Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases

In the preceding paper (Ellens et al., 1989), it was shown that liposome fusion rates are substantially enhanced under the same conditions which induce isotropic 31P NMR resonances in multilamellar dispersions of the same lipid. Both of these phenomena occur within the same temperature interval, delta TI, below the L alpha/HII phase transition temperature, TH. TH and delta TI can be extremely sensitive to the lipid composition. The present work shows that 2 mol% of diacylglycerols like those produced by the phosphatidylinositol cycle in vivo can lower TH, delta TI, and the temperature for fast membrane fusion by 15-20 degrees C. N-Monomethylated dioleoylphosphatidylethanolamine is used as a model system. These results show that physiological levels of diacylglycerols can substantially increase the susceptibility of phospholipid membranes to fusion. This suggests that, in addition to their role in protein kinase C activation, diacylglycerols could play a more direct role in the fusion event during stimulus-exocytosis coupling in vivo.     

Colour association influences honey bee choice between sucrose concentrations

Certain colours associated with floral food resources are more quickly learned by honey bees (Apis mellifera) than are other colours. But the impact of colour, and other floral cues, on bee choice behaviour has not yet been determined. In these experiments, colour association and sugar concentration of reward were varied to assess how they interact to affect bee choice behaviour. Thirty-five bees were individually given binary choices between blue and yellow artificial flowers that contained either the same rewards or rewards of different sucrose concentrations. Honey bee choice between sucrose concentrations was affected by colour association and this effect was greatest when absolute difference between rewards was the lowest. The honey bee's ability to maximize energetic profitability during foraging is constrained by floral cue effectiveness.     

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.     

Effect ofPseudomonas aeruginosa rhamnolipid on human neutrophil migration

The effect ofPseudomonas aeruginosa heat-stable hemolysin (rhamnolipid) on human neutrophil migration has been investigated. Rhamnolipid was prepared from culture filtrate and characterized by thin-layer chromatography. The lytic activity of rhamnolipid was quantitated by titration against neutrophils. Leukocyte migration response was measured using⁵¹Cr-labeled neutrophils with a double-filter technique in modified Boyden chambers. The results suggest rhamnolipid stimulated chemotaxis as well as chemokinesis. Moreover, rhamnolipid impaired a chemotactic response toN-formyl-methionyl-leucyl-phenylalanine. These effects may be important in host-parasite interactions.     

Lamellar/inverted cubic (L alpha/QII) phase transition in N-methylated dioleoylphosphatidylethanolamine

Inverted cubic (QII) phases form in hydrated N-methylated dioleoylphosphatidylethanolamine (DOPE-Me). Previous work indicated that QII phases in this and other systems might be metastable structures. Whether or not QII phases are stable has important implications for models of the factors determining the relative stability of bilayer and nonbilayer phases and of the mechanisms of transitions between those phases. Here, using X-ray diffraction and very slow scan rate differential scanning calorimetry (DSC), we show that thermodynamically stable QII phases form slowly during incubation of multilamellar samples of DOPE-Me at constant temperature. The equilibrium L alpha/QII phase transition temperature is 62.2 +/- 1 degree C. The transition enthalpy is 174 +/- 34 cal/mol, about two-thirds of the L alpha/HII transition enthalpy observed at faster scan rates. This implies that the curvature free energy of lipids in QII phases is substantially lower than in L alpha phases and that this reduction is substantial compared to the reduction achieved in the HII phase. The L alpha/QII transition is slow and is not reliably detected with DSC until the temperature scan rate is reduced to ca. 1 degrees C/h. At faster scan rates, the HII phase forms at a reproducible temperature of 66 degrees C. This HII phase is metastable until ca. 72-79 degrees C, where the equilibrium QII/HII transition seems to occur. These results, as well as the induction of QII phases in similar systems by temperature cycling (observed by others), are consistent with a theory of L alpha/QII/HII transition mechanisms proposed earlier (Siegel, 1986c).     

Adenosine 2A Receptor Antagonist Prevented and Reversed Liver Fibrosis in a Mouse Model of Ethanol-Exacerbated Liver Fibrosis

The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl₄, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl₄) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl₄, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl₄. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl_4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis.

Conclusion

Moderate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl₄. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Evaluating outcomes of restoration ecology projects on limited budgets: assessment of variation in sampling intensity and sampling frequency for four habitat types

While best practices for evaluating restoration ecology projects are emerging rapidly, budget constraints often limit postrestoration monitoring, which emphasizes the need for practical and efficient monitoring strategies. We examined the postrestoration outcome for an ENGO (Nature Conservancy of Canada) project, to assess retroactively how variation in intensity and frequency of sampling would have affected estimates of plant species composition, diversity, and richness over time. The project restored four habitat types (mesic forest, oak woodland, wet meadow, and sand barren) using sculptured seeding of tallgrass prairie and woody species. Species‐level plant cover was monitored annually for 10 years in 168 2 × 2–m quadrats. We performed randomization tests to examine estimates of species diversity and richness as a function of the number of quadrats sampled, and assessed the necessity of annual sampling for describing changes in species composition and successional trajectories. The randomization tests revealed that sampling 10–17 quadrats, depending on habitat type, was sufficient to obtain estimates of species diversity that were at least 95% of values obtained from the whole dataset. Species richness as a function of number of quadrats sampled did not plateau, which suggests that rather than increasing the number of sampling quadrats, richness could be estimated more efficiently using nonquadrat based sampling techniques. Nonmetric multidimensional scaling analysis revealed that plant species composition largely stabilized by 3–5 years postrestoration depending on habitat type. By that time, native, seeded species dominated the restoration, and the benefits of annual sampling for tracking changes in species composition diminished.     

HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I

HIV-1 Nef, which is required for the efficient onset of AIDS, enhances viral replication and infectivity by exerting multiple effects on infected cells. Nef downregulates cell-surface MHC-I molecules by an uncharacterized PI3K pathway requiring the actions of two Nef motifs-EEEE(65) and PXXP(75). We report that the Nef EEEE(65) targeting motif enables Nef PXXP(75) to bind and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K to trigger MHC-I downregulation in primary CD4+ T cells. In promonocytic cells, Nef/SFK recruits the ZAP-70 homolog Syk to downregulate MHC-I, implicating this PI3K pathway in multiple HIV-1 reservoirs. Isoform-specific PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agents to combat HIV-1. The discovery of this Nef-SFK-ZAP-70/Syk-PI3K signaling pathway explains the hierarchal role of the Nef motifs in effecting immunoevasion.