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New models of fluid transport are expected to emerge from the confinement of liquids at the nanoscale (1,2), with potential applications in ultrafiltration, desalination and energy conversion (3). Nevertheless, advancing [...]

Unsaturated clays are subject to osmotic suction gradients in geoenvironmental engineering applications and it therefore becomes important to understand the effect of these chemical concentration gradients on soil-water characteristic curves (SWCCs). This paper brings out the influence of induced osmotic suction gradient on the wetting SWCCs of compacted clay specimens inundated with sodium chloride solutions/distilled water at vertical stress of 6.25 kPa in oedometer cells. The experimental results illustrate that variations in initial osmotic suction difference induce different magnitudes of osmotic induced consolidation and osmotic consolidation strains thereby impacting the wetting SWCCs and equilibrium water contents of identically compacted clay specimens. Osmotic suction induced by chemical concentration gradients between reservoir salt solution and soil-water can be treated as an equivalent net stress component, ([p.s[pi]) that decreases the swelling strains of unsaturated specimens from reduction in microstructural and macrostructural swelling components. The direction of osmotic flow affects the matric SWCCs. Unsaturated specimens experiencing osmotic induced consolidation and osmotic consolidation develop lower equilibrium water content than specimens experiencing osmotic swelling during the wetting path. The findings of the study illustrate the need to incorporate the influence of osmotic suction in determination of the matric SWCCs. DOI: 10.1061/(ASCE)GT.1943-5606.0000389 CE Database subject headings: Clays; Unsaturated soils; Suction; Laboratory tests; Expansive soils. Author keywords: Clays; Unsaturated soils; Suction; Laboratory tests; Expansive soils.

We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural [zeta]-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a post-column reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 [micro]m (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs. 10.1021/ac1012706

Adaptation to osmotic stress can be achieved by the accumulation of compatible solutes that aid in turgor maintenance and macromolecule stabilization. The genetic regulation of solute accumulation is poorly understood, and has been described well at the molecular level only in enterobacteria. In this study, we show the importance of the alternative sigma factor RpoE2 in Sinorhizobium meliloti osmoadaptation. Construction and characterization of an S. meliloti rpoE2 mutant revealed compromised growth in hyperosmotic media. This defect was due to the lack of trehalose, a minor carbohydrate osmolyte normally produced in the initial stages of growth and in stationary phase. We demonstrate here that all three trehalose synthesis pathways are RpoE2 dependent, but only the OtsA pathway is important for osmoinducible trehalose synthesis. Furthermore, we confirm that the absence of RpoE2-dependent induction of otsA is the cause of the osmotic phenotype of the rpoE2 mutant. In conclusion, we have highlighted that, despite its low level, trehalose is a crucial compatible solute in S. meliloti, and the OtsA pathway induced by RpoE2 is needed for its accumulation under hyperosmotic conditions. DOI 10.1099/mic.0.034850-0

The osmotic technique was calibrated at various temperatures (20--40°C) using a high-capacity tensiometer. The effect of temperature on the calibration curve of the high-capacity tensiometer in the positive range has been found to be insignificant, i.e., about 0.03%/°C. The measurement at ambient temperature shows that the suction value is not significantly dependent either on the molar mass of polyethylene glycol (PEG) or on the molecular weight cutoff (MWCO) of the semipermeable cellulose membrane. On the other hand, the matric suction measured in the present work by tensiometer was lower than the total suction found in the literature measured by psychrometer. This shows that the so-called membrane effects must be characterized by not only the crossing of PEG molecules but also other complex phenomena. The calibration at controlled temperatures showed a slight suction decrease of 1%/°C. One possible explanation for this decrease is that increasing the temperature decreases the viscosity of PEG solutions, possibly accelerating the crossing of PEG molecules. It is also possible that increasing the temperature changes the physicochemical properties of the PEG solutions, resulting in a suction decrease. Key words: suction control, osmotic technique, temperature effect, high-capacity tensiometer, polyethylene glycol, calibration. La technique osmotique a ete calibreea differentes temperatures (20 a 40 °C) a l'aide d'un tensiometre a haute capacite;. L'effet de la temperature sur la courbe de calibrage s'est avere non significatif, soit environ 0,03%/°C. La mesure a temperature ambiante a demontre que la valeur de succion n'est pas significativement dependante de la masse molaire du glycol de polyethylene (GPE) ni de la masse molaire de coupure de la membrane de cellulose semi-permeable. D'un autre cote, la succion matricielle mesuree par le tensiometre dans la presente etude etait plus faible que les valeurs de succion totale mesurees avec un psychrometre reporteees dans la litterature. Ceci demontre que ce qu'on appelle couramment l'effet de membrane doit etre caracterise non seulement par le passage des molecules de GPE mais aussi par d'autres phenomenes complexes. Le calibrage a temperature controkie a permis d'observer une faible diminution de la succion de 1%/°C. Une explication possible pour cette diminution est que l'augmentation de la temperature diminue la viscosite des solutions de GPE, ce qui peut acceileerer le passage des molecules de GPE a travers la membrane. Il est aussi possible que l'augmentation de la temperature puisse modifier les proprieties physico-chimiques des solutions de GPE, ce qui entrainerait une diminution de la succion. Mots-cles: controle de la succion, technique osmotique, effet de la temperature, tensiometre a haute capacite;, glycol de polyethylene, calibrage. [Traduit par la Redaction], Introduction The effects of temperature changes on the hydromechanical behaviour of unsaturated soils are important in various geotechnical applications such as ground energy exploitation, stability of buried pipelines or power [...]

The transcription factor, tonicity-responsive enhancer binding protein (TonEBP), is involved in the adaptive response against hypertonicity. TonEBP regulates the expression of genes that catalyze the accumulation of osmolytes, and its transcriptional activity is increased by hypertonicity. The goal of the present investigation was to investigate whether cell shrinkage or high intracellular ionic concentration induced the activation of TonEBP. We designed a model system for isotonically shrinking cells over a prolonged period of time. Cells swelled in hypotonic medium and performed a regulatory volume decrease. Upon return to the original isotonic medium, cells shrank initially, followed by a regulatory volume increase. To maintain cell shrinkage, the RVI process was inhibited as follows: ethyl-isopropyl-amiloride inhibited the [Na.sup.+]/[H.sup.+] antiport, bumetanide inhibited the [Na.sup.+]-[K.sup.+]-[2Cl.sup.-] cotransporter, and gadolinium inhibited shrinkage-activated [Na.sup.+] channels. Cells remained shrunken for at least 4 h (isotonically shrunken cells). The activity of TonEBP was investigated with a Luciferase assay after isotonic shrinkage and after shrinkage in a high-NaCl hypertonic medium. We found that TonEBP was strongly activated after 4 and 16 h in cells in high-NaCl hypertonic medium, but not after 4 or 16 h in isotonically shrunken cells. Cells treated with high-NaC1 hypertonic medium for 4 h had significantly higher intracellular concentrations of both [K.sup.+] and [Na.sup.+] than isotonically shrunken cells. This strongly suggested that an increase in intracellular ionic concentration and not cell shrinkage is involved in TonEBP activation. cell volume; cell ionic strength; cell ionic concentration; NIH3T3 cells; isosmotic shrinkage; tonicity-responsive enhancer binding protein

Here we demonstrate that an impedance-based microfluidic cell volume sensor can be used to study the roles of aquaporin (AQP) in cellular water permeability and screen AQP-specific drugs. Human embryonic kidney (HEK-293) cells were transiently transfected with AQP3-or AQP4-encoding genes to express AQPs in plasma membranes. The swelling of cells in response to hypotonic stimulation was traced in real time using the sensor. Two time constants were obtained by fitting the swelling curves with a two-exponential function, a fast time constant associated with osmotic water permeability of AQP-expressing cells and a slow phase time constant associated mainly with water diffusion through lipid bilayers in the nontransfected cells. The AQP-expressing cells showed at least 10x faster osmotic water transport than control cells. Using the volume sensor, we examined the effects of [Hg.sup.2+] and [Ni.sup.2+] on the water transport via AQPs. [Hg.sup.2+] inhibited the water flux in AQP3-expressing cells irreversibly, while [Ni.sup.2+] blocked the AQP3 channels reversibly. Neither of the two ions blocked the AQP4 channels. The microfluidic volume sensor can sense changes in cell volume in real time, which enables perfusion of various reagents sequentially. It provides a convenient tool for studying the effect of reagents on the function and regulation mechanism of AQPs.

A microfluidic analogy of the electric Wheatstone Bridge has been developed for electrokinetic study of miscellaneous liquid--solid interfaces. By using an optimized glass--PDMS--glass device technology, microfluidic channels with well-controlled surface properties can be fabricated, forming an 'H' shaped fluidic network. After solving a set of linear equations, the electro-osmotic flow rate in the center channel can be deduced from indirect measurement of flow rates in the lateral channels. Experimentally, we demonstrate that the electro-osmotic mobility can be monitored every 30 s with accuracy better than 3% for a large dynamic range of electric fields. The results obtained with a borosilicate glass (D-263) and several standard biological buffers are also shown to illustrate the capability of this high throughput method.

High NaCI rapidly activates p38 MAPK by phosphorylating it, the phosphorylation presumably being regulated by a balance of kinases and phosphatases. Kinases are known, but the phosphatases are uncertain. Our initial purpose was to identify the phosphatases. We find that in HEK293 cells transient overexpression of MAPK phosphatase-1 (MKP-1), a dual-specificity phosphatase, inhibits high NaCI-induced phosphorylation of p38, and that overexpression of a dominant negative mutant of MKP-1 does the opposite. High NaCI lowers MKP-1 activity by increasing reactive oxygen species, which directly inhibit MKP-1, and by reducing binding of MKP-1 to p38. Because inhibition of p38 is reported to reduce hypertonicity-induced activation of the osmoprotective transcription factor, TonEBP/OREBP, we anticipated that MKP-1 expression might also. However, overexpression of MKP-1 has no significant effect on Ton EBP/OREBP activity. This paradox is explained by opposing effects of p38[alpha] and p38[delta], both of which are activated by high NaCI and inhibited by MKP-1. Thus, we find that overexpression of p38[alpha] increases high NaCI-induced TonEBP/ OREBP activity, but overexpression of p38[delta] reduces it. Also, siRNAmediated knockdown of p38[delta] enhances the activation of TonEBP/OREBP. We conclude that high NaCI inhibits MKP-1, which contributes to the activation of p38. However, opposing actions of p38[alpha] and p38[delta] negate any effect on TonEBP/OREBP activity. Thus, activation of p38 isoforms by hypertonicity does not contribute to activation of TonEBP/OREBP because of opposing effects of p38[alpha] and p38[delta], and effects of inhibitors of p38 depend on which isoform is affected, which can be misleading. hypertonicity | reactive oxygen species | transcription HEK293 cells | mlMCD3 cells

Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-[beta]-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most likely via a pathway independent of PDGFR-[beta] and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments, hypoosmolarity did not affect PDGFBB-induced activation of PDGFR-[beta], whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-[beta] activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand-dependent activation of PDGFR-[beta] and its downstream effectors Akt, MEK1/2, and ERK1/2 is strongly modulated (inhibited) by hyperosmotic cell shrinkage, whereas cell swelling does not seem to affect the activation of the receptor but rather to activate ERK1/2 via a different mechanism. It is thus likely that cell swelling via activation of ERK1/2 and cell shrinkage via activation of the p38 and JNK pathway and inhibition of the PDGFR signaling pathway may act as key players in the regulation of tissue homeostasis. mitogen-activated protein kinases; platelet-derived growth factor receptor-[beta]; volume sensor

This study was intended to determine the osmoadaptation strategy of Hortaea werneckii, an extremely salt-tolerant melanized ascomycetous fungus that can grow at 0-5.1 M NaCl. It has been shown previously that glycerol is the major compatible solute in actively growing H. werneckii. This study showed that the exponentially growing cells also contained erythritol, arabitol and mannitol at optimal growth salinities, but only glycerol and erythritol at maximal salinities. The latter two were both demonstrated to be major compatible solutes in H. werneckii, as their decrease correlated with the severity of hypoosmotic shock. Besides higher amounts of erythritol and lower amounts of glycerol, stationary-phase cells also contained mycosporine-glutaminol-glucoside, which might act as a complementary compatible solute. H. werneckii is constitutively melanized under various salinity conditions. Ultrastructural study showed localization of melanin in the outer parts of the cell wall as a distinct layer at optimal salinity (0.86 M NaCl), whereas cell-wall melanization diminished at higher salinities. The role of melanized cell wall in the effective retention of glycerol is already known, and was also demonstrated in H. werneckii by lower retention of glycerol in cells with blocked melanization compared to melanized cells. However, these non-melanized cells compensated for the lower amounts of glycerol with higher amounts of erythritol and arabitol. We hypothesize that H. werneckii melanization is effective in reducing the permeability of its cell wall to its major compatible solute glycerol, which might be one of the features that helps it tolerate a wider range of salt concentrations than most organisms.

Collecting duct (CD)-derived endothelin-1 (ET-1) exerts natriuretic, diuretic, and hypotensive effects. In vitro studies have implicated cyclooxygenase (COX) metabolites, and particularly PG[E.sub.2], as important mediators of CD ET-1 effects. However, it is unknown whether PG[E.sub.2] mediates CD-derived ET-1 actions in vivo. To test this, CD ET-1 knockout (KO) and control mice were studied. During normal salt and water intake, urinary PG[E.sub.2] excretion was unexpectedly increased in CD ET-1 KO mice compared with controls. Salt loading markedly increased urinary PG[E.sub.2] excretion in both groups of mice; however, the levels remained relatively higher in KO animals. Acutely isolated inner medullary collecting duct (IMCD) from KO mice also had increased PG[E.sub.2] production. The increased IMCD PG[E.sub.2] was COX-2 dependent, since NS-398 blocked all PG[E.sub.2] production. However, increased CD ET-1 KO COX-2 protein or mRNA could not be detected in inner medulla or IMCD, respectively. Inner medullary COX-1 mRNA and protein levels and IMCD COX-1 mRNA levels were unaffected by Na intake or CD ET-1 KO. KO mice on a normal or high-Na diet had elevated blood pressure compared with controls; this difference was not altered by indomethacin or NS-398 treatment. However, indomethacin or NS-398 did increase urine osmolality and reduce urine volume in KO, but not control, animals. In summary, IMCD COX-2-dependent PG[E.sub.2] production is increased in CD ET-1 KO mice, indicating that CD-derived ET-1 is not a primary regulator of IMCD PG[E.sub.2]. Furthermore, the increased PG[E.sub.2] in CD ET-1 KO mice partly compensates for loss of ET-1 with respect to maintaining urinary water excretion, but not in blood pressure control. cyclooxygenase; inner medullary; sodium

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg.[kg.sup.-1]*[day.sup -1] intraperitoneally for 14 days), an ET-1 receptor B ([ET.sub.B]) antagonist, induced a significant decrease in Gardos channel activity (1.7 [+ or -] 0.1 to 1.0 [+ or -] 0.4 mmol. 1013 [cell.sup.-1]* [h.sup.-1], n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D5o; n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (E[T.sub.A]) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of E[T.sub.B]/E[T.sub.A] antagonists but not by E[T.sub.A] antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the E[T.sub.B] receptor, leading to Gardos channel modulation in SCD. cellular dehydration; Gardos channel; transgenic sickle mice

Members of the germinal center kinase (GCK)-VI subfamily of Ste20 kinases regulate a Caenorhabditis elegans C1C anion channel and vertebrate SLCI2 cation-[Cl.sup.-] cotransporters. With no lysine (K) (WNK) protein kinases interact with and activate the mammalian GCK-VI kinases prolinealanine-rich Ste20-related kinase (PASK) and oxidative stress-responsive 1 (OSR1). We demonstrate here for the first time that GCK-VI kinases play an essential role in whole animal osmoregulation. RNA interference (RNAi) knockdown of the single C. elegans GCK-VI kinase, GCK-3, dramatically inhibits systemic volume recovery and survival after hypertonic shrinkage. Tissue-specific RNAi suggests that GCK-3 functions primarily in the hypodermis and intestine to mediate volume recovery. The single C. elegans WNK kinase, WNK-1, binds to GCK-3, and wnk-1 knockdown gives rise to a phenotype qualitatively similar to that of gck-3(RNAi) worms. Knockdown of the two kinases together has no additive effect, suggesting that WNK-1 and GCK-3 function in a common pathway. We postulate that WNK-1 functions upstream of GCK-3 in a manner similar to that postulated for its mammalian homologs. Phylogenetic analysis of kinase functional domains suggests that the interaction between GCK-VI and WNK kinases first occurred in an early metazoan and therefore likely coincided with the need of multicellular animals to tightly regulate transepithelial transport processes that mediate systemic osmotic homeostasis. cell volume regulation; osmotic stress; osmoregulation

Luminescent dinoflagellates respond to flow by the production of light. The primary mechanotransduction event is unknown, although downstream events include a calcium flux in the cytoplasm, a self-propagating action potential across the vacuole membrane, and a proton flux into the cytoplasm that activates the luminescent chemistry. Given the role of GTP-binding (G) proteins in the mechanotransduction of flow by nonmarine cells and the presence of G-proteins in dinoflagellates, it was hypothesized that flow-stimulated dinoflagellate bioluminescence involves mechanotransduction by G-proteins. In the present study, osmotic swelling of cells of the dinoflagellate Lingulodinium polyedrum was used as a drug delivery system to introduce GDP[beta]S, an inhibitor of G-protein activation. Osmotically swollen cells produced higher levels of flow-stimulated bioluminescence at a lower threshold of shear stress, indicating they were more flow sensitive. GDP[beta]S inhibited flow-stimulated bioluminescence in osmotically swollen cells and in cells that were restored to the isosmotic condition following hypoosmotic treatment with GDP[beta]S. These results provide evidence that G-proteins are involved in the mechanotransduction of flow in dinoflagellates and suggest that G-protein involvement in mechanotransduction may be a fundamental evolutionary adaptation. osmotic drug delivery; shear stress; Lingulodinium doi:10.1152/ajpregu.00649.2006

A study was carried out to analyze and model changes in apple liquid phase composition throughout equilibrium in osmotic dehydration. The fruit liquid phase overconcentration rate was observed to depend on the concentration of the osmotic solution, the processing temperature, the sample size, and shape of the cellular tissue.

For osmoprotection, Escherichia coil can synthesize glycine betaine from externally supplied choline by the Bet system (betTIBA products). The major carrier of choline is the high-affinity, proton-driven, secondary transporter BetT, which belongs to the BCCT family of transporters. Fusion proteins consisting of N-terminal fragments of BetT linked to [beta]-galactosidase (LacZ) or alkaline phosphatase (PhoA) were constructed. By analysis of 51 fusion proteins with 37 unique fusion-points, the predictions that BetT comprised 12 membrane-spanning regions and that its N- and C-terminal extensions of about 12 and 180 amino acid residues, respectively, were situated in the cytoplasm were confirmed. This is believed to represent the first experimental examination of the membrane topology of a BCCT family protein. Osmotic upshock experiments were performed with spectinomycin-treated E. coil cells that had expressed the wild-type or a mutant BetT protein during growth at low osmolality (160 mosmol [kg.sup.-1]). The choline transport activity of wild-type BetT increased tenfold when the cells were stressed with 0.4 M NaCl (total osmolality 780 mosmol [kg.sup.-1]). The peak activity was recorded 5 min after the upshock and higher or lower concentrations of NaCl reduced the activity. Deletions of 1-12 C-terminal residues of BetT caused a gradual reduction in the degree of osmotic activation from ten- to twofold. Mutant proteins with deletion of 18-101 residues displayed a background transport activity, but they could not be osmotically activated. The data showed that the cytoplasmic C-terminal domain of BetT plays an important role in the regulation of BetT activity and that C-terminal truncations can cause BetT to be permanently locked in a low-transport-activity mode.

Archaeoglobus fulgidus accumulates di-myo-inositol phosphate (DIP) and diglycerol phosphate (DGP) in response to heat and osmotic stresses, respectively, and the level of glycero-phospho-myo-inositol (GPI) increases primarily when the two stresses are combined. In this work, the pathways for the biosynthesis of these three compatible solutes were established based on the detection of the relevant enzymatic activities and characterization of the intermediate metabolites by nuclear magnetic resonance analysis. The synthesis of DIP proceeds from glucose-6-phosphate via four steps: (i) glucose-6-phosphate was converted into L-myo-inositol l-phosphate by L-myo-inositol 1-phosphate synthase; (ii) L-myo-inositol 1-phosphate was activated to CDP-inositol at the expense of CTP; this is the first demonstration of CDP-inositol synthesis in a biological system; (iii) CDP-inositol was coupled with L-myo-inositol 1-phosphate to yield a phosphorylated intermediate, 1,1'di-myo-inosityl phosphate 3-phosphate (DIPP); (iv) finally, DIPP was dephosphorylated into DIP by the action of a phosphatase. The synthesis of the two other polyol-phosphodiesters, DGP and GPI, proceeds via the condensation of CDP-glycerol with the respective phosphorylated polyol, glycerol 3-phosphate for DGP and L-myo-inositol 1-phosphate for GPI, yielding the respective phosphorylated intermediates, 1X, 1'X-diglyceryl phosphate 3-phosphate (DGPP) and 1-(1X-glyceryl) myo-inosityl phosphate 3-phosphate (GPIP), which are subsequently dephosphorylated to form the final products. The results disclosed here represent an important step toward the elucidation of the regulatory mechanisms underlying the differential accumulation of these compounds in response to heat and osmotic stresses.

Mitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) [right arrow] MAPK kinase (MKK) [right arrow] MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3-transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT 1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 [right arrow][right arrow] p38 kinase [right arrow][right arrow] TonE. osmotic stress; tonicity enhancer; betaine transporter; TonEBP

In the rat, urinary concentrating ability develops progressively during the third postnatal (P) week and nearly reaches adult level at weaning (P21) governed by a rise in circulating glucocorticoid. Elevated extracellular osmolality can lead to growth arrest of epithelial cells. We tested the hypothesis that supranormal exposure of rat pups to glucocorticoid before the endogenous surge enhances urinary concentrating ability but inhibits renomedullary cell proliferation. Proliferating-cell nuclear antigen (PCNA)-positive cells shifted from the nephrogenic zone in the first postnatal week to Tamm-Horsfall-positive thick ascending limb (TAL) cells at the corticomedullary junction at P10-14. Renal PCNA protein abundance was stable in the suckling period and decreased 10-fold after weaning. Renal PCNA protein abundance decreased in response to dexamethasone (DEXA; 100 [micro]g x [kg.sup.-1] x [day.sup.-1]. P8-12). Prolonged administration of DEXA (P1-P11) reduced selectively the area and thickness of the outer medulla and the number of PCNA-positive cells. DEXA (P8-12) increased urinary and papillary osmolality in normohydrated and water-deprived pups and led to osmotic equilibrium between interstitium and urine, whereas apoptotic and GADD153-positive cells increased in the inner medulla. TAL-associated NaC1 transporters Na-K-2C1 cotransporter, Na-K-ATPase-[[alpha].sub.1], Na/H exchanger type 3, and ROMK increased significantly at weaning and in response to DEXA. We conclude that a low level of circulating glucocorticoid is permissive for proliferation of Henle's loop and the outer medulla before weaning. A reduced papillary tonicity is a crucial factor for the reduced capacity to concentrate urine during postnatal kidney development. We speculate that supranormal exposure to glucocorticoid in the suckling period can alter kidney medullary structure and function permanently. mitosis; urea; osmolality; apoptosis

Changes in interfilament lattice spacing have been proposed as the mechanism underlying myofilament length-dependent activation. Much of the evidence to support this theory has come from experiments in which high-molecular-weight compounds, such as dextran, were used to osmotically shrink the myofilament lattice. However, whether interfilament spacing directly affects myofilament calcium sensitivity ([EC.sub.50]) has not been established. In this study, skinned isolated rat myocardium was osmotically compressed over a wide range (Dextran T500; 0-6%), and [EC.sub.50] was correlated to both interfilament spacing and [I.sub.1,1]/[I.sub.1,0] intensity ratio. The latter two parameters were determined by X-ray diffraction in a separate group of skinned muscles. Osmotic compression induced a marked reduction in myofilament lattice spacing, concomitant with increases in both [EC.sub.50] and [I.sub.1,1]/[I.sub.1,0] intensity ratio. However, interfilament spacing was not well correlated with [EC.sub.50] ([r.sup.2] = 0.78). A much better and deterministic relationship was observed between [EC.sub.50] and the [I.sub.1,1]/[I.sub.1,0] intensity ratio ([r.sup.2] = 0.99), albeit with a marked discontinuity at low levels of dextran compression; that is, a small amount of external osmotic compression (0.38 kPa, corresponding to 1% Dextran T500) produced a stepwise increase in the [I.sub.1,1]/[I.sub.1,0] ratio concomitant with a stepwise decrease in [EC.sub.50]. These parameters then remained stable over a wide range of further applied osmotic compression (up to 6% dextran). These findings provide support for a 'switch-like' activation mechanism within the cardiac sarcomere that is highly sensitive to changes in external osmotic pressure. skinned muscle; trabeculae; X-ray diffraction; dextran; osmotic pressure; myofilament length-dependent activation; regulation

While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (-200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli. cartilage; calcium signaling: actin cytoskeleton: aggrecan

The RcsCDB His-Asp phosphorelay is shown to positively regulate the bdm (biofilm-dependent modulation) and sra (stationary-phase-induced ribosome-associated protein) genes in Escherichia coli. The regulation is direct and requires an RcsB box next to the bdm -35 element. In addition, bdm is shown to be activated by osmotic shock in an Rcs-dependent way.

This study focused on perchlorate (CI[O.sup.-.sub.4]) rejection and flux-decline in bench-scale cross-flow flat-sheet filtration for two reverse osmosis (RO) and two nanofiltration (NF) membranes with a natural water, and addressed estimation of precipitative fouling/scaling with inorganic salts and characterizations of inorganic fouling and antiscalants. Thus the study considered tradeoffs between productivity (increased recovery and flux) versus CI[O.sup.-.sub.4] rejection versus membrane fouling/scaling. In this study, the rejection of water quality parameters (cations, anions, dissolved organic carbon, UV[A.sub.254], total dissolved solids) and flux-decline trends for four different membranes were investigated over a various range of operating conditions (i.e., [J.sub.0]/k ratio and recovery). Inorganic foulants on the membrane surface were analyzed by various methods (i.e., x-ray diffraction and scanning electron microscopy), and demonstrated inhibition effects of antiscalant. With increasing recovery and [J.sub.0]/k ratio, high productivity (flux) was achieved, however, the rejections of perchlorate and other water quality parameters decreased and the precipitative fouling/scaling potential of membranes increased. At the same operating conditions in the presence of an antiscalant, embodying phosphonate functional groups, flux decline trends for the four membranes indicated lower scale formation supported by the results of the fouled membrane characterizations. CE Database subject headings: Filtration; Osmosis; Fouling; Water treatment.

Hypertonicity mediated by chloride up-regulates the expression of the [gamma]-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428-6433, 2003). The purpose of this study was to examine the cellular locations and the time course of [gamma]-subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the NaK-ATPase [gamma]-subunit or the [[gamma].sub.b] splice variant. Cells grown in 300 mosmol/kg[H.sub.2]O showed no immunoreactivity for the [gamma]-subunit, whereas cells adapted to 600 or 900 mosmol/kg[H.sub.2]O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kg[H.sub.2]O with sodium chloride or choline chloride showed incorporation of [gamma] into plasma membrane 12 h after osmotic challenge and distinct membrane staining in ~40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kg[H.sub.2]O by addition of sodium acetate did not result in expression of the [gamma]-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase [gamma]-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the [gamma]-subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment. inner medullary collecting duct cells; cell culture; osmotic challenge: sodium pump subunits; immunocytochemical localization

The adaptation to hypertonicity in mammalian cells is driven by multiple signaling pathways that include p38 kinase, Fyn, the catalytic subunit of PKA, ATM, and JNK2. In addition to the well-characterized tonicity enhancer (TonE)-TonE binding protein interaction, other transcription factors (and their respective cis elements) can potentially respond to hypertonicity. This review summarizes the current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress and discusses new insights from yeast that could be relevant to the osmostress response in mammals. osmostress; tonicity enhancer/osmotic response element; tonicity enhancer binding protein; p38 kinase; ERK; JNK; osmolytes

The demand for fresh food has resulted in the development of a new category of minimally processed fruits and vegetables. The quality of minimally processed guavas influenced by modified atmosphere packaging and osmotic dehydration are evaluated.

Very little is known about how cellular osmosensors monitor changes in osmolarity of the environment. Here, we report that in yeast, Sin1 osmosensor histidine kinase monitors changes in turgor pressures. Reductions in turgor caused by either hyperosmotic stress, nystatin, or removal of cell wall activate MAPK Hog1 specifically through the SLN1 branch, but not through the SHO1 branch of the high osmolarity glycerol pathway. The integrity of the periplasmic region of Sin1 was essential for its sensor function. We found that activity of the plant histidine kinase cytokinin response 1 (Cre1) is also regulated by changes in turgor pressure, in a manner identical to that of Sln1, in the presence of cytokinin. We propose that Sln1 and Cre1 are turgor sensors, and that similar turgor-sensing mechanisms might regulate hyperosmotic stress responses both in yeast and plants.

A study examines the osmotic dehydration of red paprika by using sucrose and sodium chloride, with a view to improve its nutritional value. Diffusion model is considered for the study.

Control of electroosmosis by an external voltage in capillaries of varying geometry was examined and investigated. Controlled geometric factors included inner and outer radii, external electrode coverage area, and the method of voltage application. The behavior of the flow in response to the external voltage from earlier work and this study were compared to existing literature models. A noticeable lack of correlation between the current modeling theories and the published data is noted. In light of these results, suggestions for further areas of investigation of a description of external voltage flow control mechanism are suggested.

A new study investigates the extent of cell damage during the osmotic dehydration of plant tissues.

A new method for predicting upward solute movement in unsaturated sand soil due to evaporation is developed and tested. Laboratory experiments were conducted in an unsaturated uniformly packed sand column with a cross section of 1.20 x 0.50 m(super 2) and a constant shallow ground-water table. Evaporation was measured by a new ventilated chamber system. Solute movement from the ground water upward was monitored. Water and solute movement could be accurately reproduced by numerically solving Richards' equation and the convection-dispersion equation in one-dimension. The experimentally measured dispersivity for the unsaturated homogeneous sand agreed closely with the values which are available in the literature. This paper offers a new approach for investigating dispersion phenomena in unsaturated porous media exposed to evaporation.

Neutral solutes were used to study the effects of hydration on the activating conformational change of the receptor rhodopsin and on the acyl chain packing of the rod outer segment disk membrane. The solutes glycerol, sucrose and stachyose were found to increase the equilibrium concentration of metarhodopsin II, the conformation of photoactivated rhodopsin. Results also suggested that water molecule binding and release are essential in regulating functional conformational changes for integral membrane and soluble globular proteins.

A study was conducted to analyze an isothermal endoreversible chemical engine based on the osmosis phenomenon. The engine was driven by variations in the solvent chemical potential. Assumed to operate endoreversibly, the cycle of operation was characterized by two stages. There exists a difference in the chemical potential of the solvent between two chambers resulting from variations in the levels of the solutions.

The key physiological role of glycine betaine in the family Rhizobiaceae appears to be as an energy source, while its involvement in osmoprotection is limited to certain strains. This was gleaned from an investigation of the role of glycine betaine and choline in osmoprotection of a variety of strains from five genera of the Rhizobiaceae family. Glycine betaine and choline were found to be useful as osmoprotectants as both enhanced the growth of various rhizobial strains when externally provided. All rhizobial strains were also found to possess a choline uptake activity and can use choline as a carbon, nitrogen and energy source for growth.

A study was conducted to analyze the impact of low-osmolality nutrient media on the growth and culturability of a number of Campylobacter species. The effects of environmental temperature on the growth and survival of species were also examined. Results indicated that the osmotic threshold effect was not limited to C. jejuni NCTC 11351. Findings also suggested that declines in the culturability of C. jejuni could be correlated with losses in cytoplasmic contents resulting from exposure to low-osmolality nutrient media.

A method based on a modified version of pressure-mediated capillary electrophoresis (PreMCE) has been developed for the elimination of the chromatographic bias inherent in effective electrophoretic mobilities measured by capillary electrophoresis. This new five-band PreMCE method can be readily executed on most commercial capillary electrophoresis instruments. It yields not only precise but also accurate effective mobilities and electroosmotic flow rates, even when the analytes and electroosmotic flow markers are strongly retained on the coated fused-silica capillary wall.

We investigated the water flux through neutral carrier membranes caused by the osmotic pressure difference of the solutions on both sides of these membranes. Miniaturized thin-film ion-selective electrodes were used as their extremely small internal electrolyte volume as well as their extremely thin membrane make them specifically suitable for such studies. The dilution of the internal electrolyte due to the transmembrane water flux results in a significant shift of the ISE potentials. A model explaining the potential versus time curve of thin-film sensors was developed. The penetration coefficient of water is determined experimentally with respect to the lipophilicity of the membrane plasticizer used. The inverse of the square root of the penetration coefficient depends linearly on the logarithm of the lipophilicity for almost all investigated membranes.

Intracellular responses in hyperosmotic shock in the halotolerant organism Brevibacterium sp. strain JCM 6894 were investigated. The cells exhibited different responses to shocks in fresh culture medium and in old culture medium. In fresh culture medium, growth was stimulated and the medium became more acidic while in the old medium, growth was repressed and the medium became more alkaline. Cells in fresh culture transiently accumulated alanine and glutamine in response to the shock.

The Listeria monocytogenes rsbVW-sigB-rsbX transcription unit in L. monocytogenes is structurally similar to its counterpart in Bacillus subtilis. Its activity in L. monocytogenes is likewise responsive to many physical and environmental signals. These were concluded in an examination of the role of the sigma(super B) regulon in adaptive responses of L. monocytogenes, wherein the genes encoding homologs of sigma(super B) and several of the Rsb proteins were cloned. It was found that the sigma(super B) regulon is involved in responses to physiological and chemical signals and may function as a primary osmosensor in the bacterium.

Research was conducted to examine Fos response of fetal sheep anterior circumventricular organs to osmotic challenge in late gestation. It is hypothesized that the anterior circumventricular organs and the supraoptic and hypothalamic paraventricular nuclei would show similar activation in fetal sheep during an osmotic challenge. Results indicate that the surfornical organ, organum vasculosum of the lamina terminalis, hypothalamic supraoptic and paraventricular nuclear are regulated by osmotic challenge in fetal sheep at 130 dGA.

In the practice of 'osmotic stress,' the effect of excluded cosolvents on a biochemical equilibrium is interpreted as the number of water molecules participating in the reaction. This action is attributed to lowering of solvent water activity by the cosolvent. This concept of osmotic stress in disperse solution is erroneous: (i) A cosolvent cannot be both excluded and inert, i.e., noninteracting, because exclusion requires a positive free energy change; (ii) a decrease in water activity alone by addition of solute cannot affect an equilibrium when the reacting surface is in contact with the solvent; and (iii) osmotic stress in disperse solution is a restricted case of preferential interactions; the reaction is driven by the free energy of cosolvent exclusion, and the derived number of water molecules is solely a measure of the mutual perturbations of the chemical potentials of the cosolvent and the protein.