Control of breathing in the echidna (Tachyglossus aculeatus) during hibernation

Resting non-hibernating echidnas are characterised by low metabolic rates, but also have a very low respiratory frequency and a variable respiratory minute volume, often resulting in low levels of arterial O(2) and high CO(2). As the echidna lies at one physiological extreme among the hibernators, in terms of its large size and low metabolism and ventilatory requirement when not hibernating, a study of control of breathing during hibernation in echidnas should provide a useful test of the generality of various models. We used non-invasive techniques to study breathing patterns and the control of ventilation in 6 echidnas. Hibernating echidnas (T(b) range 7-10 degrees C) showed episodic breathing with bursts of breaths (average 36+/-16 breaths in 24+/-5 min) followed by a period of apnea (76+/-17 min) then a series (8+/-4) of slow breaths at 14+/-1 min intervals leading up to the next burst. Increasing CO(2) levels in the inspired air increased the number of breaths in a burst, eventually leading to continuous breathing. Inter burst breaths were controlled by O(2): hypoxia increased inter burst breaths, and decreased burst length, while hyperoxia abolished inter burst breaths and increased the apneic period. Overall, while CO(2) was a strong respiratory stimulus in hibernating echidnas, O(2) had little effect on total ventilation, but did have a strong effect on the breathing pattern.     

Natriuresis induced by mild hypernatremia in humans

The hypothesis that increases in plasma sodium induce natriuresis independently of changes in body fluid volume was tested in six slightly dehydrated seated subjects on controlled sodium intake (150 mmol/day). NaCl (3.85 mmol/kg) was infused intravenously over 90 min as isotonic (Iso) or as hypertonic saline (Hyper, 855 mmol/l). After Hyper, plasma sodium increased by 3% (142.0 +/- 0.6 to 146.2 +/- 0.5 mmol/l). During Iso a small decrease occurred (142.3 +/- 0.6 to 140.3 +/- 0.7 mmol/l). Iso increased estimates of plasma volume significantly more than Hyper. However, renal sodium excretion increased significantly more with Hyper (291 +/- 25 vs. 199 +/- 24 micromol/min). This excess was not mediated by arterial pressure, which actually decreased slightly. Creatinine clearance did not change measurably. Plasma renin activity, ANG II, and aldosterone decreased very similarly in Iso and Hyper. Plasma atrial natriuretic peptide remained unchanged, whereas plasma vasopressin increased with Hyper (1.4 +/- 0.4 to 3.1 +/- 0.5 pg/ml) and decreased (1.3 +/- 0.4 to 0.6 +/- 0.1 pg/ml) after Iso. In conclusion, the natriuretic response to Hyper was 50% larger than to Iso, indicating that renal sodium excretion may be determined partly by plasma sodium concentration. The mechanism is uncertain but appears independent of changes in blood pressure, glomerular filtration rate, the renin system, and atrial natriuretic peptide.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 3 isoforms by steady-state and transient kinetic analyses

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca(2+)-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca(2+)-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca(2+) in activation of the overall reaction due to a decreased E(2) to E(1)Ca(2) transition rate and an increased rate of Ca(2+) dissociation from E(1)Ca(2). At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca(2+) level (either an enhanced efflux of lumenal Ca(2+) through the pump in E(2) form or insensitivity to inhibition by lumenal Ca(2+)). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca(2+) and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E(2)P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.     

PH-induced collapse of the extracellular loops closes Escherichia coli maltoporin and allows the study of asymmetric sugar binding

LamB (maltoporin) is essential for the uptake of maltose and malto-oligosaccharides across the outer membrane of Escherichia coli. Purified LamB was reconstituted in artificial lipid bilayer membranes forming channels in the permanently open configuration at neutral pH. Almost complete channel closure was observed when the pH on both sides of the membrane was lowered to pH 4. When LamB was added to only one side of the membrane, the cis-side, and the pH was lowered at either side of the membrane, the cis- or the trans-side, the response to pH was asymmetric, suggesting preferential orientation of maltoporin channels and pH- dependent closure of only one side of the channel. In experiments with LamB mutants in which major external loops L4, L6, and/or L9 were deleted, we identified the surface-exposed loops L4 and L6 as the cause of pH-mediated closure. The pH dependence of the LamB channel is consistent with the assumption that it inserts in a preferential orientation into the lipid bilayer. About 70-80% of the reconstituted channels are oriented with the extracellular entrance toward the side to which the protein was added (the cis-side) and with the periplasmic opening on the opposite side (the trans-side). The possibility of closing the channels, which are oriented in the reverse direction by low pH at the trans-side, allowed the deduction of channel asymmetry with respect to carbohydrate binding kinetics. Whereas maltose binding was found to be almost symmetric with respect to the channel orientation, the sucrose and trehalose binding to LamB was asymmetric. The results are discussed in respect to possible physiological function of the pH-dependent closure of maltoporin.     

Impact of integrated fish farming on antimicrobial resistance in a pond environment

Integrated fish farming combines livestock production with fish farming. Animal manure is shed directly into a fish pond as fertilizer and supports the growth of photosynthetic organisms. The livestock, mainly chickens and pigs, is often fed feed containing growth promoters. In this study we investigated the impact of integrated fish farming on the levels of antimicrobial-resistant bacteria in a pond environment. One integrated broiler chicken-fish farm was studied for 2 months immediately after the start of a new fish production cycle. A significant increase over time in the resistance to six different antimicrobials was found for the indicator organism Acinetobacter spp. isolated from composite water-sediment samples. The initial resistance levels prior to the new production cycle were 1 to 5%. After 2 months the levels of resistance to oxytetracycline and sulfamethoxazole reached 100%, and the levels of resistance to ciprofloxacin were more than 80%. The long-term effects of resistance on integrated farming were studied on seven additional farms. The resistance levels were particularly high among Enterococcus spp. and were also high among Acinetobacter spp. isolated from water-sediment samples compared to the resistance levels at four control farms. In conclusion, integrated fish farming seems to favor antimicrobial-resistant bacteria in the pond environment. This could be attributed to the selective pressure of antimicrobials in the pond environment and/or to the introduction of antimicrobial-resistant bacteria from animal manure. Potential risks to human health were not addressed in this study and remain to be elucidated.     

Contributions of the free oxidized and QB-bound plastoquinone molecules to the thermal phase of chlorophyll-a fluorescence

Variable chlorophyll a (Chl a) fluorescence is composed of a photochemical and a thermal phases of similar amplitudes. The photochemical phase can be induced by a saturating single turnover flash (STF) and reflects the reduction of the Photosystem II (PS II) Q_A primary electron acceptor. The thermal phase requires multiple turnover flash (MTF) and is somehow related to the reduction of the plastoquinone (PQ) molecules. This article aimed to determine the relative contributions of the Q_B-bound and the free oxidized PQ molecules to the thermal phase of Chl a fluorescence. We thus measured the interactive effects of exogenous PQ (PQex), of an inhibitor (DCMU) acting at the Q_B site of PS II and of an artificial quencher, 2-methyl-1,4-naphtoquinone, on Chl a fluorescence levels induced by STF (F_F) and MTF (F_M) in spinach thylakoids. We observed that: (1) the incorporation of PQex in thylakoids stimulated photosynthetic electron transport but barely affected F_F and F_M in the absence of DCMU; (2) DCMU significantly increased the amplitude of F_F but slightly quenched F_M; (3) 2-methyl-1,4-naphtoquinone quenched F_M to a larger-extent than F_F; (4) DCMU increased the quenching effects of PQex on F_F and F_M and also, of methyl-1,4-naphtoquinone on F_F. These results indicate that: (1) the Q_B-bound and the free PQ molecules contribute to about 56% and 25%, respectively, to the thermal phase Chl a fluorescence in dark-adapted thylakoids; and (2) the thermal phase of Chl a fluorescence is more susceptible than the photochemical phase to the non-photochemical quenching effect of oxidized quinones. This revised version was published online in June 2006 with corrections to the Cover Date.     

Site-directed mutagenesis within the central constriction site of ScrY (sucroseporin): effect on ion transport and comparison of maltooligosaccharide binding to LamB of Escherichia coli

The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.