The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Interspecific variation in arrangement and morphology of micrasters of Tethya species (Porifera,Demospongiae)

Summary A new distinctive feature between the two Mediterranean species of Tethya, T. aurantium and T. citrina has been found in the body arrangement of different types of micrasters. Contrary to the previous assumptions, T. aurantium has two clearly distinct categories of micrasters: the chiaster-tylaster in the cortex and the larger, slender oxyaster in the choanosome. T. citrina has only slightly differentiated micraster sets in the cortex and choanosome; in the latter the shape of micrasters is close to that of oxyasters. SEM analysis shows that differences in micraster shape depend on the cylindrical or conical form of rays and on the distribution, density and strength of the microspines along their axis. The relationship between the degree of micraster differentiation and the development of the cortex in the two species is discussed.     

Role of the reticuloendothelial system in the catabolism of low density lipoprotein in the rat

The role of the reticuloendothelial system (RES) in receptor-independent catabolism of human low density lipoprotein (h-LDL) was evaluated in the rat in vivo after blockade of its phagocytic activity with gadolinium chloride (GaCl3). After blockade of the RES with GaCl3, the recovery of [125I] h-LDL in the liver of 17 alpha-ethinyl oestradiol-treated rats (EE-rats), was decreased by 37 and 16%, 15 and 60 min after h-LDL injection, respectively. This decrease did not result in a decreased LDL degradation which represented 14 and 55% of the injected dose in the two groups of rats after 15 and 60 min respectively, both on GaCl3 and control rats. Contrasting with EE-rats, the catabolism of h-LDL in untreated rats is much slower and takes place essentially through a receptor-independent mechanism. Six hours after the injection of [125I] h-LDL, 64% of the dose was degraded. This proportion decreased to 45% after blockade of the RES phagocytic activity. This 30 percent difference represents the proportion of h-LDL catabolized by receptor-independent mechanisms present in the Küpffer and endothelial cells. We conclude from our study that in the normal rat, the parenchymal cells of the liver on the one hand and the Küpffer and endothelial cells on the other hand contribute 70 and 30% respectively to the receptor-independent catabolism of low density lipoproteins in vivo.     

Isolation of apical plasma membrane in rabbit gallbladder epithelium by Percoll density gradient centrifugation

The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.     

Differential expression within a family of novel wound-induced genes in potato

Summary Wounding in higher plants leads to an increased synthesis of specific messenger RNAs. A cDNA clone complementary to a wound-induced message from potato tubers was used to isolate a lambda clone from a genomic library of Salanum tuberosum var. Maris Piper. DNA sequence analysis has shown that this single genomic clone contains two novel wound-induced genes, called win1 and win2, organised in close tandem array. The coding sequences of these two genes are highly homologous and are interrupted by a single intron. However, the sequences of the introns and flanking regions have diverged widely. Win1 and win2 encode cysteine-rich proteins of 200 and 211 amino-acids, respectively, which show striking homologies to several chitin-binding proteins. Southern analysis of genomic DNA has shown that win1 and win2 are members of a small multi-gene family which is estimated to have a minimum of five members per haploid genome of Maris Piper and appears to be conserved within the Solanaceae. We have shown by Northern analysis and S1 mapping that the two genes exhibit differential organ-specific expression after the wounding of a potato plant.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Observations on the germinability in vitro of pollen from Camellia japonica L. (Theaceae)

Pollen grains from samples of Camellia japonica living in soil (A) and in greenhouse (B) were collected daily from just-opened anthers. The pollen was sown in various liquid media and incubated at 28 C. Higher germinative ability of A in comparison with B was observed and related to the relative humidity which is higher in soil than in greenhouse. This phenomenon recurs even if the composition of the culture medium is changed. However the composition of the medium greatly influences the percentage of germination of both A and B notwithstanding the environmental conditions. The pollen collected from anthers dehiscing in the first day of the anthesis has a high germinative ability which suddenly decreases in the ripe pollen of the following days.