Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

TOR controls translation initiation and early G1 progression in yeast.

Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early G1 phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (G0). A G1 cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced G1 arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the G1 arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, G1 progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early G1 progression and growth in the absence of nutrients.     

Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.

Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The rad17 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (rad1, rad3, rad9, rad17, rad26 and hus1) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted rad17 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Rad17 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Rad17 is localized in the nucleus. Rad17 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Site-directed mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Rad17 function which is not associated with the radiation-induced G2 arrest defect seen in the rad17.d null mutant cells.     

Trypanosoma brucei: radioimmunoassay of variant surface glycoproteins from organisms grown in vitro and in vivo

Bloodstream forms of Trypanosoma brucei that were infective for mammals, when grown in vitro at 37 C for 29 days or 25 months had amounts of variant surface glycoprotein similar to the amounts from bloodstream forms isolated from infected rat blood. The amounts were measured by competition radioimmunoassays for both unique and cross-reacting determinants and the results were the same, providing evidence that a single type of variant surface glycoprotein was measured. Neither radioimmunoassay detected determinants of variant surface glycoproteins in trypanosomes transformed by culturing at 27 C to insect forms not infective for mammals.     

Evidence for reappearance of Trypanosoma brucei variable antigen types in relapse populations.

Seven out of 11 bovines infected with different clones of Trypanosoma brucei showed 2 peaks of antibody activity against the infecting clone within 7 weeks, as measured by immunofluorescence, radioimmunoassay, and neutralization of infectivity tests. Using other clones from an unrelated Stock, antibodies to these clones were not detectable, indicating that the antibodies produced were specific to the infecting organisms. These results suggest that there was a reappearance or increase in numbers of the infecting organisms or of organisms with variable surface antigens similar to those of the infecting clones. The reappearance of variable antigen types in the presence of specific antibodies would imply that antibody plays a selective rather than an inductive role in the process of antigenic variation in African trypanosomes.     

Isolation and characterization of a novel glycosyl-phosphatidylinositol-anchored glycoconjugate expressed by developing neurons.

In search of new markers for studying thymic and nervous system ontogeny, we raised rat monoclonal antibodies against glycosyl-phosphatidylinositol-anchored molecules among which larger groupings have been shown to be ectoenzymes and adhesion molecules. Two of these monoclonal antibodies (H193-4 and H194-563, IgG) were found to recognize glycosyl-phosphatidylinositol-anchored glycoconjugates of 28-33 kDa (P31) and 50-70 kDa in developing mouse brain and thymus respectively, when these tissues were analysed by immunoblot experiments. P31 antigen was found to be transiently expressed by neurons in neural primary cultures [Rougon, G., Alterman, L., Dennis, K., Guo, X. J. & Kinnon, K. (1991) Eur. J. Immunol. 21, 1397-1402]. We show in this report that, in developing mouse brain, a maximal expression occurred between embryonic day 17 and post-natal day 5, a period that corresponds to the formation of neuronal networks. P31 antigen was immunopurified and found to possess the following properties: (a) it was soluble in alkaline solvents; (b) it bound to DEAE-cellulose and was eluted by a salt gradient of 0-1 M NaCl; (c) it was sensitive to endoglycosidase F digestion; (d) it was insensitive to heparinase, hyaluronidase, chondroitinase ABC, endo-beta-galactosidase and sialidase treatment; (e) it was labile to mild acid hydrolysis without loss of immunoreactivity; (f) it contained phosphate; (g) it lost its immunoreactivity after treatment with phosphatidylinositol phospholipase C and treatment. These characteristics combine to suggest that P31 is an anionic glycoconjugate sharing similarities with Leishmania donovani lipophosphoglycan and with the heat-stable antigen recognized by J11d antibody on murine hematopo?etic cells. This last hypothesis was further confirmed by the observation that oligonucleotide probes derived from the heat-stable antigen-encoding cDNA detect, in developing brain, a 1.8-kb mRNA species similar in size to that reported for the heat-stable antigen mRNA and following the same developmental expression as P31 antigen.     

Benzoyl cellulose beads in the pure polymeric form as a new powerful sorbent for the chromatographic resolution of racemates

Cellulose-based stationary phases are known to be very efficient and versatile chiral sorbents for the chromatographic resolution of racemates. Except for microcrystalline cellulose triacetate (CTA I), basically all other cellulose-based phases have been prepared by coating of ca. 20% weight polymer on a wide pore silica gel used as a carrier. In this work we describe the preparation of benzoylcellulose (TBC) beads in the pure polymeric form (without inorganic carrier) from an emulsion of the organic polymer. The new material has been fully characterized and used as a chiral stationary phase for the resolution of various classes of racemic compounds such as benzylic alcohols or acetate derivatives of aliphatic alcohols and diols. The structural variety of the separated solutes as well as the irrational influence of the aromatic substituent in different classes of aryl compounds suggest that multiple interaction sites are involved in the complexation, making a prediction of the separation difficult. The benzoyl cellulose beads exhibit a very high loading capacity, which is particularly useful for preparative purposes as demonstrated for selected examples.     

DNA bifunctional intercalators. I. Synthesis and conformational properties of an ethidium homodimer and of an acridine ethidium heterodimer.

An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B.P., & Le Pecq, J.B.(1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acridine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and NMR spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.     

Measurement of the expected DNA lengthening caused by mono-and bisintercalating drugs using electron microscopy

PM₂ DNA molecules were treated with intercalating reagents (ethidium bromide, ethidium dimer, acridine dimer) and observed by electron microscopy. The adaptation of different electron microscopy techniques has enabled the determination of DNA lengthening upon drug intercalation. A 50% length increase was generally obtained for DNA saturated with the drugs. This result is in agreement with the intercalation model proposed by Lerman. In some cases (ethidium dimer), an increase of length larger than 50% can be obtained. Experimental conditions of DNA spreading strongly interfere with the DNA–drug interaction. In some cases it was possible to estimate the apparent binding constants and also to distinguish the mono- from the bisintercalating derivatives in their reaction with DNA.