The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.     

Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.

Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100.     

Melibiose transport of Escherichia coli.

Transport of [3H]melibiose, prepared from [3H]raffinose, was investigated in Escherichia coli. Na+ stimulated the transport of melibiose via the melibiose system, whereas Li+ inhibited it. Kinetic parameters of melibiose transport were determined. The Kt values were 0.57 mM in the absence of Na+ or Li+, 0.27 mM in the presence of 10 mM NaCl, and 0.29 mM in the presence of 10 mM LiCl. The Vmax values were 40 and 46 nmol/min per mg of protein in the absence and in the presence of NaCl and 18 nmol/min per mg of protein in the presence of LiCl. Melibiose transport via the melibiose system was temperature sensitive in a wild-type strain of Escherichia coli and was not inhibited by lactose. On the other hand, melibiose uptake via the lactose system was not temperature sensitive, was inhibited by lactose, and was not affected by Na+ and Li+. Methyl-beta-D-thiogalactoside, a substrate for both systems, inhibited the transport of melibiose via both systems.     

Calcium enhances the hemolytic action of bile salts

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.     

Analytical approach for the extraction of recombinant membrane viral glycoprotein from stably transfected Drosophila melanogaster cells

Parameters for storage, lysis and concentration of Drosophila melanogaster Schneider 2 (S2AcRVGP) cells expressing the recombinant rabies virus glycoprotein (RVGP) were studied with regard to RVGP quantification by ELISA, for productivity evaluation and future purification. Lysis buffers were formulated with Tris, NaCl, glycerol, EDTA, KCl, Na(2)PO(4), MgCl(2), PMSF and NP-40 or CHAPS. S2AcRVGP cells (10(7) cells at the exponential growth phase) were frozen at -20 degrees C as a dry pellet, suspended in buffer (B) formulations or after treatment with lysis buffer (LB) formulations. They were then thawed as cell pellets or with B formulations or PBS at 4 degrees C or at room temperature and then lysed with LB formulations. For RVGP quantification by ELISA, a protocol was chosen of cell preparation including cell freezing as dry pellet, cell thawing at 4 degrees C with B4 (Tris, NaCl, MgCl(2), PMSF) and cell lysis with the LB4 (B4 + NP-40) since it fulfilled requirements of high RVGP detection, and was easily performed with mixtures freezing quickly, and a cost-saving LB formulation could be used. Using these established conditions, we examined the optimal cell concentration for RVGP quantification by ELISA. Results showed that an increase in the RVGP detection (from 62.5 to 1083 ng/10(7) cells) paralleled a decrease in the cell number (3 x 10(7) - 10(5) cells) used. The NP-40 concentration present in the LB4 was further investigated as a function of the cell number used for sample preparation. Previous results were confirmed indicating that higher NP-40 concentrations led to a decreased detection of RVGP. Altogether our data clearly pointed out the crucial effects of cell freeze, thaw, lysis and concentration on immune detection of recombinant membrane glycoproteins and can be useful as a guideline for sample preparation for this purpose.     

Action of exogenous potassium and calcium ions on in vitro metacyclogenesis in Trypanosoma cruzi.

The cations Ca2+ and K+ and the anions Cl-, HCO3-, and PO4- were studied for their contribution to metacyclic trypomastigote formation of Trypanosoma cruzi in starvation media consisting of phosphate-buffered saline (PBS) + 10 mM proline + 10 mM sodium acetate as well as one of the following salts: 0.035% NaHCO3 (PBSNPA), 0.035% K2CO3 (PBSKPA) or 0.035% K2HPO4 (PBSPPA). Isolates CL and DM28c were activated to transform with 5% CO2 and the percent metacyclogenesis determined after incubation for 96 h in PBS starvation media. Maximal metacyclogenesis was found with CaCl2 and KCl. In the presence of K+, the percent transformation was highest with the phosphate salt, followed by the carbonate and the chloride salts. Cells incubated in PBSNPA and the cationic ionophores A23187 (5 x 10(-6) M), lasalocid (5 x 10(-6) M), and valinomycin (10(-8) M) do not survive; addition of 2 mM CaCl2 or 17 mM KCl to DM28c cells, reversed the lethal action of the ionophores permitting differentiation into metacyclic forms. The addition of CaCl2 to CL cells incubated in ionophores abrogated the lethal effect of the ionophores but transformation was significantly different than in control preparations. Adding KCl to ionophore incubated cells resulted in normal levels of transformation except in the case of valinomycin. DM28c and CL cells incubated in PBSKPA show significantly greater metacyclogenesis in the presence of 5 mM EGTA. These results indicate that exogenous concentrations of several cations and anions significantly influence T. cruzi metacyclogenesis and that the degree of response by the parasite to free ion levels may be strain dependent.     

Erythrocyte bisulfite transport

The wide range of transport rates for anions of differing chemical structure by the human erythrocyte anion transport protein (Band 3 protein) suggests that this protein is highly selective for anions that chemically resemble its natural substrate bicarbonate. To test this hypothesis, the influx of bisulfite (HSO3-), a bicarbonate analog, was compared to influxes of chloride, sulfate, and bicarbonate, as measured by the technique of colloid osmotic lysis in isotonic ammonium salt solution. The lysis time induced in chloride solution (much greater than 10 min) was markedly accelerated to 0.6 min by the addition of small amounts (5 mM) of bicarbonate, an effect characteristic of colloid osmotic lysis induced by the anion transport pathway. Lysis in bicarbonate solution was extremely rapid (0.09 min), and was markedly inhibited by acetazolamide (2.9 min). Lysis in bisulfite solution occurred spontaneously (2.2 min) but was markedly accelerated to a time similar to that of chloride (0.56 min) by addition of 5 mM bicarbonate. In contrast, sulfate induced lysis was extremely slow (less than 10% lysis at 40 min in the presence of bicarbonate). Preincubation of erythrocytes with SITS, an inhibitor of anion exchange, prevented lysis by chloride, but had no effect on lysis by bicarbonate, indicating that lysis by bicarbonate was predominantly through diffusion and not anion transport. SITS treatment of erythrocytes eliminated the catalytic effect of bicarbonate during lysis by bisulfite, indicating that anion transport of bisulfite and diffusion of the conjugate acid in the form of SO2 both contribute to the total membrane flux. When the contribution of diffusion is taken into account, the rate of bisulfite influx through the anion exchange pathway is at least 100-fold faster than that for sulfate.     

Platelet-activating factor binding to human platelet membranes

Previously reported methods for quantifying platelet-activating factor (PAF) binding to rabbit platelet membranes were modified for studies of PAF binding to human platelet membranes. The membranes were prepared by the "glycerol lysis" method and PAF binding was quantified by using polyethylene glycol precipitation to recover membrane-bound PAF. Optimal PAF binding required buffers containing 3 to 10 mm KCl and either 5 to 10 mM MgCl2 or 5 to 10 mM CaCl2. NaCl was not as effective as KCl and concentrations of NaCl greater than 3 mM strongly inhibited PAF binding. Maximal binding occurred after incubation for 60 min at 0 degree C and was reversed by the addition of excess unlabeled PAF. PAF binding was saturable. Scatchard analysis of PAF binding to 50 micrograms of membrane protein revealed 10.3 +/- 1.7 x 10(11) receptors per milligram of membrane protein and the receptors had a Kd of 7.6 +/- 1.9 nM. The calculated receptor number, binding affinity, and specificity of binding are similar to those previously calculated for PAF binding to intact human platelets, suggesting that the membrane binding site for PAF is the PAF receptor.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes.

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Lithium fluxes in Paramecium and their relationship to chemoresponse.

Paramecia respond to environmental stimuli by altering swimming behavior to disperse from or accumulate in the vicinity of the stimulus. We have found, using the T-maze assay, that treatment of paramecia with LiCl in a time- and concentration-dependent manner modifies the normal response to folate, acetate, and lactate from attraction to no response or even repulsion. Responses to NH4Cl were unaffected and to cAMP were variably affected by LiCl. Cells incubated in the presence of K+, or both Na+ and K+, but not Na+ alone reliably recovered attraction to folate. Treatment of cells with 4 mM LiCl for 1 h dramatically slowed swimming speed from about 1 mm/s in NaCl or KCl (control) to 0.18 mm/s in LiCl. Li-treated cells subsequently incubated in 4 mM NaCl, KCl or sequentially in KCl and NaCl for a total of 20 min increased their swimming speed to 0.35, 0.45 and 0.67 mm/s, respectively. Paramecia readily took up Li+ in Na(+)- and K(+)-free media reaching intracellular concentrations of 5-10 mM in 10 mM extracellular Li+. Efflux of intracellular Li+ was stimulated 35% by extracellular 10 mM NaCl and 185% by 10 mM KCl over 10 mM choline chloride. Incubation of cells in 10 mM LiCl for 1 h inhibited the rate of Ca2+ efflux by 44% compared to cells in 10 mM NaCl. This may relate to the mechanism by which Li+ perturbs chemoresponse. A mutant with defects in Ca homeostasis responds normally to NH4Cl, but not to any of the stimuli that are affected by LiCl.     

Kinetic analysis by stopped-flow radiationless energy transfer studies: effect of anions on the activity of carboxypeptidase A

We have utilized a highly sensitive radiationless energy transfer (RET) assay to investigate the effect of anions on the activity of carboxypeptidase A (CPD-A). The RET kinetic method visualizes the ES complex directly and thus enables both the mode of action of anions and the quantitation of their effect to be determined at a single substrate concentration. In marked contrast to the activating effect of anions on the closely related metalloprotease, angiotensin converting enzyme, Cl-, and other anions inhibit CPD-A catalysis. NaCl inhibits the hydrolysis of Dns-Ala-Ala-Phe throughout the pH range 6-10. Other di- and tripeptides are similarly inhibited while their ester analogues are affected only slightly. Changes in the type of cation [e.g., Na+, Li+, K+, Ca2+, and (CH3)4N+] at a constant [Cl-1] of 0.1 M showed no difference in the extent of inhibition, whereas with anion substitution the differences were marked. In all cases, the inhibition was partially competitive. At pH 5.9, the Ki values for the free enzyme are 51 (Cl-), 17 (N3-), 2.1 (SO4(2-)), and 0.21 mM (H2PO4-), and for the ES complex, the KI' values are 1000, 720, 42, and 13 mM, respectively. The other anions were shown to act at the chloride site. The results indicate that investigations of anion inhibition in 1 M NaCl, a typical assay condition, may be greatly hindered by the presence of Cl-. Thus, the competitive binding mode of phenylacetate toward peptide hydrolysis is greatly decreased by the presence of 1 M Cl- ion while its noncompetitive component is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of bphA, encoding biphenyl dioxygenase, in two polychlorinated biphenyl-degrading bacteria, psychrotolerant Pseudomonas strain Cam-1 and mesophilic Burkholderia strain LB400

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm(r) fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30 degrees C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7 degrees C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15 degrees C was approximately five times less than the level in LB400-1 grown at 30 degrees C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.     

Physiological Response of Lactobacillus plantarum to Salt and Nonelectrolyte Stress

In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K⁺ (or Na⁺) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.     

Renal palmitate transport: possible sites for interaction with a plasma membrane fatty acid-binding protein

Summary In previous studies from this laboratory [14], a mediated transport system for long chain fatty acids was observed in rat renal basolateral membrane vesicles. Transport was measured in the absence of albumin and indicated the presence of a Na⁺ independent anion exchange mechanism. The present experiments were done to characterize renal transport of fatty acids derived from fatty acid-albumin complexes. ³H-palmitate uptake by brush border (BBMV) and basolateral membrane vesicles (BLMV) isolated from rat renal cortex was determined using a rapid filtration technique. All incubation media contained 100 µM ³H-palmitate complexed to 100 µM bovine serum albumin. Up to 65% of initially bound fatty acid-albumin complexes were displaceable by washing with solution containing 0.1% albumin. Total palmitate uptake was measured as the remaining non-displaceable radioactivity. In BBMV in low ionic strength (300 mM mannitol) or ionic buffers (100 mM mannitol + 100 mM NaCl or KCl), total palmitate uptake at 15 sec did not differ from equilibrium (60 min) values of 10–11 nmoles/mg protein. Uptake was primarily due to binding. A similar pattern was seen with BLMV in 300 mM mannitol buffer: In BLMV in 100 mM NaCl or KCl buffers, equilibrium uptake was 10-fold lower than at 15 sec. This suggests binding followed by cation-dependent translocation. If a putative FABP_PM is involved in transport only, its presence should be confined to BLMV.     

Purification and characterization of a beta-galactosidase from peach (Prunus persica)

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.     

Nucleotide-activated chloride channels in lysosomal membranes.

Lysosomal membrane vesicles purified from rat liver contain a basal chloride conductance that was enhanced in the presence of ATP, non-hydrolysable ATP-analogs and, to a lesser extent, GTP. Other nucleotides, including AMP, ADP and cAMP, as well as CTP and UTP were not effective. Following fusion of the vesicles with an artificial phosphatidylethanolamine/phosphatidylserine bilayer, we found that ATP gamma S dramatically increased the incidence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS)-sensitive chloride channels with a unitary slope conductance of approx. 40 pS in 300 mM/50 mM KCl buffers and 120 pS in symmetrical 300 mM KCl buffers. Since similar results were obtained with AMP-PNP, the results indicate that lysosomes contain a chloride permeable ion channel that is activated by ATP through allosteric interaction.     

Chemosensory function of salt and water transport by the amphibian skin

Solute and water transport mechanisms of anuran skin mediate chemosensory functions that permit evaluation of ionic and osmotic properties of hydration sources in a manner similar to taste receptors in the mammalian tongue. Histochemical observations demonstrated apparent connections between spinal nerve endings and epithelial cells of the skin and we used neural and behavioral responses as measures of coupling between transport and chemosensation. The inhibition of transcellular Na+ transport by amiloride partially reduced the neural response and the avoidance of hyperosmotic NaCl but not KCl solutions. Cetylpyridinium chloride (CPC) reduced the neural response to hyperosmotic salt solutions, suggesting a chemosensory role for vanilloid receptors in the skin. Avoidance of hyperosmotic salt solutions was reduced by impermeant anions suggesting paracellular conductance is important for chemosensation. The effects of blocking the transcellular and paracellular pathways was additive but did not eliminate the avoidance of osmotically unfavorable solutions by dehydrated toads. The timing of the neural response to deionized water was similar to the onset of water absorption behavior and increased blood flow to the pelvic skin. Water absorption from 50 mM NaCl was greater than from deionized water when toads were fully immersed, but not when contact was limited to the ventral surface.     

Control of Alternaria alternata by cassia oil in combination with potassium chloride or sodium chloride

AIMS: To compare antifungal effects of cassia oil alone and in combination with potassium chloride (KCl) or sodium chloride (NaCl) against Alternaria alternata in vitro and in vivo. METHODS AND RESULTS: The inhibitory effect of cassia oil alone, or in combination with KCl and NaCl were tested in vitro. The spore germination and germ tube elongation of the pathogen was evaluated in potato dextrose broth with light microscopy analysis. The inhibitory effect of cassia oil alone, or in combination with KCl and NaCl, was determined on cherry tomatoes in vivo. The cassia oil in combination with KCl and NaCl exhibited strong antifungal effect in vivo and in vitro. CONCLUSIONS: The antifungal effect of cassia oil against Alt. alternata was enhanced significantly by combining with KCl and NaCl both in vitro and in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of cassia oil and KCl or NaCl may enhance antifungal effect of cassia oil and reduce cost.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.