Measurements of mitochondrial K+ fluxes in whole rat hearts using 87Rb-NMR

The rubidium efflux from hypothermic rat hearts perfused by theLangendorff method at 20°C was studied. At thistemperature ⁸⁷Rb-NMR efflux experiments showed theexistence of two ⁸⁷Rb pools: cytoplasmic and mitochondrial.Rat heart mitochondria showed a very slow exchange of mitochondrialRb⁺ for cytoplasmic K⁺. After washout ofcytosolic Rb⁺, mitochondria kept a stable Rb⁺level for >30 min. Rb⁺ efflux from mitochondria wasstimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmalpermeabilization and concomitant cellular energy depletion by saponin(0.01 mg/ml for 4 min) in the presence of a perfusate mimickingintracellular conditions, or by ATP-sensitive K (K_ATP)channel openers. DNP, a mitochondrial uncoupler, caused the onset ofmitochondrial Rb⁺ exchange; however, the washout was notcomplete (80 vs. 56% in control). Energy deprivation by saponin, whichpermeabilizes the sarcolemma, resulted in a rapid and completeRb⁺ efflux. The mitochondrial Rb⁺ efflux rateconstant (k) decreased in the presence of glibenclamide, aK_ATP channel inhibitor (5 µM;k = 0.204 ± 0.065 min¹; n = 8),or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM,respectively; k = 0.134 ± 0.021 min¹;n = 4) in the saponin experiments (saponin only;k = 0.321 ± 0.079 min¹; n = 3),indicating the inhibition of mitochondrial K_ATP channels. Thus hypothermia in combination with ⁸⁷Rb-NMR allowed theprobing of the mitochondrial K⁺ pool in whole heartswithout mitochondrial isolation.

     

Children’s Beliefs about Animal Minds (Child-BAM): Associations with Positive and Negative Child–Animal Interactions

Children and animals can have a great impact on each other’s lives, yet little is known about the underpinnings of these relationships. Children’s interactions with animals may be influenced by their belief in animal minds, that animals are sentient and experience thoughts and feelings. This study introduces a newly developed measure of children’s beliefs about animal minds (Child-BAM) and investigates associations between it and factors relating to positive and negative interactions with animals. Using a questionnaire-based survey of over 1,000 6- to 13-year-olds in the UK, the results show that Child-BAM was associated with higher attachment to pets (p < 0.001), compassion (p < 0.001), humane (p < 0.001) and caring behavior (p < 0.001) toward animals, emotional attachment to animals (p = 0.003), and positive attitudes toward animals (p < 0.001). Child-BAM was also associated with less acceptance of intentional animal cruelty (p = 0.001), unintentional animal cruelty (p = 0.007), and animal neglect (p = 0.01). There was a significant difference in Child-BAM between children with pets (p = 0.013), children who had a pet of their own (p = 0.016), and age group (p < 0.001). This study enhances our understanding of the psychological underpinnings of child–animal relationships and highlights the implications for animal welfare education and for preventing childhood animal cruelty.     

Practical Methodology of Cognitive Tasks Within a Navigational Assessment

This paper describes an approach for measuring navigation accuracy relative to cognitive skills. The methodology behind the assessment will thus be clearly outlined in a step-by-step manner. Navigational skills are important when trying to find symbols within a speech-generating device (SGD) that has a dynamic screen and taxonomical organization. The following skills have been found to impact children’s ability to find symbols when navigating within the levels of an SGD: sustained attention, categorization, cognitive flexibility, and fluid reasoning^1,2. According to past studies, working memory was not correlated with navigation^1,2.The materials needed for this method include a computerized tablet, an augmentative and alternative communication application, a booklet of symbols, and the Leiter International Performance Scale-Revised (Leiter-R)³. This method has been used in two previous studies. Robillard, Mayer-Crittenden, Roy-Charland, Minor-Corriveau and Bélanger¹ assessed typically developing children, while Rondeau, Robillard and Roy-Charland² assessed children and adolescents with a diagnosis of Autism Spectrum Disorder. The direct observation of this method will facilitate the replication of this study for researchers. It will also help clinicians that work with children who have complex communication needs to determine the children’s ability to navigate an SGD with taxonomical categorization.     

Genome Sequence of the Cellulolytic Gliding Bacterium Cytophaga hutchinsonii

The complete DNA sequence of the aerobic cellulolytic soil bacterium Cytophaga hutchinsonii, which belongs to the phylum Bacteroidetes, is presented. The genome consists of a single, circular, 4.43-Mb chromosome containing 3,790 open reading frames, 1,986 of which have been assigned a tentative function. Two of the most striking characteristics of C. hutchinsonii are its rapid gliding motility over surfaces and its contact-dependent digestion of crystalline cellulose. The mechanism of C. hutchinsonii motility is not known, but its genome contains homologs for each of the gld genes that are required for gliding of the distantly related bacteroidete Flavobacterium johnsoniae. Cytophaga-Flavobacterium gliding appears to be novel and does not involve well-studied motility organelles such as flagella or type IV pili. Many genes thought to encode proteins involved in cellulose utilization were identified. These include candidate endo-β-1,4-glucanases and β-glucosidases. Surprisingly, obvious homologs of known cellobiohydrolases were not detected. Since such enzymes are needed for efficient cellulose digestion by well-studied cellulolytic bacteria, C. hutchinsonii either has novel cellobiohydrolases or has an unusual method of cellulose utilization. Genes encoding proteins with cohesin domains, which are characteristic of cellulosomes, were absent, but many proteins predicted to be involved in polysaccharide utilization had putative D5 domains, which are thought to be involved in anchoring proteins to the cell surface.     

Regulation of prolactin in mice with altered hypothalamic melanocortin activity

This study used two mouse models with genetic manipulation of the melanocortin system to investigate prolactin regulation. Mice with overexpression of the melanocortin receptor (MC-R) agonist, α-melanocyte-stimulating hormone (Tg-MSH) or deletion of the MC-R antagonist agouti-related protein (AgRP KO) were studied. Male Tg-MSH mice had lower blood prolactin levels at baseline (2.9±0.3 vs. 4.7±0.7ng/ml) and after restraint stress (68±6.5 vs. 117±22ng/ml) vs. WT (p<0.05); however, pituitary prolactin content was not different. Blood prolactin was also decreased in male AgRP KO mice at baseline (4.2±0.5 vs. 7.6±1.3ng/ml) and after stress (60±4.5 vs. 86.1±5.7ng/ml) vs. WT (p<0.001). Pituitary prolactin content was lower in male AgRP KO mice (4.3±0.3 vs. 6.7±0.5μg/pituitary, p<0.001) vs. WT. No differences in blood or pituitary prolactin levels were observed in female AgRP KO mice vs. WT. Hypothalamic dopamine activity was assessed as the potential mechanism responsible for changes in prolactin levels. Hypothalamic tyrosine hydroxylase mRNA was measured in both genetic models vs. WT mice and hypothalamic dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content were measured in male AgRP KO and WT mice but neither were significantly different. However, these results do not preclude changes in dopamine activity as dopamine turnover was not directly investigated. This is the first study to show that baseline and stress-induced prolactin release and pituitary prolactin content are reduced in mice with genetic alterations of the melanocortin system and suggests that changes in hypothalamic melanocortin activity may be reflected in measurements of serum prolactin levels.     

Associations between Retinal Markers of Microvascular Disease and Cognitive Impairment in Newly Diagnosed Type 2 Diabetes Mellitus: A Case Control Study

Objective

To investigate associations between retinal microvascular changes and cognitive impairment in newly diagnosed type 2 diabetes mellitus.

Design

Case control study.

Setting

A primary care cohort with newly diagnosed type 2 diabetes mellitus.

Methods

For this analysis, we compared 69 cases with lowest decile scores (for the cohort) on the Modified Telephone Interview for Cognitive Status and 68 controls randomly selected from the remainder of the cohort. Retinal images were rated and the following measures compared between cases and controls: retinal vessel calibre, arterio-venous ratio, retinal fractal dimension, and simple and curvature retinal vessel tortuosity.

Results

Total and venular (but not arteriolar) simple retinal vessel tortuosity levels were significantly higher in cases than controls (t = 2.45, p = 0.015; t = 2.53, p = 0.013 respectively). The associations persisted after adjustment for demographic factors, retinopathy, neuropathy, obesity and blood pressure. There were no other significant differences between cases and controls in retinal measures.

Conclusions

A novel association was found between higher venular tortuosity and cognitive impairment in newly diagnosed type 2 diabetes mellitus. This might be accounted for by factors such as hypoxia, thrombus formation, increased vasoendothelial growth factor release and inflammation affecting both the visible retinal and the unobserved cerebral microvasculature.     

Spin label reduction kinetics,a procedure to study the effect of drugs on membrane permeability: The effects of monosodium urate,dimethyl sulfoxide and amphotericin B

A method is presented to study the effect of drugs on membrane permeability. It is based on the reduction of a spin label trapped in the internal aqueous compartment(s) of membranes by ascorbate ions added to the bulk aqueous phase. The decay of the electron spin resonance signal of the spin label as a function of time gives an indication of the effect of added agents on the permeability of membranes. To demonstrate the technique, the effect on model membranes of egg phosphatidylcholine of the gout-implicated compound monosodium urate, the aprotic solvent dimethyl sulfoxide and the polyene antibiotic amphotericin B were examined. Monosodium urate did not affect the permeability, casting doubt on a proposed mechanism whereby the agent disrupts the membranes via hydrogen bonding. Dimethyl sulfoxide promoted a gradual increase in rate of solute passage across cholesterol-containing model membranes. Amphotericin B had pronounced effect on the permeability of cholesterol-containing membranes, causing nearly total loss of paramagnetism immediately after addition. Some aspects of the mechanism of action of the drugs are discussed as well as the advantages and disadvantages of the method. The experiments also allow the evaluation of the effect of surface charge and cholesterol on the dimensions of model membranes.     

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.Innate immunity is the first line of immune defense and is common to all metazoans (1, 2). In this immune system, macrophages play a crucial role in the maintenance of tissue homeostasis. These cells are involved in almost every disease through their immunological and wound-healing functions (1, 2). During a pathogenic infection, trauma or neurodegeneration, macrophages are recruited and activated contributing to the phagocytosis of pathogens and the secretion of cytokines and chemokines activating other immune cells. Macrophages can develop into classically pro-inflammatory (M1) or alternatively (M2) activated macrophages. M1 macrophages are characterized by the secretion of pro-inflammatory cytokines whereas M2 macrophages secrete anti-inflammatory cytokines (3). Stimulation of macrophages with LPS activates TLR4 signaling leading to the nucleus translocation of NF-κB or IRF3 which activate genes encoding proteins involved in innate immune response (4). Many of these proteins are secreted (cytokines, chemokines…) to attract and activate other immune cells like T lymphocytes. In tumors, macrophages are oriented toward the M2 phenotype and promote cancer growth by suppressing immune cells function (5). Current research in the therapeutic field focus on ways to reactivate macrophages.Surprisingly, we have shown that during immune responses, macrophages secrete typical neuroendocrine molecules (6–8), such as neuropeptides (9) or the proprotein convertases (PC)¹ PC2 and PC1/3 and that PC1/3 is an important regulator of innate immune responses (10–12). Proprotein convertases cleave precursor proteins which can lead to the activation, inactivation or functional changes. PC2 and PC1/3 operate within the regulated secretory pathway. Their expression is not restricted to neuroendocrine tissues, they are also expressed in macrophages and lymphocytes (12). In a previous study from our group, PC1/3 knockout (KO) in mice challenged with LPS caused innate immune defects and uncontrolled cytokine secretion (10). Th1 pathway is enhanced in PC1/3 KO mice. Following LPS treatment, PC1/3 colocalized with TLR4 in the endosomal compartment (11). We concluded that PC1/3 contributes to the regulation of TLR4 signaling and the resulting cytokine secretion.The NR8383 rat pulmonary macrophage cell line was previously shown as a good model to study the role of PC1/3 in the macrophage innate immune response (13). In the present study, we developed a PC1/3-knockdown (K_D) NR8383 cell line using lentiviral-delivered shRNAs. Our aim is to understand the cellular impact of PC1/3 inhibition in macrophages and the consequences on their activation. Proteomic analysis of secreted proteins allowed us to identify pro-inflammatory cytokines and alarmins already at 24h of LPS stimulation in PC1/3-K_D secretomes which was confirmed by cytokines array. Proteomic studies of PC1/3-K_D NR8383 cellular extracts revealed an important perturbation in the intracellular trafficking machinery through the disorganization of cytoskeletal protein expression. These results were confirmed on macrophages from PC1/3 KO mice. Cytokines secretion and cytoskeleton reorganization can be linked to intracellular calcium increase in PC1/3-K_D cells. Moreover, we showed that MyD88-dependant TLR4 signaling was sustained when PC1/3 is down-regulated. We describe here that inhibition of PC1/3 induced classically activated phenotype (M1) in macrophages. The chemotactic and anti-tumor properties of the PC1/3-K_D macrophage secretome promoted the cytotoxic immune response and inhibited cancer cell viability. The down-regulation of PC1/3 could be used in cancer immunotherapy to reactivate macrophages.     

(±)-2-(N-tert-Butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone: a dopamine transporter and nicotinic acetylcholine receptor photoaffinity ligand based on bupropion (Wellbutrin, Zyban)

Towards addressing the knowledge gap of how bupropion interacts with the dopamine transporter (DAT) and nicotinic acetylcholine receptors (nAChRs), a ligand was synthesized in which the chlorine of bupropion was isosterically replaced with an iodine and a photoreactive azide was added to the 4'-position of the aromatic ring. Analog (±)-3 (SADU-3-72) demonstrated modest DAT and α4β2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4β2 neuronal nAChRs. Co-incubation of (±)-[(125)I]-3 with non-radioactive (±)-bupropion or (-)-cocaine blocked labeling of these proteins. Compound (±)-[(125)I]-3 represents the first successful example of a DAT and nAChR photoaffinity ligand based on the bupropion scaffold. Such ligands are expected to assist in mapping bupropion-binding pockets within plasma membrane monoamine transporters and ligand-gated nAChR ion channels.