Synergism between phorbol-12-myristate-13-acetate and vitamin D3 in the differentiation of U937 cells to monocytes and macrophages.

Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines, but the extent of differentiation in comparison to primary tissue macrophages is unclear. Here, we examine the morphological/phenotypic markers associated with differentiation of U937 cells into monocytes/macrophages, in response to PMA or VD3 treatment. PMA stimulus but not with VD3, induced changes in cell morphology indicative of differentiation, but did not show differentiation comparable to monocyte-derive macrophage (MDM). The cells treated with PMA+VD3 for 2 days (d) acquired morphological/phenotypic features similar to those acquired by monocytes. In contrast, U937 cells treated for 2d with PMA and VD3 followed by 6d of resting in culture without PMA but in the presence of VD3 acquired morphological and phenotypic markers similar to those of MDM; i.e. reduced nucleus/cytoplasmic ratio, high auto-fluorescence and cytoplasmic complexity. Furthermore, low expression of CD14/TLR2 and high expression of CD68/CD86 were observed. In conclusion, our results indicate a synergistic effect between PMA and VD3 in U937 cells differentiation into both monocytes or macrophages and we propose a modified PMA differentiation protocol to enhance monocyte/macrophage differentiation of U937 cells.

Letter to the Editor "Drosha, DGCR8, and Dicer mRNAs are downregulated in human cells infected with dengue virus 4" - Genet. Mol. Res. 15 (2): gmr.15027891 - Drosha, Dicer, and TRBP mRNA are downregulated in Vero cells with the 3'UTR of Dengue virus.

Dear Editor, A recent paper (Casseb et al., 2016) published in the journal Genetics and Molecular Research described the interesting concept that dengue virus (DENV)-4 infection, in the human cell line A-549, leads to the downregulation of expression of key components of microRNA (miRNA) biogenesis, such as Drosha, Dicer, and DGCR8. For this, the authors performed a time course infection of A-549 cells for 5 days. The highest viral load was observed at 3 days post-infection, which corresponded with the maximum downregulation of expression of Drosha, Dicer, and DGCR8, assayed by quantitative PCR (RT-qPCR). These results supported the recent notion of a complex interaction between DENV and the host miRNA machinery and of the host miRNA response to this particular infection. Extensive evidence has shown that DENV can take advantage of host miRNAs for its own replication (Zhu et al., 2014) and that host miRNAs can inhibit DENV replication (Wu et al., 2013).

Apoptosis as pathogenic mechanism of infection with vesicular stomatitis virus. Evidence in primary bovine fibroblast cultures.

To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resistance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of Blanco Orejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible to VSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible to VSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among the fibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for a possible relationship between resistance/susceptibility and production of factors with antiviral activity; however fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis where induced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Using morphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levels of apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosis and the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanism of VSV.

Apoptosis as pathogenic mechanism of infection with vesicular stomatitis virus: evidence in primary bovine fibroblast cultures

To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resistance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of Blanco Orejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible to VSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible to VSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among the fibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for a possible relationship between resistance/ susceptibility and production of factors wi th antiviral activity; however fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis where induced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Using morphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levels of apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosis and the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanism of VSV.

Apoptosis as pathogenic mechanism of infection with vesicular stomatitis virus: evidence in primary bovine fibroblast cultures

To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resistance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of Blanco Orejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible to VSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible to VSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among the fibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for a possible relationship between resistance/ susceptibility and production of factors wi th antiviral activity; however fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis where induced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Using morphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levels of apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosis and the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanism of VSV.(AU)

Deletion mapping of the potyviral helper component-proteinase reveals two regions involved in RNA binding.

The Potyvirus helper component-proteinase (HC-Pro) binds nonspecifically to single-stranded nucleic acids with a preference for RNA. To delineate the regions of the protein responsible for RNA binding, deletions were introduced into the full-length Potato potyvirus Y HC-Pro gene carried by an Escherichia coli expression vector. The corresponding proteins were expressed as fusions with the maltose-binding protein, purified, and assayed for their RNA-binding capacity. The results obtained by UV cross-linking and Northwestern blot assays demonstrated that the N- and C-terminal regions of HC-Pro are dispensable for RNA binding. They also revealed the presence of two independent RNA-binding domains (designated A and B) located in the central part of HC-Pro. Domain B appears to contain a ribonucleoprotein (RNP) motif typical of a large family of RNA-binding proteins involved in several cellular processes. The possibility that domain B consists of an RNP domain is discussed and suggests that HC-Pro could constitute the first example of a plant viral protein belonging to the RNP-containing family of proteins.

Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved.

Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro.