A role for TASK-1 (KCNK3) channels in the chemosensory control of breathing

Acid-sensitive K+ channels of the tandem P-domain K+-channel family (TASK-1 and TASK-3) have been implicated in peripheral and central respiratory chemosensitivity; however, because of the lack of decisive pharmacological agents, the final proof of the role of the TASK channel in the chemosensory control of breathing has been missing. In the mouse, TASK-1 and TASK-3 channels are dispensable for central respiratory chemosensitivity (Mulkey et al., 2007Go). Here, we have used knock-out animals to determine whether TASK-1 and TASK-3 channels play a role in the carotid body function and chemosensory control of breathing exerted by the carotid body chemoreceptors. Ventilatory responses to hypoxia (10% O2 in inspired air) and moderate normoxic hypercapnia (3–6% CO2 in inspired air) were significantly reduced in TASK-1 knock-out mice. In contrast, TASK-3-deficient mice showed responses to both stimuli that were similar to those developed by their wild-type counterparts. TASK-1 channel deficiency resulted in a marked reduction of the hypoxia (by 49%)- and CO2 (by 68%)-evoked increases in the carotid sinus nerve chemoafferent discharge recorded in the in vitro superfused carotid body/carotid sinus nerve preparations. Deficiency in both TASK-1 and TASK-3 channels increased baseline chemoafferent activity but did not cause a further reduction of the carotid body chemosensory responses. These observations provide direct evidence that TASK-1 channels contribute significantly to the increases in the carotid body chemoafferent discharge in response to a decrease in arterial PO2 or an increase in PCO2/[H+]. TASK-1 channels therefore play a key role in the control of ventilation by peripheral chemoreceptors

HABIT FORMATION AND DEMAND SYSTEM ESTIMATES FOR FLUID MILK IN SPAIN

Demand and Price Analysis, Livestock Production/Industries,

Electron-microscopical and light-microscopical studies in bacterial cytology

PART I. A STUDY OF THE FACTORS INFLUENCING THE PRODUCTION OF VOLUTIN IN AEROBACTER AEROENES: 1. Aero.aerogenes A3, an organism which does not produce volutin under normal conditions of culture, was found to produce volutin under three different special conditions namely (1) at the acid reaction developed during growth on a poorly -buffered sugar containing medium, (2) by growth on a medium deficient in either its nitrogen or sulphur source and (3) on transfer of phosphate- starved cells from a "First Medium" deficient in phosphate to a "Second Medium" rich in phosphate. The volutin produced under all three cultural conditions was found to react metachromatically with methylene blue - the characteristic property of the volutin found in 0.diphtheriae and Sp.volutans. Thus the volutin of Aero.aerogenes was thought to be of the same nature as the classical volutin. The volutin granules of :zero.aeroenes were also observed in unstained, wet films by the phase contrast microscope, demonstrating that the granules were not staining artefacts. 2(a) When Aero.aerógenes I3 was grown aerobically at 35°C on a 0.1 per cent glucose medium the growth was maximal when the phosphate content was 0,1 per cent or greater; the pII did not fall below 6.1 and no - 109 - volutin was produced. 'Alen the phosphate content of the medium was between 0.001 and 0.03 per cent, and no other buffer was present, the pH fell to 4.5 or less, and much volutin was produced in 24 hours. This fall in pH of the medium was found to be the determining factor for the volutin production since (1) when "citrate "or bicarbonate buffer was added to a medium containing 0.01 per cent phosphate the pH did not fall below 6.0 and volutin production was prevented, and (2) when 1.0 per cent pH 5.2 acid phosphate (ILH.2PO4) was substituted for 1.0 per cent pH 7.3 phosphate mixture the pH fell to 4.5 and abundant volutin was produced. (b) When the phosphate content of the unbuffered medium was less than 0.0003 per cent, the amount of growth was reduced even further and the pH again fell to 4.5 or less but no volutin was produced. Such cells were found to be "nuclear" indicating that they were phosphate- starved. This showed that a certain amount of phosphate was necessary for the production of volutin. (c) The substitution of various other energy sources which did not cause such a large drop in the pH of the medium resulted in a smaller volutin production. This together with the fact that - 110 - volutin only disappeared with the occurrence of cell division would show that volutin was á metabolic -product. 3(a) When the organisms were grown at 350C on a nitrogen or sulphur -deficient medium buffered at neutral pH "nuclear" volutin -containing cells were produced in 24 hours but a deficiency in the phosphate or carbon and energy source did not induce a similar production :of volutin. (b) The subculture of such nitrogen- and sulphur- deficient cells on a fresh medium resulted in the loss of the volutin only after a few hours when cell division had occurred. The transfer of the "nuclear" non -volutin containing phosphate - starved cells to a fresh phosphate containing medium resulted in abundant volutin production. 4(a) When Áero.aerogenes was grown on a series of buffered media containing 0.1 per cent glucose and various amounts of phosphate, it was found that the cells were "nuclear" in appearance and the growth was curtailed on media containing 0.0003 per cent phosphate or less. Only these phosphate- starved produced volutin on transfer to a fresh medium containing phosphate. This volutin production was apparent after 3 minutes subculture, rose to a - 111 - maximum at 2 hours and disappeared mainly within 8 hours. 'he disappearance of the volutin again appeared to be accompanied with the return of the normal staining reaction to the cells and with the occurrence of cell division. (b) If no phosphate were added to the "Second Medium" no volutin was produced on transfer of phosphate- starved cells. Therefore phosphate was required for the production of volutin. (c) Likewise the total omission of glucose from the "Second Medium" caused a complete absence of the volutin production but other components of the glycolysis cycle were found to support volutin production in the "Second Medium ". Therefore glucose or another carbon and energy source was required in the "Second Medium" for the production of volutin. (d) The presence of ammonium sulphate, potassium and magnesium ions was found to be required for maximal volutin production. 5. As phosphate seemed to be required for the production of volutin the ratio of phosphorus to nitrogen was determined for the volutin -free and volutin -rich cells produced under the different cultural conditions. In all cases it was found -112- that the ratio of total phosphorus to total nitrogen for the volutin-rich cells was almost twice that of the volutin -Free cells. .'hen the cells were fractionated and the phosphorus content of each fraction estimated, nine of the ten fractions of the volutin -rich cells were found to contain double the amount of phosphorus present in the corresponding fraction of the volutin -free cells while the remaining fraction R9 of the volutin -rich cells showed a forty -fold increase over that of the volutin -free cells. . In the phosphate- starved cells which were transferred to the phosphate containing medium the phosphorus content of the fraction R9 was found to follow the same pattern as the volutin production in that it rose to a maximum in 30 minutes and thereafter fell away to practically none at 180 minutes. 6. Volutin in Äero.aerogenes appears to be a normal metabolic product which contains phosphorus - probably metaphosphate and which is. only observed when some enzyme systems are blocked due to growth under adverse cultural conditions.PART II. A STUDY OF THE MOTILITY AND FLAGELLATION OF AEROBACTER CLOACAE AND ESCHERICHIA COLI STRAINS: 1. In all the Aero.cloacae and Esch.coli strains examined a direct correlation of the motility of young cultures as observed by the hanging drop method and the presence of flagella as seen in electron microscope preparations was obtained. Photographs are shown to illustrate the presence or absence of the flagella in some of the Aero.cloacae strains and in all of the Esch.coli A strains. 2. It was found that if a peptone water culture of Esch.coli A was incubated for 16 hours instead of 6 hours the motility was much reduced but that the flagella were still present when the organism was examined by the electron microscope. This means that the flagella become inactive in older cultures. 3. `hen the organisms were cultivated in liquid synthetic media, the flagella were still found but the motility was absent. The cause of this is not known with certainty but it may have been the lack of some essential growth factor. 4. The addition of phenol to peptone water caused loss of both motility and flagella. In synthetic liquid media the phenol had less effect. 5. ';Then the organisms were grown on solid media (peptone agar) virtually the same results were obtained for these cultures as for the peptone water cultures which were incubated for the same length of time (16 hours). Here the motility was feeble but the flagella were produced if the organis was 'motile' (i.e. motile at 6 hours in peptone water). 6. Irregular filaments were found on certain of the organisms when they were grown either in peptone water or synthetic media. 'These appendages which were named pseudoflagella were short, angular filaments of uneven diameter and were discrete in that they did not merge into one another when they crossed. They were not general features of this group of organisms as all the species did not produce them, neither were they produced due to the motility of the organism as some motile organisms did not exhibit them while some non -motile ones produced them in abundance. Similarly the pseudoflagella did not appear to be slime threads since they were not produced by all the slime - formers but were produced by some of the non - slime- formers. It was found, however, that those organisms which produced pseudoflagella were also capable of agglutinating guinea pig and fowl red blood cells and that those which did not exhibit pseudoflagella had no effect on the red blood cells. The pseudoflagella reacted to phenol in the same manner as the flagella so they may be of the same chemical nature i.e. prótein. It is suggested that the pseudoflagella might be extracellular enzyme bearing structures

Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by {approx}50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics