Anti-inflammatory activities of melatonin derivatives in lipopolysaccharide-stimulated RAW 264.7 cells and antinociceptive effects in mice.

Preclinical Research This study describes the anti-inflammatory activities of two semisynthesized melatonin (MT) derivatives, benzoyl-melatonin (BMT) and acetyl-melatonin (AMT), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7) and their antinociceptive effects in mice. The MT derivatives inhibited production of nitric oxide NO and prostaglandin E2 in LPS-stimulated RAW264.7 cells in a dose-dependent manner with IC50 values lower than those of MT. BMT produced increased tail flick latency time, decreased number of writhes, and reduced nociceptive response in mice when compared with AMT and MT. BMT and AMT had enhanced anti-inflammatory effects in LPS-stimulated RAW264.7 compared with MT. However, in mouse studies BMT exhibited the highest potency as an anti-inflammatory agent and was longer-acting as an antinociceptive compound compared with AMT or MT, suggesting that BMT has potential as an anti-inflammatory and analgesic compound.

Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation.

The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.

Bcl11b represses a mature T-cell gene expression program in immature CD4(+)CD8(+) thymocytes.

Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.

Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma.

Calorie restriction extends lifespan in organisms ranging from yeast to mammals. In yeast, the SIR2 gene mediates the life-extending effects of calorie restriction. Here we show that the mammalian SIR2 orthologue, Sirt1 (sirtuin 1), activates a critical component of calorie restriction in mammals; that is, fat mobilization in white adipocytes. Upon food withdrawal Sirt1 protein binds to and represses genes controlled by the fat regulator PPAR-gamma (peroxisome proliferator-activated receptor-gamma), including genes mediating fat storage. Sirt1 represses PPAR-gamma by docking with its cofactors NCoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptors). Mobilization of fatty acids from white adipocytes upon fasting is compromised in Sirt1+/- mice. Repression of PPAR-gamma by Sirt1 is also evident in 3T3-L1 adipocytes, where overexpression of Sirt1 attenuates adipogenesis, and RNA interference of Sirt1 enhances it. In differentiated fat cells, upregulation of Sirt1 triggers lipolysis and loss of fat. As a reduction in fat is sufficient to extend murine lifespan, our results provide a possible molecular pathway connecting calorie restriction to life extension in mammals.

Prevalence of Provocative Seizures in Persons with Epilepsy: A Longitudinal Study at Khon Kaen University Hospital, Thailand.

Background and Objective. Provocative factors are one causative factor of seizure attacks in persons with epilepsy (PWE). There are limited data of prevalence and major provocative factors in Asian populations. Methods. This study was performed at the Epilepsy Clinic, Khon Kaen University Hospital. The patients who aged 15 years or over, who had been treated at least 3 months with at least one antiepileptic drug, and who were followed up for at least one year were included. Data of seizure control and triggers were collected retrospectively from medical records. Data analysis was performed to identify independent provocative factors. Results. A total of 382 PWE met the study criteria. The mean age was 40.4 ± 0.8 years. Approximately 44% of the patients had at least one provocative factor. By multivariate analysis, the independent provocative factors with the first three highest adjusted odds ratios were sleep deprivation (adjusted OR = 8.64, 95% CI 3.73-19.99), alcohol consumption (adjusted OR = 6.76, 95% CI 1.44-31.78), and feeling stressful (adjusted OR = 2.97, 95% CI 1.29-6.86). Conclusion. Almost half of seizure attacks may be caused by provocative factors in Thai PWEs and some factors may be preventable. Avoidance of these factors should be emphasized to epilepsy patients for improving clinical outcomes and quality of life.

CTIP2 associates with the NuRD complex on the promoter of p57KIP2, a newly identified CTIP2 target gene.

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2), also known as Bcl11b, is a transcriptional repressor that functions by direct, sequence-specific DNA binding activity or by recruitment to the promoter template by interaction with COUP-TF family members. CTIP2 is essential for both T cell development and axonal projections of corticospinal motor neurons in the central nervous system. However, little is known regarding the molecular mechanism(s) by which CTIP2 contributes to either process. CTIP2 complexes that were isolated from SK-N-MC neuroblastoma cells were found to harbor substantial histone deacetylase activity, which was likely conferred by the nucleosome remodeling and deacetylation (NuRD) complex. CTIP2 was found to associate with the NuRD complex through direct interaction with both RbAp46 and RbAp48, and components of the NuRD complex were found to be recruited to an artificial promoter template in a CTIP2-dependent manner in transfected cells. Finally, the NuRD complex and CTIP2 were found to co-occupy the promoter template of p57KIP2, a gene encoding a cyclin-dependent kinase inhibitor, and identified herein as a novel transcriptional target of CTIP2 in SK-N-MC cells. Therefore, it seems likely that the NuRD complex may be involved in transcriptional repression of CTIP2 target genes and contribute to the function(s) of CTIP2 within a neuronal context.

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems.