Assessment of microbiological quality of drinking water from household tanks in Bermuda

Bermuda residents collect rainwater from rooftops to fulfil their freshwater needs. The objective of this study was to assess the microbiological quality of drinking water in household tanks throughout Bermuda. The tanks surveyed were selected randomly from the electoral register. Governmental officers visited the selected household (n = 102) to collect water samples and administer a short questionnaire about the tank characteristics, the residents' habits in terms of water use, and general information on the water collecting system and its maintenance. At the same time, water samples were collected for analysis and total coliforms and Escherichia coli were determined by 2 methods (membrane filtration and culture on chromogenic media, Colilert kit). Results from the 2 methods were highly correlated and showed that approximately 90% of the samples analysed were contaminated with total coliforms in concentrations exceeding 10 CFU/100 mL, and approximately 66% of samples showed contamination with E. coli. Tank cleaning in the year prior to sampling seems to protect against water contamination. If rainwater collection from roofs is the most efficient mean for providing freshwater to Bermudians, it must not be considered a source of high quality drinking water because of the high levels of microbial contamination.     

Glucose Accumulation Can Account for the Initial Water Flux Triggered by Na+/Glucose Cotransport

Over the last decade, several cotransport studies have led to the proposal of secondary active transport of water, challenging the dogma that all water transport is passive. The major observation leading to this interpretation was that a Na⁺ influx failed to reproduce the large and rapid cell swelling induced by Na⁺/solute cotransport. We have investigated this phenomenon by comparing a Na⁺/glucose (hSGLT1) induced water flux to water fluxes triggered either by a cationic inward current (using ROMK2 K⁺ channels) or by a glucose influx (using GLUT2, a passive glucose transporter). These proteins were overexpressed in Xenopus oocytes and assayed through volumetric measurements combined with double-electrode electrophysiology or radioactive uptake measurements. The osmotic gradients driving the observed water fluxes were estimated by comparison with the swelling induced by osmotic shocks of known amplitude. We found that, for equivalent cation or glucose uptakes, the combination of substrate accumulations observed with ROMK2 and GLUT2 are sufficient to provide the osmotic gradient necessary to account for a passive water flux through SGLT1. Despite the fact that the Na⁺/glucose stoichiometry of SGLT1 is 2:1, glucose accumulation accounts for two-thirds of the osmotic gradient responsible for the water flux observed at t = 30 s. It is concluded that the different accumulation processes for neutral versus charged solutes can quantitatively account for the fast water flux associated with Na⁺/glucose cotransport activation without having to propose the presence of secondary active water transport.     

Stimulating Effect of External Myo-Inositol on the Expression of Mutant Forms of Aquaporin 2

Myo-inositol (MI; hexahydroxycyclohexane, C₆H₆O₁₂) is a small neutral molecule used as a compatible osmolyte in the kidney medulla. At high concentrations, MI appears to act as a chemical chaperone and was shown to promote plasma membrane expression of the impaired cystic fibrosis chloride channel (Δ508-CFTR). In the present study, we measured whether MI could increase expression of two human aquaporin 2 (AQP2) mutants which were recently identified as causing nephrogenic diabetes insipidus (NDI). Both proteins (D150E and G196D) were expressed in Xenopus laevis oocytes, but only D150E displayed an increase in oocyte water permeability (P _f). Adding 5 mM MI to the bathing solution for 24 h produced a 50% increase in the D150E-associated P _f, while it had no effect on noninjected oocytes or on oocytes expressing wt-AQP2 or G196D. Western blots performed on purified plasma membrane preparations confirmed that MI increased the amount of D150E present at the plasma membrane, while G196D was always undetectable. X. laevis oocytes are remarkably impermeable to MI, and the effect of MI on D150E expression does not require the presence of intracellular MI. The effect of external MI was dose-dependent (K _0.5 was 130 μM) and specific with respect to other forms of inositols. Further studies on a second group of AQP2 mutants causing NDI showed that K228E activity was similarly stimulated by MI, while V71M, A70D and S256L were not. It is concluded that physiological concentrations of extracellular MI can stimulate the expression of a specific subgroup of AQP2 mutants.     

Real-time fluorescence-based detection of furanocoumarin photoadducts of DNA

Real-time fluorescence detection systems were adapted to identify DNA adducts formed by photogenotoxic phytochemicals. Two assays were developed: the first was based on quantitative polymerase chain reaction (qPCR) while the second used thermal denaturation and renaturation (D-R). Both assays employed yeast DNA, the fluorescent dye SYBR Green and a real-time PCR thermocycler. The furanocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), psoralen, angelicin and imperatorin, and the furanochrome khellin, were tested for adduct forming ability with up to 2 h of UVA light exposure (lambda = 320-400 nm). The known bifunctional compounds, 8-MOP, 5-MOP and psoralen, were inferred to form biadducts here based on both D-R and qPCR assays, as expected from previous research. The known monofunctional compound angelicin was used as a negative control and did not form biadducts based on either assay. Two compounds of unknown functional specificity, imperatorin and khellin, were determined to be positive and negative for biadduct activity, respectively. Detection of biadducts with 8-MOP, 5-MOP, psoralen and imperatorin, but not angelicin or khellin, was further verified by temperature gradient gel electrophoresis. The fluorescence methods improve and expand upon existing assays to monitor DNA adducts.     

Effect of inspired oxygen on periodic breathing in methy-CpG-binding protein 2 (Mecp2) deficient mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (Mecp2) that encodes a DNA binding protein involved in gene silencing. Periodic breathing (Cheyne-Stokes respiration) is commonly seen in RTT. Freely moving mice were studied with continuous recording of pleural pressure by telemetry. Episodes of periodic breathing in heterozygous Mecp2 deficient (Mecp2(+/-)) female mice (9.4 +/- 2.2 h(-1)) exceeded those in wild-type (Mecp2(+/+)) animals (2.5 +/- 0.4 h(-1)) (P = 0.010). Exposing Mecp2(+/-) animals to 40% oxygen increased the amount of periodic breathing from 118 +/- 25 s/30 min in air to 242 +/- 57 s/30 min (P = 0.001), and 12% oxygen tended to decrease it (67 +/- 29 s/30 min, P = 0.14). Relative hyperoxia and hypoxia did not affect the incidence of periodic breathing in Mecp2(+/+) animals. The ventilation/apnea ratio (V/A) was less at all levels of oxygen in heterozygous Mecp2(+/-) females compare with wild type (P = 0.003 to P < 0.001), indicating that their loop gain is larger. V/A in Mecp2(+/-) fell from 2.42 +/- 0.18 in normoxia to 1.82 +/- 0.17 in hyperoxia (P = 0.05) indicating an increase in loop gain with increased oxygen. Hyperoxia did not affect V/A in Mecp2(+/+) mice (3.73 +/- 0.28 vs. 3.5 +/- 0.28). These results show that periodic breathing in this mouse model of RTT is not dependent on enhanced peripheral chemoreceptor oxygen sensitivity. Rather, the breathing instability is of central origin.     

Identification of bacteria contaminating pulp and a paper machine in a Canadian paper mill

Over 100 bacteria from pulp and slime samples in a Canadian paper mill were identified by partial sequencing of their 16S rDNAs. Seventy-one percent of the isolates could be assigned to a bacterial genus with a high level of confidence. Another 12% exhibited at least 95% similarity within their 16S rDNA sequence with unidentified organisms that originate from warm or wet environments. Pseudomonas, Bacillus, and Pseudoxanthomonas isolates were represented at a relatively high proportion in both pulp and slime samples. This is the first time that Pseudoxanthomonas strains have been isolated from pulp and slime samples on a paper machine. Electronic Publication     

New technique for measurement of left ventricular pressure in conscious mice

Concern about the effects of anesthesia on physiological measurements led us to develop methodology to assess left ventricular (LV) pressure in conscious mice. Polyethylene-50 tubing filled with heparinized saline was implanted in the LV cavity through its apex via an abdominal approach and exteriorized to the back of the animal. This surgery was done under anesthesia with either an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg) (K+X) in 11 mice or isoflurane (ISF; 1.5 vol%) by inhalation in 14 mice. Postoperatively, mice were trained daily to lie quietly head first in a plastic cone. LV pressure, the first derivative of LV pressure (dP/dt), and heart rate (HR) in the conscious state were compared between the two groups at 3 days and 1 wk after recovery from surgery using a 1.4-Fr Millar catheter inserted into the LV through the tubing, with the mice lying quietly in the plastic cone. Acutely during anesthesia, K+X decreased HR (from 698 to 298 beats/min), LV systolic pressure (from 107 to 65 mmHg), and maximal dP/dt (dP/dt(max)) (from 15,724 to 4,445 mmHg/s), all P < 0.01. Similar but less marked negative chronotropic and inotropic effects were seen with ISF. HR and dP/dt(max) were decreased significantly in K+X mice 3 days after surgery compared with those anesthetized with ISF (655 vs. 711 beats/min, P < 0.05; 14,448 vs. 18,048 mmHg/s, P < 0.001) but increased to the same level as in ISF mice 1 wk after surgery. In ISF mice, recovery of function occurred rapidly and there were no differences in LV variables between 3 days and 1 wk. LV pressure and dP/dt can be measured in conscious mice with a micromanometer catheter inserted through tubing implanted permanently in the LV apex. Anesthesia with either K+X or, to a lesser extent, ISF, depressed LV function acutely. This depression of function persisted for 3 days after surgery with K+X (but not ISF) and did not recover completely until 1 wk postanesthesia.     

Importance of histamine in the cytokine network in the lung through H2 and H3 receptors: stimulation of IL-10 production

Histamine, a well-known inflammatory mediator, has been implicated in various immunoregulatory effects that are poorly understood. Thus, we tested the hypothesis that histamine inhibits the release of a proinflammatory cytokine, namely TNF, by stimulating the release of an anti-inflammatory cytokine, IL-10. Alveolar macrophages (AMs) from humans, Sprague Dawley rats, and the AM cell line, NR8383, were treated with different concentrations of histamine (10-5-10-7 M) for 2 h prior to their stimulation with suboptimal concentration of LPS (1 ng/ml) for 4 h. Histamine inhibited TNF release in a dose-dependent manner. This inhibition was mimicked by H2 and H3 receptor agonists, but not by H1 receptor agonist. Furthermore, we demonstrated the expression of H3 receptor mRNA in human AMs. Interestingly, treatment of AMs with anti-IL-10, anti-PGE2, or a NO synthase inhibitor (Nomega-nitro-l -arginine methyl ester) before the addition of histamine abrogated the inhibitory effect of the latter on TNF release. Histamine treatment (10-5 M) increased the release of IL-10 from unstimulated (2.2-fold) and LPS-stimulated (1. 7-fold) AMs. Unstimulated AMs, NR8383, express few copies of IL-10 mRNA, as tested by quantitative PCR, but expression of IL-10 was increased by 1.5-fold with histamine treatment. Moreover, the stimulation of IL-10 release by histamine was abrogated by pretreatment with anti-PGE2 or the NO synthase inhibitor, Nomega-nitro-l -arginine methyl ester. Thus, histamine increases the synthesis and release of IL-10 from AMs through PGE2 and NO production. These results suggest that histamine may play an important role in the modulation of the cytokine network.     

Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life

Selected topics in the respiratory response to acute hypoxia in the fetus and newborn are reviewed. Peripheral chemoreceptors acting through ionotrophic glutamate receptors play an important role in affecting the initial augmentation phase. Whether fall off in peripheral chemoreceptor activity contributes to the secondary depressive phase remains controversial. A number of approaches including permanent electrolytic and reversible cooling lesions, Fos protein activation, and double-labeling immunohistochemistry has converged to show that an area in and around the locus ceruleus in the rostral pons affects the central depression. There is evidence that this is mediated by catecholamines acting at alpha(2)-adrenergic receptors. Tonic activity in early expiratory (postinspiratory) neurons may contribute to hypoxia-induced apneic episodes in the fetus and newborn. Desensitization of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors has been demonstrated in respiratory-related neurons both in vivo and in vitro. The role that this process might play in the depressive phase of the hypoxic ventilatory response has not been established. In vitro experiments with isolated brain stem-spinal cord preparations or transverse brain stem slices usually involve anoxia, whereas whole animal experiments use 8-15% O(2). Therefore, caution must be exercised in attempting to construct a unifying framework from these two approaches.