Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice

Introduction

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim

To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods

Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results

Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion

Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.     

Muscle Carnosine Is Associated with Cardiometabolic Risk Factors in Humans

BackgroundCarnosine is a naturally present dipeptide abundant in skeletal muscle and an over-the counter food additive. Animal data suggest a role of carnosine supplementation in the prevention and treatment of obesity, insulin resistance, type 2 diabetes and cardiovascular disease but only limited human data exists.ConclusionOur data shows that higher carnosine content in human skeletal muscle is positively associated with insulin resistance and fasting metabolic preference for glucose. Moreover, it is negatively associated with HDL-cholesterol and basal energy expenditure. Intervention studies targeting insulin resistance, metabolic and cardiovascular disease risk factors are necessary to evaluate its putative role in the prevention and management of type 2 diabetes and cardiovascular disease.     

Dexamethasone induces rapid tyrosine-phosphorylation of ZAP-70 in Jurkat cells

Steroid hormones are known to mediate rapid non-genomic effects occurring within minutes, besides the classical genomic actions mediated by the nuclear translocation of the cytoplasmic glucocorticoid receptor (GR). The glucocorticoid hormone (GC) has significant role in the regulation of T-cell activation; however, the cross-talk between the GC and T-cell receptor (TcR) signal transducing pathways are still to be elucidated. We examined the rapid effects of GC exposure on in vitro cultured human T-cells. Our results showed that Dexamethasone (DX), a GC analogue, when applied at high dose (10 microM), induced rapid (within 5 min) tyrosine-phosphorylation events in Jurkat cells. Short DX pre-treatment strongly inhibited the tyrosine-phosphorylation stimulated by CD3 cross-linking. Furthermore, we also investigated the phosphorylation status of ZAP-70, an important member of tyrosine kinase mediated signalling pathway of TcR-elicited T-cell activation. Here, we demonstrate that high dose DX induced a rapid ZAP-70 tyrosine-phosphorylation in Jurkat T-cells. DX-induced ZAP-70 phosphorylation could be inhibited by RU486 (GR antagonist), suggesting that this process was GR mediated. DX-induced ZAP-70 phosphorylation did not occur in the absence of active p56-lck as examined in the p56-lck kinase-deficient Jurkat cell line JCaM1.6. Our results show that DX, at a high dose, can rapidly influence the initial tyrosine-phosphorylation events of the CD3 signalling pathway in Jurkat cells, thereby modifying TcR-derived signals. Lck and ZAP-70 represent an important molecular link between the TcR and GC signalling pathways.     

Evaluation of gene targeting by homologous recombination in ovine somatic cells

Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.     

The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins

The membrane-tethered mucins are cell surface-associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane-associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane-tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site-directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately re-create an accessible cleavage site.     

Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450

The biological mechanisms responsible for aging remain poorly understood. We propose that increases in DNA damage and mutations that occur with age result from a reduced ability to repair DNA damage. To test this hypothesis, we have measured the ability to repair DNA damage in vitro by the base excision repair (BER) pathway in tissues of young (4-month-old) and old (24-month-old) C57BL/6 mice. We find in all tissues tested (brain, liver, spleen and testes), the ability to repair damage is significantly reduced (50-75%; P<0.01) with age, and that the reduction in repair capacity seen with age correlates with decreased levels of DNA polymerase beta (beta-pol) enzymatic activity, protein and mRNA. To determine the biological relevance of this age-related decline in BER, we measured spontaneous and chemically induced lacI mutation frequency in young and old animals. In line with previous findings, we observed a three-fold increase in spontaneous mutation frequency in aged animals. Interestingly, lacI mutation frequency in response to dimethyl sulfate (DMS) does not significantly increase in young animals whereas identical exposure in aged animals results in a five-fold increase in mutation frequency. Because DMS induces DNA damage processed by the BER pathway, it is suggested that the increased mutagenicity of DMS with age is related to the decline in BER capacity that occurs with age. The inability of the BER pathway to repair damages that accumulate with age may provide a mechanistic explanation for the well-established phenotype of DNA damage accumulation with age.     

Production of SacI and SacII isoschizomers by soil streptomycetes

Five fresh soil Streptomyces spp. strains were isolated, phylogenetically characterized on the basis of 16S rDNA sequences and analyzed for the presence of restriction modification systems. Three type II site-specific endonucleases were detected and partially purified. Two isolated enzymes were isoschizomers of SacI restriction endonuclease recognizing 5′-GAGCTC-3′ sequence; the third one recognised 5′-CCGCGG-3′ sequence and it was an isoschizomer of SacII. SacII like modification was observed in other two isolates having no detectable restriction activity. The lack of correlation between restriction and modification phenotypes and phylogenetic classification of the isolates indicates efficient gene transfer mechanism in the Streptomyces genus.     

Mesenchymal Stem Cells in the Treatment of Human Spinal Cord Injury: The Effect on Individual Values of pNF-H,GFAP, S100 Proteins and Selected Growth Factors,Cytokines and Chemokines

At present, there is no effective way to treat the consequences of spinal cord injury (SCI). SCI leads to the death of neural and glial cells and widespread neuroinflammation with persisting for several weeks after the injury. Mesenchymal stem cells (MSCs) therapy is one of the most promising approaches in the treatment of this injury. The aim of this study was to characterize the expression profile of multiple cytokines, chemokines, growth factors, and so-called neuromarkers in the serum of an SCI patient treated with autologous bone marrow-derived MSCs (BM-MSCs). SCI resulted in a significant increase in the levels of neuromarkers and proteins involved in the inflammatory process. BM-MSCs administration resulted in significant changes in the levels of neuromarkers (S100, GFAP, and pNF-H) as well as changes in the expression of proteins and growth factors involved in the inflammatory response following SCI in the serum of a patient with traumatic SCI. Our preliminary results encouraged that BM-MSCs with their neuroprotective and immunomodulatory effects could affect the repair process after injury.