Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C₁ cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C₁ was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10) (m) increased furosemideinsensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanideinsensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloridedepleted cells. When cells with normal chloride content were depolarized (135 mm medium potassium + 10 m valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl^–Br^–>NO ₃ ^– >I^–. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.     

Compressibility,densimetric and calorimetric studies of hydration of carrageenans in the random form

The partial molal volume and adiabatic compressibility were measured, as well as their counterion activity, for sodium and potassium salts of three types of carrageenan (κ-, ι- and λ-components) in aqueous solutions at 25°C. Furthermore, the amount of related unfreezable water was estimated by the differential scanning calorimetry. On the basis of these results, the hydration states of carrageenans in the random form were comparatively discussed in relation to their chemical structure, counterion binding and polymer concentration. The sodium salt of each component showed a larger amount of hydration when compared with the corresponding potassium salt. The amount of hydration estimated from molal volume and compressibility data (in dilute solution) increased in the order of κ < ι < λ, while the amount of unfreezable water (in concentrated solution) decreased in the same order. These characteristics hydration behaviours of carrageenans seemed to be reasonably explained in terms of the effects of the charge density and counterion dissociation of these polyions.     

Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures

Dilute salt solutions eluted peroxidase and hydroxyproline-rich glycoproteins (HRGP's) very rapidly (60 % within 10s) from the surface of intact tomato cells grown in suspension culture. Further purification of the HRGPs based on (a) their solubility in 10% trichloroacetic acid and (b) chromatography on carboxymethyl cellulose, gave two components (P1 and P2) rich in serine, tyrosine, lysine and arabinosylated hydroxyproline. The sum of the hydroxyproline arabinoside profiles of P1 and P2 approximated that of the wall. P1, unlike P2, was histidine-rich and also contained proline. Significantly, isodityrosine (IDT) was absent from P1 and P2 but present in cell wall hydrolysates where, the Hyp:IDT molar ratio was ca 15: 1. In cells 4 days after subculture, ³H-proline pulse-chase data indicated turnover of P1 and P2 presumably resulting from covalent attachment to the wall as neither P1 nor P2 appeared in the growth medium. At day four the cell mean generation time (MGT) was 4.6 days, the cell hydr oxyproline content was 0.7 % (w/w), the half lives of P1 and P2 were both ca 12 hr, and the combined CaCl₂ elutable P1 and P2 precursor pools contained ca 400 μg Hyp/g cells (dry weight). Calculated from the MGT and Hyp content, the cell demand was 44.μg Hyp/g cells (dry weight)/hr. The precursor pool size was therefore sufficient for 9 hours growth. However the pool turnover calculated from half life and pool size was 5.6 %/hr or 22.4μg Hyp/g cells (dry weight)/hr. Thus the supply of P1 and P2 precursors met > 50 % of the cell wall demand. Corroborative experiments showed that after depletion of the P1 and P2 pools by salt elution, washed cells resuspended in growth medium repleted the precursor pools at a rate corresponding to a synthesis of 43μg Hyp/g cells (dry weight)/hr, or 98 % of the demand. These data allow us to make the following suggestions: P1 and P2 represent monomeric extensin precursor subunits. Salt elution of P1 and P2 indicates their ionic binding by pectic carboxyl groups. The rapidity of elution indicates a high diffusivity of these extended rodlike macromolecules through the cell wall. This may imply a preferred orientation for P1 and P2 perpendicular rather than parallel to the plane of the wall. The lack of IDT in P1 and P2 implies that IDT forms in muro, possibly via peroxidase. We speculate that some of these IDT residues may crosslink an extensin precursor ‘tweft’ around a cellulose microfibrillar ‘twarp’. Such formation of heteromultimeric extensin interpenetrated by microfibrils would create a mechanically coupled extensin-cellulose network.     

INFLUENCE OF CELL VOLUME ON THE GROWTH AND SIZE REDUCTION OF MARINE AND ESTUARINE DIATOMS1

The maximal growth rate (μ_max) of 19 marine and estuarine diatoms decreased with increasing cell volume (V). The relationship between log μ_max (Y) and log V (X) was calculated. Statistical analyses showed that the slope of the equation was not significantly different from those obtained by other researchers and that the 95% confidence intervals of mean μ_max at cell volumes of 10³–10⁵μm³ were not significantly different from those cited in most studies. A new regression line for diatoms was calculated as follows: log μ_max= 0.47–0.14 log V; r =–0.69. The rate of size reduction per generation of the 19 diatom species ranged from 0.03 to 0.87 μm per generation. The rate increased with increasing cell length and cell volume and with decreasing maximum division rate. Statistical analyses showed that the rate was closely related to the cell volume and to the reciprocal of the growth rate. The relationships between maximal growth rate and cell volume and between rate of size reduction and cell volume showed that a diatom with a large volume had a smaller maximal growth rate and a larger rate of size reduction than a diatom with a small volume. The estimates using the equation for the regression line between the rate of size reduction and the reciprocal of maximum division rate indicated that a diatom with a high maximum division rate would need more generation equivalents for a certain size reduction than a diatom with a low maximum division rate, but the periods required for reduction would be approximately equal irrespective of maximum division rate.     

Use of a thermal and light refugium by Daphnia and its effects on foraging pumpkinseeds

The effects of thermal stratification and light gradients on the feeding behavior of pumpkinseeds, Lepomis gibbosus, were tested in vertical aquarium columns. Successful captures, unsuccessful captures and unsuccessful searches by foraging sunfish on Daphnia pulex were recorded. Clearance and feeding rates of the sunfish were lowest when prey densities remained high, indicating that the Daphnia were occupying an area that could not be searched by the sunfish. Thermal stratification limited the searching volume and prey availability of the sunfish, while creating a refuge for the Daphnia. Light intensities ≤ 4.2 × 10^-3 W m^-2 decreased the searching and capture abilities of the sunfish under isothermal conditions. Thermal stratification had more of an effect than the light gradients, creating a refuge for the Daphnia causing them to be unavailable and less vulnerable to predation by the sunfish. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cellular volume regulation in freshwater bivalves

The effect of ambient osmolality on the height of lateral ciliated cells from the gills of two freshwater bivalve species (Dreissena polymorpha, Toxolasma texasensis) was directly observed microscopically. The addition of 1 mmol · l⁻¹ KCl to an artificial pondwater (APW) superfusion medium resulted in an increase in cell height. When the superfusion solution was made hyperosmotic (∼90 mmol · kg⁻¹ H₂O) by the addition of 45 mmol · l⁻¹ NaCl to APW, the cell height decreased by about 20–30% and there was no evidence of a regulatory volume increase over 20–30 min. In contrast, when 1 mmol · l⁻¹ KCl was added to the hyperosmotic medium the cell height always partially (40–50%) recovered. When the gill tissue was returned to APW following the hyperosmotic treatment the cells returned to the original cell height. Bivalve gills superfused with the hyperosmotic NaCl and KCl solution in the presence of 1 mmol · l⁻¹ ouabain experienced a similar 25% decrease in cell height. When the ouabain-treated tissues were returned to APW the cells swelled, overshooting the original cell height. These results indicate these freshwater bivalves have a limited ability for cellular volume regulation using inorganic ions, but depend on a suitable balance of Na⁺ and K⁺ in the environment to effect regulatory volume changes. Accepted: 17 October 1997     

Assessment of cardiac output from noninvasive determination of arterial pressure profile in subjects at rest

The stroke volume of the left ventricle (SV) was calculated from noninvasive recordings of the arterial pressure using a finger photoplethysmograph and compared to the values obtained by pulsed Doppler echocardiography (PDE). A group of 19 healthy men and 12 women [mean ages: 20.8 (SD 1.6) and 22.2 (SD 1.6) years respectively] were studied at rest in the supine position. The ratio of the area below the ejection phase of the arterial pressure wave (A _s) to SV, as obtained by PDE, yielded a calibration factor dimensionally equal to the hydraulic impedance of the system (Z _ao =A _s ·SV ^–1). TheZ _ao amounted on average to 0.062 (SD 0.018) mmHg · s · cm^–3 for the men and to 0.104 (SD 0.024) mmHg · s · cm^–3 for the women. TheZ _ao was also estimated from the equation:Z _ao = a · (d + b ·HR + c ·PP + e ·MAP)^–1, whereHR was the heart rate,PP the pulse pressure,MAP the mean arterial pressure and the coefficients of the equation were obtained by an iterating statistical package. The value ofZ _ao thus obtained allowed the calculation of SV from measurements derived from the photoplethysmograph only. The mean percentage error between the SV thus obtained and those experimentally determined by PDE amounted to 14.8 and 15.6 for the men and the women, respectively. The error of the estimate was reduced to 12.3 and to 11.1, respectively, if the factorZ _ao, experimentally obtained from a given heart beat, was subsequently applied to other beats to obtain SV from theA _s measurement in the same subject.     

Homology modeling of an immunoglobulin-like domain in the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin is expressed by cells of alpha mating type. On the basis of sequence similarities, alpha-agglutinin has been proposed to contain variable-type immunoglobulin-like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence-based alignment algorithms impossible. We have therefore developed a secondary structure-based method that allowed homology modeling of alpha-aggulutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.     

蛇毒蛋白Echistatin的C端突变基因的构建，表达及其活性研究

通过ＰＣＲ定点突变的技术,将蛇毒蛋白Ｅｃｈｉｓｔａｔｉｎ基因的Ｃ端进行了突变（Ａｌａ４８→Ａｒｇ４８→,Ｔｈｒ４９→Ｖａｌ４９）,模拟纤维蛋白Ｎ端的四肽（Ｇｌｙ－Ｐｒｏ－Ａｒｇ－Ｖａｌ）,以期增加Ｅｃｓ（Ｅｃｈｉｓｔａｔｉｎ）的活性。突变的基因重组到表达质粒ｐＪＣ２６４上,经ＩＰＴＧ诱导,以ＣｈｅＹ－Ｅｃｓ融合蛋白方式进行了表达,表达量占菌体总蛋白的１５～２０％。ＳｅｐｈａｄｅｘＧ－７５初步纯化该融合蛋白,然后用ＣＮＢｒ裂解,透析,冻干,反相ＨＰＬＣ纯化Ｃ端突变体Ｅｃｓ蛇毒蛋白,Ｎ端十个氨基酸分析与天然的相符,在ＰＲＰ（ｐｌａｔｅｌｅｔ－ｒｉｃｈｐｌａｓｍａ）测活体系中,１０μｍｏｌ／Ｌ的ＡＤＰ诱导,Ｃ端突变体Ｅｃｓ抑制血小板凝聚的活性约为野生型４倍。得到了Ｅｃｓ的Ｃ端突变后使Ｅｃｓ抑制血小板凝聚的活性提高的结果。     

Basolateral potassium membrane permeability of A6 cells and cell volume regulation

The K⁺ permeabilities (⁸⁶Rb(K) transport) of the basolateral membranes (J_bK) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation.Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Ca _i (max <1 min) and cell swelling (max at 3–5 min) followed by a regulatory volume decrease (5–30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the ⁸⁶Rb(K) transport and the SCC were partially blocked by Ba²⁺, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3–6 min) stimulation of the ⁸⁶Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca²⁺-free media or quinidine addition did not affect the initial osmotic stimulation of J_bK but prevented its secondary regulation, whereas TEA, glibenclamide and DIDS completely blocked the initial J_bK increase. Under hypo-osmotic conditions, the initial J_bK increase was enhanced by the presence of 1 mm of barium and delayed with higher concentrations (5 mm). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media.We propose that a TEA- and glibenclamide-sensitive but quinidine-insensitive increase in K⁺ permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K⁺ permeability mediated by the observed calcium rise.This work was supported by grants from the Commissariat à l'Energie Atomique and the Centre National de Recherche Scientifique URA 638.