Characterization of the membrane-destabilizing properties of different pH-sensitive methacrylic acid copolymers

The intracellular delivery of active biomacromolecules from endosomes into the cytoplasm generally requires a membrane-disrupting agent. Since endosomes have a slightly acidic pH, anionic carboxylated polymers could be potentially useful for this purpose since they can destabilize membrane bilayers by pH-triggered conformational change. In this study, five different pH-sensitive methacrylic acid (MAA) copolymers were characterized with respect to their physicochemical and membrane lytic properties as a function of pH. pH-dependent conformational changes were studied in aqueous solution by turbidimetry and spectrofluorimetry. The hydrophobic domains that formed upon a decrease in pH were found to be dependent on copolymer's composition. Hemolysis and cytotoxicity assays demonstrated that the presence of the hydrophobic ethyl acrylate monomer and/or sufficient protonation of the carboxylic acid groups were important parameters for efficient membrane destabilization. Excessive copolymer hydrophobicity was not associated with membrane destabilization, but resulted in high macrophage cytotoxicity. Overall, this study gave more insights into the structure-activity relationship of MAA copolymers with membrane bilayers. Gaining knowledge of modulation of the physicochemical properties of copolymers and the optimization of copolymer-lipid interactions may lead to the elaboration of much more efficient drug delivery systems.     

Dichotomous organization of the external globus pallidus

Different striatal projection neurons are the origin of?a?dual organization essential for basal ganglia function. We have defined an analogous division of labor in the external globus pallidus (GPe) of Parkinsonian rats, showing that the distinct temporal activities of two populations of GPe neuron in?vivo are?underpinned by distinct molecular profiles and axonal connectivities. A first population of prototypic GABAergic GPe neurons fire antiphase to subthalamic nucleus (STN) neurons, often express parvalbumin, and target downstream basal ganglia nuclei, including STN. In contrast, a second population (arkypallidal neurons) fire in-phase with STN neurons, express preproenkephalin, and only innervate the striatum. This novel cell type provides the largest extrinsic GABAergic innervation of striatum, targeting both projection neurons and interneurons. We conclude that GPe exhibits several core components of?a dichotomous organization as fundamental as?that in striatum. Thus, two populations of GPe neuron?together orchestrate activities across all basal ganglia nuclei in a cell-type-specific manner.     

Effects of Enriched Environment on COX-2, Leptin and Eicosanoids in a Mouse Model of Breast Cancer

Cyclooxygenase-2 (COX-2) and adipokines have been implicated in breast cancer. This study investigated a possible link between COX-2 and adipokines in the development of mammary tumors. A model of environmental enrichment (EE), known to reduce tumor growth was used for a syngeneic murine model of mammary carcinoma. 3-week-old, female C57BL/6 mice were housed in standard environment (SE) or EE cages for 9 weeks and transplanted orthotopically with syngeneic EO771 adenocarcinoma cells into the right inguinal mammary fat pad. EE housing influenced mammary gland development with a decrease in COX-2 expressing cells and enhanced side-branching and advanced development of alveolar structures of the mammary gland. Tumor volume and weight were decreased in EE housed mice and were associated with a reduction in COX-2 and Ki67 levels, and an increase in caspase-3 levels. In tumors of SE mice, high COX-2 expression correlated with enhanced leptin detection. Non-tumor-bearing EE mice showed a significant increase in adiponectin levels but no change in those of leptin, F₂-isoprostanes, PGF_2α, IL-6, TNF-α, PAI-1, and MCP-1 levels. Both tumor-bearing groups (SE and EE housing) had increased resistin, IL-6, TNF-α, PAI-1 and MCP-1 levels irrespective of the different housing environment demonstrating higher inflammatory response due to the presence of the tumor. This study demonstrates that EE housing influenced normal mammary gland development and inhibited mammary tumor growth resulting in a marked decrease in intratumoral COX-2 activity and an increase in the plasma ratio of adiponectin/leptin levels.     

Neuroprotectin D1 Induces Neuronal Survival and Downregulation of Amyloidogenic Processing in Alzheimer’s Disease Cellular Models

The mediator neuroprotectin D1 (NPD1) is an enzymatic derivative of the omega-3 essential fatty acid docosahexaenoic acid. NPD1 stereoselectively and specifically binds to human retinal pigment epithelium (RPE) cells and neutrophils. In turn, this lipid mediator induces dephosphorylation of Bcl-x_L in a PP2A-dependent manner and induces PI3K/Akt and mTOR/p70S6K pathways leading to RPE cell survival during oxidative stress-induced apoptosis. As a proof of principle of its systemic in vivo bioactivity, NPD1 attenuates laser-induced choroidal neovascularization in mice. Using human neural cells transfected with amyloid precursor protein (APP)sw (Swedish double mutation APP695sw, K595N, M596L), NPD1 was shown to regulate secretase-mediated production of Aβ peptide, downregulates pro-inflammatory gene expression, and promotes cell survival. In human neural cells overexpressing beta-amyloid precursor protein (βAPP), the lipid mediator suppressed Aβ42 shedding by downregulating β-secretase (BACE1) while activating the α-secretase (ADAM10), thus shifting the βAPP cleavage from the noxious amyloidogenic pathway into a non-amyloidogenic, neurotrophic pathway. Furthermore, downregulation of Aβ42 peptide release by NPD1 may be dependent upon PPARγ activation. In conclusion, NPD1 exhibits anti-inflammatory, anti-amyloidogenic, and anti-apoptotic bioactivities in human neural cells in part via PPARγ signaling and through the targeting of α- and β-secretase systems.     

Apelin and the proopiomelanocortin system: a new regulatory pathway of hypothalamic α-MSH release

Neuronal networks originating in the hypothalamic arcuate nucleus (Arc) play a fundamental role in controlling energy balance. In the Arc, neuropeptide Y (NPY)-producing neurons stimulate food intake, whereas neurons releasing the proopiomelanocortin (POMC)-derived peptide α-melanocyte-stimulating hormone (α-MSH) strongly decrease food intake. There is growing evidence to suggest that apelin and its receptor may play a role in the central control of food intake, and both are concentrated in the Arc. We investigated the presence of apelin and its receptor in Arc NPY- and POMC-containing neurons and the effects of apelin on α-MSH release in the hypothalamus. We showed, by immunofluorescence and confocal microscopy, that apelin-immunoreactive (IR) neuronal cell bodies were distributed throughout the rostrocaudal extent of the Arc and that apelin was strongly colocalized with POMC, but weakly colocalized with NPY. However, there were numerous NPY-IR nerve fibers close to the apelin-IR neuronal cell bodies. By combining in situ hybridization with immunohistochemistry, we demonstrated the presence of apelin receptor mRNA in Arc POMC neurons. Moreover, using a perifusion technique for hypothalamic explants, we demonstrated that apelin-17 (K17F) increased α-MSH release, suggesting that apelin released somato-dendritically or axonally from POMC neurons may stimulate α-MSH release in an autocrine manner. Consistent with these data, hypothalamic apelin levels were found to be higher in obese db/db mice and fa/fa Zucker rats than in wild-type animals. These findings support the hypothesis that central apelin is involved in regulating body weight and feeding behavior through the direct stimulation of α-MSH release.     

A New Method for Locating Changes in a Tree Reveals Distinct Nucleotide Polymorphism vs Divergence Patterns in Mouse Mitochondrial Control Region

A new, model-based method was devised to locate nucleotide changes in a given phylogenetic tree. For each site, the posterior probability of any possible change in each branch of the tree is computed. This probabilistic method is a valuable alternative to the maximum parsimony method when base composition is skewed (i.e., different from 25% A, 25% C, 25% G, 25% T): computer simulations showed that parsimony misses more rare → common than common → rare changes, resulting in biased inferred change matrices, whereas the new method appeared unbiased. The probabilistic method was applied to the analysis of the mutation and substitution processes in the mitochondrial control region of mouse. Distinct change patterns were found at the polymorphism (within species) and divergence (between species) levels, rejecting the hypothesis of a neutral evolution of base composition in mitochondrial DNA. Received: 15 March 1999 / Accepted: 7 October 1999     

1-Thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives as antitumor agents

A class of substituted 1-thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives was found to have potent anti-proliferative activity against a broad range of tumor cell lines. A compound from this class (14) was profiled across a broad panel of hematologic and solid tumor cancer cell lines demonstrating cell cycle arrest at the G0/G1 interphase and has potent anti-proliferative activity against a distinct and select set of cancer cell types with no observed effects on normal human cells. An example is the selective inhibition of human B-cell lymphoma cell line (BJAB). Compound 14 was orally bioavailable and tolerated well in mice. Synthesis and structure activity relationships (SAR) in this series of compounds are discussed.     

Niche partitioning of the genetic lineages of the oak powdery mildew complex

Recent molecular studies have shown that four genetic lineages, probably cryptic species, cause oak powdery mildew in Europe. We tested the hypothesis of niche differentiation between the two most frequent species (Erysiphe alphitoides and Erysiphe quercicola), by determining their relative prevalences at various times during the growing season. E. quercicola was strictly associated with the flag-shoot symptom in a 2-yr sampling campaign of 35 natural oak seedling populations. Additional sampling during Aug.–Sep. in the same stands showed that E. alphitoides predominated in most (70 %) cases. Time-course monitoring of powdery mildew in two natural regenerating oak populations confirmed the inverse patterns of relative abundance for these two species in early and late season. The coexistence of these two closely related species may be due to the use of different strategies, resulting from a trade-off between overwintering (flag-shoots vs. chasmothecia) and late-season performance.     

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.