Purification and functional reconstitution of soybean nodulin 26. An aquaporin with water and glycerol transport properties

Infection of soybean roots by nitrogen-fixing Bradyrhizobium japonicum leads to expression of plant nodule-specific genes known as nodulins. Nodulin 26, a member of the major intrinsic protein/aquaporin (AQP) channel family, is a major component of the soybean symbiosome membrane (SM) that encloses the rhizobium bacteroid. To investigate the water and solute transport characteristics of nodulin 26, we purified the protein from SMs and reconstituted it into carboxyfluorescein-loaded liposomes for transport studies using stopped-flow spectrofluorimetry. Liposomes containing nodulin 26 exhibited a high osmotic permeability (Pf = 0. 012 +/- 0.0013 cm/s), a value fivefold higher than that obtained with control liposomes. Water flux through nodulin 26 showed a low activation energy (Ea) (4.07 kcal/mol) and was reduced 70% upon addition of 1 mM HgCl2. Reconstituted nodulin 26 exhibited a single-channel conductance of 3.8 +/- 2.5 x 10(-)15 cm3/s (n = 3), a value that is lower than other characterized AQPs. Nodulin 26 proteoliposomes also facilitate glycerol transport, showing a 43-fold higher rate of glycerol flux than control liposomes. This observation was supported by expression experiments in Xenopus oocytes that showed that nodulin 26 facilitated glycerol flux in a manner indistinguishable from the Escherichia coli GlpF glycerol facilitator. Consistent with the results of water transport, glycerol transport was inhibited by HgCl2 and showed a low Ea (4.43 kcal/mol). These results indicate that nodulin 26 is a multifunctional AQP that confers water and glycerol transport to the SM, and likely plays a role in osmoregulation during legume/rhizobia symbioses.     

Cardiorespiratory and anesthetic effects of propofol and thiopental in dogs

OBJECTIVE: To compare cardiorespiratory and anesthesia effects of IV administered propofol and thiopental in dogs. ANIMALS: 6 healthy mixed-breed dogs. PROCEDURE: Each dog was anesthetized with isoflurane, then a thermistor catheter was inserted in the pulmonary artery. After a minimum of 2.5 hours of recovery, a catheter was placed in a cephalic vein for administration of lactated Ringer's solution and drugs. Propofol (8 mg/kg of body weight) or thiopental (19.4 mg/kg) was administered to each dog in a randomized crossover design study. All dogs were intubated and allowed to breathe 100% oxygen spontaneously. Heart rate and rhythm; systolic, diastolic, and mean arterial blood pressures; respiratory rate; end-tidal carbon dioxide concentration; tidal volume; and reflexes (toe web pinch, palpebral response, and jaw tone) were measured before and every 2 minutes for the first 10 minutes, then at 15, 30, and 60 minutes after drug administration. Cardiac output was determined at 0, 2, 6, 10, 15, 30, and 60 minutes, and blood samples were collected at 0, 2, 10, and 30 minutes. Time to endotracheal extubation, head lift, and ability to sit sternally and walk unaided were recorded. RESULTS: 3 of 6 dogs in each group were apneic after drug administration. Reflexes were decreased similarly for both anesthetic agents, but were not completely lost. Time to sternal position and walking unaided were significantly shorter in response to propofol. CONCLUSION: Anesthesia was rapid; however, respiratory depression and apnea were major adverse effects associated with propofol and thiopental. Propofol has the advantage of inducing rapid, coordinated anesthesia recovery.     

Functional analysis of nodulin 26, an aquaporin in soybean root nodule symbiosomes

Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome. The symbiosome membrane (SM) is a selectively permeable barrier that controls metabolite flux between the plant cytosol and the symbiotic bacterium inside. Nodulin 26 (NOD 26), a member of the aquaporin (AQP) water channel family, is a major protein component of the SM. Expression of NOD 26 in Xenopus oocytes gave a mercury-sensitive increase in osmotic water permeability (Pf). To define the biophysical properties of NOD 26 water channels in their native membranes, symbiosomes were isolated from soybean root nodules and the SM separated as vesicles from the bacteria. Permeabilities were measured using stopped-flow fluorimetry in SM vesicles with entrapped carboxyfluorescein. Osmotic water permeability (Pf) of SM was high, with a value of 0.05 +/- 0.003 cm/s observed at 20 degrees C (mean +/- S.E.; n = 15). Water flow exhibited a low activation energy, was inhibited by HgCl2 (0.1 mM), and exhibited a unit conductance of 3.2 +/- 1.3 x 10(-15) cm3/s, a value 30-fold lower than that of AQP 1, the red blood cell water channel. Diffusive water permeability (Pd) was 0.0024 +/- 0.0002 cm/s, and the resulting Pf to Pd ratio was 18.3, indicating that water crosses the SM in single file fashion via the NOD 26 water channel. In addition to high water permeability, SM vesicles also show high mercury-sensitive permeability to glycerol and formamide, but not urea, suggesting that NOD 26 also fluxes these solutes. Overall, we conclude that NOD 26 acts as a water channel with a single channel conductance that is 30-fold lower than AQP 1. Because the solutes that permeate NOD 26 are far larger than water, and water appears to cross the channel via a single file pathway, solute flux across NOD 26 appears to occur by a pathway that is distinct from that for water.     

Conditioned stimulus determinants of conditioned response form in Pavlovian fear conditioning.

Four experiments using barpress conditioned suppression in rats found that tone evoked more freezing (immobility) than did light. Still, tone and light appeared to have similar conditioned value as assessed by suppression in Experiments 1, 2, and 3, and by blocking, second-order conditioning, and overconditioning assays in Experiments 1, 2, and 3, respectively. Experiment 4 arranged for tone to evoke less suppression than light but more freezing. Results suggest that in fear conditioning, the nature of the conditioned stimulus affects the form of conditioned responding (strong vs. weak freezing). This conclusion extends one drawn by P. C. Holland (see record 1977-12147-001) on the basis of his work in appetitive conditioning. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Observing bullying at school: The mental health implications of witness status.

This study explores the impact of bullying on the mental health of students who witness it. A representative sample of 2,002 students aged 12 to 16 years attending 14 schools in the United Kingdom were surveyed using a questionnaire that included measures of bullying at school, substance abuse, and mental health risk. The results suggest that observing bullying at school predicted risks to mental health over and above that predicted for those students who were directly involved in bullying behavior as either a perpetrator or a victim. Observing others was also found to predict higher risk irrespective of whether students were or were not victims themselves. The results are discussed with reference to past research on bystander and witness behavior. (PsycINFO Database Record (c) 2010 APA, all rights reserved)