Liver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis

Background

Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests.

Methods and Findings

In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87).

Conclusions

Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is necessary to extend these concepts into clinical practice. Please see later in the article for the Editors'' Summary     

Time Space Translation: A Hox Mechanism for Vertebrate A-P Patterning

The vertebrate A-P axis is a time axis. The head is made first and more and more posterior levels are made at later and later stages. This is different to the situation in most other animals, for example, in Drosophila. Central to this timing is Hox temporal collinearity (see below). This occurs rarely in the animal kingdom but is characteristic of vertebrates and is used to generate the primary axial Hox pattern using time space translation and to integrate successive derived patterns (see below). This is thus a different situation than in Drosophila, where the primary pattern guiding Hox spatial collinearity is generated externally, by the gap and segmentation genes.     

Bioenergetic role of mitochondrial fusion and fission

Mitochondria are highly dynamic organelles. Frequent cycles of fusion and fission adapt the morphology of the mitochondrial compartment to the metabolic needs of the cell. Mitochondrial fusion is particularly important in respiratory active cells. It allows the spreading of metabolites, enzymes, and mitochondrial gene products throughout the entire mitochondrial compartment. This serves to optimize mitochondrial function and counteracts the accumulation of mitochondrial mutations during aging. Fragmented mitochondria are frequently found in resting cells, and mitochondrial fission plays an important role in the removal of damaged organelles by autophagy. Thus, mitochondrial fusion and fission both contribute to maintenance of mitochondrial function and optimize bioenergetic capacity. Multiple signalling pathways regulate the machinery of mitochondrial dynamics to adapt the shape of the mitochondrial compartment to the metabolic conditions of the cell. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

CENP-T proteins are conserved centromere receptors of the Ndc80 complex

Centromeres direct the assembly of kinetochores, microtubule-attachment sites that allow chromosome segregation on the mitotic spindle. Fundamental differences in size and organization between evolutionarily distant eukaryotic centromeres have in many cases obscured general principles of their function. Here we demonstrate that centromere-binding proteins are highly conserved between budding yeast and humans. We identify the histone-fold protein Cnn1(CENP-T) as a direct centromere receptor of the microtubule-binding Ndc80 complex. The amino terminus of Cnn1 contains a conserved peptide motif that mediates stoichiometric binding to the Spc24-25 domain of the Ndc80 complex. Consistent with the critical role of this interaction, artificial tethering of the Ndc80 complex through Cnn1 allows mini-chromosomes to segregate in the absence of a natural centromere. Our results reveal the molecular function of CENP-T proteins and demonstrate how the Ndc80 complex is anchored to centromeres in a manner that couples chromosome movement to spindle dynamics.     

The class V myosin motor protein, Myo2, plays a major role in mitochondrial motility in Saccharomyces cerevisiae

The actin cytoskeleton is essential for polarized, bud-directed movement of cellular membranes in Saccharomyces cerevisiae and thus ensures accurate inheritance of organelles during cell division. Also, mitochondrial distribution and inheritance depend on the actin cytoskeleton, though the precise molecular mechanisms are unknown. Here, we establish the class V myosin motor protein, Myo2, as an important mediator of mitochondrial motility in budding yeast. We found that mutants with abnormal expression levels of Myo2 or its associated light chain, Mlc1, exhibit aberrant mitochondrial morphology and loss of mitochondrial DNA. Specific mutations in the globular tail of Myo2 lead to aggregation of mitochondria in the mother cell. Isolated mitochondria lacking functional Myo2 are severely impaired in their capacity to bind to actin filaments in vitro. Time-resolved fluorescence microscopy revealed a block of bud-directed anterograde mitochondrial movement in cargo binding-defective myo2 mutant cells. We conclude that Myo2 plays an important and direct role for mitochondrial motility and inheritance in budding yeast.     

Nonoverlapping expression of IL10, IL12p40, and IFNgamma mRNA in the marginal zone and T cell zone of the spleen after antigenic stimulation

The differentiation of CD4(+) T cells is regulated by cytokines locally within the compartments of secondary lymphoid organs during adaptive immune responses. Quantitative data about the expression of cytokine mRNAs within the T and B cell zones of lymphoid organs are lacking. In this study, we assessed the expression of multiple cytokine genes within the lymphoid compartments of the spleen of rats after two types of stimulation. First, the spleen was stimulated directly by a blood-derived Ag. Second, the spleen was stimulated indirectly by incoming lymphocytes that had been activated and released during a proceeding immune response at a distant tissue site. Using laser microdissection, we show that the expression of cytokine mRNAs was compartment specific, transient, and preceded cell proliferation after the direct antigenic stimulation. Surprisingly, the indirect stimulation by incoming activated lymphocytes induced similar cytokines in the T cell zone. However, the nonoverlapping expression was lost and IL10 appeared as the major cytokine in all compartments. Thus, tracking two types of immune activation without disturbing the integrity of structures reveals distinct and overlapping events in the compartments of the spleen. This information adds a new dimension to the understanding of immune responses in vivo.     

NITPICK: peak identification for mass spectrometry data

Background  

The reliable extraction of features from mass spectra is a fundamental step in the automated analysis of proteomic mass spectrometry (MS) experiments.     

PA,a stress-induced short cut to switch-on ethylene signalling by switching-off CTR1?

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.¹ PA was shown to inhibit (1) CTR1''s protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.Key words: lipid signaling, phosphatidic acid, ethylene, constitutive triple response 1, plant stress signaling, protein kinase, phospholipase D