Cloning and sequencing of the cDNA encoding the major albumin of Theobroma cacao

The major albumin, a polypeptide of 21 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), has been identified and partially purified by preparative gel electrophoresis. Some N-terminal sequence was obtained, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 221-amino-acid polypeptide of M_r 24003. The existence of a precursor to the 21-kDa polypeptide of this size was confirmed by immunoprecipitation from total poly(A)⁺ RNA translation products. The polypeptide has a hydrophobic signal sequence of 26 amino acids before the mature start, and the mature polypeptide would have an M_r of 21223. The protein sequence is homologous with sequences of the Kunitz protease and -amylase inhibitor family, and the protein probably functions to defend the seed's protein reserves from the digestive enzymes of invading pests. However because the protein comprises 25–30% of the total seed protein it may itself also function as a storage protein. Electron micrographs of immunogold-labelled embryo sections show that the protein is located in membrane-enclosed organelles.Abbreviations cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies, and to Dr G. Cope, of the Biological Sciences Electron Microscopy Unit, Sheffield University, for taking the electron micrographs.To whom correspondence should be addressed.     

3-phenylacetylamino-2,6-piperidinedione inhibition of rat Nb2 lymphoma cell mitogenesis

3-Phenylacetylamino-2,6-piperidinedione (A10), an amino acid analog, has been reported to possess antineoplastic activity against certain neoplastic tissues. The antimitogenic properties of A10 were studied by determining its effect on prolactin (PRL)- and interleukin 2 (IL-2)-stimulated mitogenic responses in the rat Nb2 lymphoma cell line. The addition of A10 (1-12 mM) to PRL (0.4 ng/ml)-stimulated cells inhibited growth in a dose-dependent manner. DNA synthesis patterns studied by thymidine incorporation demonstrated that A10 was significantly inhibitory (25% at 20 hr; 50% at 40 hr, P less than 0.01). IL-2 stimulation of mitogenesis was also sensitive to A10 inhibition. The inhibition of PRL stimulated mitogenesis was reversible when A10 was removed after 24 hr of culture and A10 showed no toxicity in a chromium release assay. These data suggest that A10 effects may be cytostatic, rather than cytotoxic.     

Do plasma ACTH and cortisol concentrations in the late-gestation fetal sheep vary diurnally?

This study tested the hypothesis that fetal plasma ACTH and cortisol concentrations vary diurnally, and the mean concentration and the amplitude of the rhythm vary as a function of fetal gestational age. Nine chronically-catheterized fetal sheep were studied between 120 and 142 days' gestation. All of the fetuses were born spontaneously and alive. The pregnant ewes were maintained in a room with a regular light cycle (on at 07.30, off at 17.30). Food and water were available ad libitum. Blood samples were drawn at 4-h intervals throughout a 24-h period. There were no significant daily variations in fetal plasma ACTH, cortisol, or progesterone concentrations, except in the last 3 days of fetal life. In these fetuses ACTH and cortisol concentrations were increased in the afternoon and evening. We conclude that there is no diurnal rhythm in ovine fetal hypothalamo-pituitary-adrenal axis activity, and that the increased plasma concentrations of ACTH and cortisol in the afternoon and evening hours of the last few days of fetal life might be a response to increased uterine contraction activity.     

Co-immunolocalization of topoisomerase II and chloroplast DNA in developing,dividing and mature wheat chloroplasts

This paper describes the first localization of immunofluorescence of topoisomerase II in developing chloroplasts. In order to investigate the relationship between topoisomerase II and chloroplast DNA (ctDNA) replication during chloroplast development the 7-day-old wheat leaf was used. Topoisomerase II was immunolabelled and fluorescein tagged and the ctDNA simultaneously stained with 4,6-diamidino-2-phenylindole (DAPI) in the same sections. Topoisomerase II was detected at every stage of chloroplast development and maximal levels of topoisomerase II were found in chloroplasts at the time of ctDNA replication. Topoisomerase II was localized around the plastid periphery, exactly mirroring the position of the ctDNA. After chloroplast division both topoisomerase II and ctDNA are seen to be restricted to small discrete areas within the plastid, but at different sites. These findings strongly suggest a role for topoisomerase II in ctDNA decatenation prior to chloroplast division.     

The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS-cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.     

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate