Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Angiotensin II type 1 receptor blockade partially attenuates hypoxia-induced pulmonary hypertension in newborn piglets: relationship with the nitrergic system

The objective of this study was to observe possible interactions between the renin-angiotensin and nitrergic systems in chronic hypoxia-induced pulmonary hypertension in newborn piglets. Thirteen chronically instrumented newborn piglets (6.3 +/- 0.9 days; 2369 +/- 491 g) were randomly assigned to receive saline (placebo, P) or the AT(1) receptor (AT(1)-R) blocker L-158,809 (L) during 6 days of hypoxia (FiO(2) = 0.12). During hypoxia, pulmonary arterial pressure (Ppa; P < 0.0001), pulmonary vascular resistance (PVR; P < 0.02) and the pulmonary to systemic vascular resistance ratio (PVR/SVR; P < 0.05) were significantly attenuated in the L (N = 7) group compared to the P group (N = 6). Western blot analysis of lung proteins showed a significant decrease of endothelial NOS (eNOS) in both P and L animals, and of AT(1)-R in P animals during hypoxia compared to normoxic animals (C group, N = 5; P < 0.01 for all groups). AT(1)-R tended to decrease in L animals. Inducible NOS (iNOS) did not differ among P, L, and C animals and iNOS immunohistochemical staining in macrophages was significantly more intense in L than in P animals (P < 0.01). The vascular endothelium showed moderate or strong eNOS and AT(1)-R staining. Macrophages and pneumocytes showed moderate or strong iNOS and AT(1)-R staining, but C animals showed weak iNOS and AT(1)-R staining. Macrophages of L and P animals showed moderate and weak AT(2)-R staining, respectively, but the endothelium of all groups only showed weak staining. In conclusion, pulmonary hypertension induced by chronic hypoxia in newborn piglets is partially attenuated by AT(1)-R blockade. We suggest that AT(1)-R blockade might act through AT(2)-R and/or Mas receptors and the nitrergic system in the lungs of hypoxemic newborn piglets.

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT(1)/AT(2) receptors to activate SERCA and to promote Ca2+ mobilization

Ferrao FM, Lara LS, Axelband F, Dias J, Carmona AK, Reis RI, Costa-Neto CM, Vieyra A, Lowe J. Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT(1)/AT(2) receptors to activate SERCA and to promote Ca2+ mobilization. Am J Physiol Renal Physiol 302: F875-F883, 2012. First published January 4, 2012; doi:10.1152/ajprenal.00381.2011.-ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK1 cells on Ca2+-ATPase of the sarco(endo) plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca2+ mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca2+ spark, demonstrating a positively self-sustained stimulus of Ca2+ mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC -> DAG(PMA)-> PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca2+ stock in the reticulum to ensure a more efficient subsequent mobilization of Ca2+. This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca2+ homeostasis in renal cells and also for regulation of Ca2+-modulated fluid reabsorption in proximal tubules.

Evaluation of frozen-section analysis of surgical margins in the treatment of breast cancer

Objective: To evaluate surgical margins in cases of ductal carcinoma through a histopathological exam using frozen sections. Materials and Methods: Retrospective study encompassing 242 conservative surgeries, 179 of which included intraoperative frozensection histopathology and 63 intraoperative nonfreezing techniques (macroscopy/gross examination and cytology). The results of such analyses were compared with those of the histology processing following paraffin embedment and hematoxylin and eosin (H & E) staining. A margin was deemed free when the distance between the tumor and the surgical border was equal to or greater than two millimeters. The factors given consideration for possibly affecting the results were: age, surgical aspects (skin removal and widening of surgical margins), histopathological findings (size, affected lymph nodes, and angiolymphatic invasion), and extensive intraductal and immunohistochemical components (estrogen, progesterone, Ki-67, and HER-2 receptors). In the statistical analyses, the chi-square test was used and negative predictive values were calculated. Results: The negative predictive values were 87.1% and 79.3% for frozen and nonfrozen sections, respectively. There was no significant difference between the two groups (p = 0.14). The factors under consideration had no influence on the results of the intraoperative exam of the margins. Conclusion: The present study allowed to conclude that the intraoperative exam of the surgical margins by frozen section is not superior to a macroscopy and / or cytology exam.

Facilitation of sodium intake by combining noradrenaline into the lateral parabrachial nucleus with prazosin peripherally

Injections of noradrenaline into the lateral parabrachial nucleus (LPBN) increase arterial pressure and 1.8% NaCl intake and decrease water intake in rats treated with the diuretic furosemide (FURO) combined with a low dose of the angiotensin converting enzyme inhibitor captopril (CAP). In the present study, we investigated the influence of the pressor response elicited by noradrenaline injected into the LPBN on FURO+CAP-induced water and 1.8% NaCl intake. Male Holtzman rats with bilateral stainless steel guide-cannulas implanted into LPBN were used. Bilateral injections of noradrenaline (40 nmol/0.2 μl) into the LPBN increased FURO+CAP-induced 1.8% NaCl intake (12.2±3.5, vs., saline: 4.2±0.8 ml/180 min), reduced water intake and strongly increased arterial pressure (50±7, vs. saline: 1±1 mmHg). The blockade of the α1 adrenoceptors with the prazosin injected intraperitoneally abolished the pressor response and increased 1.8% NaCl and water intake in rats treated with FURO+CAP combined with noradrenaline injected into the LPBN. The deactivation of baro and perhaps volume receptors due to the cardiovascular effects of prazosin is a mechanism that may facilitate water and NaCl intake in rats treated with FURO+CAP combined with noradrenaline injected into the LPBN. Therefore, the activation of α2 adrenoceptors with noradrenaline injected into the LPBN, at least in dose tested, may not completely remove the inhibitory signals produced by the activation of the cardiovascular receptors, particularly the signals that result from the extra activation of these receptors with the increase of arterial pressure.

Studio elettrofisiologico di modificazioni a lungo termine della forza della trasmissione sinaptica nel sistema nervoso centrale

Il nucleo accumbens (NAc), il maggior componente del sistema mesocorticolimbico, è coinvolto nella mediazione delle proprietà di rinforzo e nella dipendenza da diverse sostanze d’abuso. Le sinapsi glutammatergiche del NAc possono esprimere plasticità, tra cui una forma di depressione a lungo termine (LTD) dipendente dagli endocannabinoidi (eCB). Recenti studi hanno dimostrato un’interazione tra le vie di segnalazione del sistema eCB e quelle di altri sistemi recettoriali, compreso quello serotoninergico (5-HT); la vasta colocalizzazione di recettori serotoninergici e CB1 nel NAc suggerisce la possibilità di un’interazione tra questi due sistemi. In questo studio abbiamo riscontrato che una stimolazione a 4 Hz per 20 minuti (LFS-4Hz) delle afferenze glutammatergiche in fettine cerebrali di ratto, induce una nuova forma di eCB-LTD nel core del NAc, che richiede l’attivazione dei recettori CB1 e 5-HT2 e l’apertura dei canali del Ca2+ voltaggio-dipendenti di tipo L. Inoltre abbiamo valutato che l’applicazione esogena di 5-HT (5 M, 20 min) induce una LTD analoga (5-HT-LTD) a livello delle stesse sinapsi, che richiede l’attivazione dei medesimi recettori e l’apertura degli stessi canali del Ca2+; LFS-4Hz-LTD e 5-HT-LTD sono reciprocamente saturanti. Questi risultati suggeriscono che la LFS-4Hz induce il rilascio di 5-HT, che si lega ai recettori 5-HT2 a livello postsinaptico incrementando l’influsso di Ca2+ attraverso i canali voltaggio-dipendenti di tipo L e la produzione e il rilascio di 2-arachidonoilglicerolo; l’eCB viaggia a ritroso e si lega al recettore CB1 a livello presinaptico, causando una diminuzione duratura del rilascio di glutammato, che risulta in una LTD. Queste osservazioni possono essere utili per comprendere i meccanismi neurofisiologici che sono alla base della dipendenza da sostanze d’abuso, della depressione maggiore e di altre malattie psichiatriche caratterizzate dalla disfunzione della neurotrasmissione di 5-HT nel NAc.

Binding sites of muscarinic and adrenergic receptors in gastrointestinal tissues of dairy cows suffering from left displacement of the abomasum

Muscarinic acetylcholine (M) and adrenergic (AR) receptors mediate gastrointestinal motility. Using radioligand binding assays and real-time polymerase chain reaction, the densities of binding sites and mRNA levels of M(2), M(3), alpha(2AD)- and beta(2)-AR were compared in muscle tissues from the abomasal fundus, pylorus, duodenum, caecum, and external loop of the spiral colon of eight cows with left displacement of abomasum (LDA), and of eight healthy cows. Specific binding of the [(3)H]-ligands to each of the four receptors was competitive and saturable. Binding sites of M(2) (all intestinal sites), M(3) (duodenum and caecum), and of alpha(2AD)-AR (abomasal fundus) were lower (P<0.05) in cows with LDA than in healthy cows. The coefficients of correlation between binding sites and mRNA transcripts of receptors were dissimilar in cows with LDA and healthy cows. The decrease in densities of M (intestine) and of alpha(2AD)-AR (abomasum) receptors suggests their implication in the impairment of motility associated with or leading to LDA.

Impact of subunit positioning on GABAA receptor function

The major isoforms of the GABAA (gamma-aminobutyric acid type A) receptor are composed of two alpha, two beta and one gamma subunit. Thus alpha and beta subunits occur twice in the receptor pentamer. As it is well documented that different isoforms of alpha and beta subunits can co-exist in the same pentamer, the question is raised whether the relative position of a subunit isoform affects the functional properties of the receptor. We have used subunit concatenation to engineer receptors of well-defined subunit arrangement to study this question. Although all five subunits may be concatenated, we have focused on the combination of triple and dual subunit constructs. We review here what is known so far on receptors containing simultaneously alpha1 and alpha6 subunits and receptors containing beta1 and beta2 subunits. Subunit concatenation may not only be used to study receptors containing two different subunit isoforms, but also to introduce a point mutation into a defined position in receptors containing either two alpha or beta subunits, or to study the receptor architecture of receptors containing unconventional GABAA receptor subunits. Similar approaches may be used to characterize other members of the pentameric ligand-gated ion channel family, including nicotinic acetylcholine receptors, glycine receptors and 5-HT3 (5-hydroxytryptamine) receptors.

Beta-caryophyllene is a dietary cannabinoid

The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB(1)) and CB(2) receptors. Although the CB(1) receptor is responsible for the psychomodulatory effects, activation of the CB(2) receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)-beta-caryophyllene [(E)-BCP] selectively binds to the CB(2) receptor (K(i) = 155 +/- 4 nM) and that it is a functional CB(2) agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB(2) receptor, showing ligand pi-pi stacking interactions with residues F117 and W258. Upon binding to the CB(2) receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB(2) receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB(2) receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis.

Adenosine signaling contributes to ethanol-induced fatty liver in mice.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

Glutamate receptors expressed in {\it Xenopus\/} oocytes: Studies in function and regulation

The amino acid glutamate is the primary excitatory neurotransmitter for the CNS and is responsible for the majority of fast synaptic transmission. Glutamate receptors have been shown to be involved in multiple forms of synaptic plasticity such as LTP, LTD, and the formation of specific synaptic connections during development. In addition to contributing to the plasticity of the CNS, glutamate receptors also are involved in, at least in part, various pathological conditions such as epilepsy, ischemic damage due to stroke, and Huntington's chorea. The regulation of glutamate receptors, particularly the ionotropic NMDA and AMPA/KA receptors is therefore of great interest. In this body of work, glutamate receptor function and regulation by kinase activity was examined using the Xenopus oocyte which is a convenient and faithful expression system for exogenous proteins. Glutamate receptor responses were measured using the two-electrode voltage clamp technique in oocytes injected with rat total forebrain RNA. NMDA elicited currents that were glycine-dependent, subject to block by Mg$\sp{2+}$ in a voltage-dependent manner and sensitive to the specific NMDA antagonist APV in a manner consistent with those types of responses found in neural tissue. Similarly, KA-evoked currents were sensitive to the specific AMPA/KA antagonist CNQX and exhibited current voltage relationships consistent with the calcium permeable type II KA receptors found in the hippocampus. There is evidence to indicate that NMDA and AMPA/KA receptors are regulated by protein kinase A (PKA). We explored this by examining the effects of activators of PKA (forskolin, 1-isobutyl-3-methylxanthine (IBMX) and 8-Br-cAMP) on NMDA and KA currents in the oocyte. In buffer where Ca$\sp{2+}$ was replaced by 2 mM Ba$\sp{2+},$ forskolin plus IBMX and 8-Br-cAMP augmented currents due to NMDA application but not KA. This augmentation was abolished by pretreating the oocytes in the kinase inhibitor K252A. The use of chloride channel blockers resulted in attenuation of this effect indicating that Ba$\sp{2+}$ influx through the NMDA channel was activating the endogenous calcium-activated chloride current and that the cAMP mediated augmentation was at the level of the chloride channel and not the NMDA channel. This was confirmed by (1) the finding that 8-Br-cAMP increased chloride currents elicited via calcium channel activation while having no effect on the calcium channels themselves and (2) the fact that lowering the Ba$\sp{2+}$ concentration to 200 $\mu$M abolished the augmentation NMDA currents by 8-Br-cAMP. Thus PKA does not appear to modulate ionotropic glutamate receptors in our preparation. Another kinase also implicated in the regulation of NMDA receptors, calcium/phospholipid-dependent protein kinase (PKC), was examined for its effects on the NMDA receptor under low Ba$\sp{2+}$ (200 $\mu$M) conditions. Phorbol esters, activators of PKC, induced a robust potentiation of NMDA currents that was blockable by the kinase inhibitor K252A. Furthermore activation of metabotropic receptors by the selective agonist trans-ACPD, also potentiated NMDA albeit more modestly. These results indicate that neither NMDA nor KA-activated glutamate receptors are modulated by PKA in Xenopus oocytes whereas NMDA receptors appear to be augmented by PKC. Furthermore, the endogenous chloride current of the oocyte was found to be responsive to Ba$\sp{2+}$ and in addition is enhanced by PKA. Both of these latter findings are novel. In conclusion, the Xenopus oocyte is a useful expression system for the analysis of ligand-gated channel activity and the regulation of those channels by phosphorylation. ^

Evidence for the role of agonist binding frequency in receptor -mediated activation of adenylate cyclase

$\beta$-adrenergic receptor-mediated activation of adenylate cyclase exhibits an agonist-specific separation between the dose/response curve (characterized by the EC$\sb{50}$) and the dose/binding curve (characterized by the K$\sb{\rm d}$). Cyclase activity can be near-maximal when receptor occupancy is quite low (EC$\sb{50}$ $\ll$ K$\sb{\rm d}$). This separation between the binding and response curves can be explained by the assumption that the rate of cyclase activation is proportional to the concentration of agonist-bound receptors, since the receptor is mobile and can activate more than one cyclase (the Collision Coupling Model of Tolkovsky and Levitzki). Here it is established that agonist binding frequency plays an additional role in adenylate cyclase activation in S49 murine lymphoma cells. Using epinephrine (EC$\sb{50}$ = 10 nM, K$\sb{\rm d}$ = 2 $\mu$M), the rate of cyclase activation decreased by 80% when a small (1.5%) receptor occupancy was restricted (by addition of the antagonist propranolol) to a small number (1.5%) of receptors rather than being proportionally distributed among the cell's entire population of receptors. Thus adenylate cyclase activity is not proportional to receptor occupancy in all circumstances. Collisions between receptor and cyclase pairs apparently occur a number of times in rapid sequence (an encounter); the high binding frequency of epinephrine ensures that discontiguous regions of the cell surface experience some period of agonist-bound receptor activity per small unit time minimizing "wasted" collisions between activated cyclase and bound receptor within an encounter. A contribution of agonist binding frequency to activation is thus possible when: (1) the mean lifetime of the agonist-receptor complex is shorter than the mean encounter time, and (2) the absolute efficiency (intrinsic ability to promote cyclase activation per collision) of the agonist-receptor complex is high. These conclusions are supported by experiments using agonists of different efficiencies and binding frequencies. These results are formalized in the Encounter Coupling Model of adenylate cyclase activation, which takes into explicit account the agonist binding frequency, agonist affinity for the $\beta$-adrenergic receptor, agonist efficiency, encounter frequency and the encounter time between receptor and cyclase. ^

The effect of protein tyrosine phosphatase SHP1 on tumorigenicity of the MDA -MB231 breast cancer cells

SHP1 is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. It is highly expressed in hematopoietic cells and expressed in normal epithelium at lower levels. While SHP1 in hematopoietic cells is thought to be a negative regulator of cellular signaling by associating with and dephosphorylating various receptors and their downstream effectors after they become activated, its precise function in epithelium remains to be understood. The potential involvement of SHP1 in human tumorigenesis has been hypothesized from the findings that SHP1 can interact with, dephosphorylate, and regulate the activity of several protein tyrosine kinases (PTKs) implicated in human cancer. These PTKs include epidermal growth factor receptor (EGFR) and Src. Such speculation is also supported by the report that SHP1 is overexpressed in human ovarian cancers. ^ Here we report, for the first time, that the levels of SHP1 expression and activity are altered in human breast cancer cells in comparison with normal breast epithelium. In particular, SHP1 expression is nearly lost in the breast cancer cell lines MDA-MB231 and MDA-MB435. After the re-introduction of SHP1 both in wild type (wt) and enzymatically inactive (dn) forms, into the MDA-MB231 cells, we observed no changes in cellular proliferation. However, the overexpression of wt SHP1 led to increased anchorage-independent growth in the MDA-MB231 cells. SHP1 phosphatase activity is essential for such an increase since the overexpression of dn SHP1 had no effect. Enhanced turnorigenicity in nude mice was also observed in the MDA-MB231 cells overexpressing wt SHP1, but not dn SHP1, suggesting the crucial function of SHP1 enzymatic activity in this process. Our observations in this study indicate that SHP1 promotes tumorigenesis by a mechanism or mechanisms apart from enchancing angiogenesis. In addition, we have found no evidence that the overexpression of SHP1 could affect metastatic potential in the MDA-MB231 cells. ^ In the MDA-MB231 cells stably transfected with either wt or dn SHP1 the peak level of EGFR tyrosine phosphorylation induced by EGF, as well as the sensitivity to EGF stimulation, was not altered. However, the overexpression of wt SHP1 led to a slight increase in the kinetics of EGFR dephosphorylation, whereas the overexpression of dn SHP1 led to slightly delayed kinetics of EGFR dephosphorylation. The overexpression of either the wt or dn SHP1 did not lead to any significant increase in Src kinase activity. ^ In NIH3T3 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by EGF or Akt activation by PDGF. In 3T3H4 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by heregulin. The transient overexpression of wt SHP1 in the MDA-MB231 cells caused an apparent increase, ranging from 10% to 20%, in the G0/G1 population of the cells with a corresponding decrease in the S phase population. ^ In order to understand the mechanisms by which SHP1 exerts its positive effect on the tumorigenic potential of the MDA-MB231 cells, we employed two-dimensional electrophoresis in an attempt to identify cellular protein(s) with significantly altered tyrosine phosphorylation level upon wt SHP1 overexpression. The overexpression of wt SHP1 but not dn SHP1, leads increased tyrosine phosphorylation of a protein with a molecular weight of approximately 40 kDa and a pI between 5.9 to 6.6. ^

Valorización de subproductos agrícolas y forestales como sustratos de cultivo en el estado de Oaxaca (México)

El presente trabajo aborda el aprovechamiento de algunos subproductos agrícolas (bagazo de maguey y fibra de coco) y forestales (corteza de pino) en el Estado de Oaxaca (Sur de México). El objetivo principal se centra en localizar, cuantificar y caracterizar estos con vistas a su aplicación como sustratos o componentes de sustratos en cultivos ornamentales, forestales y hortícolas, y a su uso como enmiendas en cultivos tipo. Así mismo se persigue reducir el uso de la turba y la tierra de monte como sustratos mayoritarios en la actualidad. Para la localización de los subproductos se utilizaron los datos de los registros parcelarios de los productores de coco para la obtención de copra (generadores de fibra de coco) de la región costa y de los productores de mezcal (generadores del residuo de bagazo de maguey) de la región valles centrales, así como las ubicaciones de los aserraderos forestales en el Estado de Oaxaca. Se emplea un Sistema de Información Geográfica (SIG) con una cartografía digitalizada de los elementos del medio (clima, geología y suelo), de los cultivos generadores (bagazo de maguey, fibra de coco y corteza de pino), de la agricultura protegida como receptora (tomate) y de la agricultura extensiva con cultivos receptores de enmienda (café, hule, limón, mango, palma de coco y maguey). La producción anual de los residuos se cartografía y cuantifica con los siguientes resultados: bagazo de maguey 624.000 t, fibra de coco 86.000 m3 y 72.000 t de corteza de pino. Mediante el estudio de las características de los suelos de los cultivos receptores y de los requerimientos de materia orgánica de cada cultivo se calcularon las necesidades totales de materia orgánica para cada suelo. Los resultados de las cantidades globales para cada cultivo en todo el Estado muestran una necesidad total de 3.112.000 t de materia orgánica como enmienda. Con los datos obtenidos y a través de un algoritmo matemático se realiza una propuesta de localización de dos plantas de compostaje (de bagazo de maguey y fibra de coco) y cuatro plantas de compostaje de corteza de pino. Con el fin de conocer los subproductos a valorizar como sustrato o componente de sustrato se caracteriza su composición física‐química, siguiendo Normas UNE‐EN, y se analizan mediante Resonancia Magnética Nuclear (RMN). Para el acondicionamiento de bagazo de maguey y la corteza de pino se realizaron ensayos de compostaje. Al final de 241 días la temperatura y la humedad de ambos procesos se encontraban en los rangos recomendados, indicando que los materiales estaban estabilizados y con calidad para ser utilizados como sustrato o componente de sustrato. Para la fibra de coco se realizó el proceso de molienda en seco de conchas de coco provenientes de la comunidad de Río Grande Oaxaca (Principal zona productora de copra en Oaxaca). Posteriormente se emplean los materiales obtenidos como componentes para sustratos de cultivo. Se estudia el compost de bagazo de maguey y siete mezclas; el compost de corteza de pino y ocho mezclas y la fibra de coco con tres mezclas. Estos sustratos alternativos permiten obtener mezclas y reducir el uso de la tierra de monte, turba, arcilla expandida y vermiculita, siendo por tanto una alternativa sostenible para la producción en invernadero. Se elaboraron mezclas especificas para el cultivo de Lilium hibrido asiático y oriental (siete mezclas), sustratos eco‐compatibles para cultivo de tomate (nueve mezclas), para la producción de planta forestal (siete mezclas) y para la producción de plántula hortícola (ocho mezclas). Como resultados más destacados del bagazo de maguey, corteza de pino y las mezclas obtenidas se resume lo siguiente: el bagazo de maguey, con volúmenes crecientes de turba (20, 30, 50 y 60 %) y la corteza de pino, con volúmenes de turba 40 y 60%, presentan valores muy recomendados de porosidad, capacidad de aireación, capacidad de retención de humedad y equilibrio agua‐aire. Para la fibra de coco, la procedente de Río Grande presenta mejor valoración que la muestra comercial de fibra de coco de Morelos. Por último se llevó a cabo la evaluación agronómica de los sustratos‐mezclas, realizando cinco experimentos por separado, estudiando el desarrollo vegetal de cultivos tipo, que se concretan en los siguientes ensayos: 1. Producción de Lilium asiático y oriental en cama para flor de corte; 2. Producción de Lilium oriental en contenedor para flor de corte; 3. Producción de plántula forestal (Pinus greggii E y Pinus oaxacana M); 4. Producción de tomate (Solanum lycopersicum L) y 5. Producción de plántula de tomate en semillero (Solanum lycopersicum L). En relación a la producción de Lilium hibrido asiático en cama los sustratos corteza de pino (CPTU 80:20 v/v), corteza de pino + sustrato comercial (CPSC 80:20 v/v) y corteza de pino+turba+arcilla expandida+vermiculita (CPTAEV2 30:40:15:15 v/v) presentan los mejores resultados. Dichos sustratos también presentan adecuados resultados para Lilium hibrido oriental con excepción de la corteza de pino + turba (CPTU 80:20 v/v). En la producción de Lilium hibrido oriental en contenedor para flor de corte, además de los sustratos de CPSC y CPTAEV2, la mezcla de corteza de pino+turba+arcilla expandida+vermiculita (CPTAEV 70:20:5:5 v/v) manifestó una respuesta favorable. En el ensayo de producción de plántulas de Pinus greggii E y Pinus oaxacana Mirov, las mezclas con corteza de pino+turba+arcilla expandida+vermiculita (CPTAEV2 30:40:15:15 v/v) y bagazo de maguey turba+arcilla expandida+vermiculita (BMTAEV2 30:60:5:5 v/v) son una alternativa que permite disminuir el empleo de turba, arcilla expandida y vermiculita, en comparación con el sustrato testigo de turba+arcilla expandida+vermiculita (TAEV 60:30:10 v/v). En la producción de tomate (Solanum lycopersicum L) frente a la utilización actual del serrín sin compostar (SSC), las mezclas alternativas de bagazo de maguey+turba (BMT 70:30 v/v), fibra de coco de Río Grande (FCRG 100v/v) y corteza de pino+turba (CPT 70:30 v/v), presentaron los mejores resultados en rendimientos. Así mismo, en la producción de plántulas de tomate las dos mezclas alternativas de bagazo de maguey+turba+ arcilla expandida+vermiculita (BMTAEV5 50:30:10:10 v/v) y (BMTAEV6 40:40:10:10 v/v) presentaron mejores resultados que los obtenidos en la mezcla comercial (Sunshine 3), mayoritariamente utilizada en México en la producción de plántula de tomate y hortícola. ABSTRACT This paper addresses the use of some agricultural products (maguey bagasse and coconut fiber) and forestry (pine bark) in the State of Oaxaca (southern Mexico). The principal purpose is to locate, quantify and characterize these with the idea of applying them as substrates or substrate components in ornamental crops, forestry, horticultural, and their use as crop amendments. On the other hand, the reduction of peat and forest soil as main substrates is pursued. For the location of the products, registry parcel data from copra producers (coconut fiber generators) of the coastal region and mescal producers (maguey bagasse residue generators) of the central valleys region, as well as the locations of forest mills in the State of Oaxaca. A Geographic Information System (GIS) with digital mapping of environmental factors (climate, geology and soil), crop generators of residues (maguey bagasse, coconut and pine bark) receptors of amendments such as protected agriculture (tomato) and extensive agriculture crops (coffee, rubber, lemon, mango, coconut and agave). The annual production of waste is mapped and quantified with the following results: 624,000t maguey bagasse, coconut fiber 72,000 m3 and 86,000 t of pine bark. Through the study of receiving crops soils properties of and organic matter requirements of each crop, total needs of organic matter for each soil were estimated. The results of the total quantities for each crop across the state show a total of 3,112,000 t of organic matter needed as amendment. Using that data and a mathematical algorithm, the location of two composting plants (agave bagasse and coconut fiber) and four composting plants pine bark was proposed. In order to know the by‐products that were going to be used as substrates or substrate components, their physical‐chemical composition was analyzed following UNE‐EN technics. Furthermore they were analyzed by Nuclear Magnetic Resonance (NMR). For conditioning of maguey bagasse and pine bark, composting essays were conducted. At the end of 241 days the temperature and humidity of both processes were at the recommended ranges, indicating that the materials were stabilized and had reached the quality to be used as a substrate or substrate component. Coconut shells from the community of Rio Grande Oaxaca (Main copra producing area in Oaxaca) were put through a process of dry milling. Subsequently, the obtained materials were used as components for growing media. We studied the maguey bagasse compost and seven mixtures; the pine bark compost and eight blends and coconut fiber with three mixtures. These alternative substrates allow obtaining mixtures and reduce the use of forest soil, peat, vermiculite and expanded clay, making it a sustainable alternative for greenhouse production. Specific mixtures were prepared for growing Lillium, Asian and eastern hybrids (seven blends), eco‐compatible substrates for tomato (nine mixtures), for producing forest plant (seven mixtures) and for the production of horticultural seedlings (eight mixtures). Results from maguey bagasse, pine bark and mixtures obtained are summarized as follows: the maguey bagasse, with increasing volumes of peat (20, 30, 50 and 60%) and pine bark mixed with 40 and 60% peat by volume, have very recommended values of porosity, aeration capacity, water retention capacity and water‐air balance. Coconut fiber from Rio Grande had better quality than commercial coconut fiber from Morelos. Finally the agronomic evaluation of substrates‐mixtures was carried out conducting five experiments separately: 1. Production of Asiatic and Eastern Lilium in bed for cut flower, 2. Production of oriental Lillium in container for cut flower, 3.Production of forest seedlings (Pinus greggii E and Pinus oaxacana M), 4. Production of tomato (Solanum lycopersicum L) and 5. Tomato seedling production in seedbed (Solanum lycopersicum L). In relation to the production of hybrid Asian Lilium in bed, pine bark substrates (CPTU 80:20 v/v), pine bark + commercial substrate (CPSC 80:20 v/v) and pine bark + peat + expanded clay + vermiculite (CPTAEV2 30:40:15:15 v/v) showed the best results. Such substrates also have adequate results for Lilium Oriental hybrid except pine bark + peat (CPTU 80:20 v / v). In the production of Lilium oriental hybrid container for cut flower, besides the CPSC and CPTAEV2 substrates, the mixture of pine bark + peat + vermiculite expanded clay (CPTAEV 70:20:5:5 v / v) showed a favorable response. In the production of Pinus greggii E and Pinus oaxacana Mirov seedlings trial, mixtures with pine bark + peat + expanded clay + vermiculite (CPTAEV2 30:40:15:15 v/v) and maguey bagasse+ peat+ expanded clay + vermiculite (BMTAEV2 30:60:5:5 v / v) are an alternative which allows reducing the use of peat, vermiculite and expanded clay in comparison with the control substrate made of peat + expanded clay+ vermiculite (60:30 TAEV: 10 v/v). In the production of tomato (Solanum lycopersicum L), alternative mixes of maguey bagasse + peat (BMT 70:30 v/v), coconut fiber from Rio Grande (FCRG 100 v / v) and pine bark + peat (CPT 70:30 v / v) showed the best results in yields versus the current use of sawdust without compost (SSC). Likewise, in the production of tomato seedlings of the two alternative mixtures maguey bagasse + peat expanded clay + vermiculite (BMTAEV5 50:30:10:10 v/v) and (BMTAEV6 40:40:10:10 v/v) had better results than those obtained in the commercial mixture (Sunshine 3), mainly used in Mexico in tomato seedling production and horticulture.

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.