High-affinity NO3 --H+ cotransport in the fungus Neurospora:Induction and control by pH and membrane voltate

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO₃ ^- challenge and to quantify transport activity. The NO₃ ^--associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO₃ ^--free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 μM NO₃ ^-. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity mensurable approx. 100 min after first exposure to NO₃ ^-; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO₃ ^- additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 μM NO₃ ^-; and it was suppressed when NH₄ ⁺, was present during the first, inductive exposure to NO₃ ^-. Voltage clamp measurements carried out immediately before and following NO₃ ^- additions showed that the NO₃ ^--evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages -400 to +100 mV). Measurements of NO₃ ^- uptake using NO₃ ^--selective macroelectrodes indicated a charge stoichiometry for NO₃ ^- transport of 1(+):1(NO₃ ^-) with common K(m) and J(max) values around 25 μM and 75 pmol NO₃ ^- cm^-2sec^-1, respectively, and combined measurements of pH(o) and [NO₃ ^-](o) showed a net uptake of approx. 1 H⁺ with each NO₃ ^- anion. Analysis of the NO₃ ^- current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pH(o) and [NO₃ ^-](o). Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pH(o) of 6.1, driving the membrane voltage from -350 to -150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO₃ ^-. By contrast, the same depolarization effected an approx. 20% fall in the K(m) for transport as a function in [H⁺](o). These, and additional results are consistent with a charge-coupling stoichiometry of 2(H⁺) per NO anion transported across the membrane, and implicate a carrier cycle in which NO binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO₃ ^- transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO₃ ^- transport; finally, they distinguish metabolite repression of NO₃ ^- transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.

The effect of soil pH on rhizosphere carbon flow of Lolium perenne

Perennial rye-grass plants were grown at 15°C in microcosms containing soil sampled from field plots that had been maintained at constant pH for the last 30 years. Six soil pH values were tested in the experiment, with pH ranging from 4.3-6.5. After 3 weeks growth in the microcosms, plant shoots were exposed to a pulse of ¹⁴C-CO₂. The fate of this label was determined by monitoring ¹⁴C-CO₂ respired by the plant roots/soil and by the shoots. The ¹⁴C remaining in plant roots and shoots was determined when the plants were harvested 7 days after receiving the pulse label. The amount of ¹⁴C (expressed as a percentage of the total ¹⁴C fixed by the plant) lost from the plant roots increased from 12.3 to 30.6% with increasing soil pH from 4.3 to 6. Although a greater percentage of the fixed ¹⁴C was respired by the root/soil as soil pH increased, plant biomass was greater with increasing soil pH. Possible reasons for observed changes in the pattern of ¹⁴C distribution are discussed and, it is suggested that changes in the soil microbial biomass and in plant nitrogen nutrition may, in particular be key factors which led to increased loss of carbon from plant roots with increasing soil pH. © 1990 Kluwer Academic Publishers.

FLAG-idarubicin and allogeneic stem cell transplantation for Ph-positive ALL beyond first remission

We describe a single centre experience of eight consecutive patients with relapsed or refractory Ph+ ALL treated with the FLAG/idarubicin regimen followed by BMT or PBSCT. Following FLAG/idarubicin, one achieved a partial response and seven CR. All patients subsequently received allogeneic transplants: one sibling BMT, three matched unrelated (MUD) BMT and four sibling PBSCT. Two patients received second transplants with PBSC from their original BM donors following FLA/Ida with no further conditioning. Three patients are alive in CR 9, 24 and 32 months after transplant. Seven of eight patients had a cytogenetic response following FLAG/Ida induction and one of seven became bcr-abl negative. All eight patients had a complete cytogenetic response following transplant. Four of five assessable patients became p190 bcr-abl negative after transplant; three of these subsequently relapsed. Both patients with the p210 bcr-abl transcript remained bcr-abl positive in CR after transplant. FLAG/Ida was well tolerated and appears to be effective in inducing remission in relapsed Ph+ ALL. The use of FDR-containing chemotherapy without further conditioning prior to PBSCT deserves further study in heavily pre-treated patients and, in patients with relapsed ALL following BMT, may be a safer option than DLI (donor lymphocyte infusion) by avoiding the associated risk of aplasia.

Sensitized terbium(iii) macrocyclic-phthalimide complexes as luminescent pH switches

Four new macrocyclic-phthalimide ligands were synthesised via the coupling of N-(3-bromopropyl)phthalimide either to cyclen (1,4,7,10- tetraazacyclododecane) itself or its carboxylate-functionalized analogues, and photophysical studies were carried out on their corresponding Tb(iii) complexes in aqueous media as a function of pH. Luminescence intensities of Tb·L^1a-Tb·L^3a were in 'switched off' mode under acidic conditions (pH < 4), and were activated on progression to basic conditions as the phthalimido functions therein were hydrolysed to their corresponding phthalamates Tb·L^1b-Tb·L^3b. Emission of phthalamate-based macrocyclic Tb(iii) complexes Tb·L ^1b-Tb·L^3b was in 'switched on' mode between pH 4 and 11, exhibiting high quantum yields (Φ) and long lifetimes (τ) of the order of milliseconds at pH ∼ 6. Tb(iii) emissions were found to decline with increasing number of chromophores. The values of Φ and τ were 46% and 2.4 ms respectively for Tb·L^1b at pH ∼ 6 when activated. This is the best pH-dependent sensor based on a Tb(iii) complex reported to date, benefiting from the macrocyclic architecture of the ligand. © 2013 The Royal Society of Chemistry.

Chloride transport and the resulting corrosion of steel bars in alkali activated slag concretes

As the relative performance of alkali activated slag (AAS) concretes in comparison to Portland cement (PC) counterparts for chloride transport and resulting corrosion of steel bars is not clear, an investigation was carried out and the results are reported in this paper. The effect of alkali concentration and modulus of sodium silicate solution used in AAS was studied. Chloride transport and corrosion properties were assessed with the help of electrical resistivity, non-steady state chloride diffusivity, onset of corrosion, rate of corrosion and pore solution chemistry. It was found that: (i) although chloride content at surface was higher for the AAS concretes, they had lower chloride diffusivity than PC concrete; (ii) pore structure, ionic exchange and interaction effect of hydrates strongly influenced the chloride transport in the AAS concretes; (iii) steel corrosion resistance of the AAS concretes was comparable to that of PC concrete under intermittent chloride ponding regime, with the exception of 6% Na2O and Ms of 1.5; (iv) the corrosion behaviour of the AAS concretes was significantly influenced by ionic exchange, carbonation and sulphide concentration; (v) the increase of alkali concentration of the activator generally increased the resistance of AAS concretes to chloride transport and reduced its resulting corrosion, and a value of 1.5 was found to be an optimum modulus for the activator for improving the chloride transport and the corrosion resistance.

Chloride induced corrosion of steel bars in alkali activated slag concretes

The studies on chloride induced corrosion of steel bars in alkali activated slag (AAS) concretes are scarcely reported in the past. In order to make this issue clearer and compare the corrosion performance of AAS with Portland cement (PC) counterpart, an investigation was carried out and the results are reported in this paper. Corrosion properties were assessed with the help of rate of corrosion, electrical resistivity and pore solution chemistry. It was found that: (i) steel corrosion resistance of the AAS concretes was comparable or in some cases even worse than that of Portland cement (PC) concrete under intermittent chloride ponding regime; (ii) the corrosion behaviour of the AAS concretes was significantly influenced by ionic exchange, carbonation and sulphide concentration; (iii) the increase of alkali concentration of the activator generally reduced chloride resulting corrosion, and a value of 1.5 was found to be an optimum modulus for the activator for improving the corrosion resistance.

Effect of high pressure processing in combination with Weissella viridescens as a protective culture against Listeria monocytogenes in ready-to-eat salads of different pH

This study explored the effect of HPP (400 MPa/1 min) and a Weissella viridescens protective culture, alone or in conjunction, against Listeria monocytogenes in ready-to-eat (RTE) salads with different pH values (4.32 and 5.59) during storage at 4 and 12 °C. HPP was able to reduce the counts of the pathogen after treatment achieving approximately a 4.0 and 1.5 log CFU/g reduction in the low and higher pH RTE salad, respectively. However, L. monocytogenes was able to recover and grow during subsequent storage. W. viridescens grew in both RTE salads at both storage temperatures, with HPP resulting in only a small immediate reduction of W. viridescens ranging from 0.50 to 1.2 log CFU/g depending on the pH of the RTE salad. For the lower pH RTE salad, the protective culture was able to gradually reduce the L. monocytogenes counts during storage whereas for the higher pH RTE salad in some cases it delayed growth significantly or exerted a bacteriostatic effect. exerted a bacteriostatic effect. The results revealed that the increased storage temperature led to an increase in the inactivation/inhibition of L. monocytogenes in the presence of W. viridescens. The combination of HPP and W. viridescens is a promising strategy to control L. monocytogenes and can increase safety even when a break in the chill chain occurs.

Comorbidity in severe asthma requiring systemic corticosteroid therapy: cross-sectional data from the Optimum Patient Care Research Database and the British Thoracic Difficult Asthma Registry

Objective: To determine the prevalence of systemic corticosteroid-induced morbidity in severe asthma.
Design: Cross-sectional observational study.Setting The primary care Optimum Patient Care Research Database and the British Thoracic Society Difficult Asthma Registry.
Participants: Optimum Patient Care Research Database (7195 subjects in three age- and gender-matched groups)—severe asthma (Global Initiative for Asthma (GINA) treatment step 5 with four or more prescriptions/year of oral corticosteroids, n=808), mild/moderate asthma (GINA treatment step 2/3, n=3975) and non-asthma controls (n=2412). 770 subjects with severe asthma from the British Thoracic Society Difficult Asthma Registry (442 receiving daily oral corticosteroids to maintain disease control).
Main outcome measures: Prevalence rates of morbidities associated with systemic steroid exposure were evaluated and reported separately for each group.
Results: 748/808 (93%) subjects with severe asthma had one or more condition linked to systemic corticosteroid exposure (mild/moderate asthma 3109/3975 (78%), non-asthma controls 1548/2412 (64%); p<0.001 for severe asthma versus non-asthma controls). Compared with mild/moderate asthma, morbidity rates for severe asthma were significantly higher for conditions associated with systemic steroid exposure (type II diabetes 10% vs 7%, OR=1.46 (95% CI 1.11 to 1.91), p<0.01; osteoporosis 16% vs 4%, OR=5.23, (95% CI 3.97 to 6.89), p<0.001; dyspeptic disorders (including gastric/duodenal ulceration) 65% vs 34%, OR=3.99, (95% CI 3.37 to 4.72), p<0.001; cataracts 9% vs 5%, OR=1.89, (95% CI 1.39 to 2.56), p<0.001). In the British Thoracic Society Difficult Asthma Registry similar prevalence rates were found, although, additionally, high rates of osteopenia (35%) and obstructive sleep apnoea (11%) were identified.

Conclusions: Oral corticosteroid-related adverse events are common in severe asthma. New treatments which reduce exposure to oral corticosteroids may reduce the prevalence of these conditions and this should be considered in cost-effectiveness analyses of these new treatments.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <or=-5.60 and <or=-8.00, respectively. Using this assay, it was found that GL-HCl permeates through human skin with a flux 1.497+/-0.42 microg cm-2 h-1, a permeability coefficient of 5.66+/-1.6x10(-6) cm h-1 and with a lag time of 10.9+/-4.6 h.