Ultrastructural changes of sarcolemma and mitochondria in the isolated rabbit heart during ischemia and reperfusion

Isolated rabbit hearts were perfused by the Langendorff technique, made ischemic and subsequently reperfused. It was found that ischemia results in: (i) aggregation of the intramembranous particles in the sarcolemma and (ii) extrusion of pure lipidic multilamellar structures (liposomes) from swollen mitochondria. Subsequent reperfusion resulted in further aggregation of the sarcolemmal intramembranous particles and disruption of the sarcolemma, which was attended by the formation of liposome-like structures. Intramembrane particle aggregation is explained in terms of lateral phase separation of the membrane lipids and a reduction of repulsive forces between the membrane proteins, both induced by a decrease in pH and an increase in Ca2+ concentration intracellularly. The formation and extrusion of the multilamellar structures are discussed in terms of destabilization of the bilayer which results in a structural blebbing-off of pure lipid.     

Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

Characterization of a transferrin-diphtheria toxin conjugate

We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis.     

Abnormal interaction of the human apolipoprotein A-I variant [Lys107----0] with high density lipoproteins

Several isoforms of apoprotein A-I [apoA-I], the major apoprotein of high density lipoproteins [HDL], have been described. We compared the in vivo and in vitro properties of normal human apoA-I with those of apoA-I [Lys107----0]. Fluorescence and circular dichroic spectra showed that deletion of Lys107 decreases apoprotein self-association. In vivo metabolic studies in the rat indicated that the interaction of apoA-I [Lys107----0] with HDL was lower than normal. We conclude that deletion of Lys107 results in a reorganization of the apoprotein structure that decreases its potential to form hydrophobic associations.     

Purification and various properties of asialoglycoprotein receptors from the mouse liver

The receptor for asialoglycoproteins was isolated from murine liver and was purified by means of biospecific chromatography on sepharose-Asialo-orosomucoid. The obtained receptor with an absorption maximum at 277 nm binds to the nonreducing terminal galactosyl residues of glycoproteins similar to the receptors from liver of other mammalians. The interaction between this receptor and desialylated glycoproteins requires the presence of calcium. The dependence of specific binding on the concentration of [125I]acialo-orosomucoid used as a ligand gives a saturating curve. The dissociation constant for the receptor-ligand complex is 0.4 X 10(-9) M. Similar to asialo-orosomucoid, the receptor binds the p-aminophenyl-beta-D-galactopyranoside derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase synthesized by us earlier. Possible use of the asialoglycoprotein receptor as a highly specific carrier transporting the modified acid alpha-glucosidase to hepatocyte lysosomes is discussed.     

The transition point for protein synthesis in late S-G2 may be delayed by genome bromosubstitution without affecting the time of entry into mitosis

Bromosubstitution for most of the S period in synchronous populations of Allium cepa L. meristematic cells resulted in a delay in the late S-G2 transition point where protein synthesis is needed for later mitotic entrance to occur. This retardation in the position of the transition point was not accompanied by the expected delay in the entrance into mitosis, suggesting that such protein synthesis is a requisite, but not a timer for prophase triggering.     

Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm. typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium , as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.     

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.