Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Identification of the dye gene product, mutational loss of which alters envelope protein composition and also affects sex factor F expression in Escherichia coli K-12

Summary The product of the dye gene of Escherichia coli, mapping at 99–100 min, is required for expression of the sex factor F, and also appears to be involved in the regulation of envelope proteins. Mutation of dye thus results in loss of expression of the F-factor (Fex^–, i.e. male sterility, and dye sensitivity (Dye^s). We have isolated a plasmid, pRB38, in which a 6 kb SalI fragment carrying the dye ⁺ gene was cloned into the plasmid pACYC184. This 6 kb SalI fragment also carries two nearby markers, chlG, involved in the synthesis of the molybdenum cofactor, and phoM, required for constitutive expression of alkaline phosphatase.Some of the polypeptides synthesised by pRB38 were identified using the maxi-cell procedure. The product of the dye gene was found to be a polypeptide of M_r=29,000. Thus derivatives of pRB38 in which the transposon was inserted into dye, resulting in a Dye^S Fex^– phenotype when these plasmids were in a dye strain, failed to produce this polypeptide and in some cases produced a truncated product. Such insertions also resulted in a Chl^r and Pho^– phenotype when the plasmid was in a (dye-chlG-phoM) phoR strain, although complementation tests suggested that the phoM ⁺ and chlG ⁺ genes were still intact. Insertions of into the promoter distal end of dye did not result in a Dye^S Fex^– phenotype, although a truncated Dye protein was synthesised, and a Chl^r Pho^– phenotype was produced.It has been suggested (Gaffney et al. 1983) that the dye (=sfrA) gene product is necessary for F-factor expression because it is required for translocation of the F-factor TraJ protein to the outer membrane. Our results suggest that the Dye protein is also required for expression of the molybdenum cofactor and of alkaline phosphatase, and could perhaps be involved in the translocation of these proteins to the membrane.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Experience with selective venous sampling in diagnosis of ACTH-dependent Cushing's syndrome.

Twenty-three patients with adrenocorticotrophic hormone-(ACTH)-dependent Cushing''s syndrome were subjected to selective venous catheterisation and sampling for ACTH on a total of 26 occasions. Out of 10 patients with pituitary-dependent disease, nine had raised ACTH concentrations in one or both high internal jugular vein samples. Eight patients had 11 proved sites of ectopic hormone production: of these, six were correctly identified by the sampling technique, and in four of them this was the only accurate method of localisation. The results of one catheterisation were misleading, and on 10 occasions they were inconclusive; five patients remained undiagnosed by any method. Overall, 15 of the 26 catheterisations provided diagnostically valuable information. Selective venous catheterisation and sampling for ACTH is effective in confirming a pituitary source of the hormone and may be valuable in locating the source of ectopic ACTH production in some cases.