The effect of osmotic and ion-osmotic stresses on the blood and urine composition and urine flow of rainbow trout (Salmo gairdneri)

1. 1. Changes in urine and plasma concentrations (sodium, potassium, magnesium, calcium and total osmotic) and urine production were determined in fish exposed to various concentrations of an ionically active substance, sodium chloride, and a non-electrolyte, mannitol, as well as freshwater.2. 2. Responses occurred for the most part over a short crisis period preceeding establishment of new stable conditions.3. 3. It was shown that plasma homeostasis was not maintained in response to changing ion-osmotic and osmotic gradients.4. 4. Urinary osmotic and ionic concentrations were unaffected and urine production was shown to be inversely related to the external concentration.5. 5. It is suggested that ionic shifts between body compartments are an important aspect of ion-osmotic adaptation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24991/1/0000418.pd

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Ionic and Osmotic Regulation in Rainbow Trout, Salmo Gairdneri.

Three experiments were performed using rainbow trout (Salmo gairdneri), to determine; (1)the feasibility of using an osmotic determinant to separate the ionic and osmotic components of ion-osmoregulation, (2)changes in urine and plasma concentrations and urine production associated with ionic and osmotic regulation and , (3)the energy expenditures involved in ionic and osmotic regulation. The separation of the ionic and osmotic components was accomplished by exposing fish to different but equal concentrations of ion-osmotic (sodium chloride) and osmotic (mannitol) solutions. In the first set of experiments fish were exposed to mannitol and sodium chloride concentrations of 400 to 700 mOsm. The 24-LC50's were calculated to be 767 and 175 mOsm for mannitol and sodium chloride, respectively. Mannitol's relatively lower toxicity indicated that it was possible to use it as an osmotic determinant in ion-osmo-regulation experiments utilizing fish. Plasma and urine regulatory changes in ionic and osmotic regulation were determined in a second set of experiments by exposing fish to mannitol and sodium chloride solutions and measuring plasma and urinary osmotic and ionic concentrations and urine production. Freshwater acclimated fish were transferred to test solutions of deionized water and mannitol and sodium chloride concentrations of 10, 250, and 500 mOsm. Control fish were held in freshwater. Plasma samples were taken prior to the transfer and after exposure periods of 1, 2, 4, and 7 days. Urine samples were also taken prior to transfer and then daily for 4 days after the exposure. In addition, short-term ion-osmotic adaptation was studied by exposing fish to 200 mOsm sodium chloride and freshwater and measuring plasma osmotic and ionic concentrations every 4 hours for 36 hours. Responses occurred for the most part over a short crisis period preceding establishment of new stable conditions. Plasma osmotic, sodium and potassium concentrations of hypo- and hyper- ion-osmotic exposed fish reached maximum levels after 8-24 hours, then decreased and stabilized after 7 days. Plasma calcium and magnesium concentrations of hypo-ion-osmotic exposed fish decreased initially and remained at reduced levels. Plasma osmotic concentrations of fish exposed to hyper-osmotic solutions also increased, however, there was no change in plasma sodium or potassium concentrations in these fish. As in the case of the hyper-ion-osmotic exposed fish plasma calcium and magnesium concentrations decreased in fish exposed to hypo- and hyper-osmotic solutions. There was no significant difference (p < 0.05) in urinary osmotic and ionic concentrations between fish exposed to hypo- and hyper-, ion-osmotic and osmotic solutions and the control fish. Urine production was shown to be inversely related to the external concentration, higher rates of urine production occurring at hypo-concentrations. Significantly (p < 0.05) lower rates of urine production were found after 4 days in fish exposed to ion-osmotic solutions than those in osmotic solutions at all concentrations. It was shown that plasma homeostasis was not maintained in response to changing ion-osmotic gradients and in the case of plasma osmotic concentration with changing osmotic gradients. Also, short-term plasma ionic homeostasis was maintained in response to osmotic adaptation. Recovery did not occur with a large hyper-ion-osmotic gradient when death resulted. It is suggested that ionic shifts between body compartments are an important aspect of ion-osmotic adaptation. In the last set of experiments the energetic costs of ionic and osmotic regulation was calculated from oxygen consumption data from fish exposed to freshwater, deionized water and mannitol and sodium chloride concentrations of 10, 250, and 500 mOsm. The results indicated that the cost of ionic regulation is significantly higher than the cost of osmotic regulation and that the cost of osmotic regulation is small.Ph.D.Animal PhysiologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/158289/1/8116275.pd

Energy expenditure for osmoregulation in rainbow trout, Salmo gairdneri

Master of ScienceResource EcologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/113960/1/39015003265850.pd