Osmotic Induction Research Articles

Evaluation of Osmotic Induction of Calcein Treatments for Marking Juvenile Walleyes

AbstractOsmotic induction of calcein was evaluated as a nonlethally detectable alternative to immersion in oxytetracycline for batch marking 42–58‐d old walleyes Sander vitreus. A preliminary laboratory study and five larger‐scale pond experiments were conducted to evaluate mark efficacy, mark retention, and tolerance to immersion in solutions of salt at 25 g/L or 50 g/L of water followed by immersion in a solution of calcein at 5 g/L of water. Walleyes became obviously stressed when immersed in the salt solutions, and mortalities immediately following the treatments ranged from 0% to 53%. All surviving walleyes possessed marks visible with a hand‐held detector. However, the intensity of marks decreased exponentially on walleyes held in ponds, and only walleyes treated by initial immersion in the salt solution of 50 g/L possessed detectable marks when harvested 17–19 weeks posttreatment. The brightest marks occurred on structures most protected from sunlight. Marks on pelvic scales removed at time of capture also remained visible via epifluorescent microscopy for at least 2 weeks when stored in a freezer. These results suggest that osmotic induction of calcein would be a suitable method of producing nonlethally detectable marks for short‐term stocking evaluations if techniques to reduce the stress induced by the marking process could be deployed.Received December 12, 2011; accepted May 1, 2012

Fos proteins are not prerequisite for osmotic induction of vasopressin transcription in supraoptic nucleus of rats

While it is well known that osmotic stimulation induces the expression of Fos family members in the supraoptic nucleus (SON), it is unclear whether the induced protein products are involved in the regulation of the gene transcription of arginine vasopressin (AVP). In the present study, we examined the in vivo correlation between changes in AVP gene transcription and expression of the various Fos family members in the SON after acute osmotic stimuli. The data demonstrated that the peak of AVP transcription (measured by intronic in situ hybridization) observed 15 min after an injection of hypertonic saline preceded the expression of Fos proteins, which became detectable at 30 min and peaked at 120 min. Electrophoretic mobility shift assay showed that the expressed Fos proteins bound to the composite AP-1/CRE-like site in the AVP promoter. These data suggest that Fos proteins in the SON induced by acute osmotic stimuli could affect AVP gene transcription by binding to the AVP promoter, but they are not prerequisite for the induction of AVP gene transcription.

The Anti-Anti-Sigma Factor BldG Is Involved in Activation of the Stress Response Sigma Factor σ H in Streptomyces coelicolor A3(2)

The alternative stress response sigma factor σ(H) has a role in regulation of the osmotic stress response and in morphological differentiation in Streptomyces coelicolor A3(2). Its gene, sigH, is located in an operon with the gene that encodes its anti-sigma factor UshX (PrsH). However, no gene with similarity to an anti-anti-sigma factor which may have a role in σ(H) activation by a "partner-switching" mechanism is located in the operon. By using a combination of several approaches, including pull-down and bacterial two-hybrid assays and visualization of the complex by native polyacrylamide electrophoresis, we demonstrated a direct interaction between UshX and the pleiotropic sporulation-specific anti-anti-sigma factor BldG. Osmotic induction of transcription of the sigHp2 promoter that is specifically recognized by RNA polymerase containing σ(H) was absent in an S. coelicolor bldG mutant, indicating a role of BldG in σ(H) activation by a partner-switching-like mechanism.

An osmotic induction method for externally marking saltwater fishes, Stigmatopora argus and Stigmatopora nigra, with calcein

Calcein marking via osmotic induction and overnight immersion was compared in saltwater fishes. Immersion in a salt solution for 10 min followed by 30 min in a 500 mg l−1 calcein solution produced the highest fluorescent mark in 40 min with no effect on survival.

Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor

Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity.

Tonicity-dependent induction of Sgk1 expression has a potential role in dehydration-induced natriuresis in rodents

In various mammalian species, including humans, water restriction leads to an acute increase in urinary sodium excretion. This process, known as dehydration natriuresis, helps prevent further accentuation of hypernatremia and the accompanying rise in extracellular tonicity. Serum- and glucocorticoid-inducible kinase (Sgk1), which is expressed in the renal medulla, is regulated by extracellular tonicity. However, the mechanism of its regulation and the physiological role of hypertonicity-induced SGK1 gene expression remain unclear. Here, we identified a tonicity-responsive enhancer (TonE) upstream of the rat Sgk1 transcriptional start site. The transcription factor NFAT5 associated with TonE in a tonicity-dependent fashion in cultured rat renal medullary cells, and selective blockade of NFAT5 activity resulted in suppression of the osmotic induction of the Sgk1 promoter. In vivo, water restriction of rats or mice led to increased urine osmolality, increased Sgk1 expression, increased expression of the type A natriuretic peptide receptor (NPR-A), and dehydration natriuresis. In cultured rat renal medullary cells, siRNA-mediated Sgk1 knockdown blocked the osmotic induction of natriuretic peptide receptor 1 (Npr1) gene expression. Furthermore, Npr1-/- mice were resistant to dehydration natriuresis, which suggests that Sgk1-dependent activation of the NPR-A pathway may contribute to this response. Collectively, these findings define a specific mechanistic pathway for the osmotic regulation of Sgk1 gene expression and suggest that Sgk1 may play an important role in promoting the physiological response of the kidney to elevations in extracellular tonicity.

Development and Evaluation of Methods for Osmotic Induction Marking of Golden Perch Macquaria ambigua with Calcein and Alizarin Red S

AbstractWe conducted a series of osmotic induction procedures for marking golden perch Macquaria ambigua with calcein and alizarin red S (ARS), evaluated the factors that influenced mark quality, and tested for any effects on fish growth and mortality. Three aspects of the marking protocols were considered: immersion time in a 5% salt solution (0, 5, or 10 min), immersion time in the fluorescent dye (5 or 10 min), and concentration of the dye (low or high). Quantitative estimates of mark intensity using photographs of marked fish were made with image analysis software. Although there were some significant interactions between factors, salt immersion was generally the primary determinant of mark intensity, followed by dye concentration and dye immersion time. Fish marked with calcein did not have higher mortality rates than unmarked fish and had significantly higher growth rates. The highest‐exposure ARS treatments resulted in higher mortality and lower growth rates than for unmarked fish. Following this result, a lower‐exposure ARS marking protocol was tested, which resulted in no detectable effects on mortality or growth rates while still producing high‐quality marks. Although further study of the long‐term retention of marks under field conditions is required to understand the limitations of the chemical marking methods examined in this study, our results suggest that the methods would greatly enhance our knowledge of the outcomes of fish stocking.

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.

Osmotic regulation of transcription in Lactococcus lactis: Ionic strength-dependent binding of the BusR repressor to the busA promoter

The busA locus of Lactococcus lactis encodes a glycine betaine uptake system. At low osmolarity, the transcription of busA is repressed by the BusR protein, which is responsible for the osmotic inducibility of the busA promoter (busAp). In this work, we investigated the mechanism of the osmo-dependent repression by BusR. We found that BusR binding to the busA promoter is dependent on the ionic strength in vitro. Using a BusR derivative carrying a phosphorylation site and the Escherichia coli RNA polymerase holoenzyme, we showed that these proteins are able to form a stable ternary complex by both binding to the same busAp fragment. The association/dissociation of BusR to the RNA polymerase–busAp complex is strictly correlated to the surrounding ionic strength. Together, these results suggest that during growth at low osmolarity BusR represses transcription from busAp at a step further the recruitment of the RNA polymerase. At high osmolarity, an elevated cytoplasmic ionic strength would dissociate BusR from busAp, resulting in the osmotic induction of the busA operon.

Success of calcein marking via osmotic induction in brown trout fry, Salmo trutta

Fisheries Management and EcologyVolume 14, Issue 3 p. 231-233 Success of calcein marking via osmotic induction in brown trout fry, Salmo trutta D. N. STUBBING, D. N. STUBBING The Game Conservancy Trust, Fordingbridge, Hampshire, UKSearch for more papers by this authorR. D. MOSS, R. D. MOSS The Game Conservancy Trust, Fordingbridge, Hampshire, UKSearch for more papers by this author D. N. STUBBING, D. N. STUBBING The Game Conservancy Trust, Fordingbridge, Hampshire, UKSearch for more papers by this authorR. D. MOSS, R. D. MOSS The Game Conservancy Trust, Fordingbridge, Hampshire, UKSearch for more papers by this author First published: 10 May 2007 https://doi.org/10.1111/j.1365-2400.2007.00525.xCitations: 8 Dominic Stubbing, The Game Conservancy Trust, Fordingbridge, Hampshire SP6 1EF, UK (e-mail: [email protected]) Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume14, Issue3June 2007Pages 231-233 RelatedInformation

Production of External Fluorescent Marks on Golden Perch Fingerlings through Osmotic Induction Marking with Alizarin Red S

AbstractOsmotic induction marking trials with alizarin red S (ARS) were conducted on golden perch Macquaria ambigua to evaluate the use of ARS as a lower‐cost alternative to osmotic induction marking with calcein for producing external fluorescent marks on hatchery‐produced fish. After a 10‐min immersion in a 5% solution of salt, experimental groups of golden perch were rinsed for 5 s in freshwater and immersed for 10 min in either a 0.1% or 0.5% solution of ARS, whereas control groups were either left untreated or immersed in a 0.1% solution of ARS for 10 min without prior immersion in the salt solution. All fish treated in the 0.5% solution of ARS died within 5 min of immersion, but no mortalities were observed in any of the controls or fish treated with the 0.1% solution of ARS. External fluorescent marks were observed on both the osmotic induction‐marked fish and the direct ARS immersion‐marked fish 100 d after treatment, but the osmotic induction‐marked fish possessed more intense marks. Clear marks remained visible on fish marked by osmotic induction 9 months after treatment. This study indicates that highly visible external marks can be produced through osmotic induction with ARS at a substantially lower cost than marks produced through osmotic induction with calcein. Although further refinement and testing are needed, we conclude that the method has considerable potential for use in the mass marking of fish.

Physiological Osmotic Induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extracellular Matrix Proteins and Fibrinogen

Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests.

Osmotic induction of stress proteins in nemerteans

Nemerteans can experience fluctuating salinity during exposure to low tides in intertidal mudflats. This study attempted to determine if stress proteins (SPs) can be induced in nemerteans exposed to fluctuating salinities under laboratory conditions. Paranemertes peregrina Coe 1901 were collected from intertidal mudflats. The ribbon worms were then acclimated at 34‰ for 3–4 days prior to the salinity stress experiments. For the hyperosmotic experiment, 39 and 44‰ were selected. Salinities of 24 and 29‰ were used for the hyposmotic experiment. Both experiments used 34‰ as the control. Three worms were sampled at 0, 2, 4, 6, 12, 18 and 24 h after incubation at each of the salinities. Weights were recorded before and after the sampling periods to determine water efflux and influx under hyperosmotic and hyposmotic conditions, respectively. SPs detected in the nemertean proteins were separated by SDS–PAGE electrophoresis and identified using Western immunoblots with specific antibodies. SP levels were then quantified by densitometry. Worms from the 39 and 44‰ treatments showed significant weight loss compared with control worms. Worms from the 24 and 29‰ treatments had significant weight gain compared with control worms. SP70 and SP90 were identified. Two‐way analysis of variance showed varying significant effects of salinity, time and the interaction between the two parameters on the induction of SP70 and SP90 in both the hyperosmotic and hyposmotic experiments. Detection of SPs demonstrates their importance in the physiological response of nemerteans to fluctuating salinities.

Genomewide identification of Sko1 target promoters reveals a regulatory network that operates in response to osmotic stress in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the ATF/CREB transcription factor Sko1 (Acr1) regulates the expression of genes induced by osmotic stress under the control of the high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway. By combining chromatin immunoprecipitation and microarrays containing essentially all intergenic regions, we estimate that yeast cells contain approximately 40 Sko1 target promoters in vivo; 20 Sko1 target promoters were validated by direct analysis of individual loci. The ATF/CREB consensus sequence is not statistically overrepresented in confirmed Sko1 target promoters, although some sites are evolutionarily conserved among related yeast species, suggesting that they are functionally important in vivo. These observations suggest that Sko1 association in vivo is affected by factors beyond the protein-DNA interaction defined in vitro. Sko1 binds a number of promoters for genes directly involved in defense functions that relieve osmotic stress. In addition, Sko1 binds to the promoters of genes encoding transcription factors, including Msn2, Mot3, Rox1, Mga1, and Gat2. Stress-induced expression of MSN2, MOT3, and MGA1 is diminished in sko1 mutant cells, while transcriptional regulation of ROX1 seems to be unaffected. Lastly, Sko1 targets PTP3, which encodes a phosphatase that negatively regulates Hog1 kinase activity, and Sko1 is required for osmotic induction of PTP3 expression. Taken together our results suggest that Sko1 operates a transcriptional network upon osmotic stress, which involves other specific transcription factors and a phosphatase that regulates the key component of the signal transduction pathway.

SNAT2 silencing prevents the osmotic induction of transport system A and hinders cell recovery from hypertonic stress

Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two siRNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress.

Osmolarity, a Key Environmental Signal Controlling Expression of Leptospiral Proteins LigA and LigB and the Extracellular Release of LigA

The high-molecular-weight leptospiral immunoglobulin-like repeat (Lig) proteins are expressed only by virulent low-passage forms of pathogenic Leptospira species. We examined the effects of growth phase and environmental signals on the expression, surface exposure, and extracellular release of LigA and LigB. LigA was lost from stationary-phase cells, while LigB expression was maintained. The loss of cell-associated LigA correlated with selective release of a lower-molecular-weight form of LigA into the culture supernatant, while LigB and the outer membrane lipoprotein LipL41 remained associated with cells. Addition of tissue culture medium to leptospiral culture medium induced LigA and LigB expression and caused a substantial increase in released LigA. The sodium chloride component of tissue culture medium was primarily responsible for the enhanced release of LigA. Addition of sodium chloride, potassium chloride, or sodium sulfate to leptospiral medium to physiological osmolarity caused the induction of both cell-associated LigA and LigB, indicating that osmolarity regulates the expression of Lig proteins. Osmotic induction of Lig expression also resulted in enhanced release of LigA and increased surface exposure of LigB, as determined by surface immunofluorescence. Osmolarity appears to be a key environmental signal that controls the expression of LigA and LigB.

Retention and Nonlethal External Detection of Calcein Marks in Rainbow Trout and Chinook Salmon

AbstractAn osmotic induction method was used to apply calcein marks to Chinook salmon Oncorhynchus tshawytscha and the Kamloops strain of rainbow trout O. mykiss (both migratory strains used for stocking into tributaries of Lake Superior). Preliminary exposure to a saline solution followed by immersion in a 0.5% calcein solution encouraged rapid infusion of calcein into fish tissues. Four‐month‐old Chinook salmon were exposed to a 1.5% or 5.0% salt solution to evaluate tolerance of different salinities and effects on mark intensity and retention. Rainbow trout were marked at three ages (2 weeks, 3.5 months, and 16 months posthatch) to evaluate the effects of age at treatment on mark intensity and long‐term retention (a 5.0% preliminary salt bath was used). External detection of marks in scales, head, and fin rays was accomplished with a hand‐held detector. The marking technique we used was well tolerated by both Chinook salmon and rainbow trout, and marks persisted into adulthood for rainbow trout treated at 3.5 months posthatch or later. The 5% salt concentration resulted in the persistence of more intense marks than did the 1.5% salt concentration. We demonstrated the potential applicability of calcein marks for instream population studies of migratory or anadromous species treated as fry or for studies involving release of post‐fry‐stage migratory or anadromous fish intended for recapture as adults.

Sgk1 mediates osmotic induction of NPR-A gene in rat inner medullary collecting duct cells.

We have shown previously that increased extracellular osmolality stimulates expression and promoter activity of the type A natriuretic peptide receptor (NPR-A) gene in rat inner medullary collecting duct (IMCD) cells through a mechanism that involves activation of p38 mitogen-activated protein kinase (MAPK). The serum and glucocorticoid inducible kinase (Sgk) is thought to participate in the regulation of sodium handling in distal tubular segments. We sought to determine whether this kinase might be involved in the osmotic stimulation of NPR-A gene promoter activity. Exposure of cultured IMCD cells to an additional 75 mmol/L NaCl in culture media (final osmolality 475 mosm/kg) resulted in an approximately 4-fold increase in Sgk1 protein levels after 7 hours. The Sgk1 induction was almost completely inhibited by the p38 MAPK inhibitor SB203580, indicating that NaCl activates Sgk1 through the p38 MAPK pathway. Transient transfection of a mouse Sgk1 expression vector along with a -1590 NPR-A luciferase reporter resulted in an approximately 3-fold increment in reporter activity, which was significantly reduced by cotransfection with a kinase-dead Sgk1 mutant. The NaCl-dependent induction was partially blocked (approximately 40% inhibition) by cotransfection of the kinase-dead Sgk1 mutant. Neither Sgk1 nor the kinase-dead mutant had any effect on endothelial nitric oxide synthase (eNOS) promoter activity, and the Sgk1 mutant and 8-bromo-cyclic guanosine monophosphate were, to some degree, additive in reducing osmotically stimulated NPR-A promoter activity. Collectively, these data imply that Sgk1 operates over an eNOS-independent, p38 MAPK-dependent pathway in mediating osmotic induction of the NPR-A gene promoter.

Producing Fluorescent Marks on Atlantic Salmon Fin Rays and Scales with Calcein via Osmotic Induction

Abstract Calcified tissues of Atlantic salmon in both larval and postscale developmental life stages became labeled with the fluorochrome dye known as calcein in 7 min by sequential immersion in salt and calcein solutions. I introduce the term “osmotic induction” to describe this process. At 47 d posttreatment, fish that had been exposed to osmotic induction of calcein as nonfeeding larvae exhibited mortality and growth equivalent to that of nonexposed fish (P < 0.05). Atlantic salmon parr (at 5 months posthatch) exposed to osmotic induction of calcein exhibited no mortality during a 120-d posttreatment period. In addition to inducing a calcein mark on fin rays of all larvae and parr, the procedure also produced a distinct calcein mark on scales of parr. A second exposure of parr to osmotic induction 90 d after the first produced a conspicuous 2-band pattern on scales which corresponded to the two separate marking events. Calcein marks are invisible to the unaided eye but were observed without sacrificing...

Isolation and Characterization of ButA, a Secondary Glycine Betaine Transport System Operating in Tetragenococcus halophila

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.