Comparative studies of seed proteins of the genusArtocarpus with respect to lectins

Proteins from two species of the genusArtocarpus (A. integrifolia L. andA. incisa L.) were compared by ammonium sulphate fractionation, molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis, with special attention to the lectins. The protein content and hemagglutinating activity were markedly different in the two seeds. The protein pattern obtained by both molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis were quite different. The only similarities found were the elution volume of the lectins in the Sephadex G-100 column and the lectin bands (11 500 and 15 000 daltons) in SDS-polyacrylamide gel electrophoresis.     

Broad-Host-Range Agrocin of Agrobacterium tumefaciens

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.     

The inhibition of nucleic acid synthesis by mycophenolic acid

1. Mycophenolic acid, an antibiotic of some antiquity that more recently has been found to have marked activity against a range of tumours in mice and rats, strongly inhibits DNA synthesis in the L strain of fibroblasts in vitro. 2. The extent of the inhibition of DNA synthesis is markedly increased by preincubation of the cells with mycophenolic acid before the addition of [(14)C]thymidine. 3. The inhibition of DNA synthesis by mycophenolic acid in L cells in vitro is reversed by guanine in a non-competitive manner, but not by hypoxanthine, xanthine or adenine. 4. The reversal of inhibition by guanine can be suppressed by hypoxanthine, 6-mercaptopurine and adenine. 5. Mycophenolic acid does not inhibit the incorporation of [(14)C]thymidine into DNA in suspensions of Landschütz and Yoshida ascites cells in vitro. 6. Mycophenolic acid inhibits the conversion of [(14)C]hypoxanthine into cold-acid-soluble and -insoluble guanine nucleotides in Landschütz and Yoshida ascites cells and also in L cells in vitro. There is some increase in the radioactivity of the adenine fraction in the presence of the antibiotic. 7. Mycophenolic acid inhibits the conversion of [(14)C]hypoxanthine into xanthine and guanine fractions in a cell-free system from Landschütz cells capable of converting hypoxanthine into IMP, XMP and GMP. 8. Preparations of IMP dehydrogenase from Landschütz ascites cells, calf thymus and LS cells are strongly inhibited by mycophenolic acid. The inhibition showed mixed type kinetics with K(i) values of between 3.03x10(-8) and 4.5x10(-8)m. 9. Evidence was also obtained for a partial, possibly indirect, inhibition by mycophenolic acid of an early stage of biosynthesis of purine nucleotides as indicated by a decrease in the accumulation of formylglycine amide ribonucleotide induced by the antibiotic azaserine in suspensions of Landschütz and Yoshida ascites cells and L cells in vitro.     

Cytological aspects of haemoglobin and chlorocruorin synthesis in polychaete annelids

Summary The ultrastructure of the haematopoietic cells in the polychaetes Neoamphitrite figulus Dalyell, Lanice conchilega (Pallas), Arenicola marina (L.), Myxicola infundibulum Renier, Megalomma vesiculusom (Montagu), Sabella penicillus L., are compared: all show similarities in having well developed Golgi, granular endoplasmic reticulum and haemoglobin or chlorocruorin in vesicles, and numerous mitochondria. The porphyrin byproducts of synthesis are combined with iron as haematins within electrondense granules built up from multi-lamellar organelles. The structure of the basal lamina which alone separates the cells from the lumen of the vessels is described and evidence is presented for the method of release of the haem into the plasma by reverse pinocytosis. The cycle of synthesis within the cell is discussed and the process of haem synthesis in annelids is reviewed. The structure of the haemoglobin-containing coelomocytes of Neoamphitrite figulus is briefly described.This work was made possible by an award from the Science Research Council. We would also like to thank the Director and Staff of the Plymouth Laboratory, where some of the work was done, for their hospitality and facilities.     

Role of extracellular signaling on endothelial cell proliferation and protein N-glycosylation

In vitro studies of angiogenic phenomenon have been limited due to nonavailability of a simple and biologically relevant model of the capillary wall. Recent development of a capillary endothelial cell line from the vascular bed of bovine adrenal medulla made us to study the effect of heparin, thrombin, thyroxine, glucagon, insulin, and phorbol myristate acetate (PMA) on the proliferative and metabolic activities such as glycosylation of asparagine-linked glycoproteins of these cells in culture. Out of six different agents studied here, only heparin, thrombin, and thyroxine reduced the doubling time of these cells by 24 hr with no observed morphological abnormality. Glucagon, showed marginal reduction in the cell doubling time. By contrast, insulin and PMA enhanced the doubling time. Insulin treatment though induced the S phase of cell cycle but it blocked the cells entry into the G2 + M phase. PMA arrested the cells in G0/G1 phase. The cellular response to protein N-glycosylation is increased in the presence of thyroxine, insulin, and thrombin and the effect is dose dependent. Further analysis on SDS-PAGE indicated that glycosylation of 80-120 kDa and 43 kDa glycoprotein species are enhanced when these cells are treated with insulin and thrombin. Glycopeptide generated from these glycoproteins suggested that they all carry "high mannose" and "complex" type oligosaccharide chains attached to their protein core.     

Intramolecularly quenched fluorogenic peptide substrates for human renin.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.