Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

Structural Characteristics of the Short-Tail Fibers of T4 Bacteriophage

The characteristics of pure preparations of short-tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43-nm fibers were the most abundant structure. During the process of purification of the short-tail fibers, the formation of aggregates was observed each time the material containing the short-tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short-tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short-tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well-defined bundles. The amino acid composition of the highly purified short-tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short-tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short-tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.     

Dehydrodieugenols from Ocotea cymbarum

The trunk wood of Ocotea cymbarum from the Amazon basin contains α-phellandrene, α-pinene, eugenol, dehydrodieugenol and its monomethyl ether, as well as the previously unknown dehydrodieugenol-B (4,5′-diallyl-2′-hydroxy-2,3′-dimethoxydiphenyl ether).     

Heavy metal composition of polysomal fractions following cadmium challenge

Endogeneous levels of zinc and copper were found to be 1.2±0.1×10⁻² and 0.3±0.1×10⁻² μg/A260 unit, respectively, in polysomal fractions from control animals; cadmium, however, was undetectable. In experimental animals (injected with cadmium) zinc, copper, and cadmium were found in polysomal fractions isolated by two different methods. One hour after a cadmium injection there was a rise in both the zinc and copper content of the polysomal fractions, which then declined steadily to below control levels by 16 h. Neither zinc nor cadmium were dialyzable from these fractions by a TRIS buffer; however, addition of 0.01M EDTA to the buffer resulted in removal of 75% of the zinc and all of the detectable cadmium. The addition of cadmium (CdCl₂) to control supernatants (adjusted to the cadmium concentration present in supernatants 6 h after in vivo exposure) resulted in metal binding to polysomal fractions in levels comparable to those observed after in vivo exposures to the metal. When cadmium was added in the form of cadmium thionein, a smaller fraction of the metal was isolated with the polysomal fraction. Cadmium bound to polysomal fractions in vivo (24 h after exposure) was sensitive to release by protease digestion, but insensitive to release by ribonuclease digestion.     

Marker rescue transformation by linear plasmid DNA in Bacillus subtilis

Although plasmid-free Bacillus subtilis cannot be transformed for markers carried by linear or nicked plasmid DNA, a resident plasmid can rescue a marker on such damaged DNA under certain conditions. Linearized chimeric plasmid DNA has been used to transform cultures carrying a resident plasmid which is homologous with a portion of the donor. This system has revealed the following properties of the marker rescue process: (1) It is recE dependent. (2) It requires the presence in the resident plasmid of sequences which are homologous to the donor. (3) When the selected marker is on a nonhomologous segment it must be flanked by segments which are homologous to the resident plasmid. (4) The efficiency of rescue varies in a regular way with the position of the linearizing cut. (5) Marker rescue is first order with respect to DNA concentration. These properties and other data are interpreted as providing a strong indication that marker rescue occurs by recombination, although an alternative explanation involving recE-dependent recircularization of the donor plasmid has not been eliminated. Our results also suggest that if the major pathway of marker rescue is by recombination, an average of 0.15 Mdal (single strand) must be removed from each donor DNA molecule or otherwise rendered unavailable for recombination and that the exchange frequency during transformational recombination is approximately 0.2 to 0.5 Mdal⁻¹.     

Interactions of cardiac glycosides with cells and membranes Properties and structural aspects of two receptor sites for ouabain in erythrocytes

The existence of two types of binding sites for ouabain in human erythrocyte membranes is described. Receptor sites designated as ‘type I’, which may be identical to the K⁺-insensitive sites of intact cells, were detected at concentrations of ouabain as low as 10⁻⁷ M. The ‘type II’ receptor sites require the inclusion of Mg²⁺ + P_i to form complexes with ouabain; they may be identical to the K⁺-sensitive sites of intact cells. These sites were saturated at approx. 5 · 10⁻⁷ M ouabain but could not be detected at higher concentrations. The range of ouabain concentrations at which ‘type I’ receptors start to predominate (i.e. 5 · 10⁻⁸–5 · 10⁻⁷ M) was termed ‘critical digitalis concentrations’. The process of binding reached equilibrium within 1 and 4 h for ‘type I’ and ‘type II’ sites, respectively. The dissociation constant for ‘type II’ receptor-ouabain complexes was 7.6 · 10⁻⁹ M.Under similar experimental conditions, rat erythrocyte membranes exhibited only non-saturable sites.Alterations in the proportions of the two types of receptors were demonstrated by preincubation of the membranes, in the presence or absence of Mg²⁺ + P_i, prior to the addition of ouabain. In the first case, ‘type II receptor-ouabain’ complexes were stabilized at about 50% of the untreated membranes and ‘type I-ouabain’ complexes slowly approached equilibrium over a period of 24 h. In the latter instance, ‘type I’ receptors were not detected, and only ‘type II-ouabain’ complexes prevailed.     

Stereological investigation on the increase in surface area due to the microtriches of the hydatid cyst in different organs and in different hosts

Bortoletti G. and Diaz G. 1978. Stereological investigation on the increase in surface area due to the microtriches of the hydatid cyst in different organs and in different hosts. International Journal for Parasitology8: 433–436. The increase in surface area of the germinal membrane due to the microtriches has been morphometrically investigated in Echinococcus granulosus cysts developed in three different intermediate hosts. The results, achieved by Stereological methods, indicate that the development of the microtriches: (a) is more or less homogeneous all over the germinal membrane of the cysts; (b) is greater in human than in pig and sheep cysts; (c) is also greater in lung than in liver cysts within the same host and it is not related to the fertility or sterility of the parasite.     

Voltage-dependent conductance induced by hemocyanin in black lipid films

When hemocyanin is added to a black lipid film, the conductance increases in discrete steps. For negative potentials the single step conductance is constant, but for positive potentials the step conductance appears to decrease as the potential increases. At high positive potentials the conductance fluctuates between several levels. These data suggest that, in lipid membranes, hemocyanin conducts ions through discrete channels. The voltage-dependent conductance observed at high levels of conductance seems to be a consequence of the properties of the conductance of the single channel.     

Steroid-receptor quantitation and characterization by electrophoresis in highly cross-linked polyacrylamide gels.

Conditions for discontinuous polyacrylamide gel electrophoresis have been defined in which progesterone receptors of chick oviduct cytosol and a variety of steroid-binding proteins from other sources are stable and amenable to quantitative analysis. The essential modifications from standard procedures include the use of (1) separation gels in which the cross-linking agent/acrylamide monomer = 15:85, (2) glycerol (10% v/v) in all phases of the Trisglycine-HCl buffer system (pH 10.2 in the separation phase during electrophoresis at 0 degrees), and (3) a layer of a charged reducing agent, thioglycolate, beneath the sample layer. Electrophoresis of untreated oviduct cytosol labeled with [3H]progesterone +/- competing steroids revealed a heterodisperse slow peak and a sharp fast peak. Both peaks displayed the steroid-binding specificity and saturability that are characteristic of intracellular receptors. Recovery of steroid from both the slow and fast components increased linearly with sample load up to 60 mul of cytosol (1.2 mg of protein)/gel (6 mm diameter). The specific progesterone binding detected by this technique was comparable to that detected by charcoal-dextran treatment or ion exchange filtration. Relative electrophoretic mobilities (Rf) of globular protein standards and steroid-protein complexes in cytosol and chick serum were measured in separation gels with total gel concentrations (T) systematically varied from 5 to 15% (w/v). Data were processed by computer programs to obtain weighted linear regressions of log Rf on T (Ferguson plots) and the joint 95% confidence limits of the slopes (-KR) and intercepts of these plots. Molecular radii (R) of the binding components and apparent molecular weights (M) were calculated from the linear correlation of R with KR 1/2 for the standards. The value of M is approximately 158,000 obtained for the cytosol fast component was independent of the length of the separation gel, the presence of a stacking gel or prior exposure of the cytosol to KCl. It was higher than expected from the sedimentation coefficient of 4.2 S in the same pH 10.2 buffer. Electrophoresis in 170-mm separation gels without stacking gels revealed that KCl extracts of protamine-precipitated cytosol contain a different receptor form, of lower net negative charge than the cytosol fast form. The results demonstrate the utility of electrophoresis in highly cross-linked gels of several concentrations to discriminate between various receptor forms and steroid-binding components of serum. This method may lead to overestimates of M for highly asymmetric receptor forms.