Biomedical subjects

R M Dean

Publications and source records attributed to R M Dean.

9 recordsLinked to original sources

PubMed2006

Do no harm.

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Dental Occlusion, Balanced↗

PubMed1999

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1996

Glucose-induced tyrosine phosphorylation of p125 in beta cells and pancreatic islets. A novel proximal signal in insulin secretion.

In this study, we demonstrate that stimulation of beta cells with carbachol and glucose causes increased tyrosine phosphorylation of a 125-kDa protein concurrently with increased insulin secretion. The effect was observed in two different insulin-secreting cell lines and in rat pancreatic islets. Tyrosine phosphorylation was largely calcium independent and occurred within 2 min after stimulation of beta cells with glucose and the muscarinic agonist carbachol. In islets, the effect of glucose was greatly diminished by the addition of mannoheptulose, a seven-carbon sugar that inhibits glucokinase, suggesting that glucose metabolism is required for tyrosine phosphorylation of the protein to occur. Neither insulin nor insulin-like growth factor I significantly increased tyrosine phosphorylation of the 125-kDa protein, suggesting that it was not an autocrine effect. Depolarization of beta cells with glyburide or 50 m potassium dramatically increased insulin secretion but had no significant effect on tyrosine phosphorylation. Addition of phorbol ester caused a less than 2-fold increase in tyrosine phosphorylation, whereas the calcium ionophore A23187 had no effect. Among the various fuel secretagogues tested, only -glucose stimulated tyrosine phosphorylation, both alone and in combination with carbachol. Finally, the tyrosine kinase inhibitor AG879 inhibited both tyrosine phosphorylation and insulin secretion in a dose-dependent manner. Taken together, these data demonstrate the presence of a novel signaling pathway in glucose-induced insulin secretion: tyrosine phosphorylation of beta cell p125, which is a proximal step in insulin secretion. Our current working hypothesis is that glucose stimulation of beta cell p125 tyrosine phosphorylation is an essential step for insulin secretion.

Animals↗

PubMed1975