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As one of the most induced genes in activated macrophages, immune-responsive gene 1 (IRG1) encodes a mitochondrial metabolic enzyme catalysing the production of itaconic acid (ITA). Although ITA has an anti-inflammatory property, the underlying mechanisms are not fully understood. Here we show that ITA is a potent inhibitor of the TET-family DNA dioxygenases. ITA binds to the same site on TET2 as the co-substrate α-ketoglutarate, inhibiting TET2 catalytic activity. Lipopolysaccharide treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals that TET2 is a major target of ITA in suppressing lipopolysaccharide-induced genes, including those regulated by the NF-κB and STAT signalling pathways. In vivo, ITA decreases the levels of 5-hydroxymethylcytosine, reduces lipopolysaccharide-induced acute pulmonary oedema as well as lung and liver injury, and protects mice against lethal endotoxaemia, depending on the catalytic activity of Tet2. Our study thus identifies ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the inflammatory responses.

This study aimed to investigate the protective effect of nicotine on dopaminergic neurons and its mechanisms in mice with Parkinson disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). C57BL/6J mice were injected with MPTP for 8 days to establish a PD model. Nicotine was given for 10 days in the nicotine therapeutic group. Animals were examined behaviorally with the pole test and traction test. Tyrosine hydroxylase (TH) and γ-aminobutyric acid (GABA) were determined by using the immunocytochemistry (ICC) method. The ultrastructural changes of the caudate nucleus (CN) were observed under electron microscopy. The results showed that pretreatment with nicotine could improve the dyskinesia of PD mice markedly. Simultaneously, TH-positive (P

The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a FADD-like interferon converting enzyme or caspase 8 (FLICE) inhibitory protein (vFLIP) that prevents death receptor-mediated apoptosis by inhibiting the recruitment and activation of FLICE. Since vFLIP physically interacts with tumor necrosis factor receptor associated factor 2 (TRAF2) and TRAF2 mediates activation of the jun NH(2)-terminal kinase (JNK)/activation protein 1 (AP1) pathway, we hypothesized that vFLIP might also activate this pathway. To evaluate this hypothesis, we transiently and stably transfected a vFLIP expression construct and performed several complementary assays to document that vFLIP activates the JNK/AP1 pathway and does so in a TRAF-dependent fashion. As vFLIP also activates the nuclear factor kappaB (NF-kappaB) signaling pathway and the NF-kappaB and JNK/AP1 pathways both modulate cellular interleukin-6 (cIL-6) expression, we postulated that vFLIP induces expression of this cytokine. We show that vFLIP induces cIL-6 expression and activates the cIL-6 promoter, and maximal activation of the cIL-6 promoter by vFLIP requires NF-kappaB and AP1 activation. In addition, vFLIP and latency-associated nuclear antigen (LANA), another KSHV-encoded latent protein, potentiate each other's ability to activate the cIL-6 promoter. Gene silencing experiments by RNA interference demonstrate that vFLIP in BCBL-1 endogenously infected primary effusion lymphoma (PEL) cells mediates JNK/AP1 activation and cIL-6 expression. Thus, we conclude that vFLIP, in addition to its known effects on NF-kappaB activation, also modulates the JNK/AP1 pathway and induces gene expression from the cIL-6 promoter in a JNK/AP1-dependent fashion.

Background: Isolated monomorphic premature ventricular contractions (PVCs) are not uncommon in the pediatric population. The degree of cardiac dysfunction caused by PVCs in children without structural heart disease is unknown. Purpose: To investigate the influence of PVCs on echocardiographic left ventricular (LV) systolic function in children without structural heart disease. Methods: Forty asymptomatic children with isolated monomorphic PVCs without structural heart disease were selected. The median age was 6 years, range of 3–12 years. The following subgroups were compared: frequent vs. infrequent PVCs (> or ≤10/min); short vs. long coupling interval (RR′/RR ratio ≤ or >0.6); and short vs. long QT interval (QT ≤ or >400 ms). Using echocardiography the left ventricular ejection fraction (LVEF in percentage) and cardiac index (CI in L/min/m2) were measured for both normal sinus beats (SB-LVEF and CI), PVCs (PVC-LVEF and CI) and the average LVEF and CI were calculated. All values were expressed as means ± SD. Results: In all children LV dimensions and the SB-LVEF and CI were within normal limits. The PVC-LVEF (48 ± 5) and PVC-CI (1.57 ± 0.19) were significantly decreased and the average CI was 2.41 ± 0.29. In 27 pts with >10 PVCs/min the average LVEF and CI decreased to 53 ± 5 and 2.08 ± 0.24 respectively. In 16 pts with PVCs and a short coupling interval (RR′/RR ≤ 0.6) the PVC-LVEF and PVC-CI was 43 ± 0.03 and 1.50 ± 0.14, which was significantly lower than in 24 pts with a long coupling interval (58 ± 4, 1.88 ± 0.11). In 11 pts with a prolonged QT interval (>400 ms) the PVC-LVEF and PVC-CI was significantly lower than in the 29 children with a shorter QT interval, 41 ± 5 vs. 55 ± 4 and 1.46 ± 0.13 vs. 1.86 ± 0.15 respectively (all p 10/min), have a short coupling interval or a prolonged QT interval.

Enzymatic digests of proteins isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS-PAGE at low picomole levels can be analyzed by the new integrated system.

The primary structure and posttranslational modifications of rabbit lens alpha-crystallins were examined using electrospray ionization mass spectrometry to determine the molecular weights of the intact proteins and fast atom bombardment mass spectrometry to analyze proteolytic digests of the alpha A- and alpha B-crystallins. The previously determined primary structure of alpha A-crystallin was confirmed. Posttranslational modifications detected included one phosphorylation site and the presence of a truncated form minus the five C-terminal residues. The previously undetermined amino acid sequence of rabbit alpha B-crystallin was determined to be the same as the bovine alpha B-crystallin sequence except at three residues: Thr 40, Thr 132, and Pro 153. Rabbit alpha B-crystallin showed evidence of phosphorylation at the same three sites as bovine alpha B-crystallin. The molecular weights of the intact proteins indicated that any one molecule had a maximum of two phosphorylations. Also, there was a truncated form which did not include the five C-terminal residues.