Genetic effects of in vitro germination and plantlet development in chilli pepper.

The objective of this study was to estimate the genetic effects on in vitro germination and development of chili peppers. For this, four genotypes (UFPB-132, -134, -137, and -390) were used as parents. They were crossed in a complete diallel scheme. The parents (4) and hybrid (12) seeds were germinated in glass bottles containing MS media. The following variables were evaluated: seed germination percentage, deformed seedlings percentage, radicle emission percentage, root length, root number, hypocotyl length, hypocotyl width, definitive leaf number, leaf length, and leaf width. The data were submitted to analysis of variance and the means were grouped by the Scott-Knott criteria (P ≤ 0.05). The diallel analysis was performed according to the Griffing method, method I, fixed model. Significant differences for all analysed variables were observed. Additive and non-additive effects were observed influencing the performance of the genotypes in relation to the evaluated variables. The genitor 132 showed the highest general combining ability for almost all evaluated characteristics, except seed germination percentage and deformed seedlings percentage. This study revealed dominance effects are responsible for genotypic variation for almost all evaluated traits. In addition, we found significant reciprocal effects for all studied characters. For the traits to which non-additive variances were important (germination percentage, deformed seedlings percentage, root number, hypocotyl length, hypocotyl width, and definitive leaf number, leaf length, and leaf width), there was an additional opportunity for developing F1 hybrid.

Estabelecimento in vitro e micropropagação de maracujá silvestre (Passiflora foetida L. ) / In vitro establishment and micropropagation of Passiflora foetida L

Entre as espécies do gênero Passiflora, a P. foetida L. apresenta a maior variabilidade genética e tem grande importância medicinal, pois é usada no tratamento de doenças como asma, icterícia, e na forma de emplastros, para as erisipelas e doenças de pele com inflamação. Portanto, são necessários estudos que visem a micropropagação e conservação. As sementes de P. foetida L. apresentam dormência e muitas vezes, levam alguns meses para germinar, produzindo mudas desuniformes e de baixo vigor. Neste sentido, a cultura de tecidos apresenta-se como uma forma alternativa a propagação. Assim, o objetivo do trabalho foi estabelecer e micropropagar P. foetida L., para formação de um banco de germoplasma. Para tanto, sementes foram escarificadas, desinfestadas e inoculadas em meio MS(½) sem reguladores de crescimento e cultivados por 66 dias. Explantes de hipocótilos obtidos de plantas germinadas in vitro, foram cultivados no mesmo meio suplementado com 1,0 mg L-1 de BAP. Na fase de estabelecimento, 45% dos explantes brotaram e formaram gemas axilares. 88,9% dos explantes de hipocótilo induziram brotação e 11,1% produziram calos. Plântulas regeneradas com 1,82 cm de altura, com raízes foram aclimatadas.

Among the species of the genus Passiflora, P. foetida L. presents highest genetic variability and also great medicinal importance. This species is used in the treatment of diseases such as asthma, jaundice, and in the form of poultices for erysipelas and skin diseases with inflammation. Therefore, studies are needed to preserve them. Its seeds present dormancy and often take several months to germinate. The tissue culture is a alternative form to propagate species. The objective was to establish and micropropagation P. foetida L., and create a germplasm bank. Seeds were scarified, disinfected, and inoculated on MS medium (½) without regulators for 66 days. Hypocotyls explants obtained of seedlings in vitro germinated were transferred and placed in the same medium supplemented with BAP (1,0 mg L-1). In the establishment phase of 45% of the explants sprouted and formed axillary buds. 88,9% of hypocotyl explants induced shoots and 11,1% produced callus. Seedling measuring 1,82 cm length and rooted were acclimatized.

Ionic transport in tall columnar epithelial (TCE) cells obtained by nasal brushing from non-cystic fibrosis (CF) individuals.

Tall columnar epithelial (TCE) cells can be obtained by a non-invasive procedure through brushing the inferior turbinate and the adjacent lateral nasal wall. Here, we present results from the functional study of epithelial cells, thus obtained by using the patch-clamp technique. By patch-clamping the sub-apical region of TCE cells, we were able to identify at least three different groups of Cl- channels, namely: a) one with large conductance, rectifying, which was the most frequently found type of Cl- channel; b) a second type of small conductance, activated by cAMP and IBMX, in excised inside-out patches and voltage independent; c) a third type with a conductance around 25 pS, voltage independent, with a linear IV relationship, that could be observed in the excised inside-out configuration. The study of CFTR Cl- channel and its role in airway cell physiology has generally been conducted in cultured cells, most of which not polarized. This experimental work using freshly obtained TCE cells from the nasal epithelium, demonstrates that such cells may be one valid tool to study Cl- channels (most probably ORCC and CFTR Cl- channels) as a model for the lower respiratory epithelium.

Activation of ionic channels by deoxycholate in frog and human cell lines.

Humans, after extensive ileal resection, frequently suffer from diarrhoea, which may be due to an increased delivery of deoxycholate (DOC) to the large intestine. In the frog skin the addition of DOC (0.5 mM) to the apical side induced the activation of amiloride-sensitive Na+ channels and an increase in the unidirectional Cl- fluxes. Here we used two established cell lines (A6 and Caco2) to study the effect of DOC on ion channels at cell and membrane level using the patch-clamp technique. In A6 cells subcultured directly on Petri dishes and studied in the whole-cell configuration, DOC induced an increase in cell conductance of 110.3 +/- 4 pS pF-1 (N = 8) which was reduced to 89 +/- 14 pS pF-1 (N = 8) by the addition of DIDS (0.5 mM), The absolute values of these two effects were not statistically different (P < 0.2). In Caco2 cells, the addition of DOC (0.5 mM) induced, after 1 min, an increase in cell conductance of 583 +/- 16 pS pF-1 (N = 8) which was reduced to 560.4 +/- 16 pS pF-1 (N = 8) by DIDS (0.5 mM) and N-phenylanthranilic acid (DPC; 0.5 mM). The two values were not statistically different (P < 0.4). In Caco2 cells subcultured under the same conditions, DOC induced an increase in cell conductance of 1710 +/- 64 pS pF-1 (N = 6). Subsequent addition of amiloride (0.1 mM) reduced the cell conductance to 1558 +/- 33 pS pF-1 (N = 6). These two mean values were statistically different allowing for an error of the second kind < 0. 05. In cells in which DOC produced a conductance increase of 1010 +/- 10 pS pF-1, gadolinium (0.5 mM) induced a fall in cell conductance of 1800 +/- 10 pS pF-1. In Caco2 cells, addition of DOC (0.5 mM) to the bath reversibly induced the appearance of or an increase in channel activity in patches studied in cell-attached and excised inside-out configuration. In inside-out experiments (N = 13) DOC (0. 5 mM) induced the appearance of channel activity with conductances and reversal potentials (Er) of 27.7 +/- 1.9 pS and 0.8 +/- 5.7 mV, respectively. In cell-attached patches (N = 13) these values were 24.9 +/- 4.4 pS and -18.1 +/- 6.4 mV. In excised inside-out patches from Caco2 cells, subjected to electrochemical gradients for Na+, K+ and Cl-, (+85, -85 and 0 mV, respectively), addition of DOC also induced an increase in the baseline conductance and a shift in the reversal potential from values around +25 mV to values around 0 mV. Bile salts activated both anionic and cationic channels and did not require the presence of intracellular factors for these effects. We suggest that they act at the membrane level.

The effect of bile acid on the activation of amiloride-sensitive sodium channels in frog skin.

The effect of the bile acid, deoxycholic acid (DOC), on the transport properties of isolated frog skin was studied under short-circuit conditions. The addition of DOC (1 mM) to the apical side of the frog skin induced a threefold increase in the short-circuit current (Iac). This effect was inhibited by amiloride. DOC also increased the conductance of the preparation by two different mechanisms. At low concentrations (< 2.5 mM) it activated amiloride-sensitive sodium channels. At higher concentrations of DOC, basolateral-apical unindirectional fluxes, measured with 22Na+, 36Cl-. [14C]mannitol and [14C]inulin, showed a selective increase in the permeability to Na+, Cl- and mannitol in relation to [14C]inulin. These data suggest that sodium and chloride ions use the same diffusional pathway across the preparation. This pathway discriminates between NaCl and mannitol, and discriminates even more in relation to inulin. The effects of DOC are additive to those of cAMP (1 mM). ADH (20 mU ml-1), prostaglandin E2 (0.1 microM) or forskolin (10 microM). It is concluded from our study that the final effect of DOC in stimulating the Isc in frog skin is through the activation of amiloride-sensitive sodium channels. However, since DOC is liposoluble, a direct activation of an adenylate cyclase or of phosphokinase A cannot be excluded.

Opening of frog skin sodium channels by phenobarbital.

This paper presents a study of the action of phenobarbital (anticonvulsant barbituric acid with pK1 = 7.3 and pK2 = 11.8) on the short-circuit current and on the transepithelial conductance of the frog isolated skin. An increase in these parameters was found after addition of the drug to the outside medium (apical). This effect is significantly higher at pHs lower than pK1, suggesting that the neutral form of this substance is the active form. All studies were performed at pH 7. Phenobarbital induces a depolarization of the apical barrier and an increase in 22Na+ radioactive tracer fluxes, which parallel the increase in the observed short-circuit current. This suggests an opening of the Na+ channels in this barrier. A dose-response curve shows that phenobarbital possibly acts on two different sites, with different affinity constants and stoichiometries. To further characterize these channels, comparative studies with oxytocin (a hormone that opens amiloride-sensitive Na+ channels in the apical side) were performed. The results show that phenobarbital causes the same percentage of increase in Isc in control and oxytocin-pretreated skins. Amiloride was also found to inhibit the phenobarbital-sensitive channels with an affinity constant [KAMIL = (60 +/- 1.78) x 10(-8) M] insensitive to the phenobarbital concentration.

Ion transport across the epithelium of the rabbit caecum.

The isolated rabbit caecum was studied in vitro. Under our experimental conditions, the rabbit caecum secreted potassium and chloride and absorbed sodium. To characterize the transport properties of the apical and the basolateral barriers, transepithelial electrical and flux (22Na, 36Cl and 86Rb) measurements and their sensitivity to transport inhibitors (furosemide, DIDS, ouabain and barium) are presented together with intracellular measurements with double-barrelled microelectrodes of intracellular electrical potentials and ionic activities. The fluxes of sodium and chloride were insensitive to DIDS and furosemide. The secretion of potassium and the absorption of sodium were both inhibited by ouabain, indicating that they are coupled through the sodium pump. Ouabain induced a slow fall in the chloride net fluxes, suggesting that these fluxes are also driven by the sodium pump, albeit indirectly. The basolateral to apical fluxes of potassium are insensitive to barium added to the apical side, but are accelerated by the replacement of chloride by gluconate on the apical side, suggesting the presence of a K+/Cl- symport in the apical barrier.

The effect of trimethoprim on sodium transport across the frog skin epithelium.

Trimethoprim, a dihydrofolate reductase inhibitor, is used as a therapeutical agent in combination with sulfamethoxazole. Studies of the interaction of trimethoprim with membrane transport are rare. This paper presents a study of the effect of trimethoprim on the short-circuit current (Isc) and on the transepithelial total conductance (Gt) of the isolated frog skin. We found a fast drop (less than 1 min) of Isc (50%) and Gt (30%) after the addition of the drug to the outside medium (mucosa). A dose-response curve of the effect of the drug has shown that trimethoprim has no effect for concentrations below 0.01 mM and reaches a half-maximum inhibition at 0.5 mM. Trimethoprim induces a decrease in the sodium radioactive tracer fluxes that parallel the decrease of the observed Isc. It induces a hyperpolarization of the mucosal barrier in the same way that amiloride does. A comparative study of the effect of trimethoprim and amiloride has shown that trimethoprim acts on the amiloride-sensitive sodium channels of the mucosal barrier and behaves as a competitive inhibitor of amiloride with a dissociation constant KTRIM of 53 x 10(-5) M while KAMIL = 46 x 10(-8) M.

The coupled movements of sodium and chloride across the basolateral membrane of frog skin epithelium.

1. When frog skin epithelium was exposed to a chloride-free solution bathing the basolateral side of the frog skin preparation the short-circuit current fell and there was a simultaneous loss of chloride and water from its cells. This effect was partially blocked by furosemide when this drug was added to the basolateral bathing solution. 2. Under control conditions and when added to the solution bathing the basolateral side of the preparation furosemide had no effect on the ion and water contents of the frog skin epithelium. 3. Furosemide but not SITS (4-acetamide-4'-isothiocyanate-stilbene-2,2'-disulphonic acid) or amiloride blocked the recovery of short-circuit current and the reuptake of chloride and water by preparations pre-incubated with chloride-free solution on the basolateral side. The recovery of the short-circuit current was also blocked by the replacement of basolateral potassium by sodium. 4. The effect on the short-circuit current of graded replacements by impermeant ions of sodium or chloride did not show saturation for concentrations of these ions up to their control values. 5. Replacement of basolateral potassium by sodium inhibited the short-circuit current and the recovery observed when potassium was reintroduced in the basolateral bathing solution was blocked by furosemide. 6. The replacement of basolateral sodium or chloride by impermeant ions induced an immediate fall in the intracellular concentrations of both sodium and chloride suggesting that the transport system coupling the movements of the two ions across the basolateral membrane is operative under control conditions. 7. It is proposed that the coupled movements of sodium and chloride across the basolateral membrane of the frog skin epithelium are mediated by a sodium-potassium-2 chloride co-transport system which under control conditions is very near equilibrium.

Effects of cell volume changes on membrane ionic permeabilities and sodium transport in frog skin (Rana ridibunda).

1. Membrane potential and conductances and short-circuit current were continuously measured with microelectrodes and conventional electrophysiological techniques in a stripped preparation of frog skin epithelium. The effects of the removal of chloride or sodium ions and the concentration or dilution of the serosal (inner) bathing solution were studied. 2. Chloride- or sodium-free solutions produced a cell depolarization of about 30 mV in parallel with a fall in the short-circuit current. Mucosal and serosal membrane conductances both decreased and the sodium permeability of the mucosal barrier was calculated to fall to about one-half its value in standard Ringer solution. The observed decrease in the short-circuit current is probably related to the combined effect of the decrease in sodium permeability and the decrease in the driving force across the mucosal membrane. 3. The removal of chloride or sodium ions reduced the depolarization caused by serosal perfusion with high-potassium solutions (50 mM-KCl). The ratio of the change in cell membrane potential under short-circuit conditions to the change in the potassium equilibrium potential (delta Ec(s.c.)/delta EK), was 0.59 in standard Ringer solution and 0.26 and 0.24 after the removal of chloride or sodium respectively. The depolarizing effect of barium-containing solutions (2 mM-BaCl2) was also markedly reduced in chloride- or sodium-free solutions, suggesting a decrease of the potassium selectivity of the serosal membrane in these conditions. 4. Increasing the osmolality of the serosal bathing solution produced similar effects, i.e. cell depolarization, fall in the short-circuit current and membrane conductances and reduction of the depolarizing effect of high-potassium and barium solutions. On the contrary, dilution of the serosal bath produced the opposite effects, consistent with an increase in the serosal permeability to potassium. 5. The effects of chloride- or sodium-free solutions were reversed by the dilution of the serosal bath. Cells repolarized when exposed to low-osmolality solutions after being in the absence of serosal chloride or sodium. The repolarization ran in parallel with the restoration of the short-circuit current and the potassium selectivity of the serosal membrane. 6. The results show that the effects produced by the removal of sodium or chloride ions from the serosal bathing solution are most probably mediated by a reduction in cell volume. Cell volume changes would lead to changes in the serosal membrane selectivity to potassium and thus to changes in cell membrane potential and sodium transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular chloride activity and membrane potential in stripped frog skin (Rana temporaria).

The regulation of cell chloride activity in frog skin was investigated using double barrelled Cl--microelectrodes to measure cell membrane potentials and chloride activity in the isolated frog epidermis. Experiments were done under short-circuit conditions, impaling cells from the serosal side. The basic electrophysiological parameters of the isolated skin were similar to those reported in the literature for whole preparations. Intracellular chloride activity was on average 21.9 mM and membrane potential was about 57 mV, implying that chloride was distributed away from its electrochemical equilibrium (i.e., concentrated inside the cells). Chloride activity decreased after removal of either Cl- or Na+ from the serosal bathing solution, with no change in membrane potential. The chloride permeability of the serosal membrane was calculated to be 2.6 X 10(-6) cm X s-1 which represents about 1/4 of the total conductance of the serosal membrane. We suggest that an electrically silent sodium-dependent uphill transport of chloride is present at the basolateral membrane of the frog skin, which accounts for the non-passive distribution of chloride.

Epithelial transport parameters: an analysis of experimental strategies.

A set of experiments was simulated on a computer version of the Koefoed-Johnsen & Ussing model for high-resistance epithelia. The results obtained were analysed according to procedures commonly applied to the analyses of experimental data and interpreted in terms of the model parameters. Although the computer model encodes a stoichiometry of 3:2 for Na-K exchange through the Na pump, the simulation of published experimental procedures yields different figures in almost every case. We show that ENa as originally defined by Ussing & Zerahn (Acta physiol. scand. 23, 110-127 (1951)) and as obtained from flux-ratio experiments has different values under different experimental conditions with unchanged system parameters and that it is distinct from ENa measured by other methods. We also show that unless the pump is saturated with internal Na an increase in the rate of pumping cannot cause a substantial increase in the rate of transepithelial Na transport.

The effect of low external pH on properties of the paracellular pathway and junctional structure in isolated frog skin.

1. Undirectional and net fluxes of Na, Cl and mannitol were measured across the isolated short-circuited frog skin when the mucosal surface was bathed with Ringer solution at normal (7.4) or low (2.5) pH. When this solution was changed from normal to low pH, there was a marked increase in both influx and backflux of Cl and mannitol. Na backflux increased markedly but Na influx did not, resulting in a substantial decrease in net flux. 2. In open-circuit conditions at low pH both undirectional fluxes of Na increased and the potential across the skin dropped by a third. 3. The total conductance, Gt and the short-circuit current, Isc increased when the mucosal solution was changed from normal to low pH. The structural integrity of the 'tight junctions' in the epithelium was disrupted by the low pH treatment and at least 50% of the junctions examined showed a separation of the two, previously apposed, membranes. 4. It has been shown previously that when a low pH solution bathes the mucosal surface the total and shunt conductance increase; the present results demonstrate that under these conditions the short-circuit current no longer provides a good estimate of the net Na flux. We present calculations to show that protons can be the carriers for the extra charge transfer. 5. Using our values for net Na flux in open circuit and published values for the solute-linked volume flow, Jv, it could be shown that the osmolarity of the absorbate decreased at low pH.

The regulation of volume and ion composition in frog skin.

1. Chemical determinations of Cl- in frog skin indicate that the intracellular concentration of this ion is above that corresponding to an equilibrium distribution. 2. Published data on Na+ efflux from the cell compartments into the two external bathing solutions, and on unidirectional fluxes across the whole preparation suggest that there are large unidirectional fluxes across the basolateral membranes. 3. Cl- uptake from the mucosal barrier is very small, and the removal of Cl- from the mucosal bathing solution does not affect the intracellular Cl- concentration. 4. Removal of Cl- from the serosal bathing solution produces a drastic decrease in cell Cl- together with a loss of water. 5. This is accompanied by a less marked effect on Na+ and K+ content of the cells. 6. The removal of Na+ from the serosal bathing solution produces also a decrease in Cl-, Na+ and K+ content of the cells with a marked loss of water. 7. It is suggested that the basolateral membrane of the frog skin is the site of mechanisms able to regulate volume and ion composition of the epithelial cells and that part of these mechanisms consists of a coupling between the movements of Na+ and Cl-.

The relationship of K+ efflux at the inner surface of the isolated frog skin epithelium to the short circuit current.

Isolated frog skins (without chorion) were incubated with 42K+ Ringers' solution, bathing the internal surface for 2 h. All the K+ contained in the frog skin was equilibrated in specific activity with external 42K+. The kinetics of the washout of 42K+ from the internal surface of the skin exhibits one fast and one slow exponential component. Amiloride reduces the release of 42K+ corresponding to both components without affecting the K+ content of the skin. Ouabain increases the loss of 42K+ of the slow component by 200%. Since the total K+ in the skin decreases to 25% of its original value both compartments are affected. The results suggest that two distinct functional compartments exist defined by two 42K+ release ratios and that because of the large K+ contents of these compartments both are intracellular. The relation with the transepithelial Na+ transport and the morphological identification of these compartments is discussed.

The mechanism of action of Cu2+ on the frog skin.

The mechanism of action of Cu2+ when applied to the external side of the frog skin preparation was investigated. Cu2+ acts most probably on the external barrier of this preparation, since it increases the transport pool of Na+ proportionally to the increase in the short circuit current (Isc). Cu2+ does not open new routes for the Na+ entry since the stimulated Isc is still completely abolished by amiloride. The Isc dependence of Na+ concentration in the external medium is modified by copper, since the Km value increases in addition to changes in V. It is suggested that copper acts at the external barrier Na channels in a way similar to that proposed by Zeiske and Lindemann ((1974) Biochim. Biophys. Acta 352, 323--326) for benzoylimidazole-2 guanidine and benzoylthiazole-2 guanidine and by Dick and Lindemann ((1975) Pflügers Arch. ges. Physiol. 355, R72) for para-chloromercuribenzenosulfonate and para-chloromercuribenzoate.

The use of 60Co-EDTA as an extracellular marker in frog skin.

60Co-EDTA was tested as an extracellular marker in isolated epithelia of frog skin. It was found to be non-toxic for frog skin and 0.1 mM EDTA proved to be enough to saturate any adsorption sites. Comparing with [14C]inulin, 60Co-EDTA marks a slightly greater extracellular space and this volume is constant between 15 min and 2 h. Furthermore it is reproducible and its use simplifies the methodology of the determination of water and electrolyte contents in the cells.

Chloride dependence of active sodium transport in frog skin: the role of intercellular spaces.

1. In agreement with previous observations the replacement of Cl by a nonpenetrating anion in the solution bathing either the outside or both sides of the frog skin causes a fall in the short-circuit current. 2. When Cl is replaced by a non-penetrating anion in the solution bathing the outside of the frog skin the Isc is still a correct measure of the net Na transport. 3. Under the same conditions both active and shunt paths seem to be affected since there is a decrease in Isc, Na influx, amiloride-dependent conductance, and initial Na uptake across the external barrier, together with a decrease in Cl-backfluxes and amiloride-independent conductance. There is also a decrease in water permeability and a reduction in size of the intercellular spaces. 4. The removal of Cl does not appear to affect the entry step of Na but may have an effect on the shunt path. This in turn may change the active Na transport.

The effect of copper on frog skin: the role of sulphydryl groups.

1. Cu2+ at a concentration of 10-4 M, when applied to the external side of the frog skin produces an increase in the short-circuit current (Isc). 2. This effect was studied in skins of Rana temporaria adapted to cold,(5 degrees C) and room temperature (20 degrees C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu2+ than skins with high Isc. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high Isc give good responses to Cu2+. 5. 5,5' -dithiobis (2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu2+, and dithiothreitol an SH-reducing agent, reverses the effect of this ion. 6. Cu2+ also induces an increase in the unidirectional K+ fluxes and unmasks a net outward potassium flux. 7. The outward K+ flux induced by Cu2+ is sensitive to ouabain. 8. It is concluded that Cu2+ increases the permeability of the external barrier of the frog skin to Na+ and K+, probably by reacting with SH groups.