Regulation of fla/che transcription in Bacillus subtilis

fla/che is the master operon for motility in Bacillus subtilis and codes for most of the flagellum components and for the alternative sigma factor sigD, the latter responsible for transcription of the flagellin gene hag and additional genes. Despite fla/che being the subject of numerous investigations over the last 20 years, there are still open questions concerning the fine tuning of its expression and the signals leading to the enhancement of its transcription, necessary for swarming motility. Two promoter sequences drive fla/che transcription: PD3(fla/che) and PA(fla/che). PD3(fla/che), transcribed by the internal SigD, is not sufficient to promote motility, while PA(fla/che) is both necessary and sufficient for fla/che expression and swimming and swarming motility. Several studies have established that fla/che operon expression is modulated by the DegS/DegU two-component system, although the role of phosphorylated DegU is still not clear. Also SwrA, a protein essential for swarming and for full swimming motility, acts as a positive regulator of fla/che, by stimulating transcription from PA(fla/che). An autoregulatory circuitry exists between SwrA and fla/che, because swrA transcription is sigD-dependent, and in turn fla/che - and thus sigD - transcription is promoted by SwrA; this loop maintains both fla/che and swrA expression ON. The molecular function of SwrA has remained thus far unknown. In fact, SwrA does not bear any resemblance to any characterized protein, nor does it contain DNA binding or other functional domains that can give a hint about its molecular role. We have addressed the impact of DegU phosphorylation and the role of SwrA on fla/che expression. We have demonstrated a cooperative effect between SwrA and DegU in enhancing PA(fla/che) activity, which depends on DegU phosphorylation. We have also evidences that DegU is a dual-function regulator, since it can also act as a repressor for PA(fla/che). Furthermore, we have observed that PD3(fla/che) and sigD constitute another positive feedback loop that contributes in maintaining fla/che expression ON. This loop is alternative to the one that is promoted by SwrA, and thus represents a sort of contingency plan for motility

Protein kinase C activation affects, via the mRNA-binding Hu-antigen R/ELAV protein, vascular endothelial growth factor expression in a pericytic/endothelial coculture model.

PURPOSE: To explore whether, following direct contact, there is mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether protein kinase C (PKC) activation, a condition associated with hyperglycemia, can affect, via the mRNA-binding Hu-antigen R (HuR)/ELAV protein, the expression of vascular endothelial growth factor (VEGF). METHODS: PC and EC were cultured separately or in direct contact (1:1 ratio), and exposed or not to phorbol esters, a PKC activator (100 nM for 15 min). Barrier integrity was evaluated by measuring endothelial electrical resistance and permeability to sodium fluorescein. Immunocytochemistry was performed to visualize EC and PC in coculture, and to evaluate phorbol 12-myristate-13-acetate (PMA)-induced HuR translocation. PKCβI/βII, HuR, and VEGF protein content was measured with western blotting, VEGF secretion in cell culture medium was evaluated with enzyme-linked immunosorbent assay (ELISA), and quantification of VEGF mRNA was performed with real-time quantitative PCR. RESULTS: In monocultures, VEGF mRNA/protein basal levels were more elevated in PC than in EC. However, the basal expression of VEGF protein, but not mRNA, in PC and EC was affected by culture conditions. In fact, physical contact with PC upregulated VEGF protein levels in the EC, while VEGF was downregulated in PC cocultured with EC. In this last condition, PKCβII and HuR protein basal levels were also decreased in monocultured PC. Moreover, in basal conditions, the amount of VEGF released from the coculture was higher than from the monocultures. Direct activation of PKCβ induced HuR translocation from the nuclear area to the cytoplasm, and increased the protein levels of the kinase itself, HuR, and VEGF in PC and EC in both culture conditions. Concerning VEGF mRNA, PKC activation induced an increase in PKC levels only in monocultured EC and, conversely, a significant decrease in the same transcript amount in cocultured PC. PMA stimulus also led to a significant increase in VEGF secretion in coculture. CONCLUSIONS: When cocultured with PC, EC form a significantly tighter barrier than the endothelial monolayer. The physical contact leads to opposite changes in VEGF protein levels in PC and EC. In particular, in basal conditions, cocultured PC seemed to downregulate their own expression of this proproliferating factor, as well as that of PKCβII and HuR, likely to maintain the 1:1 ratio with the cohabiting EC. In mono- and cocultured PC/EC, PKC direct activation led to a similar increase in PKCβI/βII, HuR, and VEGF protein levels, changes that may also occur at early stages of diabetic retinopathy. The release of VEGF in the medium was favored by physical contact between PC and EC and was further increased by PMA exposure. In contrast with the effects on VEGF protein, PKCβ activation induced modifications in VEGF mRNA content that are different in function of the cell type and the culture conditions. These findings suggest that the changes in the VEGF protein and transcript observed in PC/EC can be ascribed to distinct and concomitant pathways. Further studies on this in vitro coculture model would be useful to better understand the PC/EC interaction in physiologic and pathological conditions

Pulsed electromagnetic field (PEMF) prevents pro-oxidant effects of H2O2 in SK-N-BE(2) human neuroblastoma cells

Purpose The redox milieu, together with reactive oxygen species (ROS) accumulation, may play a role in mediating some biological effects of extremely-low-frequency electromagnetic fields (ELF-EMF). Some of us have recently reported that a pulsed EMF (PEMF) improves the antioxidant response of a drug-sensitive human neuroblastoma SH-SY5Y cell line to pro-oxidants. Since drug resistance may affect cell sensitivity to redox-based treatments, we wanted to verify whether drug-resistant human neuroblastoma SK-N-BE(2) cells respond to a PEMF in a similar fashion. Materials and methods SK-N-BE(2) cells were exposed to repeated 2 mT, 75 Hz PEMF (15 min each, repeated 3 times over 5 days), and ROS production, Mn-dependent superoxide dismutase (MnSOD)-based antioxidant protection and viability were assessed after 10 min or 30 min 1 mM hydrogen peroxide. Sham controls were kept at the same time in identical cell culture incubators. Results The PEMF increased the MnSOD-based antioxidant protection and reduced the ROS production in response to a pro-oxidant challenge. Conclusions Our work might lay foundation for the development of non-invasive PEMF-based approaches aimed at elevating endogenous antioxidant properties in cellular or tissue models

Are Hsp70 protein expression and genetic polymorphism implicated in multiple sclerosis inflammation?

Genetic and environmental factors contribute to disease Multiple Sclerosis (MS) susceptibility, the most prevalent neurological pathology affecting young individuals in Western countries. We focused our attention on HSP70-2, an inducible chaperon induced under stress conditions. Genotype analysis of HSP70-2 (+1267 A/G) polymorphism revealed a significant association between the minor allele G and presence of MS (OR:1.31, 95% CI: 1.02-1.69, P=0.039). In addition, Hsp70-2 protein content in vitro from PBMC was significantly lower in MS patients with GG genotype compared to AA genotype, indicating an implication of the G allele of HSP70-2 gene polymorphism in the development of MS

Response to oxidative stress of peripheral blood mononuclear cells from multiple sclerosis patients and healthy controls

The complex scenario of multiple sclerosis (MS) pathology involves several mechanisms, including oxidative stress response. The heat shock proteins (HSPs) are important for the protection of the cells; however, their role in MS is not clear. The present research is focused on the response of peripheral blood mononuclear cells (PBMCs) to oxidative stress and to the involvement of HSP70-2 (a protein coded by the HSPA1B gene, located in the MHC class III). To this aim, we challenged PBMCs from MS patients and healthy controls with hydrogen peroxide. Specifically, PBMCs mitochondrial activity, HSP70-2 protein expression and the production of intracellular reactive oxygen species were assessed. These parameters were also related to the HSP70-2 rs1061581 polymorphism, which is linked to the risk of developing MS. Moreover, mitochondrial activity and HSP70-2 protein levels were also related to disease severity. Overall, our results indicate that PBMCs, from both MS patients and healthy controls, may display a similar response towards an oxidative insult; within this context, HSP70-2 does not seem to be central in the protection of PBMCs. Nevertheless, the HSP70-2 rs1061581 polymorphism is related to ROS levels and appears to have a role in the different expression of HSP70-2 under oxidative stimulus