Análisis genético y molecular de propiedades fisicoquímicas del almidón y su asociación con la modificación del endospermo en maíz de calidad proteínica

Quality protein maize (QPM) was created converting the soft opaque-2 endosperm into a vitreous phenotype, but the mechanisms involved in this modification are not completely understood. Recombinant inbred lines (RILs) derived from K0326Y QPM and W64Ao2 were used to identify quantitative trait loci (QTL) associated with starch physicochemical properties. RILs contrasting in vitreousness were also used to evaluate the expression of starch biosynthesis genes. Mapping identified 5-6 QTLs for each trait that explained 66 % of the phenotypic variation. Three QTLs on bins 4.05, 5.04, and 9.03 were found close to the starch biosynthesis genes Brittle-2 (Bt2), Amylose extender-1 (Ae1), and Waxy-1 (Wx1), respectively. The expression of Wx1 was three-fold greater in K0326Y QPM than W64Ao2 and eight-fold higher in vitreous than opaque RILs, which corresponded with the greater accumulation of granule bound starch synthase I (GBSSI) and the higher amylose content observed in the vitreous lines. The increased synthesis of amylose may result in starch granules with more amorphous regions that favor their compaction. These results suggest that endosperm modification in QPM is associated with the synthesis of starch containing more amylose during kernel development, which may facilitate the packing of the starch granules resulting in the vitreous phenotype.El maíz de calidad proteínica (QPM) fue creado convirtiendo el endospermo suave opaco-2 en un fenotipo vítreo, pero los mecanismos involucrados en esta modificación no se conocen por completo. Se utilizaron líneas recombinantes puras derivadas de las líneas K0326Y QPM y W64Ao2 para identificar loci de características cuantitativas (QTL) asociados con propiedades fisicoquímicas del almidón. También se usaron RILs contrastantes en vitrosidad para evaluar la expresión de genes de biosíntesis de almidón. El mapeo identificó 5 o 6 QTL para cada característica que explicaron en promedio el 66 % de la variación fenotípica. Tres de los QTLs en los bins 4.05, 5.04 y 9.03 se encontraron cerca de los genes de síntesis de almidón Brittle 2 (Bt2), Amylose extender 1 (Ae1), y Waxy 1 (Wx1), respectivamente. La expresión de Wx1 fue tres veces mayor en K0326Y QPM que en W64Ao2 y ocho veces mayor en líneas vítreas en comparación con las opacas, lo que correspondió con la mayor acumulación de la enzima almidón sintasa unida al gránulo I (GBSSI) y el mayor contenido de amilosa observado en las líneas vítreas. El incremento en la síntesis de amilosa podría resultar en gránulos de almidón con más regiones amorfas que favorecen su compactación. Estos resultados sugieren que la modificación del endospermo en QPM está asociada con la síntesis de almidón conteniendo más amilosa durante el desarrollo del grano, lo cual podría facilitar el empaquetamiento de los gránulos de almidón resultando en el fenotipo vítreo

Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections

Lactoferrin and lactoferrin chimera inhibit damage caused by enteropathogenic Escherichia coli in HEp-2 cells

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. It produces a characteristic intestinal histopathological lesion on enterocytes known as ‘attaching and effacing’ (A/E), and these two steps are mediated by a type-III secretory system. In the present study, we evaluated the effect on the initial host cell attachment step produced by bovine lactoferrin (bLF) and three synthetic peptides: lactoferricin (LFcin), lactoferrampin (LFampin) and LFchimera. A special focus was given to the hemolytic activity and EPEC-induced actin polymerization in HEp-2 cells, as well as to the espA gene expression, which produces the protein responsible for primary contact with the host cells. Results show that EPEC attachment to HEp-2 cells was significantly suppressed by bLF and LFchimera at 125 and 40 μM, respectively. EPEC-mediated actin polymerization was blocked by bLF and LFchimera at 88 and 99%, respectively. LFchimera inhibited the attachment and A/E lesion caused by EPEC in a dose-dependent manner. In the presence of 125 μM bLF, the expression level of the espA gene was decreased by 50% compared to the untreated control. LFchimera at concentrations of 20 μM and 40 μM diminished the level of espA gene expression 100 and 1000 fold, respectively (P < 0.001). Although bLF, LFchimera, LFcin, and LFampin all significantly blocked the hemolysis produced by EPEC (P < 0.001), the two former compounds produced this effect at lower concentrations. These two compounds, bLF and LFchimera, were able to inhibit the first steps of the mechanism of the damage used by EPEC. This data suggests that LFchimera could provide protection against enteropathogens that share this mechanism

Possible involvement of orphan receptors GPR88 and GPR124 in the development of hypertension in spontaneously hypertensive rat

Hypertension (HBP) is a chronic disease characterized by increased blood pressure, which despite several treatments maintains a high morbi-mortality, which suggests that there are other mechanisms involved in this pathology, within which the orphan receptors could be candidates for the treatment of the HBP; these receptors are called orphan receptors because their ligand is unknown. These receptors have been suggested to participate in some pathologies because they are associated with various systems such as GPR88, which has been linked to the dopaminergic system, and GPR124 with angiogenesis, suggesting that these receptors could take part in HBP. Hence, the aim of this work was to study the expression of orphan receptors GPR88 and GPR124 in various tissues of normotensive and hypertensive rats. We used Wistar Kyoto (WKY) and spontaneously hypertensive rat (SHR) of 6–8 and 10–12 weeks of age and we determined systolic blood pressure (SBP), heart rate, as well as mRNA of GPR88 and GPR124 receptors by reverse transcription polymerase chain reaction (RT-PCR) in the aorta, heart, kidney, and brain. Our results showed that GPR88 and GPR124 were expressed in all analyzed tissues, but their expression is dependent on the age and development of HBP because their expression tends to be modified as HBP is established. Therefore, we conclude that GPR88 and GPR124 receptors may be involved in the development or maintenance of high blood pressure

Bactericidal effect of bovine lactoferrin, LFcin, LFampin and LFchimera on antibiotic-resistant Staphylococcus aureus and Escherichia coli

Increased prevalence of antibiotic-resistant bacteria has become a major threat to the health sector worldwide due to their virulence, limited therapeutic options and distribution in both hospital and community settings. Discovery and development of new agents to combat antibiotic-resistant bacteria is thus needed. This study therefore aimed to evaluate the ability of bovine lactoferrin (LF), peptides from two antimicrobial domains lactoferricin B (LFcin17-30) and lactoferrampin (LFampin265-284) and a chimeric construct (LFchimera) containing both peptides, as potential bactericidal agents against clinical isolates of antibiotic-resistant Staphylococcus aureus and Escherichia coli. Results in kinetics of growth show that LF chimera and peptides inhibited the growth of both bacterial species. By confocal microscopy and flow cytometry it was observed that LF and FITC-labeled peptides are able to interact with these bacteria and cause membrane permeabilization, as monitored by propidium iodide staining, these effects were decreased by preincubation with lipopolysaccharide in E. coli. By electron microscopy, a clear cellular damage was observed in bacteria after treatments with LFchimera and peptides, suggesting that interaction and membrane disruption are probably involved as a mechanism of action. In conclusion, results show that LFchimera, LF and peptides have potential as bactericidal agents in the antibiotic-resistant strains of S. aureus and E. coli and also the work strongly suggest that LFcin17-30 and LFampin265-284 acts synergistically with antibiotics against multidrug resistant EPEC and MRSA in vitro