Dynamics of protein uptake within the adsorbent particle during packed bed chromatography

Elevated osmolality and pCO(2) have been shown to alter sialylation in a protein-specific manner. In Chinese hamster ovary (CHO)MT2-l-8 cells, tPA sialylation changed only slightly from 40 to 250 mm Hg pCO(2), whereas neural cell adhesion molecule polysialic acid (NCAM PSA) content decreased by up to 70% at 250 mm Hg pCO(2), pH 7.2. NCAM PSA content also decreased with increasing NaCl or NH(4)Cl concentration. This suggests that PSA content is a sensitive indicator of conditions that may alter glycosylation. Amino acids and their derivatives have been used to protect hybridoma and CHO cell growth under hyperosmotic stress. We examined the impact of osmoprotectants on NCAM PSA content in CHO MT2-1-8 cells under hyperosmolality (up to 545 mOsm/kg) and at 195 and 250 mm Hg pCO(2). NCAM PSA content at 545 mOsm/kg was at least two-fold greater in the presence of glycine betaine or L-proline compared to that without osmoprotectant. Surprisingly, in the presence of 20 mM glycine betaine, PSA levels were 50-60% of the control level for osmolalities ranging from 320 to 545 mOsm/kg. Thus, glycine betaine inhibits NCAM polysialylation at osmolalities below 435 mOsm/kg and is beneficial at higher osmolalities. In contrast to glycine betaine, L-proline increased PSA content by 25-120% relative to the unprotected culture at < or =545 mOsm/kg. The decrease in NCAM PSA levels of CHO MT2-1-8 cells cultured at 195 mm Hg pCO(2)-435 mOsm/kg was not mitigated by the presence of 25 mM glycine betaine, glycine, or L-threonine, even though all of these compounds enhanced cell growth. At 250 mm Hg pCO(2), all osmoprotectants tested (20 mM L-threonine, L-proline, glycine, or glycine betaine) increased NCAM polysialylation, with 20 mM glycine betaine restoring NCAM PSA to near control levels. Thus, osmoprotectants may (partially) offset changes in glycosylation, as well as the inhibition of growth, in cells under environmental stress. Supernatant beta-galactosidase levels, which increase upon alkalization of acidic organelles, did not differ significantly under elevated pCO(2) and hyperosmolality from that at control conditions.     

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea, decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24 h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24 h, diminishing to FW values. Nerve FAA increase 187% within 24 h, and remain elevated. Hemolymph FAA decrease (-75%) after 24 h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (-70%) decreases. Total FAA pools contribute 10-20% to intracellular (22-70 mmol/kg) and 0.5-2.4% to hemolymph (3-7 mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.     

Free amino acids and cell volume regulation in the euryhaline ciliate Paramecium calkinsi

Paramecium calkinsi was isolated from a tidal marsh in which the salinity fluctuated widely on a daily basis. In the laboratory, this ciliate survived for days in sea water ranging in osmotic strength from 10 to 2,000 mOsm and divided in nutritive media of 1,000 mOsm or less. When transferred from 750 to 250 mOsm, cells swelled but regained 78% of the original volume within 60 min and the original volume within 1 day. Cells acclimated to 250 mOsm and transferred to 750 mOsm shrank, regained 40% of the original volume in 60 min, and regained little more volume during the next 24 hr. Free amino acids (FAA), principally proline and alanine, are osmolytes in P. calkinsi. In cells that have been acclimated for more than 1 month, Pro is undetectable at 10 mOsm but at 250 mOsm is present in substantial amounts and is still higher at 750 mOsm. Ala is found in cells at all three salinities and increases dramatically with increasing salinity. A complex pattern of amino acid changes occurs during the 4 hr following a transfer from 250 to 750 mOsm, resulting in a marked increase in Ala but no change in Pro. Thus the metabolic changes that lead to the increased FAA levels of acclimated cells are apparently long-term and complex. After transfer of cells from 750 to 250 mOsm there is a rapid and selective loss of Pro and Ala from the cells to the medium.     

Plasma thyroxine and cortisol profiles and gill and kidney Na+/K+-ATPase and SDH activities during acclimation of the catfish Heteropneustes fossilis (bloch) to higher salinity, with special reference to the effects of exogenous cortisol on hypo-osmoregulatory ability of the catfish

When the stenohaline catfish Heteropneustes fossilis was transferred from fresh water (FW) to 30% seawater (SW), the Na(+)/K(+)-ATPase activity significantly increased in the kidney, while in gills it remained more or less constant. A reverse pattern was observed for succinic dehydrogenase (SDH) activity inasmuch as it significantly increased in gills and remained unchanged in the kidney. Plasma osmolality significantly increased within 3 days of transfer to 30% SW and remained significantly higher throughout the duration of experiment. These results suggest that catfish gills may not be able to reverse their function from salt uptake in FW to salt excretion at higher salinity, and that the elimination of monovalent as well as divalent ions is performed by the kidney but not the gills. The significant decline in plasma cortisol (F) levels following transfer to higher salinity may not be due to reduced production but rather to an enhanced utilization and clearance rate, a conclusion supported by the fact that exogenous administration of cortisol acetate (FA) resulted in significant increases in branchial and renal Na(+)/K(+)-ATPase in FW and 30% SW. FA also improved the plasma osmotic regulatory ability of the catfish, possibly due to a change in branchial function from salt-absorption to salt excretion, as was evident from a significant increase in branchial Na(+)/K(+)-ATPase activity in the fish in 30% SW pretreated with FA for 5 days. Consistently higher levels of plasma thyroxine (T4) following transfer to higher salinity suggest the involvement of this hormone at higher salinity.     

Hyperosmotic pressure on HEK 293 cells during the growth phase, but not the production phase, improves adenovirus production

Hyperosmotic stress has been widely explored as a means of improving specific antibody productivity in mammalian cell cultures. In contrast, a decrease in cell-specific productivity of adenovirus production has been reported in several studies in which virus production in HEK 293 cell cultures was conducted under hyperosmotic conditions. However, production of viral vectors and, in particular, adenoviral vectors is the result of two consecutive phases: the growth phase and the virus production phase. In this study, the singular and combined effects of osmolality on the phases of cell growth and virus production were evaluated in culture media with osmolalities ranging from 250 to 410 mOsm. A two-factor, five-level full factorial design was used to investigate the effect of osmotic stress on cell physiology, as determined through the characterization of cell growth, cell metabolism, cell viability, cell cycle, cell RNA and total protein content, and total virus yield/cell-specific virus productivity. Overall, the results show that the growth of cells under hyperosmotic conditions induced favorable physiological states for viral production, and the specific virus productivity was improved by more than 11-fold when the medium's osmolality was increased from 250 to 410 mOsm during the cell growth phase. Both hypo- and hyperosmotic stresses in the virus production phase reduced virus productivity by as much as a factor of six. Optimal virus productivity was achieved by growing cells in media with an osmolality of 370 mOsm or greater, followed by a virus production phase at an osmolality of 290 mOsm. Compared to standard culture and production conditions in isotonic media, the shift from high to low osmolality between the two phases resulted in a two- to three-fold increase in virus yields. This hyperosmotic pressure effect on virus productivity was reproduced in five different commercial serum-free media.     

Effects of hypo- or hyperosmotic stress on lipid synthesis and gluconeogenic activity in tissues of the crab Neohelice granulata

The present study assesses the effects of osmotic stress on phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) activities and (14)C-total lipid synthesis from (14)C-glycine in the anterior and posterior gills, jaw muscle, and hepatopancreas of Neohelice granulata. In posterior gills, 24-h exposure to hyperosmotic stress increased PEPCK, FBPase and G6Pase activities. Increase in (14)C-lipid synthesis was associated to the decrease in PEPCK activity after 72-h exposure to hyperosmotic stress. Hypo-osmotic stress decreased PEPCK and G6Pase activities in posterior gills; however, (14)C-lipids increased after 72-h exposure to stress. In anterior gills, decreases in the G6Pase activity after 72-h of hyperosmotic stress and in (14)C-lipogenesis after 144-h were observed, while PEPCK activity increased after 144 h. Exposure to hypo-osmotic stress increased (14)C-lipid synthesis and PEPCK activity in anterior gills. Muscle G6Pase activity increased after 72-h exposure to hypo-osmotic stress; however, no significant change was observed in the lipogenesis. PEPCK decreased in muscle after 144-h exposure to hyperosmotic, coinciding with increased (14)C-lipid synthesis. In the hepatopancreas, a decrease in the (14)C-lipogenesis occurred after 24-h exposure to hyperosmotic stress, accompanied by increase in (14)C-lipid synthesis. Additionally, PEPCK activity returned to control levels. The hepatopancreatic lipogenesis from amino acids was not involved in the metabolic adjustment during hypo-osmotic stress. However, gluconeogenesis is one of the pathways involved in the adjustment of the intracellular concentration of nitrogenated compounds.     

The osmotic response of salmon louse,Lepeophtheirus salmonis (Copepoda: Caligidae), during the transition from sea water to fresh water

Summary The osmotic changes in haemolymph and body tissues of the ectoparasitic salmon louse,Lepeophtheirus salmonis, have been studied upon transfer from sea water (SW) to dilute sea water (37% SW), and then to fresh water (FW). The parasite shows osmoconformity in SW but hyperosmotic regulation in 37% SW regardless of whether it is attached to the salmon host or free swimming in the water. The same conclusion is reached by haemolymph Cl^– measurements. In FW, the osmotic tolerance and response of attached and free swimming parasites differ: Attached animals maintain steady haemolymph osmolality and Cl^– concentration and survive for at least 1 week, while free swimming parasites quickly become diluted and start to die within 8 h.Acclimation to 37% SW is accompanied by changes in body tissue water content and in the content of ninhydrin positive substances and specific amino acids which suggest the presence of cell volume regulation. Glycine is the dominating free amino acid in the cephalothorax tissues but alanine, proline and taurine also occur in high amounts. Lysine is found to increase significantly during FW acclimation of attached parasites. A breakdown of cell volume regulation is suggested to limit the survival of attached salmon louse in fresh water.Abbreviations FW fresh water - NPS ninhydrin positive substances - SW sea water     

Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress

Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40–50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.     

Osmoprotective effect of glycine betaine on thrombopoietin production in hyperosmotic Chinese hamster ovary cell culture: clonal variations

When 23 recombinant Chinese hamster ovary (rCHO) cell clones were cultivated in hyperosmolar medium resulting from NaCl addition (533 mOsm/kg), their specific thrombopoietin (TPO) productivity (q(TPO)) was increased. However, due to depressed cell growth at elevated osmolality, no enhancement in the maximum TPO titer was made in batch cultures of all 23 clones. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved TPO production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures of 23 clones were performed in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all 23 clones. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled 22 clones to grow at 542 mOsm/kg, where most clones could not grow in the absence of glycine betaine, but at a cost of reduced q(TPO). However, the relative decrease in q(TPO) varied significantly among clones. Thus, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among clones. Six out of 23 clones displayed more than a 40% increase in the maximum TPO titer in the hyperosmolar medium containing glycine betaine, compared with that in the standard medium with a physiological osmolality. Taken together, the results obtained here emphasize the importance of selection of clones for the successful use of hyperosmotic pressure and glycine betaine as an economical means to improve TPO production.     

Differential responses in gills of euryhaline tilapia, Oreochromis mossambicus, to various hyperosmotic shocks

Euryhaline tilapia (Oreochromis mossambicus) survived in brackish water (BW; 20‰) but died in seawater (SW; 35‰) within 6 h when transferred directly from fresh water (FW). The purpose of this study was to clarify responses in gills of FW tilapia to various hyperosmotic shocks induced by BW or SW. In FW-acclimated tilapia, scanning electron micrographs of gills revealed three subtypes of MR cell apical surfaces: wavy-convex (subtype I), shallow-basin (subtype II), and deep-hole (subtype III). Density of apical surfaces of mitochondrion-rich (MR) cell in gills of the BW-transfer tilapia decreased significantly within 3 h post-transfer due to disappearance of subtype I cells, but increased from 48 h post-transfer because of increasing density of subtype III cells. SW-transfer individuals, however, showed decreased density of MR cell openings after 1 h post-transfer because subtype I MR cell disappeared. On the other hand, relative branchial Na⁺/K⁺-ATPase (NKA) α1-subunit mRNA levels, protein abundance, and NKA activity of the BW-transfer group increased significantly at 6, 12, and 12 h post-transfer, respectively. In the SW-transfer group, relative mRNA and protein abundance of gill NKA α1-subunit did not change while NKA activity declined before dying in 5 h. Upon SW transfer, dramatic increases (nearly 2-fold) of plasma osmolality, [Na⁺], and [Cl⁻] were found prior to death. For the BW-transfer group, plasma osmolality was eventually controlled by 96 h post-transfer by enhancement of NKA expression and subtype III MR cell. The success or failure of NKA activation from gene to functional protein as well as the development of specific SW subtype in gills were crucial for the survival of euryhaline tilapia to various hyperosmotic shocks.     

Aminotransferase and the Production of Alanine During Hyperosmotic Stress in Paramecium calkinsi

When Paramecium calkinsi encounter hyperosmotic stress, intracellular free alanine increases. In vivo assays indicate that the reaction catalyzed by alanine aminotransferase contributes to the build up of alanine in response to hyperosmotic shock. ¹⁴C-pyruvate is converted to ¹⁴C-alanine in cells grown axenically at 200 mosm. When shifted to 600 mosm, the rate of conversion of pyruvate to alanine increases, and conversion at either 200 or 600 mosm is blocked by 1 mM aminooxyacetic acid (AOA), an inhibitor of aminotransferase. Intracellular free alanine increase is partially inhibited by AOA, and AOA prevents cells living in fresh water from acclimating to higher salinities, an indication that the increase in intracellular alanine is physiologically significant.     

Hyperosmotic stress and elevated pCO2 alter monoclonal antibody charge distribution and monosaccharide content

Medium osmolality increases with pCO2 at constant pH. Elevated pCO2 and osmolality inhibit hybridoma growth to similar extents in both serum-containing and serum-free media. The combination of osmolality and elevated pCO2 synergizes to negatively impact cell growth. IgG2a glycosylation by hybridoma cells was evaluated under elevated pCO2 (to 250 mmHg pCO2) and/or osmolality (to 476 mOsm/kg). IgG2a site occupancy did not change significantly under any of the conditions studied, which is consistent with the robust glycosylation of other antibodies produced under various environmental stresses. However, changes were observed in the IgG2a charge distribution. Changes in the isoelectric point (pI) were greater under hyperosmotic stress, increasing by 0.32 and 0.41 pH units at 435 mOsm/kg in serum-containing and serum-free medium, respectively. Hyperosmotic stress also resulted in a concomitant increase in the heterogeneity of the charge distribution. The mean pI in serum-containing medium decreased by 0.16 pH units at 250 mmHg pCO2 when osmolality was controlled at 320 mOsm/kg but increased by 0.20 pH units when the osmolality increased with pCO2 (195 mmHg pCO2-435 mOsm/kg). In serum-free medium, elevated pCO2 did not alter pI, regardless of medium osmolality. In contrast to elevated osmolality at control pCO2, elevated pCO2 did not significantly alter the IgG2a charge heterogeneity under any of the conditions studied. The IgG2a was not sialylated, so sialylation changes were not responsible for changes in the charge distribution. IgG2a galactose content decreased with elevated osmolality, as a result of either elevated NaHCO3 or NaCl. However, when osmolality was controlled at elevated pCO2, the galactose content tended to increase. The mannose content decreased with increasing stress, while the fucose content remained relatively unchanged. It is likely that the observed increases in the pI of murine IgG2a were due to increased organellar pH, which is reflected by increased specific beta-galactosidase activity in the supernatant.     

Handling of ferric iron by branchial and intestinal epithelia of climbing perch (Anabas testudineus Bloch)

With a view to test how the branchial and intestinal tissues of fish, the two sites of metal acquisition, utilize the water-borne ferric [Fe(III)] iron and whether the accumulation of this form of iron influences cellular Na/K gradient in these tissues, the gills and intestines of climbing perch adapted to freshwater (FW) and acclimated to dilute seawater (20 ppt; SW) were analyzed for ouabain-sensitive Na+, K+-ATPase activity, Fe and electrolyte contents after loading a low (8.95 microM) or high dose (89.5 microM) of Fe(III) iron in the water. The SW gills showed higher levels of total Fe after treating with 8.95 microM of Fe(III) iron which was not seen in the FW gills. Na+, K+-ATPase activity, reflecting Na/K pump activity, showed an increase in the FW gills and not in the SW gills. Substantial increase in the branchial Na and K content was observed in the SW gills, but the FW gills failed to show such effects after Fe(III) loading. The total Fe content was declined in the FW intestine but not in the SW intestine. Water-borne Fe(III) iron decreased the activity of Na+, K+-ATPase in the SW intestine while not changing its activity in the FW intestine. The Na and K content in the FW intestine did not respond to Fe(III) iron exposure but showed a reduction in its Na levels in the SW intestine. The moisture content in the gills and intestines of both the FW and SW perch remained unaffected after Fe(III) loading. In FW fish, the plasma Na levels were decreased by a low dose of Fe(III) iron, though a high dose of Fe(III) iron was required in the SW fish for such an effect. Overall, the results for the first time provide evidence that gills act as a major site for Fe(III) iron absorption and accumulation during salinity acclimation which depends on a high cellular Na/K gradient.     

Impact of transport processes in the osmotic response of Corynebacterium glutamicum

Osmoregulation, the adaptation of cells to changes in the external osmolarity, is an important aspect of the bacterial stress response, in particular for a soil bacterium like Corynebacterium glutamicum. Consequently, this organism is equipped with several redundant systems for coping with both hyper- and hypoosmotic stress. For the adaptation to hypoosmotic stress C. glutamicum possesses at least three different mechanosensitive (MS) channels. To overcome hyperosmotic stress C. glutamicum accumulates so-called compatible solutes either by means of biosynthesis or by uptake. Uptake of compatible solutes is in general preferred to de novo synthesis because of lower energy costs. Noticeable, only secondary transporters belonging to the MHS (ProP) or the BCCT-family (BetP, EctP and LcoP) are involved in the uptake of proline, betaine and ectoine. In contrast to Escherichia coli or Bacillus subtilis no ABC-transporters were found catalyzing uptake of compatible solutes. BetP was one of the first examples of the growing group of osmosensory proteins to be analyzed in detail. This transporter is characterized, besides the catalytic activity of betaine uptake, by the ability to sense osmotic changes (osmosensing) and to respond to the extent of osmotic stress by adaptation of transport activity (osmoregulation). BetP detects hyperosmotic stress via an increase in the internal K(+) concentration following a hyperosmotic shift, and thus acts as a chemosensor.     

Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea,decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24?h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24?h, diminishing to FW values. Nerve FAA increase 187% within 24?h, and remain elevated. Hemolymph FAA decrease (?75%) after 24?h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (?70%) decreases. Total FAA pools contribute 10–20% to intracellular (22–70?mmol/kg) and 0.5–2.4% to hemolymph (3–7?mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.     

Influence of the Partitioning of Osmolytes by the Cytoplasm on the Passive Response of Cells to Osmotic Loading

Due to the dense organization of organelles, cytoskeletal elements, and protein complexes that make up the intracellular environment, it is likely that membrane-permeant solutes may be excluded from a fraction of the interstitial space of the cytoplasm via steric restrictions, electrostatic interactions, and other long-range intermolecular forces. This study investigates the hypothesis that the intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery in response to hyperosmotic loading, based on prior theoretical and biomimetic experimental studies. Osmotic loading experiments are performed on immature bovine chondrocytes using culture conditions where regulatory volume responses are shown to be insignificant. Osmotic loading with membrane-permeant glycerol (92 Da) and urea (60 Da) are observed to produce partial volume recoveries consistent with the proposed hypothesis, whereas loading with 1,2-propanediol (76 Da) produces complete volume recovery. Combining these experimental results with the previous theoretical framework produces a measure for the intracellular partition coefficient of each of these solutes. At 1000 mOsm, 1,2-propanediol is the only osmolyte to yield a partition coefficient not statistically different from unity, κ^p_i = 1.00 ± 0.02. For glycerol, the partition coefficient increases with osmolarity from κ^p_i = 0.48 ± 0.19 at 200 mOsm to κ^p_i = 0.80 ± 0.07 at 1000 mOsm; urea exhibits no such dependence, with an average value of κ^p_i = 0.87 ± 0.07 for all osmolarities from 200 to 1000 mOsm. The finding that intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery under osmotic loading offers a simple method for characterizing the partition coefficient. These measurements suggest that significant partitioning may occur even for small membrane-permeant osmolytes. Furthermore, a positive correlation is observed, suggesting that a solute's cytoplasmic partition coefficient increases with increasing hydrophobicity.     

Adaptation to salinity by fish. Macromolecular changes in mitochondria and microsomes of the gill

Mitochondria isolated from the gill tissues of the fishSarothredon mossambica were analysed for their macromolecular content, following transfer from freshwater to media of higher salinity. The results suggest a breakdown of mitochondria during the initial phases of the stress and a regeneration during continued exposure. Also all the synthetic machineries, in general, seem to be triggered in gill tissue during continuous exposure to hyperosmotic media.     

Betaine aids in the osmoregulation of duodenal epithelium of broiler chicks, and affects the movement of water across the small intestinal epithelium in vitro.

In Experiment 1, the water holding capacity of broiler chick intestinal tissue was studied in vitro. The chicks were fed with corn-based diets with or without a 0.2% betaine supplementation in the drinking water. Slices from duodenum and jejunum were incubated in iso-osmotic (300 mM) or hyperosmotic saline (600 mM) with or without 10 mM betaine. The water volume of tissue slices was studied by adding tritiated water in the incubation medium while [14C]inulin was used to correct for the adherent water. After 30 min of incubation, by which time the steady-state of tritium influx had been achieved, the 3H and 14C-activities of the tissue slices were measured. The ileal and duodenal tissues incubated in the hyperosmotic saline accumulated less tritium than those incubated in iso-osmotic saline. Duodenal slices incubated in hyperosmotic saline with the presence of betaine showed a tritium content similar to slices incubated in iso-osmotic saline. The data suggest that the presence of betaine helped the duodenal, but not jejunal, epithelium to maintain water balance in hyperosmotic conditions. The dietary betaine supplementation diminished the differences between the incubation treatments in duodenal, but not in ileal tissue. In Experiment 2, the same double labeling method, but with shorter incubation times, was used to assess the rate of water flux from the incubation medium to duodenal or jejunal slices. The dietary treatments (as in Experiment 1) had little effect on the results. Betaine in the hyperosmotic saline significantly decreased the rate of tritium accumulation into the tissue slices, indicating that betaine slowed down the influx of water to the epithelium. We suggest that betaine affects the movement of water across the intestinal epithelium and has a role in the osmoregulation of small intestine of broiler chicks.     

Identification and pathway analysis of immediate hyperosmotic stress responsive molecular mechanisms in tilapia (Oreochromis mossambicus) gill

Salinity is a major environmental factor that strongly influences cellular and organismal function. We have used the euryhaline fish Oreochromis mossambicus to identify and annotate immediate hyperosmotic stress responsive molecular mechanisms and biological processes in gill epithelial cells. Using a suppression subtractive hybridization (SSH) approach, we have identified and cloned 20 novel immediate early genes whose mRNAs are induced in gill epithelial cells 4 h after transfer of fish from freshwater (FW) to seawater (SW). Full-length or partial sequences of open reading frames (ORFs) were obtained using the rapid amplification of cDNA ends (RACE) technique. Kinetics of induction was analyzed for all hyperosmotic stress-induced genes. Most genes show a robust transient increase in mRNA abundance characteristic of immediate early stress response genes with peak levels observed between 2 and 8 h after seawater transfer. The newly identified genes were classified according to their sequence similarity with other vertebrate homologs and based on their predicted functions. Pathway analysis revealed that more than half of the identified immediate hyperosmotic stress genes interact closely within a cellular stress response signaling network. Moreover, the 20 genes cluster together in six molecular processes that are rapidly activated in tilapia gills upon salinity transfer. These processes are (1) stress response signal transduction, (2) compatible organic osmolyte accumulation, (3) energy metabolism, (4) lipid transport and cell membrane protection, (5) actin-based cytoskeleton dynamics, and (6) protein and mRNA stability. Our identification and analysis of a set of novel osmo-responsive tilapia genes provides insight into critical physiological processes and pathways constituting the hyperosmotic stress adaptation program in gill epithelial cells of euryhaline fishes.