Watershed nitrogen input and riverine export on the west coast of the US

This study evaluated the sources, sinks, and factors controlling net export of nitrogen (N) from watersheds on the west coast of the US. We calculated input of new N to 22 watersheds for 1992 and 2002. 1992 inputs ranged from 541 to 11,644 kg N km⁻² year⁻¹, with an overall area-weighted average of 1,870 kg N km⁻² year⁻¹. In 2002, the range of inputs was 490–10,875 kg N km⁻² year⁻¹, averaging 2,158 kg N km⁻² year⁻¹. Fertilizer was the most important source of new N, averaging 956 (1992) and 1,073 kg N km⁻² year⁻¹ (2002). Atmospheric deposition was the next most important input, averaging 833 (1992) and 717 kg N km⁻² year⁻¹ (2002), followed by biological N fixation in agricultural lands. Riverine N export, calculated based on measurements taken at the furthest downstream USGS water quality monitoring station, averaged 165 (1992) and 196 kg N km⁻² year⁻¹ (2002), although data were available for only 7 watersheds at the latter time point. Downstream riverine N export was correlated with variations in streamflow (export = 0.94 × streamflow − 5.65, R ² = 0.66), with N inputs explaining an additional 16% of the variance (export = 1.06 × streamflow + 0.06 × input − 227.78, R ² = 0.82). The percentage of N input that is exported averaged 12%. Percent export was also related to streamflow (%export = 0.05 × streamflow − 2.61, R ² = 0.60). The correlations with streamflow are likely a result of its large dynamic range in these systems. However, the processes that control watershed N export are not yet completely understood.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Performance assessment of malolactic fermenting bacteria Oenococcus oeni and Lactobacillus brevis in continuous culture

The growth performance of malolactic fermenting bacteria Oenococcus oeni NCIMB 11648 and Lactobacillus brevis X₂ was assessed in continuous culture. O. oeni grew at a dilution rate range of 0.007 to 0.052 h⁻¹ in a mixture of 5:6 (g l⁻¹) of glucose/fructose at an optimal pH of 4.5, and L. brevis X₂ grew at 0.010 to 0.089 h⁻¹ in 10 g l⁻¹ glucose at an optimal pH of 5.5 in a simple and safe medium. The cell dry weight, substrate uptake and product formation were monitored, as well as growth kinetics, yield parameters and fermentation balances were also evaluated under pH control conditions. A comparison of growth characteristics of two strains was made, and this showed significantly different performance. O. oeni has lower maximum specific growth rate (μ_max=0.073 h⁻¹), lower maximum cell productivity (Q _x ^max=17.6 mg cell l⁻¹ h⁻¹), lower maximum biomass yield (Y _x/s ^max=7.93 g cell mol⁻¹ sugar) and higher maintenance coefficient (m _s=0.45 mmol⁻¹ sugar g⁻¹ cell h⁻¹) as compared with L. brevis X₂ (μ_max=0.110 h⁻¹; Q _x ^max=93.2 g⁻¹ cell mol⁻¹ glucose; Y _x/s ^max=22.3 g cell mol⁻¹ glucose; m _s=0.21 mmol⁻¹ glucose g⁻¹ cell h⁻¹). These data suggest a possible more productive strategy for their combined use in maturation of cider and wine.     

Metabolic rates in an anadromous clupeid, the American shad (Alosa sapidissima)

To assess the energetics of migration in an anadromous fish, adult American shad (Alosa sapidissima) were swum in a large respirometer at a range of speeds (1.0–2.3 body lengths (BL) s⁻¹, 13–24 °C). Metabolic rate (M_O2) was logarithmically related to swimming speed (Bl s⁻¹; r ² = 0.41, slope = 0.23 ± 0.037) and tailbeat frequency (beats × min⁻¹; r ² = 0.52, slope = 0.003 ± 0.0003). Temperature had a significant effect on metabolic rate (r ² = 0.41) with a Q₁₀ of 2.2. Standard metabolic rate (SMR), determined directly after immobilization with the neuroblocker gallamine triethiodide, ranged from 2.2–6.2 mmolO₂ kg⁻¹ h⁻¹ and scaled with mass (W) such that SMR = 4.0 (±0.03)W^{0.695(±0.15)}. Comparison of directly determined and extrapolated SMR suggests that swimming respirometry provides a good estimate of SMR in this species, given the differences in basal activity monitored by the two methods. Overall, American shad metabolic rates (M_O2 and SMR) were intermediate between salmonids and fast-swimming perciforms, including tunas, and may be a result of evolutionary adaptation to their active pelagic, schooling life history. This study demonstrates variability in metabolic strategy among anadromous fishes that may be important to understanding the relative success of different migratory species under varying environmental conditions. Accepted: 3 March 1999     

Short latency vestibular evoked potentials in the Japanese quail (Coturnix coturnix japonica)

Short-latency vestibular-evoked potentials to pulsed linear acceleration were characterized in the quail. Responses occurred within 8 ms following the onset of stimuli and were composed of a series of positive and negative peaks. The latencies and amplitudes of the first four peaks were quantitatively characterized. Mean latencies at 1.0 g ms⁻¹ ranged from 1265 ± 208 μs (P1, N = 18) to 4802 ± 441 μs (N4, N = 13). Amplitudes ranged from 3.72 ± 1.51 μV (P1/N1, N = 18) to 1.49 ± 0.77 μV (P3/N3, N = 16). Latency-intensity (LI) slopes ranged from −38.7 ± 7.3 μs dB⁻¹ (P1, N = 18) to −71.6 ± 21.9 μs dB⁻¹ (N3, N = 15) and amplitude-intensity (AI) slopes ranged from 0.20 ± 0.08 μV dB⁻¹ (P1/N1, N = 18) to 0.07 ± 0.04 μV dB⁻¹ (P3/N3, N = 11). The mean response threshold across all animals was −21.83 ± 3.34 dB re: 1.0 g ms⁻¹ (N = 18). Responses remained after cochlear extirpation showing that they could not depend critically on cochlear activity. Responses were eliminated by destruction of the vestibular end organs, thus showing that responses depended critically and specifically on the vestibular system. The results demonstrate that the responses are vestibular and the findings provide a scientific basis for using vestibular responses to evaluate vestibular function through ontogeny and senescence in the quail. Accepted: 18 January 1997     

Control analysis of photosynthate partitioning

Experiments were carried out to investigate the contribution of ADP-glucose pyrophosphorylase and the plastid phosphoglucosemutase to the control of starch synthesis. Mutants ofArabidopsis thaliana (L.) Heyhn. were constructed with 50% and 7% of the wild-type adenosine 5′-diphosphoglucose pyrophosphorylase (ADPGlc-PPase), or 50% and null plastid phosphoglucomutase (PGM). The changes in the steady-state rates of sucrose synthesis, starch synthesis and CO₂ fixation were measured in saturating CO₂ in low (75 μmol·m⁻²·s⁻¹) and high (600 μmol·m⁻²·s⁻¹) irradiance. In low irradiance, a 50% decrease of PGM had no significant effect on fluxes, while a 50% and 93% decrease of ADPGlc-PPase led to a 23% and 74% inhibition of starch synthesis. Decreased ADPGlc-PPase led to an increase of hexose phosphates, triose phosphates and fructose-1,6-bisphosphate. Fixation of CO₂ was not inhibited because the inhibition of starch synthesis was matched by a stimulation of sucrose synthesis. In high irradiance, a 50% decrease of PGM led to a 20% inhibition of starch synthesis. A 50% and 93% decrease of ADPGlc-PPase led to a 39% and 90% inhibition of starch synthesis. Sucrose synthesis was also inhibited, and the rate of photosynthesis was decreased. Decreased ADPGlc-PPase led to an increase of hexose phosphates but triose phosphates and fructose-1,6-bisphosphate did not increase. These results are used to estimate flux-control coefficients for these enzymes for starch synthesis. Firstly, the flux to starch is only controlled by ADPGlc-PPase in low irradiance, but control is redistributed to other enzymes in the pathway when a rapid flux is imposed, e.g. in high irradiance and CO₂. Secondly, reducing the rate of starch synthesis by decreasing the activity of enzymes in this pathway does not always lead to a compensating increase in the rate of sucrose synthesis. Thirdly, decreasing the activity of an enzyme by a factor of two compared to the remainder of the pathway often leads to it exerting very considerable control. Fourthly, each enzyme starts to exert considerable control when only a fraction of its V_max activity is being utilised in vivo, for example the maximum flux at ADPGlc-PPase never exceeded 20% of the V_max activity. The summation theory is also applied to check whether additional major control sites are required. In low irradiance, the efficiency of light harvesting will exert considerable control over the rate of starch synthesis.     

The Impact of heat exposure and repeated exercise on circulating stress hormones

To determine if heat exposure alters the hormonal responses to moderate, repeated exercise, 11 healthy male subjects [age = 27.1 (3.0) years; maximal oxygen consumption, V˙O_2max = 47.6 (6.2) ml · kg · min⁻¹; mean (SD)] were assigned to four different experimental conditions according to a randomized-block design. While in a thermoneutral (23°C) or heated (40°C, 30% relative humidity) climatic chamber, subjects performed either cycle ergometer exercise (two 30-min bouts at ≈50% V˙O_2max, separated by a 45-min recovery interval, CEx and HEx conditions), or remained seated for 3 h (CS and HS conditions). Blood samples were analyzed for various exercise stress hormones [epinephrine (E), norepinephrine (NE), dopamine, cortisol and human growth hormone (hGH)]. Passive heating did not alter the concentrations of any of these hormones significantly. During both environmental conditions, exercise induced significant (P < 0.001) elevations in plasma E, NE and hGH levels. At 23°C during bout 1: E = 393 (199) pmol · l⁻¹ (CEx) vs 174 (85) pmol · l⁻¹ (CS), NE = 4593 (2640) pmol · l⁻¹ (CEx) vs 1548 (505) pmol · l⁻¹ (CS), and hGH = 274 (340) pmol · l⁻¹ (CEx)vs 64 (112) pmol · l⁻¹ (CS). At 40°C, bout 1: E = 596 (346) pmol · l⁻¹ (HEx) vs 323 (181) pmol · l⁻¹ (HS), NE = 7789 (5129) pmol · l⁻¹ (HEx) vs 1527 (605) pmol · l⁻¹ (HS), and hGH = 453 (494) pmol · l⁻¹ (HEx) vs 172 (355) pmol · l⁻¹ (HS). However, concentrations of plasma cortisol were increased only in response to exercise in the heat [HEx = 364 (168) nmol · l⁻¹ vs HS = 295 (114) nmol · l⁻¹). Compared to exercise at room temperature, plasma levels of E, NE and cortisol were all higher during exercise in the heat (P < 0.001 in all cases). The repetition of exercise did not significantly alter the pattern of change in cortisol or hGH levels in either environmental condition. However, repetition of exercise in the heat increased circulatory and psychological stress, with significantly (P < 0.001) higher plasma concentrations of E and NE. These results indicate a differential response of the various stress hormones to heat exposure and repeated moderate exercise. Accepted: 16 April 1997     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Actual and potential rates of hydrogen photoproduction by continuous culture of the purple non-sulphur bacterium Rhodobacter capsulatus

The influence of (NH₄)₂SO₄ concentration and dilution rate (D) on actual and potential H₂ photoproduction has been studied in ammonium-limited chemostat cultures of Rhodobacter capsulatus B10. The actual H₂ production in a photobioreactor was maximal (approx. 80 ml h⁻¹l⁻¹) at D = 0.06 h⁻¹ and 4 mM (NH₄)₂SO₄. However, it was lower than the potential H₂ evolution (calculated from hydrogen evolution rates in incubation vials), which amounted to 100–120 ml h⁻¹ l⁻¹ at D = 0.03–0.08 h⁻¹. Taking into account the fact that H₂ production in the photobioreactor under these conditions was not limited by light or lactate, another limiting (inhibiting) factor should be sought. One possibility is an inhibition of H₂ production by the H₂ accumulated in the gas phase. This is apparent from the non-linear kinetics of H₂ evolution in the vials or from its inhibition by the addition of H₂; initial rates were restored in both cases after the vials had been refilled with argon. The actual H₂ production in the photobioreactor at D = 0.06 h⁻¹ was shown to increase from approximately 80 ml h⁻¹ l⁻¹ to approximately 100 ml h⁻¹ l⁻¹ under an argon flow at 100 ml min⁻¹. Under maximal H₂ production rates in the photobioreactor, up to 30% of the lactate feedstock was utilised for H₂ production and 50% for biomass synthesis. Received: 22 April 1997 / Received revision: 14 July 1997 / Accepted: 27 July 1997     

Woody debris contribution to the carbon budget of selectively logged and maturing mid-latitude forests

Woody debris (WD) is an important component of forest C budgets, both as a C reservoir and source of CO₂ to the atmosphere. We used an infrared gas analyzer and closed dynamic chamber to measure CO₂ efflux from downed coarse WD (CWD; diameter≥7.5 cm) and fine WD (FWD; 7.5 cm>diameter≥2 cm) to assess respiration in a selectively logged forest and a maturing forest (control site) in the northeastern USA. We developed two linear regression models to predict WD respiration: one based on WD temperature, moisture, and size (R ²=0.57), and the other on decay class and air temperature (R ²=0.32). WD respiration (0.28±0.09 Mg C ha⁻¹ year⁻¹) contributed only ≈2% of total ecosystem respiration (12.3±0.7 Mg C ha⁻¹ year⁻¹, 1999–2003), but net C flux from CWD accounted for up to 30% of net ecosystem exchange in the maturing forest. C flux from CWD on the logged site increased modestly, from 0.61±0.29 Mg C ha⁻¹ year⁻¹ prior to logging to 0.77±0.23 Mg C ha⁻¹ year⁻¹ after logging, reflecting increased CWD stocks. FWD biomass and associated respiration flux were ≈7 times and ≈5 times greater, respectively, in the logged site than the control site. The net C flux associated with CWD, including inputs and respiratory outputs, was 0.35±0.19 Mg C ha⁻¹ year⁻¹ (net C sink) in the control site and −0.30±0.30 Mg C ha⁻¹ year⁻¹ (net C source) in the logged site. We infer that accumulation of WD may represent a small net C sink in maturing northern hardwood forests. Disturbance, such as selective logging, can enlarge the WD pool, increasing the net C flux from the WD pool to the atmosphere and potentially causing it to become a net C source.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Motor control and cardiovascular responses during isoelectric contractions of the upper trapezius muscle: evidence for individual adaptation strategies

Ten females (25–50 years of age) performed isometric shoulder flexions, holding the right arm straight and in a horizontal position. The subjects were able to see the rectified surface electromyogram (EMG) from either one of two electrode pairs above the upper trapezius muscle and were instructed to keep its amplitude constant for 15 min while gradually unloading the arm against a support. The EMG electrodes were placed at positions representing a “cranial” and a “caudal” region of the muscle suggested previously to possess different functional properties. During the two contractions, recordings were made of: (1) EMG root mean square-amplitude and zero crossing (ZC) frequency from both electrode pairs on the trapezius as well as from the anterior part of the deltoideus, (2) supportive force, (3) heart rate (HR) and mean arterial blood pressure (MAP), and (4) perceived fatigue. The median responses during the cranial isoelectric contraction were small as compared to those reported previously in the literature: changes in exerted glenohumeral torque and ZC rate of the isoelectric EMG signal of −2.81% · min⁻¹ (P = 0.003) and 0.03% · min⁻¹ (P= 0.54), respectively, and increases in HR and MAP of 0.14 beats · min⁻² (P= 0.10) and 0.06 mmHg · min⁻¹ (P= 0.33), respectively. During the contraction with constant caudal EMG amplitude, the corresponding median responses were −2.51% · min⁻¹ (torque), 0.01% · min⁻¹ (ZC rate), 0.31 beats · min⁻² (HR), and 0.93 mmHg · min⁻¹ (MAP); P=0.001, 0.69, 0.005, and 0.003, respectively. Considerable deviations from the “isoelectric” target amplitude were common for both contractions. Individuals differed markedly in response, and three distinct subgroups of subjects were identified using cluster analysis. These groups are suggested to represent different motor control scenarios, including differential engagement of subdivisions of the upper trapezius, alternating motor unit recruitment and, in one group, a gradual transition towards a greater involvement of type II motor units. The results indicate that prolonged low-level contractions of the shoulder muscles may in general be accomplished with a moderate metabolic stress, but also that neuromuscular adaptation strategies differ significantly between individuals. These results may help to explain why occupational shoulder-neck loads of long duration cause musculoskeletal disorders in some subjects but not in others. Accepted: 1 March 1997     

Age and growth of the nototheniid fishTrematomus bernacchii Boulenger from Terra Nova Bay,Antarctica

Age and growth of the nototheniid fishTrematomus bernacchii Boulenger 1902 were estimated by reading the sagittal otoliths of 457 adult specimens caught off Terra Nova Bay (Ross Sea) in the austral summer 1990–1991. Annuli in ground and polished otoliths were examined using a dissecting microscope under reflected light. The Von Bertalanffy growth equation was L_t=273.5 [1 − e^{−0.109(t+2.10)}] for males (n=122) and L_t=422.2 [1 − e^{−0.055(t+1.92)}] for females (n=211) where L is total length in millimetres. Maximum estimated age was 21 years for females and 16 years for males. This is in agreement with the hypothesis that considers slow growth and old age as a typical feature of Antarctic fishes.     

Evaluation of the cytogenetic damage induced by the organophosphorous insecticide acephate

The organophosphorous insecticide acephate was tested for its ability to induce in vitro cytogenetic effect in human peripheral lymphocytes by using the chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) assay. The level of nuclear DNA damage of acephate was evaluated by using the comet assay. Concentrations of 12.5, 25, 50, 100 and 200 μg mL⁻¹ of acephate were used. All concentrations of acephate induced significant increase in the frequency of CAs and in the formation of MN dose dependently (r = 0.92 at 24 h, r = 0.95 at 48 h for CAs, r = 0.87 for MN). A significant increase was observed in induction of SCE at 50, 100 and 200 μg mL⁻¹ concentrations during 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) during 48 h treatment period in a dose-dependent manner (r = 0.84 at 24 h, r = 0.88 at 48 h). Acephate did not affect the replicative index and cytokinesis-block proliferation index (CBPI). However, it significantly decreased the mitotic index at all three highest concentrations (50, 100, 200 μg mL⁻¹) for 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) for 48 h treatment, dose-dependently (r = 0.94 at 24 h, r = 0.92 at 48 h). A significant increase in mean comet tail length was observed at 100 and 200 μg mL⁻¹ concentrations compared with negative control in a concentration-dependent manner (r = 0.94). The mean comet tail intensity was significantly increased at only 200 μg mL⁻¹ concentration. The present results indicate that acephate is a clastogenic, cytotoxic agent and it causes DNA damage at high concentrations in human lymphocytes in culture.     

Cooperative effect of water molecules in the self-catalyzed neutral hydrolysis of isocyanic acid: a comprehensive theoretical study

The detailed reaction mechanism for the water-assisted hydrolysis of isocyanic acid, HNCO + (n + 1) H₂O → CO₂ + NH₃ + nH₂O (n = 0−6), taking place in the gas phase, has been investigated. All structures were optimized and characterized at the MP2/6-31 + G* level of theory, and then re-optimized at MP2/6-311++G**. The seven explicit water molecules participating in the hydrolysis can be divided into two groups, one directly involved in the proton relay, and the other located in the vicinity of the substrate playing the cooperative role by engaging in hydrogen-bonding to HN = C = O. Two possible reaction pathways, the addition of water molecule across the C = N bond or across the C = O bond, are discussed, and the former is proved to be more favorable energetically. Our calculations suggest that, in the most kinetically favorable pathway for the titled hydrolysis, three water molecules are directly participating in the hydrogen transfer via an eight-membered cyclic transition state, while the other four water molecules catalyze the hydrolysis of HN = C = O by forming three eight-membered cooperative loops near the substrate. This strain-free hydrogen-bond network leads to the best estimated rate-determining activation energy of 24.9 kJ mol⁻¹ at 600 K, in excellent agreement with the gas-phase kinetic experimental result, 25.8 kJ mol⁻¹.     

Single-channel properties of a stretch-sensitive chloride channel in the human mast cell line HMC-1

A stretch-activated (SA) Cl⁻ channel in the plasma membrane of the human mast cell line HMC-1 was identified in outside-out patch-clamp experiments. SA currents, induced by pressure applied to the pipette, exhibited voltage dependence with strong outward rectification (55.1 pS at +100 mV and an about tenfold lower conductance at −100 mV). The probability of the SA channel being open (P _o) also showed steep outward rectification and pressure dependence. The open-time distribution was fitted with three components with time constants of τ_1o = 755.1 ms, τ_2o = 166.4 ms, and τ_3o = 16.5 ms at +60 mV. The closed-time distribution also required three components with time constants of τ_1c = 661.6 ms, τ_2c = 253.2 ms, and τ_3c = 5.6 ms at +60 mV. Lowering extracellular Cl⁻ concentration reduced the conductance, shifted the reversal potential toward chloride reversal potential, and decreased the P _o at positive potentials. The SA Cl⁻ currents were reversibly blocked by the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) but not by (Z)-1-(p-dimethylaminoethoxyphenyl)-1,2-diphenyl-1-butene (tamoxifen). Furthermore, in HMC-1 cells swelling due to osmotic stress, DIDS could inhibit the increase in intracellular [Ca²⁺] and degranulation. We conclude that in the HMC-1 cell line, the SA outward currents are mediated by Cl⁻ influx. The SA Cl⁻ channel might contribute to mast cell degranulation caused by mechanical stimuli or accelerate membrane fusion during the degranulation process.     

Serum heat shock protein 27 antigen and antibody levels appear to be related to the macrovascular complications associated with insulin resistance: a pilot study

Heat shock protein 27 (Hsp27) is over-expressed when cells are exposed to stressful conditions that include oxidative stress. Oxidative stress has been implicated in the pathogenesis of cardiovascular disease (CVD), diabetes and insulin resistance. We have investigated the concentrations of serum Hsp27 antigen and antibodies in subjects from different glycaemic categories, who either did or did not have established CVD. Serum Hsp27 antigen and antibody levels (immunoglobulins M and G (IgM and IgG)) were determined by enzyme-linked immunosorbent assays (ELISAs) in 68 individuals: 26 with normal glucose tolerance (NGT), 10 with (+) and 16 without (−) a history of CVD and 42 individuals with varying degrees of glucose intolerance (GI; 21 with and 21 without a history of CVD). Insulin sensitivity was determined in each subject using indices derived from the homeostasis model assessment of sensitivity and the insulin sensitivity index for glycaemia. Serum Hsp27 concentrations were significantly higher in GI (+CVD) subjects compared to GI (−CVD) subjects (p = 0.03), NGT (−CVD) subjects (p = 0.02) and NGT (+CVD) subjects (p = 0.04) and were positively correlated to fasting plasma glucose for all subjects (r = 0.28, p = 0.03). IgM antibody levels were significantly higher in GI (+CVD) subjects compared to NGT (−CVD) group (p = 0.02) and were inversely related to fasting insulin concentrations (r = −0.27, p = 0.04) and the 2-h insulin concentrations (r = −0.29, p = 0.03) for all subjects. Serum IgG antibody levels were higher in GI (+CVD) group compared to GI (−CVD) group (p = 0.06). In conclusion, Hsp27 and its antibody concentrations appear to relate to the presence of cardiovascular complications in patients with GI.     

Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease

Chemical Exchange Saturation Transfer (CEST) is an MRI approach that can indirectly detect exchange broadened protons that are invisible in traditional NMR spectra. We modified the CEST pulse sequence for use on high-resolution spectrometers and developed a quantitative approach for measuring exchange rates based upon CEST spectra. This new methodology was applied to the rapidly exchanging Hδ1 and Hε2 protons of His57 in the catalytic triad of bovine chymotrypsinogen-A (bCT-A). CEST enabled observation of Hε2 at neutral pH values, and also allowed measurement of solvent exchange rates for His57-Hδ1 and His57-Hε2 across a wide pH range (3–10). Hδ1 exchange was only dependent upon the charge state of the His57 (k _ex,Im+ = 470 s⁻¹, k _ex,Im = 50 s⁻¹), while Hε2 exchange was found to be catalyzed by hydroxide ion and phosphate base ( k_{\textOH ^-} k_{{{\text{OH}}^{ - } }}  = 1.7 × 10¹⁰ M⁻¹ s⁻¹, k_{\textHPO4^{2 -}} k_{{{\text{HPO}}_{4}^{2 - } }}  = 1.7 × 10⁶ M⁻¹ s⁻¹), reflecting its greater exposure to solute catalysts. Concomitant with the disappearance of the Hε2 signal as the pH was increased above its pK _a, was the appearance of a novel signal (δ = 12 ppm), which we assigned to Hγ of the nearby Ser195 nucleophile, that is hydrogen bonded to Nε2 of neutral His57. The chemical shift of Hγ is about 7 ppm downfield from a typical hydroxyl proton, suggesting a highly polarized O–Hγ bond. The significant alkoxide character of Oγ indicates that Ser195 is preactivated for nucleophilic attack before substrate binding. CEST should be generally useful for mechanistic investigations of many enzymes with labile protons involved in active site chemistry.     

MCP-1 −2518 A/G functional polymorphism is associated with increased susceptibility to active pulmonary tuberculosis in Tunisian patients

Monocyte chemoattractant protein-1 (MCP-1) plays crucial role in protective immunity against Mycobacterium tuberculosis (MT). In this study, we examined whether single nucleotide polymorphism (SNP) −2518 A/G (rs 1024611) of MCP-1 affect the susceptibility to active tuberculosis (TB) in Tunisian populations. Genomic DNA from patients with active TB (168 cases of pulmonary TB and 55 cases of extrapulmonary TB) and ethnically controls (150 cases) was genotyped for the MCP-1 −2518 A/G SNP by polymerase chain reaction fragment length polymorphism (PCR-RFLP). We observed that −2518 G allele and GG genotype (high MCP-1 producer) frequencies were significantly more elevated in active pulmonary TB group in comparison to control group [34 vs. 22%; P = 0.0007; 15 vs. 5%, P corrected for the number of genotypes (Pc) = 0.015; respectively]. Additionally, they were associated with increased risk development of this clinical form of TB [odds ratio (OR) = 1.83, 95% confidence intervals (CI) = 1.26–2.66; OR = 3.1, 95% CI = 1.28–7.76; respectively]. However, wild type allele −2518 A and AA genotype were over-represented in control group (78 and 62%) and seem to be protective factors against TB. Moreover, −2518 AA genotype was more frequent in control group and was associated with resistance against development of active pulmonary TB (OR = 0.56, 95% CI = 0.35–0.89, Pc = 0.03). Our findings confirm the key role of −2518 A/G SNP of MCP-1 and support its association with resistance/susceptibility to the development of active pulmonary TB in the Tunisian population.     

IL-1β (−511T/C) gene polymorphism not IL-1β (+3953T/C) and LT-α (+252A/G) gene variants confers susceptibility to visceral leishmaniasis

Lymphotoxin-α (LT-α) and interleukin-1beta (IL-1β) are proinflammatory cytokines playing important roles in immunity against Leishmania infection and the outcome of the disease. As cytokine productions are under the genetic control, this study tried to find any probable relationship between these cytokine gene polymorphisms and the susceptibility to visceral leishmaniasis in Iranian pediatric patients. Ninety-five pediatric patients involved with visceral leishmaniasis and 128 non-relative healthy people, from the same area as the patients, were genotyped for LT-α (+252A/G) and IL-1β (+3953T/C and −511T/C) gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). There was not found any significant differences in allele and genotype frequencies of LT-α (+252A/G) and IL-1β (+3953) among the study groups. However, the frequency of IL-1β −511TT genotype was higher in the controls (P = 0.0004) while the frequency of IL-1β −511CC genotype and C allele were higher in the patients (P = 0.008 and P = 0.00006, respectively). Furthermore, IL-1β CC (−511/+3953) haplotype was more frequent in VL patients compared with the controls (P = 0.0002) and the distribution of TT haplotype was higher in the controls compared with the patients (P = 0.003). In conclusion, based on the results, IL-1β −511C allele, CC genotype and CC (−511/+3953) haplotype could be considered as the susceptibility factors for visceral leishmaniasis while IL-1β −511TT genotype, T allele and TT haplotype (−511/+3953) might be counted as the influential factors for resistance to the disease.