Search

ATP depletion of erythrocytes is associated with discocyte-echinocyte transformation and accumulation of calcium. This study was undertaken to evaluate whether this shape transformation results from interaction of Ca2+, Mg2+, and ATP with structural proteins of the membrane. ATP, Mg2+, or Ca2+ were incorporated into ghosts during hypotonic hemolysis followed by restoration of isotonicity with NaCl and incubation. Reconstituted ghosts were examined by electron microscope after osmium tetroxide fixation followed by platinum and carbon shadowing and in-phase contrast microscope. Four ghost configurations were observed: (1) flat, discoidal electron-penetrable ghosts were produced when ghosts were prepared without any additives, or with ATP alone, Mg2+ alone, or EDTA; (2) biconcave and cup-shaped less electron-penetrable ghosts predominated when equimolar concentrations of Mg2+ and ATP (0.5-2 mM) were introduced into ghosts during hemolysis; (3) spherical, spiculated, or markedly deformed electron-dense ghosts of smaller volume were the prevalent shape forms when Ca2+ alone was introduced during hemolysis; (4) small, smooth, electron-dense spheroids were present when both Ca2+ and Mg ATP (2 mM) were added during hemolysis. The above shapes were independent of ouabain, monovalent cations (NaCl, KCl, or choline chloride), transmembrane osmotic gradient, and ghost volume. The shape effects of Ca2+ and Mg ATP were prevented by preincubation of red cells with SH-blocking agent N-ethylmaleimide (4 mM) and by repeated ghost washing, which was associated with removal of water-soluble fibrous proteins from membranes. It is concluded that the interaction of Ca2+, Mg2+, and ATP with membrane structural proteins exerts a significant role in the control of shape of human erythrocytes.

This paper describes a method for the extraction, separation and determination of amino acids, both free and protein-bound, which are present in tobacco. The introduction of the lyophilization process and the ''cold finger'' technique made it possible to obtain a good purification of extracts which, unlike the previous methods, have been preserved for a long time. The combined use of column chromatography and mass spectrometry enabled all amino acids present to be detected. The analyses carried out on different tobacco varieties showed certain quantitative and qualitative differences in the composition of amino acids and only small quantitative variations in the composition of protein-bound amino acids

ATP depletion of erythrocytes is associated with discocyte-echinocyte transformation and accumulation of calcium. This study was undertaken to evaluate whether this shape transformation results from interaction of Ca2+, Mg2+, and ATP with structural proteins of the membrane. ATP, Mg2+, or Ca2+were incorporated into ghosts during hypotonic hemolysis followed by restoration of isotonicity with NaCl and incubation. Reconstituted ghosts were examined by electron microscope after osmium tetroxide fixation followed by platinum and carbon shadowing and in-phase contrast microscope. Four ghost configurations were observed: (1) flat, discoidal electron-penetrable ghosts were produced when ghosts were prepared without any additives, or with ATP alone, Mg2+alone, or EDTA; (2) biconcave and cup-shaped less electron-penetrable ghosts predominated when equimolar concentrations of Mg2+and ATP (0.5-2 mM) were introduced into ghosts during hemolysis; (3) spherical, spiculated, or markedly deformed electron-dense ghosts of smaller volume were the prevalent shape forms when Ca2+alone was introduced during hemolysis; (4) small, smooth, electron-dense spheroids were present when both Ca2+and Mg ATP (2 mM) were added during hemolysis. The above shapes were independent of ouabain, monovalent cations (NaCl, KCl, or choline chloride), transmembrane osmotic gradient, and ghost volume. The shape effects of Ca2+and Mg ATP were prevented by preincubation of red cells with SH-blocking agent N-ethylmaleimide (4 mM) and by repeated ghost washing, which was associated with removal of water-soluble fibrous proteins from membranes. It is concluded that the interaction of Ca2+, Mg2+, and ATP with membrane structural proteins exerts a significant role in the control of shape of human erythrocytes.

Heterogeneity within ghosts prepared from stored erythrocytes was studied by multichannel particle analysis of size distributions of reconstituted ghosts. On restoration of isotonicity of hemolysates from fresh erythrocytes, ghosts were smaller than the corresponding red cells. After subsequent incubation for 15 min, they swelled to a final stable volume. The hemolysis of stored erythrocytes and restoration of isotonicity yielded ghosts similar to those from fresh cells, but, after subsequent incubation, a second population of small ghosts appeared. This population resulted from markedly diminished ghost swelling, that is attributed to decreased sodium permeability and increased membrane rigidity, causing resistance to changes in volume. Erythrocyte ATP depletion during storage of ACD blood was associated with an increase in the percentage of small ghosts. Similar ghosts were produced from fresh cells when CaCl2 (1 x 10-6M) was introduced into ghosts during hemolysis. The erythrocyte calcium content increased during prolonged storage from 11.3 ± 6.3 µg atoms/liter of cells (stored for 1-2 wk) to 22.0 ± 7.0 µg atoms/liter of cells, after 7-9 wk of storage. The introduction of nucleotides (2 x 10-3M) into ghosts during hemolysis of stored cells prevented the appearance of small ghosts. The inhibitory effect was greater than predicted from stability constants of calcium nucleotide complexes, indicating the specific interaction of nucleotides with the membrane. The percentage of small ghosts from red cells stored for various times agreed with the percentage of nonviable cells derived from the literature. It is concluded that the decrease in ATP and the accumulation of calcium in stored erythrocytes induce conformational changes of membrane fibrous (contractile) proteins that results in decreases in membrane elasticity and permeability, which is in turn reflected by formation of small ghosts.

Conductivity, activity coefficient, and ultraviolet absorption data have shown that an interaction occurs between deoxycholate helical complexes (DCA) and sodium benzylpenicillin (P). The mechanism of this interaction is still unknown, but a merely electrostatic interpretation can be ruled out. Experimental data seem to be consistent with the stereospecific nature of this interaction. The presence of DCA helical complexes increases benzylpenicillin resistance to the hydrolytic activity of penicillinase. No interaction and therefore no decrease in the hydrolytic activity of penicillinase has been found with different derivatives, i.e., dimethoxyphenyl-and α-aminobenzylpenicillin. At the same time dehydrocholic acid sodium salt (NaDHCA), which in no case forms helical complexes, shows no effect upon P, whereas sodium salt of cholic acid (NaCA) still exerts a protective action upon P but to a lesser degree.

A qualitative and quantitative procedure has been developed for the determination of humectants in manufactured tobacco by gas-chromatographic method with a flame ionization detector. It consists of extraction with methanol, concentration of the extract and treatment with Tri-Sil reagents. The operating gas-chromatographic conditions are set forth. Samples of tobacco containing glycerine, propylene glycol, diethylene glycol, triethylene glycol, 1-3 butylene glycol and sorbitol have been analysed with recoveries, for the first five ones, in the range of 95-104 %. In order to verify that each chromatographic peak corresponded to the relative glycol, with no interference by other silylated compounds, the mass spectra were obtained through the combination of gas-chromatography with mass spectrometry. The results achieved confirm, as far as tobacco is concerned, that the procedure is accurate and precise. The same method for the determination of humectants was extended to cigarette smoke. Even though this involves morecomplicated problems, as compared to tobacco, because of the presence of silylated compounds, it was found that, for certain glycols, the gas-chromatography of the trimethyl derivatives can be also used as a method of analysis. The mass spectra of some polyhydric alcohols are shown

Heterogeneity within ghosts prepared from stored erythrocytes was studied by multichannel particle analysis of size distributions of reconstituted ghosts. On restoration of isotonicity of hemolysates from fresh erythrocytes, ghosts were smaller than the corresponding red cells. After subsequent incubation for 15 min, they swelled to a final stable volume. The hemolysis of stored erythrocytes and restoration of isotonicity yielded ghosts similar to those from fresh cells, but, after subsequent incubation, a second population of small ghosts appeared. This population resulted from markedly diminished ghost swelling, that is attributed to decreased sodium permeability and increased membrane rigidity, causing resistance to changes in volume. Erythrocyte ATP depletion during storage of ACD blood was associated with an increase in the percentage of small ghosts. Similar ghosts were produced from fresh cells when CaCl2(1 × 10−6M) was introduced into ghosts during hemolysis. The erythrocyte calcium content increased during prolonged storage from 11.3 ± 6.3 μg atoms/liter of cells (stored for 1-2 wk) to 22.0 ± 7.0 μg atoms/liter of cells, after 7-9 wk of storage. The introduction of nucleotides (2 × 10−3M) into ghosts during hemolysis of stored cells prevented the appearance of small ghosts. The inhibitory effect was greater than predicted from stability constants of calcium nucleotide complexes, indicating the specific interaction of nucleotides with the membrane. The percentage of small ghosts from red cells stored for various times agreed with the percentage of non- viable cells derived from the literature. It is concluded that the decrease in ATP and the accumulation of calcium in stored erythrocytes induce conformational changes of membrane fibrous (contractile) proteins that results in decreases in membrane elasticity and permeability, which is in turn reflected by formation of small ghosts.

Acetaldehyde was found to be a product of the catabolism of deoxyinosine and deoxyribose-5-P in human erythrocyte ghosts. The simultaneous production of tri-osephosphates indicates the presence in erythrocytes of deoxyribosephosphate aldolase.

The stability of the polymer-like structure displayed by deoxycholic acid gels under certain conditions has been investigated. The influence of cation dimensions, hydration, and hydrogen bonds is shown. A peculiar behavior of deoxycholic acid quaternary ammonium salts is discussed. Some biological implications are mentioned briefly.