Rafael Salgado-Garciglia

Antibacterial Activity of Defensin PaDef from Avocado Fruit (Persea americana var. drymifolia) Expressed in Endothelial Cells against Escherichia coli and Staphylococcus aureus

Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americana var. drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensin PaDef cDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 μg/mL of total protein from clones (>55%). Also, S. aureus viability was inhibited from 50 μg/mL total protein (27–38%) but was more evident at 100 μg/mL (52–65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

Effects of diabetes on oxidative and nitrosative stress in kidney mitochondria from aged rats

Diabetes mellitus (DM) is characterized by chronic hyperglycemia resulting from defects in the secretion and/or action of insulin. Diabetic nephropathy (DN) develops in diabetic patients and is characterized by a progressive deterioration of renal function. The mitochondrial electron transport chain (ETC) produces most of the reactive oxygen species (ROS) that are involved in diabetic nephropathy. Due to the high incidence of DM in the elderly, the aim of this study was to evaluate oxidative and nitrosative stress in kidney mitochondria from aged rats. We evaluated lipid peroxidation (LPO), nitric oxide (NO•) production, S-nitrosylation profiles, glutathione levels, and glutathione reductase and aconitase activities under streptozotocin (STZ)-induced experimental diabetes in kidney mitochondria from aged rats. The results showed an increase in LPO, NO• production, and S-nitrosylated proteins in rats with STZ-induced diabetes. A decrease in glutathione (GSH) levels and glutathione reductase (GR) and aconitase activities in the rats that received the STZ-induced diabetes treatment was also observed, when compared with the age-related controls. The data suggest that oxidative and nitrosative stresses promote mitochondrial oxidative dysfunction in the more advanced age rat kidney in STZ-induced diabetes.

Mitochondrial response to oxidative and nitrosative stress in early stages of diabetes

Diabetes mellitus (DM) is associated with increased production of reactive oxygen and nitrogen species; consequently, an increase in lipid peroxidation and a decrease in antioxidants resulting in mitochondrial dysfunction. Using a rat model of DM induced by streptozotocin, we show the opposite: an increase in NO levels, S-nitrosylation, aconitase activity, and total glutathione and a decrease in lipid peroxidation at early stages of diabetes. These data imply that the decrease in lipid peroxidation is a vital early response to hyperglycemia to prevent escalation of ROS generation in mitochondria. These results also suggest a need for novel therapeutic targets to prevent the neurological consequences of diabetes.

Hypolipidemic Activity of Eryngium carlinae on Streptozotocin-Induced Diabetic Rats

Diabetes mellitus (DM) is a significant risk factor for the development of cardiovascular complications. This study was undertaken to investigate the effect of chronic administration of ethanolic extract of Eryngium carlinae on glucose, creatinine, uric acid, total cholesterol, and triglycerides levels in serum of streptozotocin- (STZ-) induced diabetic rats. Triglycerides, total cholesterol, and uric acid levels increased in serum from diabetic rats. The treatment with E. carlinae prevented these changes. The administration of E. carlinae extract reduced the levels of creatinine, uric acid, total cholesterol, and triglycerides. Thus administration of E. carlinae is able to reduce hyperlipidemia related to the cardiovascular risk in diabetes mellitus.

Oxidative stress and antioxidant response in a thermotolerant yeast

Stress tolerance is a key attribute that must be considered when using yeast cells for industrial applications. High temperature is one factor that can cause stress in yeast. High environmental temperature in particular may exert a natural selection pressure to evolve yeasts into thermotolerant strains. In the present study, three yeasts (Saccharomyces cerevisiae, MC4, and Kluyveromyces marxianus, OFF1 and SLP1) isolated from hot environments were exposed to increased temperatures and were then compared with a laboratory yeast strain. Their resistance to high temperature, oxidative stress, and antioxidant response were evaluated, along with the fatty acid composition of their cell membranes. The SLP1 strain showed a higher specific growth rate, biomass yield, and biomass volumetric productivity while also showing lower duplication time, reactive oxygen species (ROS) production, and lipid peroxidation. In addition, the SLP1 strain demonstrated more catalase activity after temperature was increased, and this strain also showed membranes enriched in saturated fatty acids. It is concluded that the SLP1 yeast strain is a thermotolerant yeast with less oxidative stress and a greater antioxidant response. Therefore, this strain could be used for fermentation at high temperatures.

The defensin from avocado (Persea americana var. drymifolia) PaDef induces apoptosis in the human breast cancer cell line MCF-7.

Antimicrobial peptides (AMPs) are cytotoxic to cancer cells; however, mainly the effects of AMPs from animals have been evaluated. In this work, we assessed the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on the MCF-7 cancer cell line (a breast cancer cell line) and evaluated its mechanism of action. PaDef inhibited the viability of MCF-7 cells in a concentration-dependent manner, with an IC50=141.62μg/ml. The viability of normal peripheral blood mononuclear cells was unaffected by this AMP. Additionally, PaDef induced apoptosis in MCF-7 cells in a time-dependent manner, but did not affect the membrane potential or calcium flow. In addition, PaDef IC50 induced the expression of cytochrome c, Apaf-1, and the caspase 7 and 9 genes. Likewise, this defensin induced the loss of mitochondrial Δψm and increased the phosphorylation of MAPK p38, which may lead to MCF-7 apoptosis by the intrinsic pathway. This is the first report of an avocado defensin inducing intrinsic apoptosis in cancer cells, which suggests that it could be a potential therapeutic molecule in the treatment of cancer.

Acmella oppositifolia micropropagation by single-node culture

Acmella oppositifolia plantlet formation was achieved by subculturing single-node explants on Murashige and Skoog medium without growth regulators. The explants from 1-month-old in vitro plantlets produced shoots over a 7-day culture period. From these in vitro cultured nodes readily rooted shoots elongated on auxin-free MS medium. Plants produced were easily acclimatized and subsequently flowered in a greenhouse. This species is of medicinal value in tropical America from Mexico to Colombia.

Expression of Mycobacterium leprae HSP65 in tobacco and its effectiveness as an oral treatment in adjuvant-induced arthritis

Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recominant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.

PaDef defensin from avocado ( Persea americana var. drymifolia) is cytotoxic to K562 chronic myeloid leukemia cells through extrinsic apoptosis

Plant defensins, a group of antimicrobial peptides, show selective cytotoxicity toward cancer cells. However, their mechanisms of action remain poorly understood. Here, we evaluated the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on K562 chronic myeloid leukemia cells and analyzed the pathway involved in the induction of cell death. The defensin PaDef was not cytotoxic against human PBMCs; however, it was cytotoxic for K562 cell line (IC50 = 97.3 μg/ml) activating apoptosis at 12 h. PaDef did not affect the mitochondrial membrane potential (ΔΨm), neither the transmembranal potential or the release of intracellular calcium. Also, PaDef induced gene expression of caspase 8 (∼2 fold), TNF-α (∼4 fold) and TNFR1 (∼10 fold). In addition, the activation of caspase 8 was detected at 24 h, whereas caspase 9 activity was not modified, suggesting that the extrinsic apoptosis pathway could be activated. In conclusion, PaDef induces apoptosis on K562 cells, which is related to the activation of caspase 8 and involves the participation of TNF-α, which is a novel property for a plant defensin.

Identification and control of pathogenic fungi in neotropical valued orchids (Laelia spp.)

Laelia species are valued orchids for ornamental and ritual uses. Since their natural populations are at risk, a suitable alternative is cultivation. However, the fungi affecting orchid cultivars are largely unknown. In this study, pathogenic fungi of Laelia autumnalis, L. rubescens, and L. speciosa in cultures of Morelia city orchid collections were identified using molecular and morphological methods. Pathogenicity tests were conducted using in vitro L. speciosa seedlings and a bioassay with three fungicides. Thirty-nine strains belonging to 10 taxa were isolated: Aspergillus fumigatus, Colletotrichum boninense, C. karstii, Fusarium bactridioides, F. oxysporum, F. proliferatum, F. solani, Fusarium sp., Xylaria feejeensis, and an Ascomycete strain. The most common pathogens in adult plants were F. proliferatum and A. fumigatus. The most aggressive fungus was F. solani. The Ascomycete, A. fumigatus and X. feejeensis, are reported herein as opportunistic orchid pathogens for the first time under in vitro conditions. The fungicide thiabendazole significantly inhibited the growth of F. proliferatum and F. solani, while benomyl inhibited the growth of A. fumigatus and F. bactridioides after five days. All tested fungicides did not inhibit the growth of C. boninense, F. oxysporum, Fusarium sp., and the Ascomycete strain. Our results will be useful to improve management recommendations for these endangered orchid species.