A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.     

A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis

As the first step towards correlating structure and function of tubulin in the slime mold Physarum polycephalum we have elucidated the nucleotide sequence of a cDNA that appears to code for all but the last 25 to 30 C-terminal amino acids of a plasmodial alpha-tubulin. Differences in amino acid sequence from those of other alpha-tubulins are distributed fairly evenly throughout the sequence, although a relatively extensive conserved region is found in position 396 to 426 near the C terminus. A small region in position 298 to 307 contains a cluster of amino acid residues unique to Physarum alpha-tubulin. The sequence is 70% homologous to two yeast alpha-tubulins and about 83% homologous to five animal alpha-tubulins. A comparison of the homologies of all the known alpha-tubulins indicates that a large decrease in the accepted point mutation rate has occurred during the evolution of the metazoa, suggesting a major functional specialization of microtubules.     

Synergistic action, on the masculinization of female chick embryo gonads, of a graft of embryonic testis and administration of an anti-estrogenic substance (tamoxifen)

The capability of an embryonic testis graft to induce a male gonadal sex differentiation in genetically female chick embryos was significantly enhanced by the administration of a antiestrogenic drug (tamoxifen). This observation reinforces the view that the experimentally-induced testis differentiation depends chiefly on a inhibition of estrogen influence.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.     

Experimentally induced true hermaphroditism in genetically female fowl

Summary Two types of hermaphroditism were experimentally induced in genetically female fowls by grafting of embryonic testes in embryos. Of the 27 hermaphrodites observed during the 8 months after hatching, 20 possessed a right testis and a left ovary and 7 a right testis and a left ovotestis. The testes and ovotestes contained seminiferous tubules with a more or less developed germ cell complement, attaining in many cases the early spermatid stage. The interstitial tissue was poorly functional, as shown by the absence of male secondary sex characters. The ovary or ovarian part of the ovotestes possessed numerous small ovarian follicles. The female arrangement of the plumage and the absence of spurs demonstrated the secretion of oestrogens. A mechanism is proposed for explaining this partial masculinization of genetically female gonads, a phenomenon which occurs during the period of embryonic sex differentiation, and is responsible for this experimental true hermaphroditism.     

Lec15 cells transfer glucosylated oligosaccharides to protein.

B4-2-1 cells (Lec15 cells) are Chinese hamster ovary cells deficient in mannosylphosphoryldolichol synthase activity. They synthesize the truncated lipid intermediate Man5GlcNAc2-P-P-dolichol rather than the Glc3Man9GlcNAc2-P-P-dolichol synthesized by wild-type cells. In this report we present evidence that these cells did synthesize glucosylated Man5GlcNAc2-P-P-dolichol, but this species represented only a minor fraction of the labeled oligosaccharide-lipid. On the other hand, glucosylated oligosaccharides were a major species transferred to protein in these cells, showing that in vivo, glucosylated oligosaccharides are preferentially transferred to protein. The truncated oligosaccharides found in B4-2-1 cells were removed from the protein by N-glycanase treatment, since they were resistant to both endo-beta-N-acetylglucosaminidase H and F activity. B4-2-1 cells processed the glucosylated, truncated oligosaccharides transferred to G protein of vesicular stomatitis virus, leading to infectious virus.     

Enhancement of cadmium effects on growth and nutrient composition of birch (Betula pendula) by buthionine sulphoximine (BSO)

Binding of Cd to non-specific metal-binding peptides (phytochelatins)in birch roots has been suggested as an explanation for toleranceto Cd toxicity in birch (Betula pendula). In the present study,the tolerance of birch roots to Cd was further investigatedby using buthionine sulphoximine (BSO) as an inhibitor of phytochelatinsynthesis. Birch seedlings, grown in nutrient solution at pH4.2, were exposed to 0 or 2 µM CdCl₂ combined with 0 or0.1 mM BSO for 6 d. Plant growth (fresh weight increase andshoot to root dry weight ratio) and the nutrient compositionin fine roots, whole roots and shoots were determined. The effectsof Cd on growth confirms the results of earlier studies on birch,suggesting a reduced shoot growth, but preserved or stimulatedroot growth. When Cd and BSO were combined, overall plant growthwas severely reduced. BSO was also shown to aggravate Cd-inducedreductions of root and shoot concentrations of K, Ca and Mgbut to impede the accumulation of Cd. The results suggest that phytochelatins participate in protectingthe root against Cd interferences with growth, possibly by restrictingCd-induced changes in the nutrient composition of the plant. Key words: Betula pendula, buthionine sulphoximine, cadmium, phytochelatins, roots, tolerance     

The ultrastructure of the cuticle of Nematoda

 The ultrastructure of the body cuticle in species of six of seven representative genera of Stilbonematinae (Eubostrichus, Catanema, Laxus, Robbea, Leptonemella, and Stilbonema) was investigated using SEM and TEM techniques. Additionally, one species of Spirinia (Spiriniinae) and one of Desmodora (Desmodorinae) were studied for outgroup comparison. The body cuticle of all investigated stilbonematids shows a consistent pattern composed of specific elements in a characteristic arrangement to each other. This pattern does not occur in Stilbonematinae alone, but also in Desmodorinae and Spiriniinae. Furthermore, a comparison within the Desmodorida reveals that this cuticular pattern apparently is present in the cuticle of representatives of Monoposthiidae, Epsilonematidae, and Draconematidae. The present results lead to the following conclusions: (1) the cuticle of Stilbonematinae contains no autapomorphic characters for this taxon, (2) there is a common cuticular pattern within the Desmodorida, and (3) this pattern is an autapomorphic character for the order Desmodorida. Accepted: 4 February 1996     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994