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.     

Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.     

Failure to detect platelet-activating factor using the splenectomized mouse bioassay

The ability of purified preparations of platelet-activating factor (PAF), from three different suppliers, to induce thrombocytopaenia in mice after splenectomy and to activate mouse platelets in vitro was examined. Although the PAF preparations were potent activators of horse and cow platelets in vitro, injections of up to 1 microgram PAF failed to elicit thrombocytopaenia responses in either CD1 or Swiss Webster random-bred mice. However, when thrombin was injected into Swiss Webster mice, a dose-dependent decrease in the concentration of platelets was observed. Furthermore, isolated platelets from these strains and from 3 inbred lines (C3H/He, BALB/c, C57BL/6) of mice, were not aggregated by PAF in vitro but were sensitive to adenosine diphosphate and thrombin. No change in circulating platelet concentrations was observed over the initial 7 days of gestation in intact Swiss Webster and C57BL/6 or splenectomized C57BL/6 mice, suggesting either an absence of PAF production during early pregnancy in these strains or insensitivity of their platelets to PAF. These results suggest that many mouse strains are unsuitable for the bioassay of PAF.     

The major albumin protein from pea (Pisum sativum L.)

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.Abbreviation PMA pea major albumin protein     

Key developmental stages of winter wheat,Triticum aestivum

Winter wheat,Triticum aestivum, is important as both a forage and grain crop in the central and southern Great Plains of the United States. A simpler system of describing winter wheat developmental stages than those currently being used is needed. Ten key developmental stages are described: 1) germination and emergence, 2) tillering, 3) leaves strongly erect, 4) node formation, 5) boot, 6) heading, 7) flowering, 8) grain filling, 9) ripening, and 10) maturity. All stages can be visually identified in the field with a minimum of training and are important with respect to grazing management, fertilizer applications, pest control, forage yield, and grain harvesting. Often no time factors can be placed on the occurrence of these stages due to environmental and cultivar differences. A discussion of stress influences on grain and forage yield is included.     

Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen-stigma interactions

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6–8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene ( PCP1 ) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea , Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F₂ segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus .     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.