Lactulose and mannitol intestinal permeability detected by capillary electrophoresis

Aim of this study was to set up a method by capillary electrophoresis to detect lactulose and mannitol in urine after an oral load, and to estimate the intestinal permeability in controls and in type I diabetes patients. The underivatized carbohydrates were monitored by indirect UV detection using sorbate, cetyltrimethylammonium bromide and LiOH as background electrolyte. Urines were purified by solid phase extraction, shaken with cation exchange resin, filtered and analysed. Carbohydrates migrated in <10 min in relation to their pK(a) and M(r). Controls (n = 33) and patients (n = 23) had an excretion ratio lactulose/mannitol 0.025 (0.018-0.051) and 0.067 (0.050-0.127), respectively (p < 0.01, median, interquartile range).     

Long-term trends of epilimnetic and hypolimnetic bacteria and organic carbon in a deep holo-oligomictic lake

We analysed the long-term dynamics (1980–2007) of hypolimnetic and epilimnetic bacterial abundances and organic carbon concentrations, both dissolved (DOC) and particulate (POC), in the deep holo-oligomictic Lake Maggiore, included in the Southern Alpine Lakes Long-Term Ecological Research (LTER) site. During the 28 years of investigation, bacterial abundance and POC concentrations did not decrease with declining phosphorus concentrations, while DOC concentrations showed a pronounced decrease in the epi- and hypolimnion. We used the annual mean total lake heat content and total annual precipitation as climate-related variables, and in-lake total phosphorus as a proxy for trophic state. The model (forward stepwise regression, FSR) showed that reduced anthropogenic pressure was more significant than climate change in driving the trend in DOC concentrations. Bacterial dynamics in the hypolimnion mirrored the fluctuations observed in the epilimnion, but average cell abundance was three times lower. The FSR model indicates that bacterial number variability was dependent on POC in the epilimnion and DOC in the hypolimnion. In the hypolimnion, cell biovolumes for rod and coccal morphotypes were significantly larger than in the epilimnion.     

Minimizing false positives in kinase virtual screens

In spite of recent improvements in docking and scoring methods, high false-positive rates remain a common issue in structure-based virtual screening. In this study, the distinctive features of false positives in kinase virtual screens were investigated. A series of retrospective virtual screens on kinase targets was performed on specifically designed test sets, each combining true ligands and experimentally confirmed inactive compounds. A systematic analysis of the docking poses generated for the top-ranking compounds highlighted key aspects differentiating true hits from false positives. The most recurring feature in the poses of false positives was the absence of certain key interactions known to be required for kinase binding. A systematic analysis of 444 crystal structures of ligand-bound kinases showed that at least two hydrogen bonds between the ligand and the backbone protein atoms in the kinase hinge region are present in 90% of the complexes, with very little variability across targets. Closer inspection showed that when the two hydrogen bonds are present, one of three preferred hinge-binding motifs is involved in 96.5% of the cases. Less than 10% of the false positives satisfied these two criteria in the minimized docking poses generated by our standard protocol. Ligand conformational artifacts were also shown to contribute to the occurrence of false positives in a number of cases. Application of this knowledge in the form of docking constraints and post-processing filters provided consistent improvements in virtual screening performance on all systems. The false-positive rates were significantly reduced and the enrichment factors increased by an average of twofold. On the basis of these results, a generalized two-step protocol for virtual screening on kinase targets is suggested.     

PI3K-dependent lysosome exocytosis in nitric oxide-preconditioned hepatocytes

We investigated the signal mediators and the cellular events involved in the nitric oxide (NO)-induced hepatocyte resistance to oxygen deprivation in isolated hepatocytes treated with the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])diazen-1-ium-1,2-diolate (NOC-9). NOC-9 greatly induced PI3K activation, as tested by phosphorylation of PKB/Akt. This effect was prevented by either 1H-(1,2,4)-oxadiazolo-(4,3)-quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC), or KT5823, an inhibitor of cGMP-dependent kinase (cGK), as well as by farnesyl protein transferase inhibitor, which blocks the function of Ras GTPase. Bafilomycin A, an inhibitor of the lysosome-type vacuolar H+-ATPase, cytochalasin D, which disrupts the cytoskeleton-dependent organelle traffic, and wortmannin, which inhibits the PI3K-dependent traffic of lysosomes, all abolished the NOC-9-induced hepatocyte protection. The treatment with NOC-9 was associated with the PI3K-dependent peripheral translocation and fusion with the plasma membrane of lysosomes and the appearance at the cell surface of the vacuolar H+-ATPase. Inhibition of sGC, cGK, and Ras, as well as the inhibition of PI3K by wortmannin, prevented the exocytosis of lysosomes and concomitantly abolished the protective effect of NOC-9 on hypoxia-induced pHi and [Na+]i alterations and cell death. These data indicate that NO increases hepatocyte resistance to hypoxic injury by activating a pathway involving Ras, sGC, and cGK that determines PI3K-dependent exocytosis of lysosomes.     

Non-secretory exocytoses in the brain.

Regulated exocytosis, the process by which the membrane of specific cytoplasmic organelles fuse with the plasma membrane in response to adequate stimulation, is most often considered to serve only for the discharge of secretory products, in the brain especially neurotransmitters and peptides. Growing evidence demonstrates however that non-secretory exocytoses, aimed at the insertion at the cell surface of the organelle membrane, are of great physiological importance and may also have critical roles in specific diseases. Recently, two groups of non-secretory exocytoses have been identified: those aimed at the transfer to the cell surface of specific proteins, that we have proposed to be called the protein-exposing exocytoses; and those aimed at the enlargement of the surface itself, the expansive exocytoses. Here we present the existing knowledge about three types of non-secretory exocytoses that occur in the brain: the protein-exposing exocytoses that transfer ionic receptors to the postsynaptic membrane, the best known example being that of the glutamatergic AMPA receptor, a main actor of synaptic plasticity; the expansive exocytosis necessary for the growth of nerve fibres; and the rapid exocytosis of enlargeosomes, that can induce considerable expansion of the cell surface area in a variety of cells types, including the astrocytes.     

Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation

Type I IFN-producing cells (IPC) are sentinels of viral infections. Identification and functional characterization of these cells have been difficult because of their small numbers in blood and tissues and their complex cell surface phenotype. To overcome this problem in mice, mAbs recognizing IPC-specific cell surface molecules have been generated. In this study, we report the identification of new Abs specific for mouse IPC, which recognize the bone marrow stromal cell Ag 2 (BST2). Interestingly, previously reported IPC-specific Abs 120G8 and plasmacytoid dendritic cell Ag-1 also recognize BST2. BST2 is predominantly specific for mouse IPC in naive mice, but is up-regulated on most cell types following stimulation with type I IFNs and IFN-gamma. The activation-induced promiscuous expression of BST2 described in this study has important implications for the use of anti-BST2 Abs in identification and depletion of IPC. Finally, we show that BST2 resides within an intracellular compartment corresponding to the Golgi apparatus, and may be involved in trafficking secreted cytokines in IPC.