Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales.

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo/maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and ß-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria.

Cannabis Extraction Technologies: Impact of Research and Value Addition in Latin America.

The Cannabis genus of plants has been widely used in different cultures for various purposes. It is separated into three main species: sativa, indica, and ruderalis. In ancient practices, the plant was used as a multipurpose crop and valued for its fiber, food, and medicinal uses. Since methodologies for the extraction, processing, and identification of components have become available, medical, and food applications have been increasing, allowing potential development in the pharmaceutical and healthy functional food industries. Although the growing legalization and adoption of cannabis for the treatment of diseases are key factors pushing the growth of its market, the biggest challenge is to obtain higher-quality products in a time- and cost-effective fashion, making the process of extraction and separation an essential step. Latin American countries exhibit great knowledge of extraction technologies; nevertheless, it is still necessary to verify whether production costs are economically profitable. In addition, there has been an increase in commercial cannabis products that may or may not be allowed, with or without quality fact sheets, which can pose health risks. Hence, legalization is mandatory and urgent for the rest of Latin American countries. In this article, the phytochemical compounds (cannabinoids, terpenes, and phenolic compounds), the current status of legalization, extraction techniques, and research advances in cannabis in Latin America are reviewed.

Zinc Oxide Nanoparticles Induce an Adverse Effect on Blood Glucose Levels Depending On the Dose and Route of Administration in Healthy and Diabetic Rats.

Different studies in experimental diabetes models suggest that zinc oxide nanoparticles (ZnONPs) are useful as antidiabetic agents. However, this evidence was performed and measured in long-term treatments and with repeated doses of ZnONPs. This work aimed to evaluate the ZnONPs acute effects on glycemia during the next six h after an oral or intraperitoneal administration of the treatment in healthy and diabetic rats. In this study, the streptozotocin-nicotinamide intraperitoneal administration in male Wistar rats were used as a diabetes model. 10 mg/kg ZnONPs did not modify the baseline glucose in any group. Nevertheless, the ZnONPs short-term administration (100 mg/kg) induced a hyperglycemic response in a dose and route-dependent administration in healthy (130 ± 2 and 165 ± 10 mg/dL with oral and intraperitoneal, respectively) and diabetic rats (155 ± 2 and 240 ± 20 mg/dL with oral, and intraperitoneal, respectively). The diabetic rats were 1.5 fold more sensitive to ZnONPs effect by the intraperitoneal route. In conclusion, this study provides new information about the acute response of ZnONPs on fasting glycemia in diabetic and healthy rat models; these data are essential for possible future clinical approaches.

Capsaicin Causes Vasorelaxation of Rat Aorta through Blocking of L-type Ca2+ Channels and Activation of CB1 Receptors.

The aim of this work was to determine whether Capsaicin may exert a vascular regulation through the activation of CB1 and/or CB2 receptors causing vasorelaxation in the rat aorta. Our results show the location of TRPV1 mainly in the endothelial and smooth muscle cells membrane. Nevertheless, Capsaicin caused vasorelaxation of this artery through a mechanism independent of TRPV1, since the specific antagonists Capsazepine and SB-366791 did not block the effect of Capsaicin. Because the significant expression of CB1 and CB2 receptors has been previously reported in the rat aorta, we used antagonists for these two receptors prior to the addition of Capsaicin. In these experiments, we found that the inhibition of CB1 using AM281, decreases the vasorelaxant effect caused by Capsaicin. On the other hand, the vasorelaxant effect is not altered in the presence of the CB2 receptor antagonist AM630. Furthermore, a partial decrease of the effect of Capsaicin was also seen when L-type calcium channels are blocked. A complete block of Capsaicin-induced vasorelaxation was achieved using a combination of Verapamil and AM281. In accordance to our results, Capsaicin-induced vasorelaxation of the rat aorta is neither dependent of TRPV1 or CB2 receptors, but rather it is strongly suggested that a tandem mechanism between inactivation of L-type calcium channels and the direct activation of CB1 receptors is involved. These findings are supported by CB1 docking simulation which predicted a binding site on CB1 receptors for Capsaicin.

Revealing mechanisms of salinity tissue tolerance in succulent halophytes: A case study for Carpobrotus rossi.

Efforts to breed salt tolerant crops could benefit from investigating previously unexplored traits. One of them is a tissue succulency. In this work, we have undertaken an electrophysiological and biochemical comparison of properties of mesophyll and storage parenchyma leaf tissues of a succulent halophyte species Carpobrotus rosii ("pigface"). We show that storage parenchyma cells of C. rossii act as Na+ sink and possessed both higher Na+ sequestration (298 vs. 215 mM NaCl in mesophyll) and better K+ retention ability. The latter traits was determined by the higher rate of H+ -ATPase operation and higher nonenzymatic antioxidant activity in this tissue. Na+ uptake in both tissues was insensitive to either Gd3+ or elevated Ca2+ ruling out involvement of nonselective cation channels as a major path for Na+ entry. Patch-clamp experiments have revealed that Caprobrotus plants were capable to downregulate activity of fast vacuolar channels when exposed to saline environment; this ability was higher in the storage parenchyma cells compared with mesophyll. Also, storage parenchyma cells have constitutively lower number of open slow vacuolar channels, whereas in mesophyll, this suppression was inducible by salt. Taken together, these results provide a mechanistic basis for efficient Na+ sequestration in the succulent leaf tissues.

Cholinergic Machinery as Relevant Target in Acute Lymphoblastic T Leukemia.

Various types of non-neuronal cells, including tumors, are able to produce acetylcholine (ACh), which acts as an autocrine/paracrine growth factor. T lymphocytes represent a key component of the non-neuronal cholinergic system. T cells-derived ACh is involved in a stimulation of their activation and proliferation, and acts as a regulator of immune response. The aim of the present work was to summarize the data about components of cholinergic machinery in T lymphocytes, with an emphasis on the comparison of healthy and leukemic T cells. Cell lines derived from acute lymphoblastic leukemias of T lineage (T-ALL) were found to produce a considerably higher amount of ACh than healthy T lymphocytes. Additionally, ACh produced by T-ALL is not efficiently hydrolyzed, because acetylcholinesterase (AChE) activity is drastically decreased in these cells. Up-regulation of muscarinic ACh receptors was also demonstrated at expression and functional level, whereas nicotinic ACh receptors seem to play a less important role and not form functional channels in cells derived from T-ALL. We hypothesized that ACh over-produced in T-ALL may act as an autocrine growth factor and play an important role in leukemic clonal expansion through shaping of intracellular Ca(2+) signals. We suggest that cholinergic machinery may be attractive targets for new drugs against T-ALL. Specifically, testing of high affinity antagonists of muscarinic ACh receptors as well as antagomiRs, which interfere with miRNAs involved in the suppression of AChE expression, may be the first choice options.

Mechanosensitive Ca²âº-permeable channels in human leukemic cells: pharmacological and molecular evidence for TRPV2.

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²âº and Mg²âº. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³âº (10 µM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 µM) and SKF 96365 (250 µM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 µM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 µM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²âº signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.

Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed.

Differential activity of plasma and vacuolar membrane transporters contributes to genotypic differences in salinity tolerance in a Halophyte Species, Chenopodium quinoa.

Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd.), a facultative C3 halophyte species, can efficiently control the activity of slow (SV) and fast (FV) tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013) Plant Physiology). This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i) a higher rate of Na+ exclusion from leaf mesophyll; (ii) maintenance of low cytosolic Na+ levels; (iii) better K+ retention in the leaf mesophyll; (iv) a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v) the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

Reduced tonoplast fast-activating and slow-activating channel activity is essential for conferring salinity tolerance in a facultative halophyte, quinoa.

Halophyte species implement a "salt-including" strategy, sequestering significant amounts of Na(+) to cell vacuoles. This requires a reduction of passive Na(+) leak from the vacuole. In this work, we used quinoa (Chenopodium quinoa) to investigate the ability of halophytes to regulate Na(+)-permeable slow-activating (SV) and fast-activating (FV) tonoplast channels, linking it with Na(+) accumulation in mesophyll cells and salt bladders as well as leaf photosynthetic efficiency under salt stress. Our data indicate that young leaves rely on Na(+) exclusion to salt bladders, whereas old ones, possessing far fewer salt bladders, depend almost exclusively on Na(+) sequestration to mesophyll vacuoles. Moreover, although old leaves accumulate more Na(+), this does not compromise their leaf photochemistry. FV and SV channels are slightly more permeable for K(+) than for Na(+), and vacuoles in young leaves express less FV current and with a density unchanged in plants subjected to high (400 mm NaCl) salinity. In old leaves, with an intrinsically lower density of the FV current, FV channel density decreases about 2-fold in plants grown under high salinity. In contrast, intrinsic activity of SV channels in vacuoles from young leaves is unchanged under salt stress. In vacuoles of old leaves, however, it is 2- and 7-fold lower in older compared with young leaves in control- and salt-grown plants, respectively. We conclude that the negative control of SV and FV tonoplast channel activity in old leaves reduces Na(+) leak, thus enabling efficient sequestration of Na(+) to their vacuoles. This enables optimal photosynthetic performance, conferring salinity tolerance in quinoa species.

K(bg) and Kv1.3 channels mediate potassium efflux in the early phase of apoptosis in Jurkat T lymphocytes.

Microelectrode ion flux estimation (MIFE) and patch-clamp techniques were combined for noninvasive K(+) flux measurements and recording of activities of the dominant K(+) channels in the early phases of apoptosis in Jurkat cells. Staurosporine (STS, 1 microM) evoked rapid (peaking around 15 min) transient K(+) efflux, which then gradually decreased. This transient K(+) efflux occurred concurrently with the transient increase of the K(+) background (K(bg)) TWIK-related spinal cord K(+) channel-like current density, followed by a drastic decrease and concomitant membrane depolarization. The Kv1.3 current density remained almost constant. Kv1.3 activation was not altered by STS, whereas the inactivation was shifted to more positive potentials. Contribution of K(bg) and Kv1.3 channels to the transient and posttransient STS-induced K(+) efflux components, respectively, was confirmed by the effects of bupivacaine, predominantly blocking K(bg) current, and the Kv1.3-specific blocker margatoxin. Channel-mediated K(+) efflux provoked a substantial cellular shrinkage and affected the activation of caspases.

TRESK-like potassium channels in leukemic T cells.

In this study, we present patch-clamp characterization of the background potassium current in human lymphoma (Jurkat cells), generated by voltage-independent 16 pS channels with a high ( approximately 100-fold) K+/Na+ selectivity. Depending on the background K+ channels density, from few per cell up to approximately 1 open channel per microm2, resting membrane potential was in the range of -40 to -83 mV, approaching E (K) = -88 mV. The background K+ channels were insensitive to margotoxin (3 nM), apamine (3 nM), and clotrimazole (1 microM), high-affinity blockers of the lymphocyte Kv1.3, SKCa2, and IKCa1 channels. The current depended weakly on external pH. Arachidonic acid (20 microM) and Hg2+ (0.3-10 microM) suppressed background K+ current in Jurkat cells by 75-90%. Background K+ current was weakly sensitive to TEA+ (IC50 = 14 mM), and was efficiently suppressed by externally applied bupivacaine (IC50 = 5 microM), quinine (IC50 = 16 microM), and Ba2+ (2 mM). Our data, in particular strong inhibition by mercuric ions, suggest that background K+ currents expressed in Jurkat cells are mediated by TWIK-related spinal cord K+ (TRESK) channels belonging to the double-pore domain K+ channel family. The presence of human TRESK in the membrane protein fraction was confirmed by Western blot analysis.

Methyl-beta-cyclodextrin reversibly alters the gating of lipid rafts-associated Kv1.3 channels in Jurkat T lymphocytes.

The voltage-dependent Kv1.3 potassium channels mediate a variety of physiological functions in human T lymphocytes. These channels, along with their multiple regulatory components, are localized in cholesterol-enriched microdomains of plasma membrane (lipid rafts). In this study, patch-clamp technique was applied to explore the impact of the lipid-raft integrity on the Kv1.3 channel functional characteristics. T lymphoma Jurkat cells were treated for 1 h with cholesterol-binding oligosaccharide methyl-beta-cyclodextrin (MbetaCD) in 1 or 2 mM concentration, resulting in depletion of cholesterol by 63 +/- 5 or 75 +/- 4%, respectively. Treatment with 2 mM MbetaCD did not affect the cells viability but retarded the cell proliferation. The latter treatment caused a depolarizing shift of the Kv1.3 channel activation and inactivation by 11 and 6 mV, respectively, and more than twofold decrease in the steady-state activity at depolarizing potentials. Altogether, these changes underlie the depolarization of membrane potential, recorded in a current-clamp mode. The effects of MbetaCD were concentration- and time-dependent and reversible. Both development and recovery of the MbetaCD effects were completed within 1-2 h. Therefore, cholesterol depletion causes significant alterations in the Kv1.3 channel function, whereas T cells possess a potential to reverse these changes.