Non-natural amino acids into LfcinB-derived peptides: effect in their (i) proteolytic degradation and (ii) cytotoxic activity against cancer cells.

The dimeric peptide 26[F]: (RRWQWRFKKLG)2-K-Ahx has exhibited a potent cytotoxic effect against breast cancer cell lines, with position 26 (F) being the most relevant for anti-cancer activity. In this investigation, six analogues of the 26[F] peptide were synthesized in which the 26th position was replaced by non-natural hydrophobic amino acids, finding that some modifications increased the resistance to proteolytic degradation exerted by trypsin or pepsin. Additionally, these modifications increased the cytotoxic effect against breast cancer cells and generated cell death mediated by apoptosis pathways, activating caspases 8 and 9, and did not compromise the integrity of the cytoplasmic membrane. Finally, it was found that the modified peptides have a broad spectrum of action, since they also have a cytotoxic effect against the HeLa human cervical cancer cell line. Peptide 26[F] was inoculated in mice by ip administration and the lethal dose 50 (LD50) was between 70 and 140 mg kg-1. While for the 26[1-Nal]: (RRWQWR-1-Nal-KKLG)2-K-Ahx peptide, a dose-response test was performed, and the survival rate was 100%. These results suggested that these peptides are safe in this animal model and could be considered as promissory to develop a treatment against breast cancer.

The plant defensins PaDef and γ-thionin inhibit the endothelial cell response to VEGF.

Angiogenesis is involved in wound repair and tissue maintenance but is associated with diverse diseases. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) regulate this process. Therefore, searching for treatments to inhibit or promote angiogenesis is attractive. Reports from our group showed that plant antimicrobial peptides (PAPs) PaDef from avocado and γ-thionin from habanero pepper are cytotoxic on cancer cells. However, their functions as angiogenic regulators are unknown. In this work, we evaluate the effect of PaDef and γ-thionin on the angiogenic processes of two different endothelial cell lines: bovine endothelial cells (BUVEC) and the human endothelial cell line EA.hy926. The results showed that VEGF (10 ng/mL) stimulated the BUVEC (40 ± 7 %) and EA.hy926 cell proliferation (30 ± 9 %); however, peptides (5-500 ng/mL) reverted this effect. Besides, VEGF increased the migration of BUVEC (20 ± 8 %) and EA.hy926 cells (50 ± 6 %), but both PAPs (5 ng/mL) inhibited the VEGF stimulus (100 %). Furthermore, DMOG 50 µM (an inhibitor of HIF-hydroxylase) was used in BUVEC and EA.hy926 cells to determine the effect of hypoxia on VEGF and peptide activities. The DMOG reverted the inhibitory action of both peptides (100 %), indicating that peptides act through a HIF-independent pathway. Also, the PAPs do not affect the tube formation but decrease it in EA.hy926 cells stimulated with VEGF (100 %). Additionally, docking assays showed a possible interaction between PAPs and the VEGF receptor. These results suggest that plant defensins PaDef and γ-thionin are potential angiogenic modulators of the VEGF activity on endothelial cells.

Changes in Length and Positive Charge of Palindromic Sequence RWQWRWQWR Enhance Cytotoxic Activity against Breast Cancer Cell Lines.

Breast cancer is one of the main causes of premature death in women; current treatments have low selectivity, generating strong physical and psychological sequelae. The palindromic peptide R-1-R (RWQWRWQWR) has cytotoxic activity against different cell lines derived from cancer and selectivity against noncancerous cells. To determine if changes in the charge/length of this peptide increase its activity, six peptides were obtained by SPPS, three of them with addition of Arg at the N, C-terminal or both and three with deletion of Arg at the N, C-terminal or both. The cytotoxic and selective activities were evaluated against MCF-7, MDA-MB-231, and MCF-12 cell lines and fibroblast primary cell culture, evidencing that the RR-1-R peptide with the inclusion of Arg in the N-terminal end maintained selectivity and increased cytotoxicity against lines derived from breast cancer. The effect of this addition regarding the type of induced cell death was evaluated by flow cytometry, showing very low rates of necrosis and a significant majority of apoptotic events with activation of both Caspase 8 and Caspase 9. This work allowed us to find a modification that generates a peptide with greater cytotoxic effects and can be considered a promising molecule for other approaches to improve anticancer peptides.

Antifungal Activity of Avocado Seed Recombinant GASA/Snakin PaSn.

The avocado fruit (Persea americana) has become a significant fruit in the human diet for its nutritional properties. However, the seed is a source of bioactive molecules and has been poorly utilized. Previously, we reported that the PaSn gene is expressed in the avocado seeds, a cysteine-rich antimicrobial peptide (GASA/Snakin), and demonstrated its antibacterial activity. In this work, we report the recombinant production of PaSn in the Escherichia coli system and evaluate its antifungal activity against plant and human pathogen fungi. The recombinant peptide showed antifungal activity at 200 µg/mL against phytopathogens Colletotrichum gloeosporioides and Fusarium oxysporum and human pathogens Candida albicans and C. glabrata. Our results demonstrate the usefulness of a prokaryotic expression system for avocado antimicrobial peptide production. In conclusion, the snakin PaSn could be helpful in the control of postharvest avocado and other fruits' fungal diseases and human fungal pathogens.

Prolactin regulates H3K9ac and H3K9me2 epigenetic marks and miRNAs expression in bovine mammary epithelial cells challenged with Staphylococcus aureus.

Epigenetic mechanisms are essential in the regulation of immune response during infections. Changes in the levels of reproductive hormones, such as prolactin, compromise the mammary gland's innate immune response (IIR); however, its effect on epigenetic marks is poorly known. This work explored the epigenetic regulation induced by bovine prolactin (bPRL) on bovine mammary epithelial cells (bMECs) challenged with Staphylococcus aureus. In this work, bMECs were treated as follows: (1) control cells without any treatment, (2) bMECs treated with bPRL (5 ng/ml) at different times (12 or 24 h), (3) bMECs challenged with S. aureus for 2 h, and (4) bMECs treated with bPRL at different times (12 or 24 h), and then challenged with S. aureus 2 h. By western blot analyses of histones, we determined that the H3K9ac mark decreased (20%) in bMECs treated with bPRL (12 h) and challenged with S. aureus, while the H3K9me2 mark was increased (50%) in the same conditions. Also, this result coincided with an increase (2.3-fold) in HDAC activity analyzed using the cellular histone deacetylase fluorescent kit FLUOR DE LYS®. ChIP-qPCRs were performed to determine if the epigenetic marks detected in the histones correlate with enriched marks in the promoter regions of inflammatory genes associated with the S. aureus challenge. The H3K9ac mark was enriched in the promoter region of IL-1ß, IL-10, and BNBD10 genes (1.5, 2.5, 7.5-fold, respectively) in bMECs treated with bPRL, but in bMECs challenged with S. aureus it was reduced. Besides, the H3K9me2 mark was enriched in the promoter region of IL-1ß and IL-10 genes (3.5 and 2.5-fold, respectively) in bMECs challenged with S. aureus but was inhibited by bPRL. Additionally, the expression of several miRNAs was analyzed by qPCR. Let-7a-5p, miR-21a, miR-30b, miR-155, and miR-7863 miRNAs were up-regulated (2, 1.5, 10, 1.5, 3.9-fold, respectively) in bMECs challenged with S. aureus; however, bPRL induced a down-regulation in the expression of these miRNAs. In conclusion, bPRL induces epigenetic regulation on specific IIR elements, allowing S. aureus to persist and evade the host immune response.

Cytotoxicity of a Lipid-Rich Extract from Native Mexican Avocado Seed (Persea americana var. drymifolia) on Canine Osteosarcoma D-17 Cells and Synergistic Activity with Cytostatic Drugs.

Osteosarcoma is the most common malignant bone tumor in both children and dogs. It is an aggressive and metastatic cancer with a poor prognosis for long-term survival. The search for new anti-cancer drugs with fewer side effects has become an essential goal for cancer chemotherapy; in this sense, the bioactive compounds from avocado have proved their efficacy as cytotoxic molecules. The objective of this study was to determine the cytotoxic and antiproliferative effect of a lipid-rich extract (LEAS) from Mexican native avocado seed (Persea americana var. drymifolia) on canine osteosarcoma D-17 cell line. Also, the combined activity with cytostatic drugs was evaluated. LEAS was cytotoxic to D-17 cells in a concentration-dependent manner with an IC50 = 15.5 µg/mL. Besides, LEAS induced caspase-dependent cell apoptosis by the extrinsic and intrinsic pathways. Moreover, LEAS induced a significant loss of mitochondrial membrane potential and increased superoxide anion production and mitochondrial ROS. Also, LEAS induced the arrest of the cell cycle in the G0/G1 phase. Finally, LEAS improved the cytotoxic activity of cisplatin, carboplatin, and in less extension, doxorubicin against the canine osteosarcoma cell line through a synergistic effect. In conclusion, avocado could be a potential source of bioactive molecules in the searching treatments for osteosarcoma.

Bioactive Molecules From Native Mexican Avocado Fruit (Persea americana var. drymifolia): A Review.

Avocado (Persea americana Mill.) is a tree native from central and eastern México that belongs to the Lauraceae family. Avocado has three botanical varieties known as Mexican (P. americana var. drymifolia), West Indian (P. americana var. americana), and Guatemalan (P. americana var. guatemalensis). It is an oil-rich fruit appreciated worldwide because of its nutritional value and the content of bioactive molecules. Several avocado molecules show attractive activities of interest in medicine. Avocado fatty acids have beneficial effects on cardiovascular disease risk factors. Besides, this fruit possesses a high content of carotenoids and phenolic compounds with possible antifungal, anti-cancer and antioxidant activities. Moreover, several metabolites have been reported with anti-inflammatory effects. Also, an unsaponifiable fraction of avocado in combination with soybean oil is used for the treatment of osteoarthritis. The Mexican variety is native from México and is characterized by the anise aroma in leaves and by small thin-skinned fruits of rich flavor and excellent quality. However, the study of the bioactive molecules of the fruit has not been addressed in detail. In this work, we achieved a literature review on the inflammatory, immunomodulatory and cytotoxic properties of long-chain fatty acids and derivatives from Mexican avocado seed. Also, the antioxidant and anti-inflammatory properties of the oil extracted from the avocado seed are referred. Finally, the antimicrobial, immunomodulatory, and cytotoxic activities of some antimicrobial peptides expressed in the fruit are reviewed.

Two Oleosins Expressed in the Mesocarp of Native Mexican Avocado, Key Genes in the Oil Content.

Intracellular lipid droplets (LD) provide the oil storage mechanism of plants. They are found within seeds as individual structures, even under conditions of cold stress and dehydration, due to the protein that covers them. This protein, called oleosin, is found exclusively in plants and has been widely studied in seeds. Avocado fruits (Persea americana Mill.) are rich in oil, which is stored in the mesocarp, not in the seeds. The presence of oleosin in the mesocarp tissue of avocadoes has been reported, but its physiological role is still unknown. In this study, we identify two genes that code for oleosin in the mesocarp of the native Mexican avocado. These sequences are very different from those of seed oleosins. Both genes are expressed during fruit ripening, while one, PaOle1, has the highest expression in the green fruit stage. The protein of PaOle1 is stable during the fruit ripening process and covers all the mesocarp LDs. The expression of PaOle1 gene and protein is organ specific to avocado mesocarp. Among avocadoes varieties oleosin abundance is directly related to oil content.

Prolactin and Estradiol are Epigenetic Modulators in Bovine Mammary Epithelial Cells during Staphylococcus aureus Infection.

Changes in the levels of reproductive hormones compromise the bovine innate immune response (IIR). Changes in 17ß-estradiol (E2) and prolactin (bPRL) levels affect the IIR of bovine mammary epithelial cells (bMECs), the target tissue of these hormones. In this work, we explored the effect of the combined hormones on bMEC IIR during Staphylococcus aureus infection, and if they can modulate epigenetic marks. By gentamicin protection assays, we determined that combined hormones (bPRL (5 ng/mL) and E2 (50 pg/mL)] decrease S. aureus internalization into bMECs (~50%), which was associated with a reduction in integrin α5ß1 membrane abundance (MA) (~80%) determined by flow cytometry. Additionally, combined hormones increased Toll-like receptor 2 (TLR2) MA (25%). By RT-qPCR, we showed that combined hormones induce the expression of pro- and anti-inflammatory cytokine genes, as well as up-regulate antimicrobial peptide gene expression. The combined hormones induced H3K9Ac at 12 h of treatment, which coincides with the reduction in histone deacetylase (HDAC, 15%) activity. In addition, hormones increased the H3K9me2 mark at 12 h, which correlates with a reduction in the expression of KDM4A. In conclusion, bPRL and E2 modulate the IIR of bMECs, an effect that can be related to the regulation of histone H3 modifications such as H3K9Ac and H3K9me2.

Lipid-Rich Extract from Mexican Avocado Seed (Persea americana var. drymifolia) Reduces Staphylococcus aureus Internalization and Regulates Innate Immune Response in Bovine Mammary Epithelial Cells.

Bovine mammary epithelial cells (bMECs) are capable of initiating an innate immune response (IIR) to invading bacteria. Staphylococcus aureus is not classically an intracellular pathogen, although it has been shown to be internalized into bMECs. S. aureus internalizes into nonprofessional phagocytes, which allows the evasion of the IIR and turns antimicrobial therapy unsuccessful. An alternative treatment to control this pathogen is the modulation of the innate immune response of the host. The Mexican avocado (Persea americana var. drymifolia) is a source of molecules with anti-inflammatory and immunomodulatory properties. Hence, we analyze the effect of a lipid-rich extract from avocado seed (LEAS) on S. aureus internalization into bMECs and their innate immunity response. The effects of LEAS (1-500 ng/ml) on the S. aureus growth and bMEC viability were assessed by turbidimetry and MTT assays, respectively. LEAS did not show neither antimicrobial nor cytotoxic effects. S. aureus internalization into bMECs was analyzed by gentamicin protection assays. Interestingly, LEAS (1-200 ng/ml) decreased bacterial internalization (60-80%) into bMECs. This effect correlated with NO production and the induction of the gene expression of IL-10, while the expression of the proinflammatory cytokine TNF-α was reduced. These effects could be related to the inhibition of MAPK p38 (∼60%) activation by LEAS. In conclusion, our results showed that LEAS inhibits the S. aureus internalization into bMECs and modulates the IIR, which indicates that avocado is a source of metabolites for control of mastitis pathogens.

Antioxidant Activity of the Essential Oil and its Major Terpenes of Satureja macrostema (Moc. and Sessé ex Benth.) Briq.

BACKGROUND: The aim of this study was to investigate the in vitro antioxidant activity of Satureja macrostema (Moc. and Sessé ex Benth.) Briq. (Lamiaceae) essential oil, a Mexican medicinal plant known as nurite. MATERIALS AND METHODS: Fresh aerial parts of S. macrostema plants cultivated in greenhouse for 3 months were subjected to hydrodistillation in a Clevenger apparatus to obtain essential oil. Volatile compounds were identified by gas chromatography (GC) and GC/mass spectrometry. Antioxidant effectiveness of essential oil and its major terpenes of S. macrostema was examined by three different radical scavenging methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and total antioxidant capacity (TAC). The concentrations tested were 0.001, 0.01, 0.1, and 1 mg/mL. RESULTS: The major volatile compounds were caryophyllene, limonene, linalool, pulegone, menthone, and thymol. S. macrostema essential oil showed the highest free radical scavenging activity with DPPH and ABTS methods (53.10% and 92.12%, respectively) at 1 mg/mL and 98% with TAC method at 0.1 mg/mL. Thymol exerted the highest antioxidant capacity with 0.1 mg/mL, reaching 83.38%, 96.96%, and 98.57% by DPPH, ABTS, and TAC methods. Caryophyllene, limonene, linalool, pulegone, and menthone exhibited an antioxidant capacity <25% with the DPPH and ABTS methods; however, limonene showed a TAC of 85.41% with 0.01 mg/mL. CONCLUSION: The essential oil of S. macrostema and thymol showed a free radical scavenging activity close to that of the synthetic butylated hydroxytoluene. SUMMARY: The major volatile compounds of essential oil of Satureja macrostema were caryophyllene, limonene, linalool, pulegone, menthone and thymolThe essential oil of S. macrostema showed a high free radical scavengingThymol exerted the highest antioxidant capacity by DPPH, ABTS and TAC methods. Abbreviations used: GC: Gas Chromatography; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; TAC: Total antioxidant capacity.

Arabidopsis thaliana sucrose phosphate synthase (sps) genes are expressed differentially in organs and tissues, and their transcription is regulated by osmotic stress.

Sucrose is synthesized from UDP-Glc and Fru-6-phosphate via the activity of sucrose-phosphate synthase (SPS) enzymes, which produce Suc-6-phosphate. Suc-6-phosphate is rapidly dephosphorylated by phosphatases to produce Suc and inorganic phosphate. Arabidopsis has four sps genes encoding SPS enzymes. Of these enzymes, AtSPS1F and AtSPS2F have been grouped with other dicotyledonous SPS enzymes, while AtSPS3F and AtSPS4F are included in groups with both dicotyledonous and monocotyledonous SPS enzymes. In this work, we generated Arabidopsis thaliana transformants containing the promoter region of each sps gene fused to gfp::uidA reporter genes. A detailed characterization of expression conferred by the sps promoters in organs and tissues was performed. We observed expression of AtSPS1F, AtSPS2F and AtSPS3F in the columella roots of the plants that support sucrose synthesis. Hence, these findings support the idea that sucrose synthesis occurs in the columella cells, and suggests that sucrose has a role in this tissue. In addition, the expression of AtSPS4F was identified in embryos and suggests its participation in this developmental stage. Quantitative transcriptional analysis of A. thaliana plants grown in media with different osmotic potential showed that AtSPS2F and AtSPS4F respond to osmotic stress.

Nonprofessional phagocytic cell receptors involved in Staphylococcus aureus internalization.

Staphylococcus aureus is a successful human and animal pathogen. The majority of infections caused by this pathogen are life threatening, primarily because S. aureus has developed multiple evasion strategies, possesses intracellular persistence for long periods, and targets the skin and soft tissues. Therefore, it is very important to understand the mechanisms employed by S. aureus to colonize and proliferate in these cells. The aim of this review is to describe the recent discoveries concerning the host receptors of nonprofessional phagocytes involved in S. aureus internalization. Most of the knowledge related to the interaction of S. aureus with its host cells has been described in professional phagocytic cells such as macrophages. Here, we showed that in nonprofessional phagocytes the α 5 ß 1 integrin host receptor, chaperons, and the scavenger receptor CD36 are the main receptors employed during S. aureus internalization. The characterization and identification of new bacterial effectors and the host cell receptors involved will undoubtedly lead to new discoveries with beneficial purposes.

Effects of dopamine on leptin release and leptin gene (OB) expression in adipocytes from obese and hypertensive patients.

BACKGROUND: A reduction of dopaminergic (DAergic) activity with increased prolactin levels has been found in obese and hypertensive patients, suggesting its involvement as a pathophysiological mechanism promoting hypertension. Similarly, leptin action increasing sympathetic activity has been proposed to be involved in mechanisms of hypertension. The aim of this study was to analyze the effects of DA, norepinephrine (NE), and prolactin on leptin release and leptin gene (OB) expression in adipocytes from obese and hypertensive patients. METHODS: Leptin release and OB gene expression were analyzed in cultured adipocytes from 16 obese and hypertensive patients treated with DA (0.001, 0.01, 0.1, and 1.0 µmol/L), NE (1.0 µmol/L), insulin (0.1 µmol/L), and prolactin (1.0 µmol/L), and from five nonobese and normotensive controls treated with DA (1 µmol/L), NE (1 µmol/L), insulin (0.1 µmol/L), and prolactin (1.0 µmol/L). RESULTS: A dose-related reduction of leptin release and OB gene messenger ribonucleic acid expression under different doses of DA was observed in adipocytes from obese hypertensive patients. Whereas prolactin treatment elicited a significant increase of both leptin release and OB gene expression, NE reduced these parameters. Although similar effects of DA and NE were observed in adipocytes from controls, baseline values in controls were reduced to 20% of the value in adipocytes from obese hypertensive patients. CONCLUSION: These results suggest that DAergic deficiency contributes to metabolic disorders linked to hyperleptinemia in obese and hypertensive patients.

Bacterial resistance to cationic antimicrobial peptides.

Naturally occurring cationic antimicrobial peptides (CAMPs) have been considered as promising candidates to treat infections caused by pathogenic bacteria to animals and humans. This assumption is based on their mechanism of action, which is mainly performed through electrostatic membrane interactions. Unfortunately, the rise in the reports that describe bacterial resistance to CAMPs has redefined their role as therapeutic agents. In this review, we describe the state of the art of the most common resistance mechanisms developed by bacteria to CAMPs, making special emphasis on resistance selection. Considering most of the resistance mechanisms here reviewed, the emergence of resistance is unlikely in the short term, however we also described evidences that show the evolution of resistance to CAMPs, reevaluating their use as good antibacterial agents. Finally, the knowledge related to the description of CAMP resistance mechanisms may provide useful information for improving strategies to control infections.

Association between angiotensin-1 converting enzyme gene polymorphism and the metabolic syndrome in a Mexican population.

Metabolic Syndrome (MS) is recognized as a cluster of cardiovascular risk factors. All components of MS have a genetic base. Genes of the renin angiotensin system are potential candidate genes for MS. We investigated whether angiotensin converting enzyme (ACE) gene polymorphism increases susceptibility to MS as an entity in a Mexican population. In a cross-sectional study, 514 individuals were studied including 245 patients with MS and 269 subjects without MS criteria. ACE gene polymorphism was detected using PCR. MS was defined according to The National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) criteria, except that the raised fasting plasma glucose

[Angiotensin-1 converting enzyme insertion/deletion gene polymorphism in a Mexican population with diabetic nephropathy]. / Polimorfismo inserción/deleción del gen de la enzima de conversión de la angiotensina en una población mexicana con nefropatía diabética.

BACKGROUND AND OBJECTIVE: Diabetic nephropathy is a polygenic and multifactorial disease. Studies of familial clustering and genetic predisposition suggest that genetic factors are involved. Among candidate genes, the coding for angiotensin-converting enzyme (ACE) polymorphism has been described. Our objective was to investigate the association ACE gene insertion/deletion (I/D) polymorphism with the development of diabetic nephropathy in a Mexican population. PATIENTS AND METHOD: In a cross-sectional study, we evaluated 204 patients with type 2 diabetes: 43 with incipient diabetic nephropathy, 45 with established diabetic nephropathy and 116 without diabetic nephropathy. Diabetic nephropathy was defined according the American Diabetes Association criteria. The ACE I/D gene polymorphism was detected by polymerase chain reaction. RESULTS: Patients with type 2 diabetes with both incipient diabetic nephropathy and established diabetic nephropathy significantly differed from controls with respect to variables that determined kidney damage (P<.0001) and serum lipids ( P<.01). The genotype DD was strongly associated with the development of incipient diabetic nephropathy (odds ratio [OR] adjusted = 2.83; 95% confidence interval, 1.74-4.59; P<.0001) and established diabetic nephropathy (OR adjusted = 2.95; 95% CI, 1.83-4.73; P<.0001). CONCLUSIONS: The ACE DD genotype is associated with the development of incipient diabetic nephropathy and established diabetic nephropathy in a Mexican population.

Polimorfismo inserción/deleción del gen de la enzima de conversión de la angiotensina en una población mexicana con nefropatía diabética / Angiotensin-1 converting enzyme insertion/deletion gene polymorphism in a Mexican population with diabetic nephropathy

Fundamento y objetivo: La nefropatía diabética es una enfermedad de etiología poligénica y multifactorial. Estudios de ligamiento familiar indican que están involucrados factores genéticos. Un gen candidato es el que codifica la enzima de conversión de la angiotensina (ECA), en el que se ha identificado un polimorfismo inserción/deleción (I/D). El objetivo del presente trabajo ha sido analizar la asociación del polimorfismo I/D del gen de la ECA con la nefropatía diabética incipiente y establecida en una población mexicana. Pacientes y método: Se estudió a 204 pacientes con diabetes mellitus tipo 2, de los que 43 presentaban además nefropatía diabética incipiente y 45 nefropatía diabética establecida; los 116 restantes no tenían nefropatía diabética (controles). La nefropatía diabética se definió de acuerdo con los criterios de la American Diabetes Association. El polimorfismo I/D del gen de la ECA se detectó por reacción en cadena de la polimerasa. Resultados: Los pacientes con diabetes mellitus tipo 2 y nefropatía diabética incipiente o establecida presentaron diferencias significativas respecto a los controles en las variables que determinan el daño renal (p < 0,0001) y en los lípidos (p < 0,01). El genotipo DD se asoció estrechamente con el desarrollo de nefropatía diabética tanto incipiente (odds ratio [OR] ajustada = 2,83; intervalo de confianza [IC] del 95%, 1,74-4,59; p < 0,0001) como establecida (OR ajustada = 2,95; IC del 95%, 1,83-4,73; p < 0,0001). Conclusiones: El genotipo DD del gen de la ECA se asoció con el desarrollo de nefropatía diabética incipiente y nefropatía diabética establecida en una población mexicana

Background and objective: Diabetic nephropathy is a polygenic and multifactorial disease. Studies of familial clustering and genetic predisposition suggest that genetic factors are involved. Among candidate genes, the coding for angiotensin-converting enzyme (ACE) polymorphism has been described. Our objective was to investigate the association ACE gene insertion/deletion (I/D) polymorphism with the development of diabetic nephropathy in a Mexican population. Patients and method: In a cross-sectional study, we evaluated 204 patients with type 2 diabetes: 43 with incipient diabetic nephropathy, 45 with established diabetic nephropathy and 116 without diabetic nephropathy. Diabetic nephropathy was defined according the American Diabetes Association criteria. The ACE I/D gene polymorphism was detected by polymerase chain reaction. Results: Patients with type 2 diabetes with both incipient diabetic nephropathy and established diabetic nephropathy significantly differed from controls with respect to variables that determined kidney damage (P< .0001) and serum lipids (P<.01). The genotype DD was strongly associated with the development of incipient diabetic nephropathy (odds ratio [OR] adjusted = 2.83; 95% confidence interval, 1.74-4.59; P<.0001) and established diabetic nephropathy (OR adjusted = 2.95; 95% CI, 1.83-4.73; P<.0001). Conclusions: The ACE DD genotype is associated with the development of incipient diabetic nephropathy and established diabetic nephropathy in a Mexican population

Molecular characterization of Bacillus thuringiensis strains from Argentina.

Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.