Cellular morphogenesis and the formation of marginal bands in amphibian splenic erythroblasts

The spleen of Ambystoma mexicanum (axolotl) larvae develops as a closed sac containing differentiating nucleated erythrocytes, and is typically isolated from the general circulation for about 10 days post-hatching. Beginning 3-4 days posthatching, it can be removed intact for examination of the morphology and cytoskeletal structure of the erythropoietic cells. In the smallest (earliest) spleens, spheroidal cells predominate, while older ones contain a preponderance of cells exhibiting the flattened elliptical morphology typical of all non-mammalian vertebrate erythrocytes. Most striking in the splenic erythroid population are cells with singly or doubly pointed morphology. Though common in the developing spleen and circulation of young larvae, pointed cells are less frequently encountered in the circulation of older larvae, indicating that they are intermediate stages in the differentiation of spheroids to flattened ellipsoids. This is supported by structural observations on cytoskeletons prepared from the splenic cells. Incomplete singly and doubly pointed marginal bands of microtubules are observed, many of which contain a pair of centrioles within or close to a pointed end, suggestive of organizing center function. The observations are consistent with a sequence of changes in cell morphology from spherical to doubly pointed to singly pointed to flattened ellipse, causally linked to stages of marginal band biogenesis.     

Hemolysis of human red blood cells induced by the combination of diethyldithiocarbamate (DDC) and divalent metals: modulation by anaerobiosis, certain antioxidants and oxidants.

The objective of the present communication is to describe the role played by combinations between diethydithiocarbamate (DDC) and divalent metals in hemolysis of human RBC. RBC which had been treated with DDC (10-50 microM) were moderately hemolyzed (about 50%) upon the addition of subtoxic amounts of Cu2+ (50 microM). However, a much stronger and a faster hemolysis occurred either if mixtures of RBC-DDC were immediately treated either by Co2+ (50 microM) or by a premixture of Cu2+ and Co2+ (Cu:Co) (50 microM). While Fe2+ and Ni2+, at 50 microM, initiated 30-50% hemolysis when combined with DDC (50 microM), on a molar basis, Cd2+ was at least 50 fold more efficient than any of the other metals in the initiation of hemolysis by DDC. On the other hand, neither Mn2+ nor Zn2+, had any hemolysis-initiating effects. Co2+ was the only metal which totally blocked hemolysis if added to DDC prior to the addition of the other metals. Hemolysis by mixtures of DDC + (Cu:Co) was strongly inhibited by anaerobiosis (flushing with nitrogen gas), by the reducing agents glutathione, N-acetyl cysteine, mercaptosuccinate, ascorbate, TEMPO, and alpha-tocopherol, by the PLA2 inhibitorbromophenacylbromide (BrPACBr), by tetracycline as well as by phosphatidyl choline, cholesterol and by trypan blue. However, TEMPO, BrPACBr and PC were the only agents which inhibited hemolysis induced by DDC: Cd2+ complexes. On the other hand, none of the classical scavengers of reactive oxygen species (ROS) employed e.g dimethylthiourea, catalase, histidine, mannitol, sodium benzoate, nor the metal chelators desferal and phenanthroline, had any appreciable inhibitory effects on hemolysis induced by DDC + (Cu:Co). DDC oxidized by H2O2 lost its capacity to act in concert either with Cu2+ or with Cd2+ to hemolyze RBC. While either heating RBC to temperatures greater than 37 degrees C or exposure of the cells to glucose-oxidase-generated peroxide diminished their susceptibility to hemolysis, exposure to the peroxyl radical from AAPH, enhanced hemolysis by DDC + (Cu:Co). The cyclovoltammetry patterns of DDC were drastically changed either by Cu2+, Co2+ or by Cd2+ suggesting a strong interaction of the metals with DDC. Also, while the absorbance spectrum of DDC at 280 nm was decreased by 50% either by Co2+, Cd2+ or by H2O2, a 90% reduction in absorbance occurred if DDC + H2O2 mixtures were treated either by Cu2+ or by Co2+, but not by Cd2+. Taken together, it is suggested that DDC-metal chelates can induce hemolysis by affecting the stability and the integrity of the RBC membrane, and possibly also of the cytoskeleton and the role played by reducing agents as inhibitors might be related to their ability to deplete oxygen which is also supported by the inhibitory effects of anaeobiosis.     

Experimental modifications of the circadian rythm of Plasmodium vinckei petteri following cryopreservation; probable resistance of the merozo?te to thawing

Plasmodium vinckei petteri, in white mice, is a particularly useful strain for studies on the circadian rythm of Plasmodium. An experimental model was set up: it showed that the rythm of asexual schizogony in the blood varies with the time and the mode of inoculation (cryopreserved blood or syringe passage). When frozen blood is injected, the time of schizogony depends on the time of injection; on the contrary, when the passage is by syringe from mouse to mouse, the rythm of schizogony is the same in the donor and the receptor mouse, regardless of the time of injection. The only possible explanation is that all intracellular parasites are destroyed by the thawing of blood and the free merozo?te is the only resistant stage.     

On the binding of L-S- and L-R-diastereoisomers of the substrate analog L-methionine sulfoximine to glutamine synthetase from Escherichia coli

Active site ligand interactions with dodecameric glutamine synthetase from Escherichia coli were studied spectrally, using the resolved L-S- and L-R-diastereoisomers of the substrate analog L-methionine-SR-sulfoximine. direct measurements of the reversible binding of the S-isomer to unadenylylated manganese-enzyme show a stoichiometry of 1 eq/subunit and negative cooperativity with a Hill coefficient of 0.7. The affinity of this enzyme complex is greatest for the S-isomer alone ([S]0.5 = 35 microM), least with the R-isomer alone ([S]0.5 = 0.38 mM), and intermediate (but closer to that for the S-isomer) for an equimolar mixture of S- and R-isomers ([S]0.5 = 61 microM). The affinity for the S-isomer is enhanced greater than 35-fold by ADP and is decreased approximately 3-fold by adenylylation of the enzyme. Shrake, A., Whitley, E. J. Jr., and Ginsburg, A. ((1980) J. Biol. Chem. 255, 581-589) reported that UV spectral perturbations markedly differ for binding commercial L-methionine-SR-sulfoximine to unadenylylated and adenylylated manganese enzymes. However, essentially the same saturating protein difference spectrum is produced by binding the resolved S- and R-diastereoisomers, and equimolar mixture of S- and R-isomers, and the commercial S- and R-isomeric mixture to a particular enzyme complex. Since neither the subunit interactions that give rise to the observed negative cooperativity of binding nor the affinity differences in binding the S- and R-isomers are reflected in protein difference spectra, spectral perturbations derive from a conformational change that is solely a marked for the occupancy of the single subunit site by either isomer.     

Antibody 624H12, which detects lung cancer at early stages, recognizes a sugar sequence in the glycosphingolipid difucosylneolactonorhexaosylceramide (V3FucIII3FunLc6Cer)

Immunocytochemical staining of cells in sputum by rat monoclonal antibody 624H12 detects lung cancer 2 years prior to its detection by conventional diagnostic techniques. The antigen recognized by antibody 624H12 is a sugar sequence in the glycosphingolipid difucosylneolactonorhexaosylceramide (V3FucIII3FucnLc6Cer) whose structure is (formula see; text) Both fucosyl residues are required for high affinity binding by the antibody. The antigen was expressed in 35 of 45 specimens of cancer tissue from patients with early stage non small cell lung cancer. There was no correlation between antigen expression and patient survival.