Behavioural analyses of quinine processing in choice, feeding and learning of larval Drosophila

Gustatory stimuli can support both immediate reflexive behaviour, such as choice and feeding, and can drive internal reinforcement in associative learning. For larval Drosophila, we here provide a first systematic behavioural analysis of these functions with respect to quinine as a study case of a substance which humans report as "tasting bitter". We describe the dose-effect functions for these different kinds of behaviour and find that a half-maximal effect of quinine to suppress feeding needs substantially higher quinine concentrations (2.0 mM) than is the case for internal reinforcement (0.6 mM). Interestingly, in previous studies (Niewalda et al. 2008, Schipanski et al 2008) we had found the reverse for sodium chloride and fructose/sucrose, such that dose-effect functions for those tastants were shifted towards lower concentrations for feeding as compared to reinforcement, arguing that the differences in dose-effect function between these behaviours do not reflect artefacts of the types of assay used. The current results regarding quinine thus provide a starting point to investigate how the gustatory system is organized on the cellular and/or molecular level to result in different behavioural tuning curves towards a bitter tastant.     

Dynamic regulation of the cerebral cavernous malformation pathway controls vascular stability and growth

Cardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that?enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralog, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development, as well as postnatal vessel growth.     

Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli

Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: calcitonin like receptor (CLR), receptor activity modifying protein (RAMP1) and receptor component protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here, we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a maltose binding protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While His tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function.     

Antioxidant treatment protects diabetic rats from cardiac dysfunction by preserving contractile protein targets of oxidative stress

BackgoundAnimal studies suggest that reactive oxygen species (ROS) play an important role in the development of diabetic cardiomyopathy.HypothesisMatrix metalloproteinase-2 (MMP-2) is activated by ROS and contributes to the acute loss of myocardial contractile function by targeting and cleaving susceptible proteins including troponin I (TnI) and α-actinin.MethodsUsing the streptozotocin-induced diabetic rat model, we evaluated the effect of daily in vivo administration of sodium selenate (0.3 mg/kg; DMS group), or a pure omega-3 fish oil with antioxidant vitamin E (omega-3E; 50 mg/kg; DMFA group), which has antioxidant-like effects, for 4 weeks on heart function and on several biochemical parameters related to oxidant stress and MMP-2.ResultsAlthough both treatments prevented the diabetes-induced depression in left ventricular developed pressure (LVDP) as well as the rates of changes in developed pressure (±dP/dt) (P<.001), the improvement in LVDP of the DMS group was greater compared to that of the DMFA group (P<.001). Moreover, these treatments reduced the diabetes-induced increase in myocardial oxidized protein sulfhydryl and nitrite concentrations (P<.001). Gelatin zymography and Western blot data indicated that the diabetes-induced changes in myocardial levels of MMP-2 and tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) and the reduction in TnI and α-actinin protein levels were improved in both the DMS and DMFA groups (P<.001).ConclusionsThese results suggest that diabetes-induced alterations in MMP-2 and TIMP-4 contribute to myocardial contractile dysfunction by targeting TnI and α-actinin and that sodium selenate or omega-3E could have therapeutic benefits in diabetic cardiomyopathy.     

Effect of intraluminal pillars on particle motion in bifurcated microchannels

A central feature of intussusceptive angiogenesis is the development of an intravascular pillar that bridges the opposing sides of the microvessel lumen. In this report, we created polydimethyl siloxane (PDMS) microchannels with geometric proportions based on corrosion casts of the colon microcirculation. The structure of the PDMS microchannels was a bifurcated channel with an intraluminal pillar in the geometric center of the bifurcation. The effect of the intraluminal pillar on particle flow paths was investigated using an in vitro perfusion system. The microchannels were perfused with fluorescent particles, and the particle movements were recorded using fluorescence videomicroscopy. We found that the presence of an intravascular pillar significantly decreased particle velocity in the bifurcation system (p < 0.05). In addition, the pillar altered the trajectory of particles in the center line of the flow stream. The particle trajectory resulted in prolonged pillar contact as well as increased residence time within the bifurcation system (p < 0.001). Our results suggest that the intravascular pillar not only provides a mechanism of increasing resistance to blood flow but may also participate in spatial redistribution of cells within the flow stream. Supported in part by NIH Grants HL47078, HL75426, HL054885, HL070542 and HLO74022.     

Interstitial Lung Disease in Coppersmiths in High Serum Copper Levels

Coppersmith is a worker who uses copper most commonly for the production of kitchen appliances in Turkey. This is an ancient occupation practiced for centuries in Turkey. Our objective was to investigate the prevalence of parenchymal lung diseases among coppersmiths in Kahramanmaras city in Turkey. Thirty coppersmiths were included to the study, and they all signed an informed consent. Demographics, spirometric test results and high-resolution computed tomography (HRCT) scans, and blood samples were obtained. Laboratory analysis of the serum samples showed that serum copper levels of the subjects were 0.93 ± 0.14 mg/L. Serum copper level in control group was found as 0.70 ± 0.14 mg/L, and it was significantly different between the two groups (p < 0.05). Of 30 coppersmiths, 17 HRCT findings are abnormal and seen with diffuse parenchymal interstitial lung disease pattern—ten (58.8%) respiratory bronchiolitis interstitial lung disease, five (29.4%) nonspecific interstitial pneumonia, and two (11.8%) usual interstitial pneumonia. The most prevalent HRCT pattern was micronodular pattern in workers. This is the first field study reporting the radiologic findings of coppersmiths and effect of the occupation on lung diseases.     

Effects of brassinosteroids on barley root growth,antioxidant system and cell division

Homobrassinolide (HBR), which is one of the most biologically active forms of Brassinosteroids (BRs), was used to examine the potential effects of hormone on root germination, antioxidant system enzymes and cell division of barley (Hordeum vulgare L.). Seeds were germinated between filter papers in 0.1, 0.5 and 1.0 μM HBR-supplemented distilled water for 48 h at dark with their controls. HBR application increased especially the primary root growth significantly with increasing concentrations when compared with the control materials and reached two fold increase in 1.0 μM HBR treated material. Treated and untreated control group roots were fixed in 1:3 aceto-alcohol and aceto-orcein preparations were made. Roots treated with HBR showed more mitotic activity, mitotic abnormalities and significant enlargements at the root tips when compared with control material. HBR application decreased total soluble protein content, superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.11) activities significantly at 1.0 μM HBR concentration. Data presented here is one of the first detailed analyses of HBR effect on barley root development.     

Should chronic obstructive pulmonary disease outpatients be routinely evaluated for trace elements?

We searched for serum concentrations of trace elements and correlated them to malondialdehyde (MDA), which is an indirect marker of oxidative stress, in order to clarify if routine evaluation is necessary in chronic obstructive pulmonary disease (COPD) outpatients. Serum concentrations of copper (Cu), zinc (Zn), and magnesium (Mg) were determined by atomic absorption spectrophotometry and iron (Fe) by a ILLab 1800 autoanalyzer with ILLab test kits. Serum MDA concentrations were detected in terms of TBARS (thiobarbituric acid reactive substances) spectrophotometrically. Serum Cu, Zn, Mg, Fe, and MDA concentrations in patient and control groups were all in the normal reference range. The results respectively were as follows: Cu:123±29.2 and 122.2±23.4 μg/dL; Zn: 87.8±17.8 and 96.9 ± 12.9 μg/dL; Mg: 2.3±0,5 and 2.04±0.28 mg/dL; Fe: 73.8±35.5 and 80.7±51.2 μg/dL; MDA: 1.09±0.11 and 0.95±0.06 nmol/L. MDA was not correlated to Cu, Zn, Mg, or Fe (p>0.05 for all). The serum Zn concentration of COPD group was lower than the control group (p=0.042), whereas the Mg concentration was higher (p=0.021). There was no statistical difference in other study parameters. Oxidative stress was not increased in clinically stable, regularly treated COPD patients. Although there was no deficiency in trace elements (Cu, Fe, Mg, and Zn), serum Zn was close to the lower limit of the reference value. There is no need for routine evaluation of trace elements in clinically stable, regularly treated COPD outpatients.