Biomedical subjects

R Laprade

Publications and source records attributed to R Laprade.

At least 37 records · Page 2Linked to original sources

PubMed1999

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.

Animals↗

PubMed1999

Two types of K(+) channels at the basolateral membrane of proximal tubule: inhibitory effect of taurine.

The cell-attached configuration of the patch-clamp technique was used to investigate the effects of taurine on the basolateral potassium channels of rabbit proximal convoluted tubule. In the absence of taurine, the previously reported ATP-blockable channel, K(ATP), was observed in 51% of patches. It is characterized by an inwardly rectifying current-voltage curve with an inward slope conductance of 49 +/- 5 pS (n = 15) and an outward slope conductance of 13 +/- 6 pS (n = 15). The K(ATP) channel open probability (P(o)) is low, 0.15 +/- 0.06 (n = 15) at a -V(p) = -100 mV (V(p) is the pipette potential), and increases slightly with depolarization. The gating kinetics are characterized by one open time constant (tau(o) = 5.0 +/- 1.9 ms, n = 6) and two closed time constants (tau(C1) = 5. 2 +/- 1.5 ms, tau(C2) = 140 +/- 40 ms; n = 6). In 34% of patches, a second type of potassium channel, sK, with distinct properties was recorded. Its current-voltage curve is characterized by a sigmoidal shape, with an inward slope conductance of 12 +/- 2 pS (n = 4). Its P(o) is voltage independent and averages 0.67 +/- 0.03 (n = 4) at -V(p) = -80 mV. Both its open time and closed time distributions are described by a single time constant (tau(o) = 96 +/- 19 ms, tau(C) = 10.5 +/- 3.6 ms; n = 4). Extracellular perfusion of 40 mM taurine fails to affect sK channels, whereas K(ATP) channel P(o) decreases by 75% (from 0.17 +/- 0.06 to 0.04 +/- 0.02, n = 7, P < 0.05). In conclusion, the absolute basolateral potassium conductance of rabbit proximal tubules is the resulting combination of, at least, two types of potassium channels of roughly equal importance: a high-conductance low-open probability K(ATP) channel and a low-conductance high-open probability sK channel. The previously described decrease in the basolateral absolute potassium conductance by taurine is, however, mediated by a single type of K channel: the ATP-blockable K channel.

Adenosine Triphosphate↗

PubMed1998

Cry1Ac, a bacillus thuringiensis toxin, triggers extracellular Ca2+ influx and Ca2+ release from intracellular stores in Cf1 cells

Intracellular Ca2+ concentration was measured in single Cf1 cells (Choristoneura fumiferana, spruce budworm) loaded with Fura-2, a Ca2+-sensitive fluorescent probe. Cf1 cells displayed Ca2+ surges in response to Cry1Ac and Cry1C proteins, two Cf1-toxic Bacillus thuringiensis products, but not to Cry1Aa and Cry3A, which are not toxic to Cf1 cells. In the presence of extracellular Ca2+, the toxin-induced Ca2+ response was insensitive to methoxyverapamil, a voltage-dependent Ca2+ channel blocker, but was abolished by lanthanum, a general inhibitor of Ca2+ transport. In the absence of external Ca2+, Cry1Ac induced a small intracellular Ca2+ transient which was inhibited by TMB-8, a blocker of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive pools. Under these conditions, thapsigargin, which inhibits intracellular Ca2+-ATPases, elicited a Ca2+ surge when applied alone. However, subsequent addition of Cry1Ac failed to induce a Ca2+ signal, indicating a depletion of intracellular Ca2+ pools. In Cf1 cells, therefore, bioactive B. thuringiensis toxins triggered intracellular Ca2+ surges which were mainly due to the influx of extracellular Ca2+ through toxin-made pores, as confirmed by planar lipid bilayer experiments. Furthermore, TMB-8- and thapsigargin-sensitive Ca2+ stores contributed to the Cry1Ac-induced Ca2+ signal.

Journal Article↗

PubMed1998

Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells.

The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique. Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner. In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect. Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose. On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0-1.2 nm. In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low.

Animals↗

PubMed1997

Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors.

A purified, GPI-linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33-1.7 nM) than in receptor-free membranes. The non-toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CrylAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current-voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.

Animals↗

PubMed1997

Hypertonicity decreases basolateral K+ and Cl- conductances in rabbit proximal convoluted tubule.

Collapsed proximal convoluted tubules (PCT) shrink to reach a volume 20% lower than control and do not exhibit regulatory volume increase when submitted to abrupt 150 mOsm/kg hypertonic shock. The shrinking is accompanied by a rapid depolarization of the basolateral membrane potential (VBL) of 8.4 +/- 0.5 mV, with respect to a control value of -54.5 +/- 1.9 mV (n = 15). After a small and transient hyperpolarization, VBL further depolarizes to reach a steady depolarization of 19.5 +/- 1.5 mV (n = 15) with respect to control. In the post-control period, VBL returns to -55.8 +/- 1.5 mV. The basolateral partial conductance to K+ (tK) which is 0.17 +/- 0.01 (n = 5) in control condition, decreases rapidly to nonmeasurable values during the hypertonic shock and returns to 0.23 +/- 0.03 in the post-control period. The basolateral partial conductance to Cl- (tCl), which is 0.05 +/- 0.02 (n = 5) in control, also decreases in hypertonicity to a nonmeasurable value and returns to 0.03 +/- 0.01 in post control. The partial conductance mediated by the Na-HCO3 cotransporter (tNaHCO3), which is 0.48 +/- 0.06 (n = 5) in control condition, remains the same at 0.44 +/- 0.05 (n = 5) during the hypertonic period. Similarly, the membrane absolute conductance mediated by the Na-HCO3 cotransporter (GNa-HCO3) does not vary appreciably. Concomitant with cell shrinkage, intracellular pH (pHi) decreases from a control value of 7.26 +/- 0.01 to 7.13 +/- 0.02 (n = 12) and then remains constant. Return to control solution brings back pHi to 7.28 +/- 0.03. From these results, we conclude that in collapsed PCT, a sustained decrease in cellular volume leads to cell acidification and to inhibition of K+ and Cl- conductances.

Animals↗

PubMed1997

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.

Amino Acid Sequence↗

PubMed1997

Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera).

The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium.

Journal Article↗

PubMed1997

Effect of Bacillus thuringiensis toxins on the membrane potential of lepidopteran insect midgut cells.

To test whether the ability of Bacillus thuringiensis toxins to form pores in the midgut epithelial cell membrane of susceptible insects correlates with their in vivo toxicity, we measured the effects of different toxins on the electrical potential of the apical membrane of freshly isolated midguts from gypsy moth (Lymantria dispar) and silkworm (Bombyx mori) larvae. In the absence of toxin, the membrane potential, measured with a conventional glass microelectrode, was stable for up to 30 min. It was sensitive to the K+ concentration and the oxygenation of the external medium. Addition of toxins to which L. dispar is highly [CryIA(a) and CryIA(b)] or only slightly [CryIA(c) and CryIC] sensitive caused a rapid, irreversible, and dose-dependent depolarization of the membrane. CryIF, whose toxicity towards L. dispar is unknown, and CryIE, which is at best poorly active in vivo, were also active in vitro. In contrast, CryIB and CryIIIA, a coleopteran-specific toxin, had no significant effect. The basolateral-membrane potential was unaffected by CryIA(a) or CryIC when the toxin was applied to the basal side of the epithelium. In B. mori midguts, the apical-membrane potential was abolished by CryIA(a), to which silkworm larvae are susceptible, but CryIA(b) and CryIA(c); to which they are resistant, had no detectable effect. Although the technique discriminated between active and inactive toxins, the concentration required to produce a given effect varied much less extensively than the sensitivity of gypsy moth larvae, suggesting that additional factors influence the toxins' level of toxicity in vivo.

Animals↗

PubMed1996

Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis.

The major diffusion channel in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; Mr 37 782). The Hib porin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti-Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.

Animals↗

PubMed1996

Temperature dependence of endocytosis in renal epithelial cells in culture.

Temperature dependence of fluid-phase endocytosis was determined in two renal epithelial cell lines, MDCK cells and LLC-PK1 cells, using Lucifer Yellow or horseradish peroxidase as markers. For both cell lines, grown on solid support as a confluent monolayer, biphasic curves of marker uptake vs. temperature were obtained. The changes in slope occurred around 27 degrees C, a critical temperature at which the lipids of the plasma membrane of MDCK cells enter in the gel state. Activation energies were significantly higher above 27 degrees C (15-22 kcal/mol) than below that critical temperature (9-12 kcal/mol). These data indicate that changes in membrane physical state have marked effects on endocytic processes. They suggest that two mechanisms, with different activation energies are involved in the fluid phase endocytosis by renal epithelial cells in culture.

Animals↗

PubMed1996

Volume regulation and ion transport in renal cells.

Volume regulatory mechanisms are reviewed for both short- and long-term adaptation of renal cells to anisotonic media. Within minutes after exposure to hypotonic solutions a common feature of many renal cells is the increase in plasma membrane potassium and chloride conductances. Although extrusion of intracellular potassium certainly contributes to a regulatory volume decrease, the role of chloride efflux itself is probably modest, given the relatively low intracellular chloride concentration. Indeed, other intracellular osmolytes such as taurine and other amino acids are transported out of the cell to achieve a regulatory volume decrease. On a longer-term basis cells from the renal medulla have to adapt to an extracellular milieu which can become extremely hypertonic under certain conditions. In tissue culture models, cells exposed to hypertonic media react by actively taking up small molecules, such as betaine, taurine and myo-inositol, and by synthesizing more sorbitol and glycerophosphocholine. For each of these osmolytes hypertonicity was shown to activate the transcription of a specific enzyme or transporter gene, the expression of which reaches a peak within 18 h of hypertonicity.

Animals↗

PubMed1996

Inhibition of basolateral potassium conductance by taurine in the proximal convoluted tubule.

The effect of taurine on the electrophysiological properties of the basolateral membrane of the rabbit proximal convoluted tubule was examined. Short-duration isosmotic pulses of 40 mM taurine in the bath solution induced basolateral membrane depolarizations (delta Vbl) of 6.44 +/- 0.5 mV, which were reduced by 58% in absence of Na+. In presence of barium and quinine, delta Vbl values were reduced by 55% in Na(+)-containing bath solutions and were completely abolished in Na(+)-free solutions. Continuous addition of taurine into the bath solutions for a period of 5 min induced 1) a decrease in the partial conductance of the basolateral membrane to K+ (tK) from 0.39 to 0.23; 2) an increase in the partial conductance to Cl- (tcl) from 0.055 to 0.172; 3) a rapid and transient increase of the partial conductance mediated by the Na-HCO3 cotransporter (tNaHCO3) from 0.37 to 0.52, followed by a progressive decrease to 0.29; and 4) a depolarization of the basolateral membrane of 16 mV. The absolute membrane conductance mediated by the Na-HCO3 cotransporter was not initially affected by taurine, whereas that to K (GK) initially decreased by a maximal factor of 2 after 1 min, followed by a partial recovery after 5 min, and that to Cl (GCl) increased by a factor of 4. Addition of taurine after a hypotonicity-induced cell swelling produced an inhibition of GK comparable to the one observed under isotonic conditions. These results demonstrate the presence of an electrogenic Na-dependent transport of taurine and indicate that taurine inhibits GK.

Animals↗

PubMed1996

Cell volume increases of physiologic amplitude activate basolateral K and CI conductances in the rabbit proximal convoluted tubule.

The effects of increases in cell volume (CV) of physiologic amplitude, induced either hypotonically or isotonically, were studied on the three major basolateral conductances of rabbit isolated proximal convoluted tubules. CV increases were produced by a 40 mosmol/kg H2O hypotonic shock or by the isotonic replacement of mannitol by 40 mM glucose or alanine. The hypotonic shock led to an increase in CV of 17 +/- 3% (N = 8), whereas additions of glucose and alanine led to increases in CV of 22.6 +/- 2.5% (N = 7) and 28.3 +/- 3.5 (N = 5), respectively. Under all of these conditions, the absolute conductance mediated by the NaHCO3 cotransporter did not vary appreciably. This allowed determination of the variations of the absolute conductances to potassium (GK) and chloride (GCl) from their measured partial conductances. All three protocols induced significant increases in GK by factors of 2.37 +/- 0.3, 1.43 +/- 0.16, and 1.69 +/- 0.40, and in GCI by factors of 3.32 +/- 0.57, 3.68 +/- 0.75, and 3.90 +/- 1.0 during the hypotonic, glucose, and alanine protocols, respectively. These increases in GK and GCl occurred with a delay compared with the variations in CV, indicating a more elaborate signaling mechanism than stretch-activation of channels that are known to activate channels within seconds. Intracellular pH increased from 7.19 +/- 0.03 to 7.23 +/- 0.03, 7.17 +/- 0.02 to 7.20 +/- 0.02, and 7.13 +/- 0.01 to 7.16 +/- 0.01 after the hypotonic shock and the glucose and alanine additions, respectively. The study presented here demonstrates that there is a close relationship between CV and GK and GCl, independent of the means used (hypotonically or isotonically) to increase CV in rabbit proximal convoluted tubules. CV activation of GK is proposed to account for part of the increase in GK reported previously during activation of transepithelial transport.

Animals↗

PubMed1995

Endogenous K+/H+ exchange activity in the Sf9 insect cell line.

The fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to investigate changes in the intracellular pH (pHi) of individual Sf9 cells in response to changes in the composition of the external medium. Under standard conditions, the resting pHi was 0.2-0.3 unit lower than the extracellular pH (6.5). The extracellular concentration of K+ had a major influence on pHi. Removal of K+ from the medium resulted in a rapid but reversible acidification of the cells. The buffer capacity of the cells was a U-shaped function of pHi with a minimum at about pHi = 6.2. In the presence of K+, a change in the pH of the medium was followed by an equivalent change in pHi. In its absence, however, changes in the external pH had little effect on the pH of the cells. Following removal of K+ from the medium, the cells realkalinized at an initial rate which increased with increasing concentration of added K+. This cation was about 30 times more effective in promoting realkalinization of the cells than Li+ Na+, Rb+, and Cs+. The apparent Km for K(+)-dependent H+ efflux was about 12 mM and was slightly modulated by extracellular pH. These results strongly suggest that, in Sf9 cells, a K+/H+ antiporter plays a key role in the movement of protons across the cell membrane.

Animals↗

PubMed1995

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells.

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pHi) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K+ and Na+ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pHi depends strongly on the activity of a K+/H+ exchanger. In the absence of toxin, removal of K+ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pHi equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pHi. B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H+, K+ and Na+.

Animals↗

PubMed1995

Factors affecting the stability of the renal sodium/phosphate symporter during its solubilization and reconstitution.

Phosphate is reabsorbed across the brush-border membrane of the proximal tubule by a specific sodium-dependent symporter. Like the other brush-border membrane transport proteins of the kidney, the phosphate carrier remains to be isolated in a functional state. To establish a set of parameters that allow to preserve its biological activity, the phosphate carrier was solubilized under systematically varied conditions and reconstituted into proteoliposomes. Successful reconstitution was achieved only when the extraction buffer contained lipids extracted from the renal brush-border membrane. Glycerol, an osmolyte which reduces the water activity of the solution, was also required. It could however be replaced by 150 mM sodium or potassium phosphate. Below this concentration and in the presence of glycerol, the ionic strength of the solution had little effect on the stability of the transporter, but sodium phosphate could not be replaced by sodium chloride. Phosphate transport in reconstituted vesicles depended on the concentration of detergent and pH of the extraction buffer. Finally, transport activity was increased when solubilization was carried out in the presence of a reducing agent, dithiothreitol. These results should be helpful during the purification and further characterization of the renal phosphate symporter.

Animals↗

PubMed1995

Hypotonicity increases basolateral taurine permeability in rabbit proximal convoluted tubule.

The permeabilities of the basolateral membrane of rabbit proximal convoluted tubule (PCT) to taurine (PTau) and glucose (PGlc) were estimated under control and hypotonic conditions using the initial rate of increase in cellular volume (CV) induced on isotonic replacement of 40 mM mannitol by one or the other of these substrates. Under control conditions, addition of taurine led to an increase in CV at an initial rate of 7.1 +/- 1.7%/min, leading to a cell swelling of 30.2 +/- 4.8% after 5 min (n = 6). Addition of glucose led to an increase in CV at an initial rate of 30.0 +/- 3.8%/min, leading to a cell swelling of 25.7 +/- 3.1% after 5 min (n = 7). After a period of recovery of 5 min in the absence of taurine or glucose, a 40 mosmol/kg hypotonic shock induced a cell swelling of 14.2 +/- 1.3 and 16.1 +/- 5.2%, respectively, followed by an almost complete volume regulatory decrease after 5 min. At that time, addition of taurine under continuous hypotonicity induced an increase in CV at an initial rate 2.57 +/- 0.17 times larger than that observed under the isotonic condition (P < 0.005), while addition of glucose induced an initial increase in CV identical to that observed under the isotonic condition. The increases in CV observed on addition of taurine were completely abolished in the absence of sodium under both isotonic and hypotonic conditions. The permeability to K+ was also estimated, in the absence of sodium, using the initial rate of increase in CV induced on isotonic replacement of 40 mM N-methyl-D-glucamine by K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗