Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae

Summary The B subunit (CTB) of cholera toxin (CT) can be used as a carrier protein for conjugate vaccines designed to elicit antipolysaccharide antibodies. A defined medium, AGM4, was designed to grow a high-producing mutant of Vibrio cholerae expressing only the B subunit of CT: V. cholerae 0395-NI. AGM4 contains four amino acids, asparagine, glutamic acid, arginine and serine, salts and a trace element solution. The carbon source is glucose. The fermentations performed in AGM4 indicated that CTB production paraleled the growth of the organism but that there was a maximal release of CTB during the stationary phase. There was a clear optimum of productivity at pH 8.0 and 30°C. The pH had an influence on CTB production and not only on its release. Analysis of the amino acids present in the medium showed a correlation between their consumption rates and CTB productivity. Offprint requests to: J. Shiloach     

Morphology of proteoliposomes containing fluorescein-phosphatidylethanolamine reconstituted with native and subunit III-depleted cytochromec oxidase

Beef heart cytochromec oxidase was reconstituted in asolectin liposomes containing the pH indicator fluorescein-phosphatidylethanolamine (FPE) by the cholate-dialysis procedure. The influence of PFE on the asolectin liposome size and of the removal of subunit III from the complex on its incorporation into liposomes was analyzed by freeze-fracture electron microscopy. Samples were frozen without the addition of cryoprotectants. The vesicle size distribution of native enzyme reconstituted into asolectin liposomes was homogenous, 84% of the population having a diameter of 14–37 ± 7.5 mm. The preparation containing FPE had a similar vesicle size distribution, but with bigger diameter range (20–50 nm). In all three different types of proteoliposome preparations the majority of particles containing vesicles was found to have 1 particle (42–81%). The absence of subunit III did not influence the incorporation of the enzyme into the liposomes and was as good as the preparation with native enzyme (>99%). Therefore we conclude that the suppression of the proton pump activity was due to the intrinsic properties of subunit III and not to defective incorporation into artificial membrane systems.Dedicated to the memory of Dr. R. P. Casey.     

D1 and D2 Dopamine Receptors in Caudate-Putamen of Nonhuman Primates (Macaca fascicularis)

D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 +/- 0.027 nM) with a density (Bmax) of 35.7 +/- 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 +/- 0.007 nM and the density was 25.7 +/- 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151-2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.     

Facilitation of tree seedling establishment in a sand dune succession

Abstract. A nucleated pattern of establishment by pine seedlings of Pinus strobus and P. resinosa around pre-established oak trees of Quercus rubra in a sand dune succession in Ontario, Canada was examined using field observations, seed planting and habitat manipulation. Densities of young pines beneath oak canopies were approximately six times greater than in treeless areas, and densities on the north sides of the trees were significantly greater than on the south sides. However, oaks younger than 35 yr showed no preferential establishment beneath them, while the pine population structures beneath older oaks indicated single periods of successful recruitment. Pine seed planted beneath and beyond oak canopies of three sizes germinated primarily beneath the canopies of medium and large-sized oaks, but subsequent survivorship of seedlings over two growing seasons was poor. Several micro-environmental conditions were changed by oak canopies, but only shade showed a pattern closely corresponding to that of seedling establishment. A habitat manipulation experiment confirmed the primary role of shade in facilitating pine seedling establishment beneath oaks. Failure of pine to continue recruiting successfully beneath facilitating trees is tentatively attributed to intraspecific competition among pine individuals.     

Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica

Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.Abbreviations DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - FS freeze-substitution - MT microtubule - MF microfilament - PM plasma membrane     

Induction of differentiation in human HL-60 cells by 4-hydroxynonenal, a product of lipid peroxidation

4-Hydroxynonenal (HNE) is the major diffusible toxic product generated by lipid peroxidation of cellular membranes. The level of lipid peroxidation and, consequently, the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. In the present paper the effects of HNE on the proliferation and differentiation of the HL-60 human promyelocytic cell line have been investigated. Repeated treatment at 45-min intervals with HNE (1 microM) was performed to maintain the cells in the presence of the aldehyde for 7 1/2 or 9 h. The effect of HNE on cell proliferation and differentiation was compared with dimethyl sulfoxide (DMSO)-treated cells. HNE causes a strong inhibition of cell growth without affecting cell viability. Moreover, HL-60 cells acquire the capability to produce chemiluminescence after soluble (phorbol myristate acetate) or corpuscolate (zymosan) stimulation. The phagocytic ability has also been calculated by counting the number of cells that phagocytize opsonized zymosan. Values were 43 and 55% after 10 or 12 HNE treatments, respectively, and 88% in DMSO-treated cells. Myeloperoxidase activity, 5 days after treatment, decreased by 85% in either HNE- or DMSO-treated cells while acid phosphatase activity increased with respect to untreated cells. Results obtained indicate that HNE at concentrations close to those found in the normal tissues can induce inhibition of proliferation and induction of differentiation in the HL-60 cell line.     

Benzimidazole derivatives endowed with potent antileishmanial activity

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC₅₀ values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles.