Knockdown of Dystrophin Dp71 Impairs PC12 Cells Cycle: Localization in the Spindle and Cytokinesis Structures Implies a Role for Dp71 in Cell Division

The function of dystrophin Dp71 in neuronal cells remains to be established. Previously, we revealed the involvement of this protein in both nerve growth factor (NGF)-induced neuronal differentiation and cell adhesion by isolation and characterization of PC12 neuronal cells with depleted levels of Dp71. In this work, a novel phenotype of Dp71-knockdown cells was characterized, which is their delayed growth rate. Cell cycle analyses revealed an altered behavior of Dp71-depleted cells, which consists of a delay in G0/G1 transition and an increase in apoptosis during nocodazole-induced mitotic arrest. Dp71 associates with lamin B1 and β-dystroglycan, proteins involved in aspects of the cell division cycle; therefore, we compared the distribution of Dp71 with that of lamin B1 and β-dystroglycan in PC12 cells at mitosis and cytokinesis by means of immunofluorescence and confocal microscopy analysis. All of these three proteins exhibited a similar immunostaining pattern, localized at mitotic spindle, cleavage furrow, and midbody. It is noteworthy that a drastic decreased staining in mitotic spindle, cleavage furrow, and midbody was observed for both lamin B1 and β-dystroglycan in Dp71-depleted cells. Furthermore, we demonstrated the interaction of Dp71 with lamin B1 in PC12 cells by immunoprecipitation and pull-down assays, and importantly, we revealed that knockdown of Dp71 expression caused a marked reduction in lamin B1 levels and altered localization of the nuclear envelope protein emerin. Our data indicate that Dp71 is a component of the mitotic spindle and cytokinesis multi-protein apparatuses that might modulate the cell division cycle by affecting lamin B1 and β-dystroglycan levels

Social and Economic Analysis of the Production of Maradol Papaya (Carica papaya L.): Case study in the coast of Oaxaca, Mexico

Objective: To analyze social, productive and profitability indicators of the conventional agribusiness that produces Maradol papaya in the Coast region of Oaxaca. Methodology: The present research was perform in the agribusiness “Productores de la Costa Posa Verde, S.P.R. de R.L”, in the period June 2020 to August 2021. Semi-structured interviews to the producer and family members were carried out. The economic analysis was implemented using the methodology of budgets by activities. Results: The studied agribusiness is a family type, managed by the head of the family, who is 47 years old, with a high school education and 35 years of experience in managing papaya cultivation. During the studied period, the production cost was $365,190.01 ha-1. Of the total variable costs, the highest cost per cultivated ha were harvesting (38.2$117,633.6 ha-1 and a profitability of 24.4% were observed, and the equilibrium point was 31,268.86 kg. Conclusions: The papaya agribusiness studied is profitable. In addition, papaya production is an important source of job, it contributes to improving the quality of life of the inhabitants of the region. Keywords: Economic indicators, production cost, production profitability.  Objective: To analyze the social, production, and profitability indicators of a conventional agribusiness producing Maradol papaya in the coastal region of Oaxaca. Methodology: This research was conducted at the agribusiness “Productores de la Costa Posa Verde, S.P.R. de R.L” from June 2020 to August 2021. Semi-structured interviews with the producer and his family were conducted. The economic analysis was done using the activity-based budgeting method. Results: The studied company is a family agribusiness run by the head of the family, a 47-year-old man with a high school education and approximately 35 years of experience in the cultivation of papaya. During the period of study, the production cost was $365,190.01 ha-1. From the total variable costs, the largest expenditures per cultivated ha went to harvesting (38.2$117,633.6 ha-1 was observed, together with a profitability of 24.4%, and an equilibrium point of 31,268.86 kg. Conclusions: This papaya farming agribusiness is profitable. Moreover, papaya farming is an important source of work, which contributes to improving the quality of life among the region’s inhabitants

Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons

220th ENMC workshop: Dystroglycan and the dystroglycanopathies Naarden, The Netherlands, 27–29 May 2016

Highlights • Review of clinical phenotypes associated with the dystroglycanopathies. • Discussion of current animal models and their contribution to understanding the disease process. • New insight into the glycosylation of alpha dystroglycan and the role of LARGE. • Structural information on the LARGE glycan

Analysis of the GFP-labelled β-dystroglycan interactome in HEK-293 transfected cells reveals novel intracellular networks

Highlights•HEK-293 cells were transfected with β-dystroglycan fused to GFP and the protein predominantly localized at the nuclear envelope.•Protein complexes bound to β-dystroglycan were investigated using immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis.•Novel potential interaction networks involving β-dystroglycan were identified in cytoskeleton, endoplasmatic reticulum, mitochondria and nucleus.•Lamin-Associated Polypeptide-1B (LAP1B) was identified as a novel protein that interacts with β-dystroglycan.AbstractDystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, α-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane β-DG whose cytodomain binds to the actin cytoskeleton. Glycosylation of α-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of α-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. β-DG acts as a scaffold for several cytoskeletal and nuclear proteins and very little is known about the fine regulation of some of these intracellular interactions and how they are perturbed in diseases.To start filling this gap by identifying uncharacterized intracellular networks preferentially associated with β-DG, HEK-293 cells were transiently transfected with a plasmid carrying the β-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing β-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with α-DG.Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis, led to the identification of an interactome formed by 313 exclusive protein matches for β-DG binding. A series of already known β-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel β-DG interactors and their related networks, were identified in diverse subcellular compartments such as cytoskeleton, endoplasmic reticulum/Golgi, mitochondria, nuclear membrane and the nucleus itself. Of particular interest amongst the novel identified matches, Lamin-Associated Polypeptide-1B (LAP1B), an inner nuclear membrane protein whose mutations are known to cause nuclear envelopathies characterized by muscular dystrophy, was found to interact with β-DG in HEK-293 cells. This evidence was confirmed by immunoprecipitation, Western blotting and immunofluorescence experiments. We also found by immunofluorescence experiments that LAP1B looses its nuclear envelope localization in C2C12 DG-knock-out cells, suggesting that LAP1B requires β-DG for a proper nuclear localization.These results expand the role of β-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy