Enhanced neurite outgrowth in PC12 cells mediated by connexin hemichannels and ATP

Gap junctions have traditionally been described as transmembrane channels that facilitate intercellular communication via the passage of small molecules. Connexins, the basic building blocks of gap junctions, are expressed in most mammalian tissues including the developing and adult central nervous system. During brain development, connexins are temporally and spatially regulated suggesting they play an important role in the proper formation of the central nervous system. In the current study, connexins 32 and 43 were overexpressed in PC12 cells to determine whether connexins are involved in neuronal differentiation. Both connexin 32 and 43 were appropriately trafficked to the cell membrane following overexpression and resulted in the formation of functional gap junctions. Connexin overexpression was found to cause enhanced neurite outgrowth in PC12 cells treated with nerve growth factor to initiate neuritogenesis. Surprisingly, however, enhanced neurite outgrowth was found to be the consequence of functional hemichannel formation as opposed to traditional intercellular communication. Additional analysis revealed that ATP was released into the media likely through hemichannels and acted on purinergic receptors to cause enhanced neurite outgrowth. Collectively, the results of the current study suggest that connexins may play an important role in neuronal differentiation by non-traditional mechanisms.     

Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.     

<Superscript>19</Superscript>F labelled glycosaminoglycan probes for solution NMR and non-linear (CARS) microscopy

Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of ¹⁹F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing ¹⁹F NMR spectroscopy is followed. Furthermore, the ability of ¹⁹F labelled GAGs to be imaged using CARS microscopy is demonstrated. ¹⁹F labelled GAGs enable both ¹⁹F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.     

The mechanism of antimalarial action of the ruthenium(II)–chloroquine complex [RuCl2(CQ)]2

The mechanism of antimalarial action of the ruthenium-chloroquine complex [RuCl(2)(CQ)](2) (1), previously shown by us to be active in vitro against CQ-resistant strains of Plasmodium falciparum and in vivo against P. berghei, has been investigated. The complex is rapidly hydrolyzed in aqueous solution to [RuCl(OH(2))(3)(CQ)](2)[Cl](2), which is probably the active species. This compound binds to hematin in solution and inhibits aggregation to beta-hematin at pH approximately 5 to a slightly lower extent than chloroquine diphosphate; more importantly, the heme aggregation inhibition activity of complex 1 is significantly higher than that of CQ when measured at the interface of n-octanol-aqueous acetate buffer mixtures under acidic conditions modeling the food vacuole of the parasite. Partition coefficient measurements confirmed that complex 1 is considerably more lipophilic than CQ in n-octanol-water mixtures at pH approximately 5. This suggests that the principal target of complex 1 is the heme aggregation process, which has recently been reported to be fast and spontaneous at or near water-lipid interfaces. The enhanced antimalarial activity of complex 1 is thus probably due to a higher effective concentration of the drug at or near the interface compared with that of CQ, which accumulates strongly in the aqueous regions of the vacuole under those conditions. Furthermore, the activity of complex 1 against CQ-resistant strains of P. falciparum is probably related to its greater lipophilicity, in line with previous reports indicating a lowered ability of the mutated transmembrane transporter PfCRT to promote the efflux of highly lipophilic drugs. The metal complex also interacts with DNA by intercalation, to a comparable extent and in a similar manner to uncomplexed CQ and therefore DNA binding does not appear to be an important part of the mechanism of antimalarial action in this case.     

Clinical and preclinical evidence of sex-related differences in anthracycline-induced cardiotoxicity

Anthracyclines are very effective chemotherapeutic agents that are widely used to treat pediatric and adult cancer patients. Unfortunately, the clinical utility of anthracyclines is limited by cardiotoxicity. There are several established risk factors for anthracycline-induced cardiotoxicity (AIC), including total cumulative dose, very young and very old age, concomitant use of other cardiotoxic agents, and concurrent mediastinal radiation. However, the role of sex as a risk factor for AIC is not well defined. In pediatric cancer patients, most studies support the notion that female sex is a significant risk factor for AIC. Conversely, there is anecdotal evidence that female sex protects against AIC in adult cancer patients. The lack of consistency in study designs and the different definitions of cardiotoxicity preclude reaching consensus regarding the role of sex as a risk factor for AIC in both pediatric and adult cancer patients. Therefore, more clinical research using reliable techniques such as cardiac magnetic resonance imaging is needed to determine if there truly are sex differences in AIC. In adult preclinical rodent studies, however, there is unequivocal evidence that female sex confers significant protection against AIC, with a possible protective effect of female sex hormones and/or a detrimental role of the male sex hormones. Although findings of these rodent studies may not perfectly mirror the clinical scenario in adult anthracycline-treated cancer patients, understanding the mechanisms of this significant sexual dimorphism may reveal important cardioprotective mechanisms that can be therapeutically targeted.     

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa^−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.     

An Extended Phosphate Linkage: Synthesis,Hybridization and Modeling Studies of Modified Oligonucleotides

Abstract

Novel stretched oligonucleotides (A-D) containing a 3′-α-C-methylene phosphodiester bridge (5-atoms long) have been synthesized on an automated synthesizer utilizing phosphoramidite chemistry. The key building-block 1-[3′-O-β-cyanoethyldiisopropylaminophosphiryl-2,3-dideoxy-5-O-dimethoxytriphenylmethyl-3-C-(hydroxymethyl)-β-D-erythro-pentofuranosyl]thymine (21) was prepared in a stereoselective manner from thymidine. Hybridization studies indicated a drop (1.8–3.0°C/mod.) in affinity for the complementary RNA and DNA targets. Molecular modeling results indicated that the 5-atom modified backbone had a different geometry around the phosphodiester linkage compared to the natural phosphodiester linkage. The stretched backbone may not be useful for antisense or triplex constructs, however it may find applications in biochemical/enzyme studies.

     

Spatial pattern of habitat quality modulates population persistence in fragmented landscapes

Habitat quality is one of the important factors determining population dynamics and persistence, yet few studies have examined the effects of spatial heterogeneity in within-patch habitat quality. In this paper, we use a spatially explicit agent-based model to investigate how habitat fragmentation and spatial pattern of within-patch habitat quality affect population dynamics and long-term persistence. We simulate three levels of habitat fragmentation (ranges from continuous to highly fragmented) and three types of spatial patterns in habitat quality within patches (i.e., negatively autocorrelated, randomly distributed, and positively autocorrelated). Hypothetical species differ in their niche specialization. The results demonstrate explicitly that the spatial pattern of within-patch habitat quality plays an important role in modulating the effects of habitat fragmentation on populations. Populations become less variable in size, and experience lower probability of extinction in landscapes with positively autocorrelated within-patch habitat quality. Specifically, specialized species are more vulnerable to habitat fragmentation, but this vulnerability is greatly mitigated by positively autocorrelated habitat quality within patches, in other words, exhibiting higher resistance to habitat fragmentation. The findings of this study suggest that managing habitat quality in existing habitat remnants is important to preserve species in habitats undergoing fragmentation, particularly for those with specialized habitat requirements.     

Synthesis and SAR of aminothiazole fused benzazepines as selective dopamine D2 partial agonists

Dopamine (D₂) partial agonists (D2PAs) have been regarded as a potential treatment for schizophrenia patients with expected better side effect profiles than currently marketed antipsychotics. Herein we report the synthesis and SAR of a series of aminothiazole fused benzazepines as selective D₂ partial agonists. These compounds have good selectivity, CNS drug-like properties and tunable D₂ partial agonism. One of the key compounds, 8h, has good in vitro/in vivo ADME characteristics, and is active in a rat amphetamine-induced locomotor activity model.