Alejandro Martínez-Martínez

Biochemical and spectroscopic studies of the response of Convolvulus arvensis L. to chromium(III) and chromium(VI) stress

The objective of the present study was to determine the oxidative stress caused by hexavalent chromium (Cr[VI]), the chromium (Cr) uptake, and the Cr speciation in Convolvulus arvensis L. plants grown in hydroponics media containing either Cr(VI) or Cr(III). The results demonstrated that C. arvensis plants exposed to Cr(VI) concentrations ranging from 0 to 40 mg/L expressed higher ascorbate peroxidase specific activity in roots than in shoots. On the other hand, catalase activity monitored in plants exposed to 2 mg/L of Cr(VI) for 24 h increased in roots after a few hours of exposure. However, catalase activity in shoots revealed a decrement almost immediately after treatment was initiated. The results from x-ray absorption spectroscopic studies indicated that the oxidation state of the supplied Cr(III) remained the same in plant tissues. The supplied Cr(VI), however, was reduced to the trivalent form in plant tissues. The results of inductively coupled plasma/optical emission spectroscopy demonstrated that after 5 d, the roots of plants exposed to 40 mg/L of Cr(III) or Cr(VI) accumulated approximately 25,000 and 3,500 mg/kg dry weight of Cr, respectively. Nevertheless, shoots concentrated 1,500 and 2,000 mg/kg dry weight of Cr from Cr(III) and Cr(VI), respectively, which indicated that Cr moved faster into C. arvensis plants when supplied as Cr(VI).

Lead toxicity in alfalfa plants exposed to phytohormones and ethylenediaminetetraacetic acid monitored by peroxidase, catalase, and amylase activities

This manuscript describes the toxicity of lead in alfalfa plants treated with ethylenediaminetetraacetic acid (EDTA) and the phytohormones indole-3-acetic-acid (IAA), gibberellic acid (GA), and kinetin (KN), on catalase (CAT), ascorbate peroxidase (APOX), and total amylase activity (TAA). In all cases Pb was used at 40 mg/L; EDTA at 0.2 mM (equimolar to Pb); and IAA, GA, and KN at 1, 10, and 100 microM, respectively. An experiment containing Pb at 40 mg/L, 0.2 mM EDTA, and IAA and KN at 100 microM each was performed to determine changes in TAA. A control (plain nutrient solution) also was used for comparison. In all cases the treatments were performed in triplicate. Standard procedures were followed to determine the activity of the respective enzymes. After 10 d of exposure to the treatments, the leaves were harvested, homogenized, and centrifuged, and the supernatants were analyzed for CAT, APOX, and TAA. All determinations were performed in triplicate. The results demonstrated that CAT was reduced significantly (p < 0.05) by all treatments containing Pb, IAA, and GA at 10 and 100 microM. However, only the treatments Pb/EDTA/KN at 1, 10, and 100 microM reduced the APOX. The TAA in leaves of alfalfa plants was increased significantly (p < 0.05) by all treatments. Overall, the results suggest that the CAT tests showed no lead toxicity to the alfalfa seedlings. However IAA at 10 and 100 muM revealed toxicity to the CAT enzyme. In addition, the APOX tests exhibited no toxicity to the peroxidase enzyme with the exception of Pb/EDTA/KN treatments. Finally, the TAA tests showed high Pb/EDTA/phytohormone toxicity to the amylase enzyme in alfalfa seedlings.

Coordination and speciation of cadmium in corn seedlings and its effects on macro- and micronutrients uptake.

The effect of cadmium (Cd) on both the absorption of important nutrients and the synthesis of low molecular weight thiols (LMWTs) was investigated in corn plants. The inductively coupled plasma-optical emission spectroscopy results demonstrated that the concentration of Cd in tissues (mainly in roots) increased as the concentration in the medium increased. In addition, the concentration of phosphorus increased in roots of Cd treated plants but remained at normal concentration in shoots. On the other hand, the uptake of sulfur (S) followed a similar trend as the Cd uptake. The concentration of S and the production of LMWT were found to increase significantly upon exposure to Cd. The results of the X-ray absorption spectroscopy analyses indicated that Cd within tissues was bound to S ligands with interatomic distances of 2.51-2.52 A. These results confirm a strong linkage between S uptake and the production of LMWT upon exposure to Cd.

The expression of protein disulfide isomerase from Litopenaeus vannamei hemocytes is regulated by bacterial inoculation

A partial clone encoding a member of the protein disulfide isomerase (PDI) was isolated from a Litopenaeus vannamei hemocyte cDNA library. The 5?-end sequence was obtained by RACE. The complete sequence encodes for a 502-residues protein that contains two thioredoxin domains and the typical endoplasmic reticulum retention KDEL motif. Shrimp PDI is highly similar to the homologue protein described in both vertebrates and invertebrates. Changes in the shrimp PDI mRNA expression were observed after injection of Vibrio alginolyticus, suggesting that PDI is implicated in the immune defense system. This is the first report of a PDI in crustaceans.

Insulin Stimulated-Glucose Transporter Glut 4 Is Expressed in the Retina

The vertebrate retina is a very metabolically active tissue whose energy demands are normally met through the uptake of glucose and oxygen. Glucose metabolism in this tissue relies upon adequate glucose delivery from the systemic circulation. Therefore, glucose transport depends on the expression of glucose transporters. Here, we show retinal expression of the Glut 4 glucose transporter in frog and rat retinas. Immunohistochemistry and in situ hybridization studies showed Glut 4 expression in the three nuclear layers of the retina: the photoreceptor, inner nuclear and ganglionar cell layers. In the rat retina immunoprecipitation and Western blot analysis revealed a protein with an apparent molecular mass of 45 kDa. 14C-glucose accumulation by isolated rat retinas was significantly enhanced by physiological concentrations of insulin, an effect blocked by inhibitors of phosphatidyl-inositol 3-kinase (PI3K), a key enzyme in the insulin-signaling pathway in other tissues. Also, we observed an increase in 3H-cytochalasin binding sites in the presence of insulin, suggesting an increase in transporter recruitment at the cell surface. Besides, insulin induced phosphorylation of Akt, an effect also blocked by PI3K inhibition. Expression of Glut 4 was not modified in retinas of a type 1 diabetic rat model. To our knowledge, our results provide the first evidence of Glut4 expression in the retina, suggesting it as an insulin- responsive tissue.

Preliminary studies of the effects of psychological stress on circulating lymphocytes analyzed by synchrotron radiation based-Fourier transform infrared microspectroscopy.

Psychological stress is a condition that not only generates behavioral disorders but also disrupts homeostasis and immune activity that can exacerbate or lead to inflammatory diseases. The aim of this work was to study biochemical changes in circulating immune cells from rats under psychological stress by using vibrational spectroscopy. A stress model was used, where exposure to a stressor was repeated for 5 days. Subsequently, circulating lymphocytes were examined for their biomolecular vibrational fingerprints with synchrotron radiation based-Fourier transform infrared microspectroscopy. The results showed an increased absorption at the ester lipid region (1720-1755 cm(-1)) in lymphocytes from stressed rats, suggesting lipid peroxidation. Statistical significant changes in wavenumber peak position and absorbance in the nucleic acid region were also observed (915-950 cm(-1) Z-DNA, 1090-1150 cm(-1) symmetric stretching of P-O-C, 1200-1260 cm(-1) asymmetric PO2 and 1570-1510 cm(-1) methylated nucleotides) which suggest a reduction of transcriptional activity in lymphocytes from stressed rat. These results unravel part of the mechanisms by which psychological stress may affect the immune system leading to systemic consequences.

Infrared Spectroscopy as a Tool to Study the Antioxidant Activity of Polyphenolic Compounds in Isolated Rat Enterocytes.

The protective effect of different polyphenols, catechin (Cat), quercetin (Qc) (flavonoids), gallic acid (GA), caffeic acid (CfA), chlorogenic acid (ChA) (phenolic acids), and capsaicin (Cap), against H2O2-induced oxidative stress was evaluated in rat enterocytes using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Fourier Transform Infrared Microspectroscopy (FTIRM), and results were compared to standard lipid peroxidation techniques: conjugated dienes (CD) and Thiobarbituric Acid Reactive Substances (TBARS). Analysis of ATR-FTIR and FTIRM spectral data allowed the simultaneous evaluation of the effects of H2O2 and polyphenols on lipid and protein oxidation. All polyphenols showed a protective effect against H2O2-induced oxidative stress in enterocytes, when administered before or after H2O2. Cat and capsaicin showed the highest protective effect, while phenolic acids had weaker effects and Qc presented a mild prooxidative effect (IR spectral profile of biomolecules between control and H2O2-treated cells) according to FTIR analyses. These results demonstrated the viability to use infrared spectroscopy to evaluate the oxidant and antioxidant effect of molecules in cell systems assays.

Chronic Psychological Distress as an Inducer of Microglial Activation and Leukocyte Recruitment into the Area Postrema

Background: Chronic psychological distress can cause neuroinflammation, but the involvement of leukocytes in this inflammatory response remains unclear. The area postrema (AP) is considered a neural-immune interface because it lacks a blood-brain barrier and a site for leukocyte recruitment in neuroinflammatory conditions induced by immunological insults, but its role in chronic psychological distress has not been explored. Objective: To determine leukocyte recruitment to the AP after chronic psychological distress. Methods: Rats were exposed to cat odor for 5 consecutive days to induce distress, and, on the 6th day, their brains were dissected to perform immunohistofluorescence studies of the AP. Immune cells were identified and quantified with CD45 and CD11b markers. The distribution of neurons and immune cells was determined using TrkA and CD45 markers, respectively. Results: Distress induced a significant increase in CD45+ and CD11b+ cells in the AP. Three immunophenotypes were determined in the control and distress groups: CD45+/CD11b-, CD45+/CD11b+ and CD45-/CD11b+. CD expression, morphology and fluorescence intensity enabled the identification of different immune cell types: starting from longitudinal ramified microglia (mainly in the control group) to amoeboid microglia, monocytes and lymphocytes (mostly in the distressed group). TrkA and CD45 expression in the AP revealed the proximity between soma neurons and leukocytes. Interestingly, some CD45+ cells expressed TrkA, with increased expression in the distressed group. Conclusions: The identification of microglial activation, leukocyte recruitment and the close proximity between neurons and leukocytes in the AP after chronic psychological distress exposure suggests the AP as a site for distress-induced immune responses and engraftment of leukocytes infiltrating the CNS.

Oxidant/antioxidant effects of chronic exposure to predator odor in prefrontal cortex, amygdala, and hypothalamus

The incidence of anxiety-related diseases is increasing these days, hence there is a need to understand the mechanisms that underlie its nature and consequences. It is known that limbic structures, mainly the prefrontal cortex and amygdala, are involved in the processing of anxiety, and that projections from prefrontal cortex and amygdala can induce activity of the hypothalamic–pituitary–adrenal axis with consequent cardiovascular changes, increase in oxygen consumption, and ROS production. The compensatory reaction can include increased antioxidant enzymes activities, overexpression of antioxidant enzymes, and genetic shifts that could include the activation of antioxidant genes. The main objective of this study was to evaluate the oxidant/antioxidant effect that chronic anxiogenic stress exposure can have in prefrontal cortex, amygdala, and hypothalamus by exposition to predator odor. Results showed (a) sensitization of the HPA axis response, (b) an enzymatic phase 1 and 2 antioxidant response to oxidative stress in amygdala, (c) an antioxidant stability without elevation of oxidative markers in prefrontal cortex, (d) an elevation in phase 1 antioxidant response in hypothalamus. Chronic exposure to predator odor has an impact in the metabolic REDOX state in amygdala, prefrontal cortex, and hypothalamus, with oxidative stress being prevalent in amygdala as this is the principal structure responsible for the management of anxiety.

Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans

Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,2′-dipyridyl disulphide, DPS), suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a “hinge” located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.