Ultrastructure ofCatharanthus roseus cells culturedin vitro and exposed to conditions for alkaloid accumulation

Summary Periwinkle (Catharanthus roseus) cells cultured in 1-B 5 medium display the ultrastructure of parenchyma cells. The parenchyma character remained unchanged when cells were exposed to any one of three different conditions effecting alkaloid accumulation. Transfer of cells to alkaloid production medium for 2 weeks (condition 1) accorded two special features,i.e., unusually big lipid droplets in the cytoplasm and, upon fixation, one or several electron-dense droplets of spongy precipitate in vacuoles. Among hormone-autotrophic cultures (condition 2) some cells showed a fine electron-dense vacuolar precipitate. Addition ofPhythium homogenate (fungal elicitor) to cells cultured in 1-B 5-medium for 10 days (condition 3), cells showed a frequent appearance of singular big lipid droplets in the cytoplasm, whereas vacuoles remained devoid of precipitate. The appearance of big lipid droplets and of vacuolar precipitate is interpreted as progressing cytodifferentiation, but is coincidental with alkaloid accumulation.NRCC no. 24524.     

Cladocera in space and time: Analysis of lake sediments

Shells of Bosminidae and Chydoridae are quantitatively preserved in lake sediments. The chronological deposition of these remains provides the means for longterm observation of these Cladocera, both in terms of species and communities. Chydorid analysis, as based on subfossil assemblages, is an analysis of community and provides direct observation of community dynamics over extended periods of time. It has proved to be a valuable method to obtain information on the influence of environmental factors and time on community characteristics. Morphological variation inBosmina (Eubosmina) has been followed for some thousand years. This is of special interest for the evaluation of taxonomic rank (species, forms) if closely related taxa have co-existed. Bosmina successions, as well as shifts in the chydorid fauna, are related to environmental change. Thus, cladoceran analysis of lake sediments provides information on the developmental history of lakes and allows observation of the effects of longterm environmental changes, such as climatic changes and eutrophication.     

Light-regulated methylation of chloroplast proteins

Protein carboxyl methyltransferases, which catalyze transfer of methyl groups from S-adenosyl-L-methionine to the free carboxyl groups of acidic amino acids in proteins, can be divided into two classes based on several characteristics, such as the stoichiometry of substrate protein methylation, base stability of the incorporated methyl group, specificity for substrate, and participation in a regulatory system with which methylesterases are associated. The presence of such an enzyme in a photosynthetic system was demonstrated in the present work. The extent of methylation of chloroplast proteins was stimulated 30% by light and then decreased by the same amount in the presence of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1', 1'-dimethylurea or uncouplers of phosphorylation, indicating a dependence of the methyltransferase activity on photosynthetic electron transport and the trans-membrane delta pH. The light-independent, as well as the light-dependent, activity is probably of chloroplast origin since the extent of light stimulation in the purified thylakoid membranes and the stromal fraction was similar, and at low concentrations of S-adenosyl-L-methionine the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was found to be the predominant substrate. The labeling pattern of chloroplast proteins and labeling of an exogenous nonchloroplast protein indicated that the methyltransferase activity was not substrate-specific, although at low concentrations of the methyl donor, the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was labeled almost exclusively. Based on the low stoichiometry (less than 100 pmol/mg protein) of the methylation, its base lability, irreversibility, and the lack of substrate specificity except at very low concentrations of methyl donor, it was inferred that the chloroplast methyltransferase is best classified as a class II system that may function as part of a repair mechanism to replace racemized amino acids.     

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.     

A new infrageneric classification and phylogenetic trends in the genusRhododendron (Ericaceae)

Recent studies have improved the infrageneric classification ofRhododendron, including my own investigations on flavonoids and anthocyanins as chemosystematic markers. From a synoptical comparison of morphological, anatomical and phytochemical characters a new system for the genus is proposed. Phylogenetic character progressions and relationships among subgenera, sections and subsections are discussed and illustrated. Key positions for subg.Candidastrum between chori subgenerumRhododendron andNomazalea, and for subg.Choniastrum between chori subgenerumHymenanthes andNomazalea are suggested.     

Importance of Hydrogen Sulfide, Thiosulfate, and Methylmercaptan for Growth of Thiobacilli during Simulation of Concrete Corrosion

Biogenic sulfuric acid corrosion of concrete surfaces caused by thiobacilli was reproduced in simulation experiments. At 9 months after inoculation with thiobacilli, concrete blocks were severely corroded. The sulfur compounds hydrogen sulfide, thiosulfate, and methylmercaptan were tested for their corrosive action. With hydrogen sulfide, severe corrosion was noted. The flora was dominated by Thiobacillus thiooxidans. Thiosulfate led to medium corrosion and a dominance of Thiobacillus neapolitanus and Thiobacillus intermedius. Methylmercaptan resulted in negligible corrosion. A flora of heterotrophs and fungi grew on the blocks. This result implies that methylmercaptan cannot be degraded by thiobacilli.     

High-Efficiency Conversion of Pyruvate to Acetoin by Lactobacillus plantarum during pH-Controlled and Fed-Batch Fermentations

The influence of pH on the type and concentration of metabolites produced from pyruvate by Lactobacillus plantarum ATCC 8014 was examined in pH-controlled fermentors at pH values of 4.5 to 6.5. Specific growth rates, cell dry weights, and diacetyl concentrations were highest at pH 5.5, with values of 0.78 h⁻¹, 190 mg/liter, and 1.2 mM, respectively. While the conversion efficiency (millimoles of acetoin formed per millimoles of pyruvate utilized) was highest (94.6%) at pH 4.5, acetoin levels were similar (20 mM) between pH 4.5 and 5.5. Feeding stationary-phase cells exogenous pyruvate increased acetoin levels to 78 mM.     

Transposon Mutants of Bradyrhizobium japonicum Altered in Attachment to Host Roots

Transposon mutants of Bradyrhizobium japonicum 110 ARS were produced and screened for changes in attachment ability. Mutant CFK4 produced twice as many piliated cells, attached in 2.5-fold-higher numbers to soybean root segments, and colonized roots in about 2-fold-higher numbers than did the parental strain, 110 ARS. Mutants CFK35 and CFK38 were reduced in their attachment about 2-fold and 3.5-fold, respectively. This corresponded to reductions in piliated cells in their populations, reduced reaction with anti-pilus antiserum, and reduced hydrophobic attachment. Mutants CFK4 and CFK38 nodulated soybeans at about the same level as the parent strain, but CFK35 induced only pseudonodules. Two-dimensional gel analyses of the proteins from the mutants showed relatively few changes in proteins.     

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.