Chemotaxis towards sugars inChlamydomonas reinhardtii

Chemotaxis ofChlamydomonas reinhardtii 137c cells towards maltose and sucrose was observed by capillary assay. The threshold concentration was approximately 10^–5 m for maltose and 10^–3 m for sucrose. The peak accumulation of cells occurred at 10^–3 m for maltose and 10^–2 m for sucrose. A selection procedure for chemotaxis mutants was developed. Mutant strain CHE-1 was not attracted by maltose; mutant strain CHE-2 was not attracted by sucrose. Addition of attractant fully inhibited photoaccumulation of cells. After a period of time the photoresponse of cells recovered. Under the conditions of our experiments the period of adaptation lasted 15–20 min in wild-type cells and 4–5 min in mutant cells on the given sugar. Glucose and acetate did not attract cells ofChlamydomonas. Added to the medium, these compounds had no effect on the photoaccumulation of cells.     

d-Glucose does not catabolite repress a transketolase-deficientd-ribose-producingBacillus subtilis mutant strain

WhenBacillus subtilis strain ATCC 21951, a transketolase-deficientd-ribose-producing mutant, was grown ond-glucose plus a second substrate which is metabolized via the oxidative pentose phosphate cycle (d-gluconic acid,d-xylose,l-arabinose ord-xylitol),d-glucose did not catabolite repress metabolism of the second carbon source. Thed-ribose yield obtained with the simultaneously converted carbon substrates, significantly exceeded that when onlyd-glucose was used. In addition, the concentration of glycolytic by-products and the fermentation time significantly decreased. Based on these findings, a fermentation process was developed withB. subtilis strain ATCC 21951 in whichd-glucose (100 g L^–1) andd-gluconic acid (50 g L^–1) were converted into 45 g L^–1 ofd-ribose and 7.5 g L^–1 of acetoin. A second process, based ond-glucose andd-xylose (100 g L^–1 each), yielded 60 g L^–1 ofd-ribose and 4 g L^–1 of acetoin plus 2,3-butanediol. Both mixed carbon source fermentations provide excellent alternatives to the less efficientd-glucose-based processes used so far.     

Fed-batch production of <Emphasis Type="SmallCaps">d</Emphasis>-ribose from sugar mixtures by transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SPK1

d-Ribose, a five-carbon sugar, is used as a key intermediate for the production of various biomaterials, such as riboflavin and inosine monophosphate. A high d-ribose-producing Bacillus subtilis SPK1 strain was constructed by the chemical mutation of the transketolase-deficient strain, B. subtilis JY1. Batch fermentation of B. subtilis SPK1 with 20 g l^–1 xylose and 20 g l^–1 glucose resulted in 4.78 g l^–1 dry cell mass, 23.0 g l^–1d-ribose concentration, and 0.72 g l^–1 h^–1 productivity, corresponding to a 1.5- to 1.7-fold increase when compared with values for the parental strain. A late-exponential phase was chosen as the best point for switching to a fed-batch process. Optimized fed-batch fermentation of B. subtilis SPK1, feeding a mixture of 200 g l^–1 xylose and 50 g l^–1 glucose after the late-exponential phase reduced the residual xylose and glucose concentrations to less than 7.0 g l^–1 and gave the best results of 46.6 g l^–1d-ribose concentration and 0.88 g l^–1 h^–1 productivity which were 2.0- and 1.2-fold higher than the corresponding values in a simple batch fermentation.     

Effect of prey stage on life-table attributes of a genetically manipulated strain ofMetaseiulus occidentalis (Acari: Phytoseiidae)

Metaseiulus occidentalis females from the carbaryl-organophosphate-sulfur resistant strain (COS) lived longer (25.3 days versus 19.7 days), had a higher total fecundity (43.8 eggs female^–1 versus 33.6 eggs female^–1) and a higher daily fecundity rate (2.4 eggs female^–1 day^–1 versus 2.0 eggs female^–1 day^–1), and exhibited a higher intrinsic rate of increase (0.243 individuals female^–1 day^–1 versus 0.182 individuals female^–1 day^–1) and shorter generation time (13.9 days versus 17.0 days), at 24–28°C, 47–56%rh under continuous fluorescent light, when reared on a diet of 0–48-h-old eggs rather than a diet of mixed actives ofTetranychus pacificus McGregor on bean leaf disks. The sexratio of the progeny was female-biased for both diets, 2.1 females to 1 male forM. occidentalis reared on eggs and 2.0:1 : forM. occidentalis reared on mixed actives, suggesting that diet influences sex-ratio in some unknown way.There was no significant difference in oviposition rates for repeatedly-mated and once-matedM. occidentalis females reared on a diet of younger (0–24-h old) eggs compared to a diet of older (72–96-h old) eggs ofT. pacificus.The COS strain ofM. occidentalis exhibited life-table parameters comparable to the other strains reported in the literature, suggesting that the reproductive attributes of this acarine predator were not reduced as a result of artificial laboratory selection. Diet, a biotic factor, produced substantial differences in life-table parameters, suggesting that this factor can influence conclusions regarding the potential efficacy of biological control agents.     

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana Pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.Reprinted from The Journal of Membrane Biology, Vol. 54, No. 3, pages 191–202. Our apologies for deleting the author's names on the original version.     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Homoeotische flügelbildung durch dasloboid-allel “ophthalmoptera” (ld oph ) beiDrosophila melanogaster

In awhite-apricot (w^a) stock, a new mutant was found which reduces the eyes and produces wing-structures in the eyes. This homeotic effect can occur with a high frequency, depending on temperature and genetic background. The mutant is an allele ofloboid (ld, 3–102±) and ofeyesreduced (eyr, 3–103±); the designationloboid-ophthalmoptera (ld^oph) is proposed. The varying contours of the reduced eye inld ^oph fit into a pattern which is quite similar to the cell-lineage pattern established byBecker (1957) for the normal eye. This indicates that inld ^oph-eyes one or more cell-lineage sectors are missing because the corresponding cell families are not competent to differentiate into ommatidia. Genal and orbital bristle rows are frequently doubled, and isolated clusters of bristles occur on the gena. In sectors devoid of ommatidia, “allotypic” wing structures are differentiated. These consist of wing membrance and fragments of anterior and posterior wing border in haphazard combination, and together form a balloon filled with hemolymph. The striking similarity between the homeotic mutation and transdetermination of cultured imaginaldisks (Hadorn, 1966) is discussed.      

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

Leclercia adecarboxylata Gen. Nov., Comb. Nov., formerly known asEscherichia adecarboxylata.

The nameLeclercia adecarboxylata is proposed for a group of the family Enterobacteriacae previously known asEscherichia adecarboxylata. Leclercia adecarboxylata can be phenotypically differentiated from all other species of Enterobacteriaceae. The members of this species are positive for motility, indole production, methyl red, growth in the presence of KCN, malonate, beta-galactosidase, beta-xylosidase, esculin hydrolysis, gas production fromd-glucose, and acid production fromd-cellobiose,d-lactose, melibiose,l-rhamnose, adonitol,d-arabitol, dulcitol, and salicin; the strains were negative for Voges-Proskauer, citrate (Simmons), H₂S (Kligler), lysine and ornithine decarboxylases, arginine dihydrolase, phenylalanine deaminase, gelatinase, DNase, Tween-80 hydrolysis, and acid production from myoinositol and alpha-methyl-d-glucoside. Fermentation ofd-raffinose,d-sucrose, andd-sorbitol is variable with strains. DNA relatedness of 11 strains ofL. adecarboxylata to three strains including the type strain of this species averaged 80% in reactions at 65°C. DNA relatedness to other species in Enterobacteriaceae was 2%–32%, indicating that this species was placed in a new genusLeclercia gen. nov. The type strain ofL. adecarboxylata is ATCC 23216.     

Pulmonary diffusing capacity in reptiles (Relations to temperature and O2-uptake)

Summary Pulmonary CO-diffusing capacity (D l _CO), lung volume, pulmonary perfusion and O₂-uptake were measured by non-invasive techniques in the lizardsVaranus exanthematicus andTupinambis teguixin (mean body weight 2.2 kg for both species).The CO-diffusing capacity was at 25–27°C 0.059 mlstpd·kg^–1·min^–1·Torr^–1 inVaranus, which is 47% greater than the value of 0.040 mlstpd·kg^–1·min^–1·Torr^–1 inTupinambis. The lung volume ofVaranus was 36 ml·kg^–1 and that ofTupinambis 20 ml·kg^–1. At 35–37°C the diffusing capacity of lizard lungs are about 25% of those for mammals of comparable size.InVaranus pulmonary CO-diffusing capacity increased with temperature from 0.027 mlstpd·kg^–1·min^–1·Torr^–1 at 17–19 °C to 0.075 mlstpd·kg^–1·min^–1·Torr^–1 at 35–37 °C. This change closely matched a concomitant increase of O₂-uptake. Pulmonary perfusion increased from 27 ml·kg^–1·min^–1 to 55 ml·kg^–1·min^–1 within this temperature range.The study emphasizes that pulmonary diffusing capacity cannot be fully evaluated without information on pulmonary perfusion and O₂-uptake. In reptiles and other ectotherms diffusing capacity must be reported at specified body temperature.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

Diffusion in immobilized-cell agar layers: influence of microbial burden and cell morphology on the diffusion coefficients ofl-malic acid and glucose

Summary The diffusivities ofl-malic acid and glucose in an agar membrane entrapping small amounts ofEscherichia coli orRhodospirillum rubrum whole cells were measured using time lag (TL) and steady state (SS) methods. Diffusivities were overestimated by the SS method. For concentrations of immobilizedR. rubrum cells ranging between 10⁴ and 10⁹ organisms cm^–3 agar (20 ng-2 mg dry weight cm^–3 agar), the diffusion coefficient ofl-malic acid, determined by both methods, was related to the logarithm of the membrane cell content by a decreasing linear relationship. The diffusion coefficient of glucose obtained by TL analysis was not significantly affected by the presence in the membrane of 3 ng-0.3 mg dry wt.E. coli cm^–3 agar. However, values arising from the SS method decreased linearly as a function of the amount of immobilized organisms. Membranes containingR. rubrum cells offered higher diffusional resistance tol-malic acid and glucose than those loaded with the same amount ofE. coli cells.     

Comparative studies into two isolates of Alternaria affecting umbelliferae

Summary 1. A new species ofAlternaria isolated from the diseased plants ofCarum copticum L.Peucedanum graveolens Benth. &Hook f. andFoeniculum vulgare Mill. is described and compared with other species affecting Umbelliferae.2. TheCoriandrum isolate described by the writer asAlternaria poonensis Raghunath is a shy sporulator in artificial culture with long beaked muriform spores formed singly at the end of comparatively short conidiophores.3. The isolate from the other three host plantsCarum copticum L.,Peucedanum graveolens Benth. &Hook f. andFoeniculum vulgare Mill. is named asAlternaria umbellifericola sp. nov. and is a profusely sporulating species, forming long chains of spores with rudimentary beak.4. TheCoriandrum isolate (Alternaria poonensis) has a narrower temperature range (25–30° C) than theCarum isolate (20–35° C) for optimum sporulation.5. TheCoriandrum isolate is pathogenic toCarum but not toDaucus carota L.6. TheCarum isolate is pathogenic toDaucus carota but does not infectCoriandrum sativum L.7. TheCoriandrum isolate has a wider host range among the Umbelliferae than theCarum isolate.Part of the M.Sc. thesis submitted to the Poona University, 1964.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Therlt11 andraec1 mutants ofArabidopsis thaliana lack the activity of a basic-amino-acid transporter

The concentration dependence of the influx ofl-lysine in excised roots ofArabidopsis thaliana seedlings was analyzed for the wild-type (WT) and two mutants,rlt11 andraec1, which had been selected as resistant to lysine plus threonine, and to S-2-aminoethyl-l-cysteine, respectively. In the WT three components were resolved: (i) a high-affinity, low-capacity component [K _m = 2.2 M;V _max = 23 nmol·(g FW)^–1·h^–1]; (ii) a low-affinity, high-capacity component [K _m = 159 M;V _max = 742 nmol·(g FW)^–1·h^–1]; (iii) a component which is proportional to the external concentration, with a constant of proportionalityk = 104 nmol·(g FW)^–1 h^–1];·mM^–1. The influx ofl-lysine in the mutants was lower than in the WT, notably in the concentration range 0.1–0.4 mM, where it was only 7% of that in the WT. In both mutants the reduced influx could be fully attributed to the absence of the low-affinity (high-K _m ) component. This component most likely represents the activity of a specific basic-amino-acid transporter, since it was inhibited by several other basic amino acids (arginine, ornithine, hydroxylysine, aminoethylcysteine) but not byl-valine. The high-affinity uptake ofl-lysine may be due to the activity of at least two general amino acid transporters, as it was inhibitable byl-valine, and could be further dissected into two components with a high affinity (K _i = 1–5 M; and a low affinity (K _i = 0.5–1mM) forl-valine, respectively. Therlt11 andraecl mutant have the same phenotype and the corresponding loci were mapped on chromosome 1, but it is not yet clear whether they are allelic.Abbreviations AEC S-2-aminoethyl-l-cysteine - K _i equilibrium constant - WT wild-type     

Dependence of cell membrane conductances on bathing solution HCO 3 − /CO2 inNecturus gallbladder

Summary The effects of bathing solution HCO ₃ ^– /CO₂ concentrations on baseline cell membrane voltages and resistances were measured inNecturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO ₃ ^– /CO₂ Ringer's solutions (2.4mm HCO ₃ ^– /air or 1mm HEPES/air) or a high HCO ₃ ^– /CO₂ Ringer's (10mm HCO ₃ ^– /1% CO₂). The principal finding of these studies was that the apical membrane fractional resistance (fR _a) was higher in tissues bathed in the 10mm HCO ₃ ^– /CO₂ Ringer's, averaging 0.87±0.06, whereasfR _a averaged 0.63±0.07 and 0.48±0.08 in 2.4mm HCO ₃ ^– and 1mm HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical (R _a) and basolateral membrane (R _b) resistances in tissues bathed in 10mm HCO ₃ ^– /1% CO₂ Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO ₃ ^– /CO₂ Ringer's, the higher value offR _a was found to be due to both an increase inR _a and a decrease inR _b. The higher values offR _a and lower values ofR _b confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K⁺ and Cl^– substitutions. The results of these studies suggest that an electrodiffusive Cl^– transport mechanism exists at the basolateral membrane of tissues bathed in a 10mm HCO ₃ ^– /1% CO₂ Ringer's, which can explain in part the fall inR _b. The above observations are discussed in terms of a stimulatory effect of solution [HCO ₃ ^– /PCO₂ on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes.     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Intracellular chloride and potassium ions in relation to excitability ofChara membrane

Summary Internodal cells ofChara australis were made tonoplast-free by replacing the cell sap with EGTA-containing media; then the involvement of internal Cl^– and K⁺ in the excitation of the plasmalemma was studied.[Cl^–] _i was drastically decreased by perfusing the cell interior twice with a medium lacking Cl^–. The lowered [Cl^–] _i was about 0.01mm. Cells with this low [Cl^–] _i generated action potential and showed anN-shapedV–I curve under voltage clamped depolarization like Cl^–-rich cells containing 13 or 29mm Cl^–.E _m at the peak of the action potential was constant at [Cl^–] _i between 0.01 and 29mm. The possibility that the plasmalemma becomes as permeable to other anions as to Cl^– during excitation is discussed.At [Cl^–] _i higher than 48mm, cells were inexcitable. When anions were added to the perfusion medium to bring the K⁺ concentration to 100mm, NO ₃ ^– , F^–, SO ₄ ^2– , acetate, and propionate inhibited the generation of action potentials like Cl^–, while methane sulfonate, PIPES, and phosphate did not inhibit excitability.The duration of the action potential depended strongly on the intracellular K⁺ concentration. It decreased as [K⁺] _i (K-methane sulfonate) increased. Increase in [Na⁺] _i (Na-methane sulfonate) also caused its decrease, although this effect was weaker than that of K⁺. The action of these monovalent cations on the duration of the action potential is the opposite of their action on the membrane from the outside (cf. Shimmen, Kikuyama & Tazawa, 1976,J. Membrane Biol. 30:249).