Overwintering and migration behaviour of post-spawned Atlantic salmon Salmo salar in a large hydropower-regulated river and reservoir

Tracking 47 post-spawned adult Atlantic salmon Salmo salar L. in a hydropower-regulated river through autumn, winter and spring revealed that winter survival was 56% and 75% in two study years, respectively, with higher mortality of males than females (50% vs. 33% and 100% vs. 13%, respectively). Some kelts (n = 7) displayed nondirected movements that were interpreted as a reconditioning period for an average of 9–17 days prior to directed downstream movements indicating the initiation of migration. Survival after the initiation of migration in spring was 83% and 94% to the hydropower dam in the first and second study years, and decreased to 60 and 63%, respectively, after dam passage. There were no further losses in the downriver reach in the second year, with the first year having a cumulative survival estimate of 53% to the river mouth. Kelts approached the dam when the spillway gates were available as a passage option most of the time (64%–75%), but some kelts arrived at the dam or had not yet passed when spillways were closed (n = 6) and the only remaining passage option was restricted to the turbines. However, all but one kelt that must have passed via turbine were successful in reaching the river mouth. Migratory delay presumably due to searching behaviour caused by low water flow was estimated at approximately 6 days as migration rates were significantly slower in the reservoir (median ± s.e. 8.5 ± 2.5 km day⁻¹) than up- (29.7 ± 5.0 km day⁻¹) or downriver (22.1 ± 3.1 km day⁻¹). The proportion of time (median 30%) that kelts spent swimming upstream (searching behaviour) in the reservoir was a significant variable for migration success.     

Indirect activation of the epithelial Na+ channel by trypsin

We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.     

ENaC-membrane interactions: regulation of channel activity by membrane order

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459-6463). As these changes of temperature affect membrane lipid order and lipid-protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order-dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2 degrees C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.     

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of αENaC using the serine protease subtilisin. Trimeric channels formed with αFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ∼10-fold decrease in amiloride-sensitive whole cell conductance as compared with αWT (2.2 versus 21.2 microsiemens (μS)). Both αWT and αFM were activated to similar levels by subtilisin cleavage. Channels formed with αFD, a construct that deleted the segment between the two furin sites (Δ175–204), exhibited an intermediate conductance of 13.2 μS. More importantly, αFD retained the ability to be activated by subtilisin to 108.8 ± 20.9 μS, a level not significantly different from that of subtilisin activated αWT (125.6 ± 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of α was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with αLD, a construct that extended the deleted sequence of αFD by 17 amino acids (Δ175–221). Channels with αLD were uncleaved, exhibited low baseline activity (4.1 μS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.     

Respiratory Symptoms and Airway Obstruction in HIV-Infected Subjects in the HAART Era

Background

Prevalence and risk factors for respiratory symptoms and airway obstruction in HIV-infected subjects in the era of highly active antiretroviral therapy (HAART) are unknown. We evaluated respiratory symptoms and measured airway obstruction to identify the impact of HAART and other risk factors on respiratory symptoms and pulmonary function.

Methodology/Principal Findings

Two hundred thirty-four HIV-infected adults without acute respiratory symptoms were recruited from an HIV clinic. All subjects were interviewed and performed spirometry. Multivariate linear and logistic regressions were performed to determine predictors of respiratory symptoms, forced expiratory volume in one second (FEV₁) percent predicted, and FEV₁/forced vital capacity (FEV₁/FVC). Thirty-one percent of subjects reported at least one respiratory symptom. Smoking status (current or former versus never) (odds ratio [OR] = 2.7, 95% confidence interval [CI] = 1.41–5.22, p = 0.003), higher log plasma HIV viral levels (OR = 1.12, 95%CI = 1.02–1.24, p = 0.02), and lower FEV₁/FVC (OR = 1.06 for every 0.01 decrease in FEV₁/FVC, 95%CI = 1.02–1.14, p = 0.001) were independent predictors of respiratory symptoms. Age (p = 0.04), pack-year smoking history (p<0.001), previous bacterial pneumonia (p = 0.007), and HAART use (p = 0.04) were independent predictors of decreased FEV₁/FVC.

Conclusions/Significance

Respiratory symptoms remain common in HIV-infected subjects, especially in those with a smoking history. Subjects who were older, had a greater pack-year history of smoking, or previous bacterial pneumonia had lower FEV₁/FVC ratios. Interestingly, use of HAART was independently associated with a decreased FEV₁/FVC, possibly secondary to an immune response to subclinical infections, increased autoimmunity, or other factors associated with HAART use.     

Algae as Bio-fertilizers: Between current situation and future prospective

Bio-fertilization is a sustainable agricultural practice that includes using bio-fertilizers to increase soil nutrient content resulting in higher productivity. Soil micro-flora has been exposed to improve soil fertility and increase biomass productivity and identified as a correct environmentally friendly bio-based fertilizer for pollution-free agricultural applies. The majority of cyanobacteria can fix nitrogen from the atmosphere and several species including Anabaena sp., Nostoc sp., and Oscillatoria angustissima is known to be effective cyanobacterial based bio fertilizers. Acutodesmus dimorphus, Spirulina platensis Chlorella vulgaris, Scenedesmus dimorphus, Anabaena azolla, and Nostoc sp. are some of the green microalgae and cyanobacteria species that have been successfully used as bio fertilizers to boost crop growth. Also, Chlorella vulgaris is one of the most commonly used microalgae in bio fertilizer studies. The addition of seaweed species that are Sargassum sp. and Gracilaria verrucosa leads to chemical changes as a soil fertility indicator on clay and sandy soils, and the addition of seaweed conditioner to soil can improve its organic content, return pH to normal, and reduce C/N ratio in both sandy and clay soil. This review provides an effective approach to increase soil fertility via environmentally friendly bio-based fertilizer using micro and macro algae. Instead of the usage of inorganic and organic fertilizers that have polluted impacts to soil as aggregation of heavy metals, in addition to there their human carcinogenic effects.     

Nonselective cation transport in native esophageal epithelia

Rabbit esophageal epithelia actively transport Na(+) in a manner similar to that observed in classic electrically tight Na(+)-absorbing epithelia, such as frog skin. However, the nature of the apical entry step is poorly understood. To address this issue, we examined the electrophysiological and biochemical nature of this channel. Western blotting experiments with epithelial Na(+) channel (ENaC) subunit-specific antibodies revealed the presence of all three ENaC subunits in both native and immortalized esophageal epithelial cells. The amino acid sequence of the rabbit alpha-ENaC cloned from native rabbit esophageal epithelia was not significantly different from that of other published alpha-ENaC homologs. To characterize the electrophysiological properties of this native apical channel, we utilized nystatin permeabilization to eliminate the electrical contribution of the basolateral membrane in isolated native epithelia mounted in Ussing-type chambers. We find that the previously described apical Na(+) channel is nonselective for monovalent cations (Li(+), Na(+), and K(+)). Moreover, this channel was not blocked by millimolar concentrations of amiloride. These findings document the presence of a nonselective cation channel in a native Na(+) transporting epithelia, a finding that hereto has been thought to be limited to artificial culture conditions. Moreover, our data are consistent with a potential role of ENaC subunits in the formation of a native nonselective cation channel.     

Regulation of the Epithelial Na+ Channel by Membrane Tension

The sensitivity of αβγ rat epithelial Na⁺ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.     

Multidrug-resistant phenotype and isolation of a Novel SHV- beta-Lactamase variant in a clinical isolate of Enterobacter cloacae

Background

ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species.

Results

E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a bla_SHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the bla_SHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum β-lactam and the change of the isoelectric point of the protein.

Conclusion

These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities.     

Methods for stable recording of short-circuit current in a Na+-transporting epithelium

Epithelial Na(+) transport as measured by a variety of techniques, including the short-circuit current technique, has been described to exhibit a "rundown" phenomenon. This phenomenon manifests as time-dependent decrease of current and resistance and precludes the ability to carry out prolonged experiments aimed at examining the regulation of this transport. We developed methods for prolonged stable recordings of epithelial Na(+) transport using modifications of the short-circuit current technique and commercial Ussing-type chambers. We utilize the polarized MDCK cell line expressing the epithelial Na(+) channel (ENaC) to describe these methods. Briefly, existing commercial chambers were modified to allow continuous flow of Ringer solution and precise control of such flow. Chamber manifolds and associated plumbing were modified to allow precise temperature clamp preventing temperature oscillations. Recording electrodes were modified to eliminate the use of KCl and prevent membrane depolarization from KCl leakage. Solutions utilized standard bicarbonate-based buffers, but all gasses were prehydrated to clamp buffer osmolarity. We demonstrate that these modifications result in measurements of current and resistance that are stable for at least 2 h. We further demonstrate that drifts in osmolarity similar to those obtained before prior to our modifications can lead to a decrease of current and resistance similar to those attributed to rundown.