The exosome of Trypanosoma brucei.

The yeast exosome is a complex of at least 10 essential 3'-5' riboexonucleases which is involved in 3'-processing of many RNA species. An exosome-like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli. The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution. We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex. Accordingly, the T.brucei exosome is smaller than that of yeast. Both free and complex-associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation. We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.     

Low responsiveness of six-rowed genotypes to androgenesis in barley does not have a pleiotropic basis.

A heterozygous mutant for the two- and six-rowed character was isolated in the barley cultivar Igri through application of sodium azide to isolated microspore cultures and posterior regeneration. Six-rowed and two-rowed homozygotic plants were subsequently identified in the self-pollinated M2 progenies of the original heterozygous M1. Detailed molecular markers confirmed the isogenic nature of this recovered mutant and the original cultivar Igri. A comparative study of the anther culture response of this six-rowed induced mutant vs. diploid 'Igri' was performed to assess whether the two- or six-rowed gene influences anther culture response in barley through a pleiotropic effect or via linkage disequilibrium. No significant differences for any of the recorded variables throughout the in vitro regeneration process were detected between the 'Igri' six-rowed mutant and any of their two-rowed isogenic lines. This suggests that row-type association with anther culture response in barley cultivars is due to the effect of a tight linkage with other genes directly responsible for androgenic response.     

Complexation of copper(II) by a peptide hybrid of bleomycin and amsacrine. Circular dichroism and electron spin resonance studies

A model incorporating the metal chelating moiety of bleomycin and an anilinoacridine ring able to intercalate in DNA has been synthesized. The copper(II) complex of that molecule has been studied using circular dichroism and electron spin resonance by comparison with bleomycin. The introduction of the anilinoacridine ring involves a modification in the geometry of the complex. A distortion of the square-pyramidal form (type II complex) gives rise to a type I complex in which the metallic atom is drawn out of the plane of the four square-planar ligands and displaced slightly towards the fifth ligand.     

Enzyme distribution and transport activities in electrophoretically isolated fractions of the muscle sarcotubular system

A simple electrophoretic method is introduced allowing to isolate five fractions of skeletal muscle ST-system vesicles. In a previous study differences in lipid content, 3H-ouabain binding and in presence of triads in individual fractions (Lehotsky et. al. 1986) were analysed. In the present study biochemical characterization was extended, and (in accordance with previous results) major differences were observed to exist between fraction 1 and fractions 3 and 4. SDS-PAGE showed that fractions 3 and 4 were enriched in a protein with m.w. 100 kD, these fractions showing the highest specific activities of (Mg2+ + Ca2+)-ATPase and oxalate-supported Ca2+-uptake; activities of Mg2+-ATPase and surface membrane marker enzymes were the lowest in these fractions. On the other hand, in fraction 1 the highest activities of Mg2+-ATPase and marker enzymes of the surface membrane were observed together with a decreased content of the 100 kD protein and activities of Ca2+ transport. It could be concluded that the method is suitable to differentiate between relatively pure SR (fractions 3 and 4) and fractions rich in sarcolemma or T-tubules components (fractions 1 and 5).     

Serum catalase enzyme activity in liver diseases

Serum catalase activity was moderately increased in fatty liver, acute alcoholic hepatitis and in the decompensated form of cardiac circulatory failure. It showed significant increase in acute yellow atrophy and in toxic hepatitis while no changes were detected in liver cirrhosis and viral hepatitis. Serum catalase activity showed a good correlation (r = 0.820) with the serum glutamate dehydrogenase activity. In accordance with our results, the inexpensive assay of serum catalase activity is suggested for the detection of severe liver cell damage.     

Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin

In the preceding paper (Paris, S., and Pouysségur J. (1987) J. Biol. Chem. 262, 1970-1976), AlF4- and vanadate have been shown to induce inositol phosphate formation in resting hamster fibroblasts (CCL39). In this study, we show that these two phosphate analogs are good tools to explore the causal relationship between phosphoinositide breakdown and early mitogenic events. AlF4- can activate, very similarly to the mitogen alpha-thrombin: the amiloride-sensitive Na+/H+ antiport, the bumetanide-sensitive Na+/K+/Cl- co-transport, and the expression of c-myc mRNA. The link between phospholipase C activation and these early events of the mitogenic response is demonstrated by the similarity of all dose-response curves for NaF and AlCl3 and by the common sensitivity of the four events to pertussis toxin. Vanadate likewise stimulates the Na+/H+ antiport through a pertussis toxin-sensitive pathway. On longer incubations, both fluoride and vanadate were found to be toxic and failed to induce DNA synthesis. Therefore, we have used pertussis toxin to investigate the link between phospholipase C activation and commitment to DNA synthesis. We show that pertussis toxin strikingly inhibits thrombin-induced reinitiation of DNA synthesis but does not affect the stimulation by the epidermal or fibroblast growth factors, two mitogens that do not stimulate phosphoinositide breakdown in CCL39 cells. In conclusion, these studies demonstrate that activation of phospholipase C, if not an obligatory step in the action of all growth factors, plays a crucial role in the mitogenic signaling pathway of alpha-thrombin.     

Is the binding of magnesium (II) to calmodulin significant? An investigation by magnesium-25 nuclear magnetic resonance

Previous reports on the interaction between calmodulin (CaM) and Mg2+ range from no binding to a binding constant of 10(4) M-1 [for a summary, see Cox, J. A., Comte, M., Malnoe, A., Berger, D., & Stein, E. A. (1984) Met. Ions Biol. Syst. 17, 215-273]. In order to resolve the controversy, we used 25Mg NMR to study the binding of Mg2+ to apo-CaM, CaM.Ca2(2)+ (in which sites III and IV are occupied by Ca2+), CaM.La2(3)+ (in which sites I and II are occupied by La3+), and the two tryptic fragments of calmodulin, TR1C (containing sites I and II of CaM) and TR2C (containing sites III and IV of CaM). In each system, a "titration set" and a "temperature set" were obtained, and the spectral data were analyzed by total band-shape analysis to calculate the association constant (Ka) and off-rate (koff). As in the case of Ca2+ binding, sites I and II and sites III and IV were treated as two sets of equivalent sites, and a Ca2+/Mg2+ competition experiment suggested that Mg2+ competes with Ca2+ for the same sites. For both CaM.Ca2(2)+ and TR1C, moderately large Ka (2000 and 3500 M-1, respectively) and moderate off-rates (koff = 2300 and 3000 s-1, respectively, at 25 degrees C) were observed. For both CaM.La2(3)+ and TR2C, binding of Mg2+ was weaker by a factor of ca. 10 (Ka = 300 and 200 M-1, respectively) while the off-rates were also moderate (koff = 3500 and 2200 s-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Interaction between the Ca2(+)-ATPase and the proteolipid in artificial membranes

The interaction between the Ca2+ transport ATPase and the proteolipid of rabbit sarcoplasmic reticulum was analyzed by fluorescence energy transfer, using the following donor: acceptor combinations: Ca2(+)-ATPase tryptophan----IAEDANS-proteolipid; IAEDANS-ATPase----IAF-proteolipid; IAEDANS-proteolipid----IAF-ATPase. The observed energy transfer may indicate weak interaction between the Ca2(+)-ATPase and proteolipid, but collisional energy transfer definitely contributes. The energy transfer was abolished by deoxycholate or sodium dodecylsulfate at concentrations sufficient to solubilize the membrane. In view of the low proteolipid content of sarcoplasmic reticulum and the weak interaction suggested by the energy transfer, at best only a small fraction of ATPase molecules could exist in the form of ATPase-proteolipid complexes.     

Tridimensional analysis of the formation of secretory vesicles in the Golgi apparatus of absorptive columnar cells of the mouse colon

The tridimensional structure of the Golgi apparatus has been studied in the absorptive cells of the mouse colon by means of reduced osmium postfixation and phosphatase cytochemistry. In thick sections of tissue impregnated with osmium tetroxide or treated with a technique to demonstrate TPPase activity, the Golgi formed a continuous ribbon-like structure capping the upper pole of the nucleus. Along the longitudinal axis of this ribbon, compact zones made up of superposed flattened saccules alternated with less compact zones which consisted of highly perforated saccules or bridging anastomosed tubules. In the cis-trans axis, the following elements were observed: (1) a cis element consisting of a continuous osmiophilic tubular network; (2) two or three subjacent elements selectively perforated by wells; (3) a trans compartment made up of two or three TPPase-reactive sacculotubular elements, some showing a "peeling-off" configuration. In some regions, the first flattened saccule of this trans compartment displayed discrete ovoid dilatations, located in compact zones and containing a dense granulofibrillar material; in the subjacent elements this material was seen concentrated in nodular swellings, at the intersection of the meshes of anastomosed membranous tubules. 100-300 nm vesicles containing a similar dense granulofilamentous material were observed in the trans Golgi zone and interspersed in the supranuclear cytoplasm between the Golgi zone and the apical surface of the cell. Smaller vesicles 80-100 nm in diameter containing a fine dusty material were also seen in proximity. These morphological observations suggested that at least two kinds of material were segregated in the saccules of the trans compartment and packaged in vesicles of two class sizes that detached from the Golgi stack on its trans aspect.