Oncogenic K-Ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

Liver Glucokinase A456V induces potent hypoglycemia without dyslipidemia through a paradoxical induction of the catalytic subunit of glucose-6-phosphatase

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GKA456V, described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulation in vivo. GKA456V was overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affected in vivo, demonstrating impaired GKRP regulation. GKA456V overexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GKA456V-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy.

Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

Computational toolbox towards evolutionary domain mapping of membrane proteins

Membrane proteins account for about 20% to 30% of all proteins encoded in a typical genome. They play central roles in multiple cellular processes mediating the interaction of the cell with its surrounding. Over 60% of all drug targets contain a membrane domain. The experimental difficulties of obtaining a crystal structural severely limits our ability or understanding of membrane protein function. Computational evolutionary studies of proteins are crucial for the prediction of 3D structures. In this project, we construct a tool able to quantify the evolutionary positive selective pressure on each residue of membrane proteins through maximum likelihood phylogeny reconstruction. The conservation plot combined with a structural homology model is also a potent tool to predict those residues that have essentials roles in the structure and function of a membrane protein and can be very useful in the design of validation experiments.

Botulinum neurotoxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release,was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods.To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.

Non-invasive diagnostic and prognostic evaluation of liver cirrhosis and portal hypertension.

Cirrhosis is the final stage of most of chronic liver diseases, and is almost invariably complicated by portal hypertension, which is the most important cause of morbidity and mortality in these patients. This review will focus on the non-invasive methods currently used in clinical practice for diagnosing liver cirrhosis and portal hypertension. The first-line techniques include physical examination, laboratory parameters, transient elastography and Doppler-US. More sophisticated imaging methods which are less commonly employed are CT scan and MRI, and new technologies which are currently under evaluation are MR elastography and acoustic radiation force imaging (ARFI). Even if none of them can replace the invasive measurement of hepatic venous pressure gradient and the endoscopic screening of gastroesophageal varices, they notably facilitate the clinical management of patients with cirrhosis and portal hypertension, and provide valuable prognostic information.

Oxidized oils and dietary zinc and alpha-tocopheryl acetate supplementation: effects on rabbit plasma, liver and meat fatty acid composition and meat Zn, Cu, Fe and Se content

The effects of the addition of heated oils to feeds (3%, w/w) and the dietary supplementation with a-tocopheryl acetate (TA; 100 mg/kg) and Zn (200 mg/kg) on rabbit tissue fatty acid (FA) composition and on the Zn, Cu, Fe and Se content in meat were assessed. Heating unrefined sunflower oil (SO) at 558C for 245 h increased its content in primary oxidation products and reduced its a-tocopherol content. However, this did not significantly affect tissue FA composition. Heating SO at 1408C for 31 h increased its content in secondary oxidation products and in some FA isomers asc9,t11-CLA and di-trans CLA. This led to increases in di-trans CLA in liver and in t9,c12-18:2 in meat. The c9,t11-CLA was the most incorporated CLA isomer in tissues. The dietary supplementation with a-TA did not affect the FA composition of plasma, liver or meat. The cooking of vacuum-packed rabbit meat at 788C for 5 min reduced significantly but slightly its polyunsaturated FA content. The dietary supplementation with Zn did not modify the content of Zn, Fe or Se in meat, but it reduced its Cu content. On the other hand, it increased the content of some FAs in meat when SO heated at 1408C for 31 h was added to feeds.

A new formula for calculating standard liver volume for living donor liver transplantation without using body weight.

BACKGROUND & AIMS: The standard liver volume (SLV) is widely used in liver surgery, especially for living donor liver transplantation (LDLT). All the reported formulas for SLV use body surface area or body weight, which can be influenced strongly by the general condition of the patient. METHODS: We analyzed the liver volumes of 180 Japanese donor candidates and 160 Swiss patients with normal livers to develop a new formula. The dataset was randomly divided into two subsets, the test and validation sample, stratified by race. The new formula was validated using 50 LDLT recipients. RESULTS: Without using body weight-related variables, age, thoracic width measured using computed tomography, and race independently predicted the total liver volume (TLV). A new formula: 203.3-(3.61×age)+(58.7×thoracic width)-(463.7×race [1=Asian, 0=Caucasian]), most accurately predicted the TLV in the validation dataset as compared with any other formulas. The graft volume for LDLT was correlated with the postoperative prothrombin time, and the graft volume/SLV ratio calculated using the new formula was significantly better correlated with the postoperative prothrombin time than the graft volume/SLV ratio calculated using the other formulas or the graft volume/body weight ratio. CONCLUSIONS: The new formula derived using the age, thoracic width and race predicted both the TLV in the healthy patient group and the SLV in LDLT recipients more accurately than any other previously reported formulas.

Anatomy of liver arteries for interventional radiology.

The availability of intra-arterial hepatic therapies (radio and/or chemo-embolisation, intra-arterial hepatic chemotherapy) has convinced radiologists to perfect their knowledge of the anatomy of the liver arteries. These sometimes, complex procedures most often require selective arterial catheterization. Knowledge of the different arteries in the liver and the peripheral organs is therefore essential to optimize the procedure and avoid eventual complications. This paper aims to describe the anatomy of the liver arteries and the variants, applying it to angiography images, and to understand the implications of such variations in interventional radiological procedures.

Endocrine causes of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the industrialized world. The prevalence of NAFLD is increasing, becoming a substantial public health burden. NAFLD includes a broad spectrum of disorders, from simple conditions such as steatosis to severe manifestations such as fibrosis and cirrhosis. The relationship of NAFLD with metabolic alterations such as type 2 diabetes is well described and related to insulin resistance, with NAFLD being recognized as the hepatic manifestation of metabolic syndrome. However, NAFLD may also coincide with endocrine diseases such as polycystic ovary syndrome, hypothyroidism, growth hormone deficiency or hypercortisolism. It is therefore essential to remember, when discovering altered liver enzymes or hepatic steatosis on radiological exams, that endocrine diseases can cause NAFLD. Indeed, the overall prognosis of NAFLD may be modified by treatment of the underlying endocrine pathology. In this review, we will discuss endocrine diseases that can cause NALFD. Underlying pathophysiological mechanisms will be presented and specific treatments will be reviewed.

Targeted γ-Secretase Inhibition To Control the Notch Pathway in Renal Diseases.

Notch is a membrane inserted protein activated by the membrane-inserted γ-secretase proteolytic complex. The Notch pathway is a potential therapeutic target for the treatment of renal diseases but also controls the function of other cells, requiring cell-targeting of Notch antagonists. Toward selective targeting, we have developed the γ-secretase inhibitor-based prodrugs 13a and 15a as substrates for γ-glutamyltranspeptidase (γ-GT) and/or γ-glutamylcyclotransferase (γ-GCT) as well as aminopeptidase A (APA), which are overexpressed in renal diseases, and have evaluated them in experimental in vitro and in vivo models. In nondiseased mice, the cleavage product from Ac-γ-Glu-γ-secretase inhibitor prodrug 13a (γ-GT-targeting and γ-GCT-targeting) but not from Ac-α-Glu-γ-secretase inhibitor prodrug 15a (APA-targeting) accumulated in kidneys when compared to blood and liver. Potential nephroprotective effects of the γ-secretase inhibitor targeted prodrugs were investigated in vivo in a mouse model of acute kidney injury, demonstrating that the expression of Notch1 and cleaved Notch1 could be selectively down-regulated upon treatment with the Ac-γ-Glu-γ-secretase-inhibitor 13a.

Outcomes After Liver Resection for Hepatic Alveolar Echinococcosis: A Single-Center Cohort Study.

BACKGROUND: Switzerland is a region in which alveolar echinococcosis (AE) is endemic. Studies evaluating outcomes after liver resection (LR) for AE are scarce. The aim of this study was to assess the short- and long-term outcomes of AE patients after LR in a single tertiary referral center. METHODS: We retrospectively analyzed data pertaining to all patients with liver AE who were treated with LR at our institution between January 1992 and December 2013. Patient demographics, intraoperative data, extent of LR procedures (major vs. minor LR), postoperative outcomes, and negative histological margin (R0) resection rate were recorded in a database. Recurrence rates after LR were analyzed. RESULTS: LR was performed in 59 patients diagnosed with hepatic AE (56 complete surgeries, 3 reduction surgeries). Postoperative morbidity and mortality were observed in 34 % (25 % grade I-II, 9 % grade III-IV) and 2 % of the patients, respectively. R0 (complete) resection rate was 71 % (n = 42), and R1/R2 resection rate was 29 % (n = 17). Extra-hepatic recurrence occurred in 1 case (lung) after R0 resection. In cases of R1/R2 resection, 7 intra-hepatic disease progressions occurred with a median time of 10 months (IQR 6-11 months). Long-term (more than 1 year) benzimidazole treatment stabilized the disease in 64 % (9/14) of patients with R1 status. The overall survival rate was 97 %. CONCLUSIONS: Liver AE can be safely and definitively treated with LR, provided that R0 resection is achieved. In cases of R1 resection, benzimidazole therapy seems to be effective in stabilizing the intra-hepatic disease and preventing extra-hepatic recurrence.

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.

Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.