CWs were able to remove COD in a range of 86-90%, PO4-P 22-33%, NH4-N in 84-90%, NO3-N 25-28% and NO2-N 38-42%. At medium density, removals of 79-81%, 26-32, 80-82%, 24-26%, and 39-41%, were observed, whereas in CWs with low density, the detected removals were 65-68%, 20-26%, 79-80%, 24-26% and 31-40%, respectively. These results revealed that higher COD and ammonia were removed at high plant density than at medium or low densities. Other pollutants were removed similarly in all plant densities (22-42%), indicating the necessity of hybrid CWs to increase the elimination of PO4-P, NO3-N and NO2-N. Moreover, high density favored 10 to 20% more the removal of pollutants than other plant densities. In addition, in cells with high density of plants and smaller planting distance, the development of new plant shoots was limited. Thus, it is suggested that the appropriate distance for this type of polyculture plants should be from 40 to 50 cm in expansion to real-scale systems in order to take advantage of the harvesting of species in these and allow species of greater foliage, favoring its growth and new shoots with the appropriate distance to compensate, in the short time, the removal of nutrients.

Caspase-3 Activation Correlates With the Initial Mitochondrial Membrane Depolarization in Neonatal Cerebellar Granule Neurons.

In this study we evaluated the effect of the reduction in the endoplasmic reticulum calcium concentration ([Ca2+]ER), changes in the cytoplasmic calcium concentration ([Ca2+]i), alteration of the mitochondrial membrane potential, and the ER stress in the activation of caspase-3 in neonatal cerebellar granule cells (CGN). The cells were loaded with Fura-2 to detect changes in the [Ca2+]i and with Mag-fluo-4 to measure variations in the [Ca2+]ER or with TMRE to follow modifications in the mitochondrial membrane potential in response to five different inducers of CGN cell death. These inducers were staurosporine, thapsigargin, tunicamycin, nifedipine and plasma membrane repolarization by switching culture medium from 25 mM KCl (K25) to 5 mM KCl (K5). Additionally, different markers of ER stress were determined and all these parameters were correlated with the activation of caspase-3. The different inducers of cell death in CGN resulted in three different levels of activation of caspase-3. The highest caspase-3 activity occurred in response to K5. At the same time, staurosporine, nifedipine, and tunicamycin elicited an intermediate activation of caspase-3. Importantly, thapsigargin did not activate caspase-3 at any time. Both K5 and nifedipine rapidly decreased the [Ca2+]i, but only K5 immediately reduced the [Ca2+]ER and the mitochondrial membrane potential. Staurosporine and tunicamycin increased the [Ca2+]i and they decreased both the [Ca2+]ER and mitochondrial membrane potential, but at a much lower rate than K5. Thapsigargin strongly increased the [Ca2+]i, but it took 10 min to observe any decrease in the mitochondrial membrane potential. Three cell death inducers -K5, staurosporine, and thapsigargin- elicited ER stress, but they took 30 min to have any effect. Thapsigargin, as expected, displayed the highest efficacy activating PERK. Moreover, a specific PERK inhibitor did not have any impact on cell death triggered by these cell death inducers. Our data suggest that voltage-gated Ca2+ channels, that are not dihydropyridine-sensitive, load the ER with Ca2+ and this Ca2+ flux plays a critical role in keeping the mitochondrial membrane potential polarized. A rapid decrease in the [Ca2+]ER resulted in rapid mitochondrial membrane depolarization and strong activation of caspase-3 without the intervention of the ER stress in CGN.

The Trans Golgi Region is a Labile Intracellular Ca2+ Store Sensitive to Emetine.

The Golgi apparatus (GA) is a bona fide Ca2+ store; however, there is a lack of GA-specific Ca2+ mobilizing agents. Here, we report that emetine specifically releases Ca2+ from GA in HeLa and HL-1 atrial myocytes. Additionally, it has become evident that the trans-Golgi is a labile Ca2+ store that requires a continuous source of Ca2+ from either the external milieu or from the ER, to enable it to produce a detectable transient increase in cytosolic Ca2+. Our data indicates that the emetine-sensitive Ca2+ mobilizing mechanism is different from the two classical Ca2+ release mechanisms, i.e. IP3 and ryanodine receptors. This newly discovered ability of emetine to release Ca2+ from the GA may explain why chronic consumption of ipecac syrup has muscle side effects.

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells.

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H3 receptor (hH3R445 and hH3R365) on [35S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) and depolarization-evoked [3 H]-dopamine release. Maximal specific binding (Bmax) of [3 H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells, respectively, with similar dissociation constants (Kd, 0.86 nM and 0.81 nM). The mRNA of the hH3R365 isoform was 40.9 ± 7.9% of the hH3R445 isoform. No differences in receptor affinity were found for the H3R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [35S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH3R445 cells ([35S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH3R365 cells; cAMP accumulation, -74.0 ± 4.9% versus -43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [3 H]-dopamine release evoked by 100 mM K+ (-18.9 ± 3.0% and -20.5 ± 3.3%, for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells), or the inhibition of depolarization-induced increase in [Ca2+]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH3R445 cells 0.639 ± 0.049, SH-SY5Y-hH3R365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH3R445 and hH3R365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.

Histamine H3 receptor activation stimulates calcium mobilization in a subpopulation of rat striatal neurons in primary culture, but not in synaptosomes.

The histamine H3 receptor (H3R) is abundantly expressed in the Central Nervous System where it regulates several functions pre and postsynaptically. H3Rs couple to Gαi/o proteins and trigger or modulate several intracellular signaling pathways, including the cAMP/PKA pathway and the opening of N- and P/Q-type voltage-gated Ca2+ channels. In transfected cells, activation of the human H3R of 445 amino acids (hH3R445) results in phospholipase C (PLC) stimulation and release of Ca2+ from intracellular stores. In this work we have studied whether H3R activation induces Ca2+ mobilization from intracellular stores in native systems, either isolated nerve terminals (synaptosomes) or neurons in primary culture. In rat striatal synaptosomes H3R activation induced inositol 1,4,5-trisphosphate (IP3) formation but failed to increase the intracellular calcium concentration ([Ca2+]i). In striatal primary cultures H3R activation resulted in IP3 formation and increased the [Ca2+]i in 18 out of 70 cells that responded with an elevation in the [Ca2+]i to membrane depolarization with KCl (100 mM) as evaluated by microfluorometry. Confocal microscopy studies corroborated the increase in [Ca2+]i induced by H3R activation in a fraction of those cells that were responsive to membrane depolarization. These results indicate that H3R activation stimulates the PLC/IP3/Ca2+ pathway but only in a subpopulation of striatal neurons.

Acidic intracellular Ca(2+) stores and caveolae in Ca(2+) signaling and diabetes.

Acidic Ca(2+) stores, particularly lysosomes, are newly discovered players in the well-orchestrated arena of Ca(2+) signaling and we are at the verge of understanding how lysosomes accumulate Ca(2+) and how they release it in response to different chemical, such as NAADP, and physical signals. Additionally, it is now clear that lysosomes play a key role in autophagy, a process that allows cells to recycle components or to eliminate damaged structures to ensure cellular well-being. Moreover, lysosomes are being unraveled as hubs that coordinate both anabolism via insulin signaling and catabolism via AMPK. These acidic vesicles have close contact with the ER and there is a bidirectional movement of information between these two organelles that exquisitely regulates cell survival. Lysosomes also connect with plasma membrane where caveolae are located as specialized regions involved in Ca(2+) and insulin signaling. Alterations of all these signaling pathways are at the core of insulin resistance and diabetes.