Nuclear membranous whorls

Summary Membranous whorls have been seen in the nuclei of peritoneal and testicular cells which had been subjected to various experimental manoeuvres. It seems likely that this is an early manifestation of cell degeneration which is demonstrated readily only by glutaraldehyde fixation, and to that extent can be regarded as a glutaraldehyde artifact. Acknowledgements. This work was supported by grants from the Medical Research Council, and the University of Sheffield Tuberculosis Research Fund, and by a grant to the Department from Unilever Ltd.I am grateful to Professor R. Barer for his advice and criticism, to Dr. G. A. Meek for guidance on electron microscopy, to Dr. E. J. Clegg for permission to use material from joint experiments. Technical and photographic assistance was provided by Messrs. P. GarLick and L. Murgatroyd and by Miss M. Tune.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

Identification of Two Distinct Isoforms of the Guanine Nucleotide Binding Protein Go in Neuroblastoma × Glioma Hybrid Cells: Independent Regulation During Cyclic AMP-Induced Differentiation

Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the alpha-subunit of the (pertussis toxin-sensitive) guanine nucleotide binding protein G0 were used in two-dimensional immunoblots of membranes of neuroblastoma X glioma (NG108-15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108-15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8-bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of G0 alpha, as has previously been noted in one-dimensional immunoblots. Two-dimensional analysis demonstrated that the cAMP-induced increases in levels of G0 alpha were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin-catalysed ADP-ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of G0 alpha could be achieved in one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of G0 from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of G0 from the cells. In agreement with the data from two-dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP-induced differentiation of NG108-15 cells. Of these two forms of "G0," the acidic species is equivalent to G0 from brain, but the basic form is not identical with G0*, which has been purified from bovine brain.     

Studies on the biosynthesis and metabolism of the phytoalexin lubimin and related compounds in Datura stramonium L.

Arachidonic acid, cellulase, CuSO₄, a sonicate of Phytophthora infestans mycelium and a spore suspension of Penicillium chrysogenum all elicited the formation of the sesquiterpenoid phytoalexins lubimin, 3-hydroxylubimin and rishitin in fruit cavities of Datura stramonium. 3-Hydroxylubimin was the predominant phytoalexin formed after treatment of the fruits with arachidonic acid, cellulase and the P. infestans preparation. Copper sulphate was a potent elicitor of lubimin but not 3-hydroxylubimin. The fungus P. chrysogenum metabolized lubimin and 3-hydroxylubimin to 15-dihydrolubimin and 3-hydroxy-15-dihydrolubimin respectively, both in fruit cavities inoculated with spores of this fungus and in pure culture. The 15-dihydrolubimin formed in the fruits by the fungus was further metabolized (by the fruits) to both isolubimin and 3-hydroxy-15-dihydrolubimin. The precursor-product relationships between all of the subject compounds was investigated by feeding experiments with ³H-labelled compounds. 2-Dehydro-[15-³H₁]lubimin was rapidly and efficiently incorporated into lubimin and may be the direct precursor of lubimin in planta. 3-Hydroxy[2-³H₁]lubimin was incorporated into the nor-eudesmane rishitin but 10-epi-3-hydroxy[2-³H₁]lubimin was not. An updated scheme for the biosynthesis and metabolism of lubimin and related compounds in infected tissues of solanaceous plants is presented.We thank Mr Vic Swetez for the provision of plant material, Mrs Margaret Huffee for technical assistance, Dr David Ewing for help with obtaining NMR spectra, and the Agricultural and Food Research Council for financial support.