Changes in cytoplasmic and lysosomal enzyme activities in cultured rat heart cells: The relationship to cell differentiation and cell population in culture

Summary Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or by the replating technique. [³H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function of the culture’s age, of seeding density, and replating. It was shown that (a) replating maintains predominance of myocyte population for at least 2 wk in culture; (b) heavy seeding density allows homogeneous myocyte population for the 1st wk in culture; and (c) the enzyme profile of the culture may serve as an indicator for the type of cell population in culture and its state of differentiation. This study was done as partial fulfilment of the M.Sc. thesis in Biochemistry (SY). Supported by grants from The Chief Scientist, Ministry of Health, State of Israel; The Ministry of Education and Sciences, State of Niedersachssen (FRG); and The Foundation for Heart Research from Mr. and Mrs. D. Vidal-Madjar, Paris, France.     

Einige neue oder bemerkenswerte Flechten mit gyalectoiden Apothecien von Nord-Indien und Nepal

This paper presents the results of taxonomic investigation of 8 species of lichens with gyalectoid apothecia collected in North-India and Nepal. Four species are recognized as new:Dimerella isidiata (sp. nova),D. nepalensis (sp. nova),Gyalideopsis lithophila (sp. nova) andRamonia nepalensis (sp. nova). Four species are reported for the first time from Nepal:Coenogonium moniliforme, Gyalectidium caucasicum, Gyalidea lecideopsis andG. scutellaris.     

Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.     

Effects of electron donors on Ca2+-dependent K+ transport in one-step inside-out vesicles from the human erythrocyte membrane

The interactions between reducing agents and Ca2+ in the activation of Ca2+-dependent K+ transport have been studied in one-step inside-out vesicles. The artificial electron donor system ascorbate + phenazine methosulphate increases the apparent sensitivity to Ca2+ by about 5-times over control values (half activation constant, about 5 X 10(-8) M) while oxidized cytochrome c decreases the sensitivity to about 1/3 of the controls. Using redox buffers at a fixed pCa it is shown that the shift from the low to the high-affinity state can be accounted by the reduction of a membrane component accepting two electrons and with an apparent standard redox potential (pH 7.5) of 47 mV. The electrons can be transferred directly from reduced PMS or to oxidized cytochrome c, but not from ascorbate, NADH or reduced glutathione.     

The use of lectins for determination of absolute configurations of small amounts of sugars eluted from chromatograms

A simple procedure for the determination of the absolute configuration (i.e., assignment to the D- or L-enantiomeric series) of glucose, mannose, galactose, fucose, arabinose, and rhamnose is described, based on inhibition by these sugars of 125I-labeled lectin binding to the glycoconjugates immobilized on the wells of plastic microculture plates. The method works well with 10 to 100-micrograms amounts of the sugars isolated after paper chromatography of the glycoprotein or polysaccharide hydrolysates.     

Histidine residues in elongation factor EF-tu from Escherichia coli protected by aminoacyl-tRNA against photo-oxidation

Complexes of Escherichia coli elongation factor EF-Tu with GTP or GTP and aminoacyl-tRNA were photo-oxidized by irradiation with visible light in the presence of rose bengal dye. EF-Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high-performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl-tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC). Aminoacyl-tRNA, bound to EF-Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo-oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF-Tu X GTP X aminoacyl-tRNA complex with those from the irradiated EF-Tu X GTP complex revealed that histidine-containing peptides T12 and T15 as well as methionine-containing peptide T14 were in the ternary complex markedly protected against the photo-oxidative degradation. This finding suggests that their histidines, i.e. His-66 and His-118 respectively and at least one of the methionines (Met-91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF-Tu for aminoacyl-tRNA and could be involved in the interaction between aminoacyl-tRNA and the factor.     

The use of a cyanine dye in measuring membrane potential in yeast

An attempt was made to use 3,3'-dipropylthiacarbocyanine as a membrane potential probe in yeast by following both its fluorescence changes and its uptake by the cells under different conditions. It was found that the uptake of the dye into the cytoplasmic compartment was translated into an increased fluorescence, and the uptake by the mitochondria produced a quenching of the fluorescence. The experiments to measure uptake showed that a large amount of the dye was taken up by the cells under "deenergized" conditions. The uptake of the cyanine, however, was significantly reduced by the omission of the substrate, by deenergization of the mitochondria, or by the addition of K+, but not by Na+. This cyanine seems to be a good, qualitative indicator of the potential of the plasma membrane and of the mitochondria of the cells, with a faster response than those probes used before in yeast.     

Effect of theophylline and theobromine on cell respiration and calcium transport in isolated rat liver mitochondria

The influence of theophylline and theobromine on cellular respiration and on membrane transport of calcium has been studied in isolated rat liver mitochondria, using oxygen and Ca2+ selective electrodes. A linear decrease in respiratory coefficients, in the total amount and rate of "extra" oxygen consumption induced by ADP is observed with drug concentration. Theobromine does not show any appreciable effect on these respiratory parameters, but this result is similar to that observed with theophylline for the same concentration range. Calcium uptake coupled to respiration is inhibited by both drugs depending on their concentrations. Theobromine is more effective than theophylline. Calcium saturation of the mitochondria takes place in all cases after 36 +/- 2 s but only a 20% of the maximum calcium uptake observed in the absence of the drugs is determined in the presence of 15 mM theophylline or only 1.8 mM theobromine. Comparative studies show direct correlation between the pharmacological activities as stimulants of caffeine, theophylline and theobromine and their behaviour as inhibitors of calcium uptake coupled to respiration by mitochondria.