Unsaturated fatty acid regulation of cytochrome P450 expression via a CAR-dependent pathway

The liver is responsible for key metabolic functions, including control of normal homoeostasis in response to diet and xenobiotic metabolism/detoxification. We have shown previously that inactivation of the hepatic cytochrome P450 system through conditional deletion of POR (P450 oxidoreductase) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triacylglycerol (triglyceride) homoeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR (constitutive androstane receptor) and, to a lesser degree, PXR (pregnane X receptor). To our knowledge this is the first direct evidence that P450s play a major role in controlling unsaturated fatty acid homoeostasis via CAR. The regulation of P450s involved in xenobiotic metabolism by this mechanism has potentially significant implications for individual responses to drugs and environmental chemicals.     

CDK13, a Kinase Involved in Pre-mRNA Splicing,Is a Component of the Perinucleolar Compartment

The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human.     

The induction of preterm labor in rhesus macaques is determined by the strength of immune response to intrauterine infection

Intrauterine infection/inflammation (IUI) is a major contributor to preterm labor (PTL). However, IUI does not invariably cause PTL. We hypothesized that quantitative and qualitative differences in immune response exist in subjects with or without PTL. To define the triggers for PTL, we developed rhesus macaque models of IUI driven by lipopolysaccharide (LPS) or live Escherichia coli. PTL did not occur in LPS challenged rhesus macaques, while E. coli–infected animals frequently delivered preterm. Although LPS and live E. coli both caused immune cell infiltration, E. coli–infected animals showed higher levels of inflammatory mediators, particularly interleukin 6 (IL-6) and prostaglandins, in the chorioamnion-decidua and amniotic fluid (AF). Neutrophil infiltration in the chorio-decidua was a common feature to both LPS and E. coli. However, neutrophilic infiltration and IL6 and PTGS2 expression in the amnion was specifically induced by live E. coli. RNA sequencing (RNA-seq) analysis of fetal membranes revealed that specific pathways involved in augmentation of inflammation including type I interferon (IFN) response, chemotaxis, sumoylation, and iron homeostasis were up-regulated in the E. coli group compared to the LPS group. Our data suggest that the intensity of the host immune response to IUI may determine susceptibility to PTL.     

Effect of two different long-sprint training regimens on sprint performance and associated metabolic responses

The purpose of this study was to analyze 2 different long-sprint training programs (TPs) of equal total work load, completed either with short recovery (SR) or long recovery (LR) between sets and to compare the effects of 6 long-sprint training sessions (TSs) conducted over a 2-week period on a 300-m performance. Fourteen trained subjects performed 3 pretraining maximal sprints (50-, 100-, and 300-m), were paired according to their 300-m performance, and randomly allocated to an LR or SR group, which performed 6 TSs consisting of sets of 150, 200, or 250 m. The recovery in the LR group was double that of the SR group. During the third TS and the 300-m pretest and posttest, blood pH, bicarbonate concentration ([HCO??]), excess-base (EB), and lactate concentration were recorded. Compared with a similar TS performed with SR, the LR training tends to induce a greater alteration of the acid-base balance: pH: 7.09 ± 0.08 (LR) and 7.14 ± 0.05 (SR) (p = 0.10), [HCO??]: 7.8 ± 1.9 (LR) and 9.6 ± 2.7 (SR) (p = 0.04), and EB: -21.1 ± 3.8 (LR) and -17.7 ± 2.8 (SR) (p = 0.11). A significant improvement in the 300-m performance between pre-TP and post-TP (42.45 ± 2.64 vs. 41.52 ± 2.45, p = 0.01) and significant decreases in pH (p < 0.01), EB (p < 0.001) and increase in [La] (p < 0.001) have been observed post-TP compared with those pre-TP. Although sprint training with longer recovery induces higher metabolic disturbances, both sprint training regimens allow a similar 300-m performance improvement with no concomitant significant progress in the 50- and 100-m performance.     

Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.