Osmotic properties of the sealed tubular system of toad and rat skeletal muscle

A method was developed that allows conversion of changes in maximum Ca2+-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount Of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca2+ from the sealed t-system of toad fibers Occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by

Local osmotic gradients drive the water flux associated with Na+/glucose cotransport

It recently was proposed [Loo, D. D. F., Zeuthen, T., Chandy, G. & Wright, E. M. (1996) Proc. Natl. Acad. Sci. USA 93, 13367–13370] that SGLT1, the high affinity intestinal and renal sodium/glucose cotransporter carries water molecules along with the cosubstrates with a strict stoichiometry of two Na+, one glucose, and ≈220 water molecules per transport cycle. Using electrophysiology together with sensitive volumetric measurements, we investigated the nature of the driving force behind the cotransporter-mediated water flux. The osmotic water permeability of oocytes expressing human SGLT1 (Lp ± SE) averaged 3.8 ± 0.3 × 10−4 cm⋅s−1 (n = 15) and addition of 100 μM phlorizin (a specific SGLT1 inhibitor) reduced the permeability to 2.2 ± 0.2 × 10−4 cm⋅s−1 (n = 15), confirming the presence of a significant water permeability closely associated with the cotransporter. Addition of 5 mM α-methyl-glucose (αMG) induced an average inward current of 800 ± 10 nA at −50 mV and a water influx reaching 120 ± 20 pL cm−2 ⋅s−1 within 5–8 min. After rapidly inhibiting the Na+/glucose cotransport with phlorizin, the water flux remained significantly elevated, clearly indicating the presence of a local osmotic gradient (Δπ) estimated at 16 ± 2 mOsm. In short-term experiments, a rapid depolarization from −100 to 0 mV in the presence of αMG decreased the cotransport current by 94% but failed to produce a comparable reduction in the swelling rate. A mathematical model depicting the intracellular accumulation of transported osmolytes can accurately account for these observations. It is concluded that, in SGLT1-expressing oocytes, αMG-dependent water influx is induced by a local osmotic gradient by using both endogenous and SGLT1-dependent water permeability.

Osmotic Water Permeability of Isolated Protoplasts. Modifications during Development1

A transference chamber was developed to measure the osmotic water permeability coefficient (Pos) in protoplasts 40 to 120 μm in diameter. The protoplast was held by a micropipette and submitted to a steep osmotic gradient created in the transference chamber. Pos was derived from the changes in protoplast dimensions, as measured using a light microscope. Permeabilities were in the range 1 to 1000 μm s−1 for the various types of protoplasts tested. The precision for Pos was ≤40%, and within this limit, no asymmetry in the water fluxes was observed. Measurements on protoplasts isolated from 2- to 5-d-old roots revealed a dramatic increase in Pos during root development. A shift in Pos from 10 to 500 μm s−1 occurred within less than 48 h. This phenomenon was found in maize (Zea mays), wheat (Triticum aestivum), and rape (Brassica napus) roots. These results show that early developmental processes modify water-transport properties of the plasma membrane, and that the transference chamber is adapted to the study of water-transport mechanisms in native membranes.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume- activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of similar to 75 mOsm. These currents display the typical biophysical signature of volume- activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(-)>Cl(-)>F(-)). Staurosporine (200 nM) did not block the activation of I(Cl), swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 mu M), SITS (200 mu M), ATP (500 mu M), pyridoxalphosphate-6- azophenyl-2`,4`-disulfonate (PPADS; 100 mu M), and Suramin (10 mu M) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(d)(0) and fractional distance of the binding site (delta) of 334 mu M and 47%, 880 mu M and 35%, 2,100 mu M and 49%, 188 mu M and 27%, and 66.5 mu M and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that ICl, swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line.

Water balance is achieved through the ability of the kidney to control water reabsorption in the connecting tubule and the collecting duct. In a mouse cortical collecting duct cell line (mCCD(c11)), physiological concentrations of arginine vasopressin increased both electrogenic, amiloride-sensitive, epithelial sodium channel (ENaC)-mediated sodium transport measured by the short-circuit current (Isc) method and water flow (Jv apical to basal) measured by gravimetry with similar activation coefficient K(1/2) (6 and 12 pM, respectively). Jv increased linearly according to the osmotic gradient across the monolayer. A small but highly significant Jv was also measured under isoosmotic conditions. To test the coupling between sodium reabsorption and water flow, mCCD(c11) cells were treated for 24 h under isoosmotic condition with either diluent, amiloride, vasopressin or vasopressin and amiloride. Isc, Jv, and net chemical sodium fluxes were measured across the same monolayers. Around 30% of baseline and 50% of vasopressin-induced water flow is coupled to an amiloride-sensitive, ENaC-mediated, electrogenic sodium transport, whereas the remaining flow is coupled to an amiloride-insensitive, nonelectrogenic sodium transport mediated by an unknown electroneutral transporter. The mCCD(c11) cell line is a first example of a mammalian tight epithelium allowing quantitative study of the coupling between sodium and water transport. Our data are consistent with the 'near isoosmotic' fluid transport model.

Electrical parameters and water permeability properties of monolayers formed by T84 cells cultured on permeable supports

T84 is an established cell line expressing an enterocyte phenotype whose permeability properties have been widely explored. Osmotic permeability (P OSM), hydraulic permeability (P HYDR) and transport-associated net water fluxes (J W-transp), as well as short-circuit current (I SC), transepithelial resistance (R T), and potential difference (deltaV T) were measured in T84 monolayers with the following results: P OSM 1.3 ± 0.1 cm.s-1 x 10-3; P HYDR 0.27 ± 0.02 cm.s-1; R T 2426 ± 109 omega.cm², and deltaV T 1.31 ± 0.38 mV. The effect of 50 µM 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a "net Cl- secretory agent", on T84 cells was also studied. We confirm the reported important increase in I SC induced by DCEBIO which was associated here with a modest secretory deltaJ W-transp. The present results were compared with those reported using the same experimental approach applied to established cell lines originating from intestinal and renal epithelial cells (Caco-2, LLC-PK1 and RCCD-1). No clear association between P HYDR and R T could be demonstrated and high P HYDR values were observed in an electrically tight epithelium, supporting the view that a "water leaky" barrier is not necessarily an "electrically leaky" one. Furthermore, the modest secretory deltaJ W-transp was not consistent with previous results obtained with RCCD-1 cells stimulated with vasopressin (absorptive fluxes) or with T84 cells secreting water under the action of Escherichia coli heat stable enterotoxin. We conclude that, while the presence of aquaporins is necessary to dissipate an external osmotic gradient, coupling between water and ion transport cannot be explained by a simple and common underlying mechanism.

Estudos clínico, laboratorial e eletrocardiográfico dos efeitos da solução hipertônica de cloreto de sódio a 7,5%, intravenosa, em cães clinicamente sadios

Pós-graduação em Medicina Veterinária - FMVZ

Quali-quantitative study of the phenolic and polyphenolic compounds and their antioxidant capacity in vegetal matrix processed with different technologies

Food technologies today mean reducing agricultural food waste, improvement of food security, enhancement of food sensory properties, enlargement of food market and food economies. Food technologists must be high-skilled technicians with good scientific knowledge of food hygiene, food chemistry, industrial technologies and food engineering, sensory evaluation experience and analytical chemistry. Their role is to apply the modern vision of science in the field of human nutrition, rising up knowledge in food science. The present PhD project starts with the aim of studying and improving frozen fruits quality. Freezing process in very powerful in preserve initial raw material characteristics, but pre-treatment before the freezing process are necessary to improve quality, in particular to improve texture and enzymatic activity of frozen foods. Osmotic Dehydration (OD) and Vacuum Impregnation (VI), are useful techniques to modify fruits and vegetables composition and prepare them to freezing process. These techniques permit to introduce cryo-protective agent into the food matrices, without significant changes of the original structure, but cause a slight leaching of important intrinsic compounds. Phenolic and polyphenolic compounds for example in apples and nectarines treated with hypertonic solutions are slightly decreased, but the effect of concentration due to water removal driven out from the osmotic gradient, cause a final content of phenolic compounds similar to that of the raw material. In many experiment, a very important change in fruit composition regard the aroma profile. This occur in strawberries osmo-dehydrated under vacuum condition or under atmospheric pressure condition. The increment of some volatiles, probably due to fermentative metabolism induced by the osmotic stress of hypertonic treatment, induce a sensory profile modification of frozen fruits, that in some way result in a better acceptability of consumer, that prefer treated frozen fruits to untreated frozen fruits. Among different processes used, a very interesting result was obtained with the application of a osmotic pre-treatment driven out at refrigerated temperature for long time. The final quality of frozen strawberries was very high and a peculiar increment of phenolic profile was detected. This interesting phenomenon was probably due to induction of phenolic biological synthesis (for example as reaction to osmotic stress), or to hydrolysis of polymeric phenolic compounds. Aside this investigation in the cryo-stabilization and dehydrofreezing of fruits, deeper investigation in VI techniques were carried out, as studies of changes in vacuum impregnated prickly pear texture, and in use of VI and ultrasound (US) in aroma enrichment of fruit pieces. Moreover, to develop sensory evaluation tools and analytical chemistry determination (of volatiles and phenolic compounds), some researches were bring off and published in these fields. Specifically dealing with off-flavour development during storage of boiled potato, and capillary zonal electrophoresis (CZE) and high performance liquid chromatography (HPLC) determination of phenolic compounds.

Analytical and numerical study of the electro-osmotic annular flow of viscoelastic fluids

In this work we present semi-analytical solutions for the electro-osmotic annular flow of viscoelastic fluids modeled by the Linear and Exponential PTT models. The viscoelastic fluid flows in the axial direction between two concentric cylinders under the combined influences of electrokinetic and pressure forcings. The analysis invokes the Debye-Hückel approximation and includes the limit case of pure electro-osmotic flow. The solution is valid for both no slip and slip velocity at the walls and the chosen slip boundary condition is the linear Navier slip velocity model. The combined effects of fluid rheology, electro-osmotic and pressure gradient forcings on the fluid velocity distribution are also discussed.

High gradient magnetic separation (HGMS) of erythrocytes infected with plasmodium falciparum

This thesis was undertaken to explore possible applications of high gradient magnetic separation (HGMS) for the separation of RBCs infected with Plasmodium falciparum, with the dual aim of establishing a novel and superior method for isolating late-stage infected cells, and of obtaining synchronized cell cultures.rnThe presented work presents protocols for HGMS of parasitized RBCs that fulfil these aims. Late-stage parasitized cell can be isolated essentially devoid of contamination with non-infected and ring-stage infected cells. Such an easy method for a highly quantitative and qualitative purification has not yet been reported. Synchronous cultures can be obtained both following depletion of late-stage infected cells, and following isolation of the latter. The quality of synchronization cultures matches that of sorbitol lysis, the current standard method for malaria culture synchronization. An advantage of HGMS is the avoidance of osmotic stress for RBCs. The new methods further have the appeal of high reproducibility, cost-effectiveness, and simple protocol.rnIt should be possible to take the methods beyond Plasmodium infected RBCs. Most magnetic separation techniques in the sector of biomedical research employ columns with a hydrophilic polymer-coated matrix. Our procedure employs an optimized buffer system. Polymer coating becomes unnecessary and uncoated columns are available at a fraction of the cost.

Plasma osmolyte concentrations and rectal gland mass of bull sharks Carcharhinus leucas, captured along a salinity gradient

Bull sharks (Carcharhinus leucas) were captured across a salinity gradient from freshwater (FW) to seawater (SW). Across all salinities, C leucas were hyperosmotic to the environment. Plasma osmolarity in FW-captured animals (642 +/- 7 mosM) was significantly reduced compared to SW-captured animals (1067 +/- 21 mosM). In FW animals, sodium, chloride and urea were 208 +/- 3, 203 +/- 3 and 192 +/- 2 mmol l(-1), respectively. Plasma sodium, chloride and urea in SW-captured C leucas were 289 +/- 3, 296 +/- 6 and 370 +/- 10 mmol l(-1), respectively. The increase in plasma osmolarity between FW and SW was not linear. Between FW (3 mosM) and 24%o SW (676 mosM), plasma osmolarity increased by 22% or 0.92% per 1parts per thousand rise in salinity. Between 24%o and 33parts per thousand, plasma osmolarity increased by 33% or 4.7% per 1 parts per thousand rise in salinity, largely due to a sharp increase in plasma urea between 28parts per thousand and 33parts per thousand. C. leucas moving between FW and SW appear to be faced with three major osmoregulatory challenges, these occur between 0-10parts per thousand, 11-20parts per thousand and 21-33parts per thousand. A comparison between C leucas captured in FW and estuarine environments (20-28%o) in the Brisbane River revealed no difference in the mass of rectal glands between these animals. However, a comparison of rectal gland mass between FW animals captured in the Brisbane River and Rio San Juan/Lake Nicaragua showed that animals in the latter system had a significantly smaller rectal gland mass at a given length than animals in the Brisbane River. The physiological challenges and mechanisms required for C leucas moving between FW and SW, as well as the ecological implications of these data are discussed. (C) 2004 Elsevier Inc. All rights reserved.

Polyol transport in yeast: studying transport proteins and manipulating osmotic responses

The Saccharomyces cerevisiae MIP channel Fps1p plays an important role in yeast osmoregulation by exporting glycerol. Glycerol accumulates in the cell as a compatible osmolyte during hyperosmotic conditions and is exported once conditions become hypotonic. A gpd1 gpd2 mutant is unable to produce glycerol and is therefore very sensitive to high concentrations of polyols in the growth medium. The sensitivity to C3, C4 and C5, but not C6 polyols, is suppressed by expression of truncated, hyperactive Fps1p. This is because the polyols can then equilibrate over the membrane and hence the concentration gradient collapses. This experiments reveals the substrate spectrum of Fps1p. The system can be used in different ways. For instance, growth assays on different polyols elucidate the substrate range of heterologous channels such as that of the rat aquaglyceroporin AQP9. In addition, the same system is used to search for novel hyperactive mutants of Fps1p, which provide additional information on the mechanism underlying channel regulation. Finally we illustrate that the gpd1 gpd2 double mutant expressing hyperactive Fps1p can be used to manipulate activation and deactivation of the HOG pathway, contributing to our understanding of the control of this osmoregulatory system.

Numerical simulation of mass transfer during the osmotic dehydration of biological tissues

In this paper a mathematical model based on mass transfer in plant tissues is developed. The model takes into account the diffusion and convection of each constituent within the tissue. The driving force for the convection is assumed to be the gradient of hydrostatic pressure. The mass balance equation for the transport of each constituent is established separately for intracellular and extracellular volumes but taking into account the mass exchange across the cell membrane between the intracellular and extracellular volumes. The mass transfer results in not only the change of intracellular and extracellular volumes but also the shrinkage of whole tissue. The model allows us to quantitatively simulate the time evolution of intracellular and extracellular volumes, which was observed in histological sections under the microscope. © 2005 Elsevier B.V. All rights reserved.

Ant community richness and composition across a gradient from Eucalyptus plantations to secondary Atlantic Forest

Secondary forests and exotic tree plantations are expanding across tropical landscapes. However, our current understanding of the value of these human-dominated forest landscapes for invertebrate biodiversity conservation is still very poor. In this paper, we use the leaf-litter ant fauna to assess invertebrate diversity in one commercially managed Eucalyptus plantation (four years old), two abandoned plantations of different regeneration ages (16 and 31 years), and one neighboring secondary Atlantic Forest in Southeastern Brazil. There was a clear gradient in species richness from the secondary forest to the managed Eucalyptus plantation; richness and diversity peaked in secondary forest and in the older regenerating Eucalyptus plantation. Significantly more species were recorded in secondary forest samples than in Eucalyptus plantations, but Eucalyptus plantations had a similar level of richness. Furthermore, a non-metric multidimensional scaling analysis revealed clear differences in species composition between the younger managed Eucalyptus plantation (understory absent) and habitats with sub-developed or developed understory. Eucalyptus plantations were characterized by an assemblage of widespread, generalist species very different from those known to occur in core forest habitats of southeastern Brazil. Our results indicate that while older regenerating Eucalyptus plantations can provide habitat to facilitate the persistence of generalist ant species, it is unlikely to conserve most of the primary forest species, such as specialized predators, Dacetini predators, and nomadic species.

Epilithic community development on artificial reefs deployed along a cross-shelf environmental gradient off Paraná state, southern Brazil

Concrete modules were deployed on the bottom of the 11, 18 and 30 meters isobaths along a cross-shelf hydrographic gradient off Paraná State, Southern Brazil, with the purpose of studying the colonization of sessile epilithic macroinvertebrates on artificial surfaces. After one year of submersion a total of 63 species of epilithic organisms were identified, dominated by Ostrea puelchana, Chthamalus bisinuatus, Balanus cf spongicola, Astrangia cf rathbuni, Didemnum spp, poryphers and bryozoans. Diversity index and percent cover at reef stations placed at 11, 18 and 30 meters isobaths were respectively 2.28 and 66.7%, 2.79 and 96.6% and 1.66 and 77.4%. Differences of general community structure among the three assemblages were not clearly related to the general environmental conditions at the bottom layers near the reef stations. Turbidity and larval abundance are discussed as important factors affecting colonization processes. Results indicate that depths between 15-20 meters are more suitable for the implementation of large scale artificial reef systems in the inner shelf off Paraná and, possibly, throughout the inner shelves off southern Brazil with similar hydrographic conditions.