L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.     

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

The nitric-oxide synthases (NOS, EC 1.14.13.39) are modular enzymes containing attached flavoprotein and heme (NOSoxy) domains. To generate nitric oxide (NO), the NOS FMN subdomain must interact with the NOSoxy domain to deliver electrons to the heme for O₂ activation during catalysis. The molecular basis and how the interaction is regulated is unclear. We explored the role of eight positively charged residues that create an electropositive patch on NOSoxy in enabling the electron transfer by incorporating mutations that neutralized or reversed their individual charges. Stopped-flow and steady-state experiments revealed that individual charges at Lys⁴²³, Lys⁶²⁰, and Lys⁶⁶⁰ were the most important in enabling heme reduction in nNOS. Charge reversal was more disruptive than neutralization in all cases, and the effects on heme reduction were not due to a weakening in the thermodynamic driving force for heme reduction. Mutant NO synthesis activities displayed a complex pattern that could be simulated by a global model for NOS catalysis. This analysis revealed that the mutations impact the NO synthesis activity only through their effects on heme reduction rates. We conclude that heme reduction and NO synthesis in nNOS is enabled by electrostatic interactions involving Lys⁴²³, Lys⁶²⁰, and Lys⁶⁶⁰, which form a triad of positive charges on the NOSoxy surface. A simulated docking study reveals how electrostatic interactions of this triad can enable an FMN-NOSoxy interaction that is productive for electron transfer.     

Variations in the locomotor activity of the Mexican wolf (Canis lupus baileyi) respect to moon periodicity: a study in an outdoor enclosure

It is assumed that the response of the regulatory system of mammal activity depends on the changes in light intensity throughout the 24-h cycle. The aim of this study was to determine whether the moon luminosity cycle exerts an effect on the locomotor activity of the Mexican wolf (C. lupus baileyi). Data collection was carried out with the actimetry, of 11 individuals were analyzed using ANOVA to determine the effect of the lunar cycle. Significant differences were encountered between moon phases (p = 0.001), with a decrement of activity during new and full moon. However, effects were dependent also on the age of the individuals and the daylight period. On the other hand, it is a possible regulation of the activity pattern by the effect of lunar periodicity. This periodicity needs a more detailed examination to determine its adaptive function.     

Comparison of DAS-ELISA and qRT-PCR for the detection of cucurbit viruses in seeds

The importance of seeds as virus vehicles for long-distance dissemination makes essential the availability of adequate methods of analysis to guarantee the quality of seed lots. To improve the repertoire of sensitive methods for seed diagnosis, we have developed quantitative real-time RT-PCR assays (RT-qPCR) based on the TaqMan technology to detect three viruses which are seed transmitted in cucurbits, namely, cucumber green mottle mosaic virus (CGMMV), squash mosaic virus (SqMV) and melon necrotic spot virus (MNSV), and compared these assays with DAS-ELISA, the main method used for virus detection in seeds. The estimated RT-qPCR limits of detection were 96, 97 and 740 RNA target molecules for CGMMV, SqMV and MNSV, respectively. The estimated RT-qPCR analytical sensitivity (highest dilution capable of generating a detectable amplification signal) ranged between 10 and 1 pg/μL for the three viruses. Using RT-qPCR, we could reliably detect a single SqMV- or CGMMV-contaminated seed among 999 uncontaminated seeds in a seed lot, and sensitivities were 1,000 and 10,000 times of those provided by DAS-ELISA for SqMV and CGMMV, respectively. Our RT-qPCR assays have proved to be highly suitable for the analysis of seed lots, and the possibility of their implementation into certification programme should be taken into consideration.     

Producción de fosfolipasa y proteinasa en cepas de Malassezia pachydermatis aisladas de perros con otitis y sin otitis

BackgroundMalassezia pachydermatis is part of the skin microbiota of dogs and cats. M. pachydermatis has been associated with external otitis and seborrhoeic dermatitis, reported more often in dogs than in cats. When the physical, chemical or immunological mechanisms of the skin are altered, M. pachydermatis could act as a pathogen. Thus, several virulence factors, such as the ability to produce esterase, lipase, lipoxygenase, protease, chondroitin sulphatase, and hyaluronidase, have been studied.AimsIn the present study, we aim to identify the phospholipase activity measured at pH 6.3, and the proteinase activity measured at pH 6.3 and pH 6.8 (pH from ears of dogs with external otitis) of M. pachydermatis strains isolated from dogs with and without external otitis.MethodsThe phospholipase activity was measured using a semi-quantitative method with egg yolk, and the proteinase activity with a semi-quantitative method using bovine serum albumin agar. The study was performed on 96 isolates of M. pachydermatis, 43 isolated from dogs without clinical symptoms of otitis, and 52 isolated from dogs with otitis.ResultsIn our study, 75.8% of the isolates showed phospholipase activity at pH 6.3, and 81 and 97.9% of them showed proteinase activity measured at pH 6.3 and 6.8, respectively. A higher phospholipase activity was detected in strains isolated from dogs with otitis. The proteinase activity was increased at a pH of 6.8 (97.9%) in comparison to a pH of 6.3 (81%).ConclusionsOur results suggest that the phospholipase activity may play an important role in the invasion of host tissues in chronic canine otitis cases. The proteinase activity results obtained in this study suggest that a reduction in the pH of the treatment may improve its efficacy in the resolution of M. pachydermatis otitis.     

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

This article presents a research study on carbonate formation in solid and liquid media by Thalassospira sp., Halomonas sp., Bacillus pumilus, and Pseudomonas grimontii, four bacterial strains isolated from sediments and deep seawater. As part of this study, we analyzed carbonic anhydrase activity, pH, adsorption of calcium and magnesium ions, and total organic and inorganic carbon. The geochemical program PHREEQC was also used to calculate the mineral saturation indexes in all the cultures. The minerals formed were studied with X-ray diffraction, X-ray dispersive energy microanalysis, and scanning electron microscopy. In addition, all four bacterial strains were found to induce carbonate precipitation and to have carbonic anhydrase activity. Sterile control experiments did not precipitate carbonate. In solid M1 and B4 media, all of the strains precipitated magnesium calcite, whereas in the liquid media, they precipitated different percentages of magnesium calcite, aragonite, and monohydrocalcite. In both cases, small amounts of amorphous precipitates were also produced. This article discusses carbonate formation and the possible role played by metabolic activity, bacterial surfaces and carbonic anhydrase in this process. Finally, the results obtained lead to a hypothesis regarding the importance of carbonate precipitation for the survival of bacteria populations in certain habitats.     

Antihypertensive and Antioxidant Properties from Whey Protein Hydrolysates Produced by Encapsulated Bacillus subtilis Cells

International Journal of Peptide Research and Therapeutics - The proteolytic action from encapsulated Bacillus subtilis cells into polymeric system based in sodium alginate-chitosan-poly (ethylene...     

Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma: Implications for Genome Stability

Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired and hyperactive double strand break (DSB) repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR)-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ), and Rad51, involved in homologous recombination (HR). Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ) were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.