Procainamide [HRP]

Global DNA hypoinethylation in CD4+ T cells has been detected in systemic lupus erythematosus (SLE), and it seems to be linked to its pathogenesis. We investigated the relationship between overall DNA methylation and the expression of two methyl CpG-binding domain (MBD) proteins. DNA deoxymethylcytosine (d(m)C) content of purified CD4+ T cells from 29 SLE patients and 30 healthy controls was measured by means of an ELISA. Transcript levels of two methyl CpG-binding proteins (MBD2 and MBD4) were quantified by real-time RT-PCR. Association studies were also carried out with several laboratory parameters, as well as with the patients' clinical manifestations. SLE patients had significantly less CD4+ T cell DNA d(m)C content than controls (0.802 +/- 0.134 vs. 0.901 +/- 0.133; P=0.007). MBD2 and MBD4 mRNA levels were considerably higher in the patients' group: 0.975 +/- 0683 versus 0.604 +/- 0.614 (P=0.004) and 0.359 +/- 0.330 versus 0.092 +/- 0.169, respectively (P < 0.0005). It is interesting that SLE patients showed a negative correlation between methylation indices and MBD2 (r=-0.609, P < 0.0005) and MBD4 (r=-0.395, P=0.034) transcript levels. MBD2 and MBD4 transcript overexpression and inverse correlations with DNA methylation indices indicate that both enzymes may really have a direct and active role on the genome-wide DNA hypomethylation observed in CD4+ T cells from SLE patients.

In our current study, we examined the mechanism underlying neuronal cell injuries caused by norephedrine in SH-SY5Y human neuroblastoma cells. Norephedrine was found to induce cytoplasmic vacuolation and a resultant loss of cell viability In the cells treated with norephedrine also, an autophagic marker LC3 was converted to its LC3-II activated form, suggesting the induction of autophagy. In cells transfected with RFP-LC3 and GFP-LAMP1, a punctate patterning of LC3 expression and colocalization of LAMP1 with the formed vacuoles were observed, highlighting the lysosomal nature of the vacuoles and their association with autophagosomes. An autophagic flux assay using tfLC3 (mRFP-GFP-LC3) indicated the formation of autophagosomes and autolysosomes by norephedrine stimulation at an early timepoint (similar to 3 h). However, at a later timepoint (similar to 6 h), both the dilation of autolysosomes/lysosomes and the neutralization of the vacuolar pH were also observed. These results thus indicate that norephedrine induces autophagy at an early timepoint and cell death with lysosomal dysfunction and autophagy disruption at a later timepoint. (c) 2012 Elsevier B.V. All rights reserved.