Discovery of a new class of glucosylceramide synthase inhibitors

A novel series of potent inhibitors of glucosylceramide synthase are described. The optimization of biochemical and cellular potency as well as ADME properties led to compound 23c. Broad tissue distribution was obtained following oral administration to mice. Thus 23c could be another useful tool compound for studying the effects of GCS inhibition in vitro and in vivo.     

Contribution of inter-subunit interactions to the thermostability of <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> citrate synthase

Using citrate synthase from the hyperthermophile Pyrococcus furiosus (PfCS) as our test molecule, we show through guanidine hydrochloride-induced unfolding that the dimer separates into folded, but inactive, monomers before individual subunit unfolding takes place. Given that forces across the dimer interface are vital for thermostability, a robust computational method was derived that uses the University of Houston Brownian Dynamics (UHBD) program to calculate both the hydrophobic and electrostatic contribution to the dimerisation energy at 100°C. The results from computational and experimental determination of the lowered stability of interface mutants were correlated, being both of the same order of magnitude and placing the mutant proteins in the same order of stability. This computational method, optimised for hyperthermophilic molecules and tested in the laboratory, after further testing on other examples, could be of widespread use in the prediction of thermostabilising mutations in other oligomeric proteins for which dissociation is the first step in unfolding.     

Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance

Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance.     

The involvement of voltage-operated calcium channels in somato-dendritic oxytocin release

Magnocellular neurons of the supraoptic nucleus (SON) secrete oxytocin and vasopressin from axon terminals in the neurohypophysis, but they also release large amounts of peptide from their somata and dendrites, and this can be regulated both by activity-dependent Ca(2+) influx and by mobilization of intracellular Ca(2+). This somato-dendritic release can also be primed by agents that mobilise intracellular Ca(2+), meaning that the extent to which it is activity-dependent, is physiologically labile. We investigated the role of different Ca(2+) channels in somato-dendritic release; blocking N-type channels reduced depolarisation-induced oxytocin release from SONs in vitro from adult and post-natal day 8 (PND-8) rats, blocking L-type only had effect in PND-8 rats, while blocking other channel types had no significant effect. When oxytocin release was primed by prior exposure to thapsigargin, both N- and L-type channel blockers reduced release, while P/Q and R-type blockers were ineffective. Using confocal microscopy, we found immunoreactivity for Ca(v)1.2 and 1.3 channel subunits (which both form L-type channels), 2.1 (P/Q type), 2.2 (N-type) and 2.3 (R-type) in the somata and dendrites of both oxytocin and vasopressin neurons, and the intensity of the immunofluorescence signal for different subunits differed between PND-8, adult and lactating rats. Using patch-clamp electrophysiology, the N-type Ca(2+) current density increased after thapsigargin treatment, but did not alter the voltage sensitivity of the channel. These results suggest that the expression, location or availability of N-type Ca(2+) channels is altered when required for high rates of somato-dendritic peptide release.     

The HIV-1 Vpu viroporin inhibitor BIT225 does not affect Vpu-mediated tetherin antagonism

Among its many roles, the HIV-1 accessory protein Vpu performs a viroporin function and also antagonizes the host cell restriction factor tetherin through its transmembrane domain. BIT225 is a small molecule inhibitor that specifically targets the Vpu viroporin function, which, in macrophages, resulted in late stage inhibition of virus release and decreased infectivity of released virus, a phenotype similar to tetherin-mediated restriction. Here, we investigated whether BIT225 might mediate its antiviral function, at least in part, via inhibition of Vpu-mediated tetherin antagonism. Using T-cell lines inducible for tetherin expression, we found that BIT225 does not exert its antiviral function by inhibiting Vpu-mediated tetherin downmodulation from the cell surface, the main site of action of tetherin activity. In addition, results from a bioluminescence resonance energy transfer (BRET) assay showed that the Vpu-tetherin interaction was not affected by BIT225. Our data provide support for the concept that tetherin antagonism and viroporin function are separable on the Vpu transmembrane and that viroporin function might be cell-type dependent. Further, this work contributes to the characterization of BIT225 as an inhibitor that specifically targets the viroporin function of Vpu.     

Validation of lactose[15N,15N]ureide as a tool to study colonic nitrogen metabolism

In vitro experiments have shown that fermentation of carbohydrates prevents accumulation of nitrogen in the colon. Variable results have been obtained on modulation of dietary intakes in vivo. Lactose[15N,15N]-labeled ureide has been proposed as a tool to study colonic nitrogen metabolism. However, on oral administration of the marker, different urinary excretion patterns of the 15N label have been found. In this study, 50 mg lactose[15N,15N]ureide was directly instilled in the colon through an orocecal tube to investigate the colonic handling of this molecule in a direct way. In basal conditions, 42% (range, 37-48%) of labeled nitrogen administered as lactose[15N,15N]ureide was retrieved in urine after 72 h. A substantial variability in total urinary excretion of the label was found, but the urinary excretion pattern of the label was similar in all volunteers. When inulin, a fermentable carbohydrate, was administered together with the labeled marker, a significant decrease in urinary excretion of 15N after 72 h was found, to 29% (range, 23-34%). The effect of a smaller dose of inulin (250 mg) on colonic handling of lactose[15N,15N]ureide (50 mg), was investigated in another group of volunteers, and this time, fecal excretion of the marker was also evaluated. The results seem to indicate that fermentation of inulin causes an increased fecal excretion of the marker, thereby reducing urinary excretion but not retention in the human nitrogen pool. This instillation study shows that lactose[15N,15N]ureide is a tool with good properties to investigate the effect of different types of carbohydrates on nitrogen metabolism in the proximal colon in vivo.     

p38gamma regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP

Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38gamma MAP kinase (SAPK3/p38gamma) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38gamma-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular-junctional complexes, and cell shape and volume in response to osmotic stress.     

Maturation of escape circuit function during the early adulthood of cockroaches Periplaneta americana

During postembryonic development of insects, sensorimotor pathways, which generate specific behaviors, undergo maturational changes. It is less clear whether such pathways are typically stable, or undergo further maturation, during the adult stage. In the present study, we have examined this issue by multilevel analysis of a simple model system, the escape behavior of the cockroach, from identified synapses to behavior. We show that the escape system is highly responsive immediately after the molt to adulthood, but that the latency of escape responses was not at its typical value immediately after the molt to adult. The latency of escape behavior increased over the first 30 days of adult life, perhaps indicating maturational adjustments of the escape sensorimotor pathway. The first station in the escape circuitry is the synaptic connections between the cercal wind receptors and the giant interneurons. We measured unitary excitatory synaptic potentials between single sensory neurons and an identified giant interneuron (GI(2)). We found a decrease in the synaptic strength between identified cercal hairs from a single column and GI(2) over the first month after the adult molt. Consequently, the latency and the number of action potentials of GI(2) in response to natural stimuli increased and decreased respectively during this time. Thus, we show that both behavioral performance and the wind sensitivity of GI(2) decreased over the first month after molt. We conclude that the cockroach escape system undergoes further sensorimotor maturation over a period of 1 month, and that cellular changes correlate with, or predict, some changes in behavioral performance.     

Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein

Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein. Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 mug/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.