Cloning and characterization of peter pan, a novel Drosophila gene required for larval growth.

We identified a new Drosophila gene, peter pan (ppan), in a screen for larval growth-defective mutants. ppan mutant larvae do not grow and show minimal DNA replication but can survive until well after their heterozygotic siblings have pupariated. We cloned the ppan gene by P-element plasmid rescue. ppan belongs to a highly conserved gene family that includes Saccharomyces cerevisiae SSF1 and SSF2, as well as Schizosaccharomyces pombe, Arabidopsis, Caenorhabditis elegans, mouse, and human homologues. Deletion of both SSF1 and SSF2 in yeast is lethal, and depletion of the gene products causes cell division arrest. Mosaic analysis of ppan mutant clones in Drosophila imaginal disks and ovaries demonstrates that ppan is cell autonomous and required for normal mitotic growth but is not absolutely required for general biosynthesis or DNA replication. Overexpression of the wild-type gene causes cell death and disrupts the normal development of adult structures. The ppan gene family appears to have an essential and evolutionarily conserved role in cell growth.

Human ARF protein interacts with topoisomerase I and stimulates its activity.

The ARF gene (p19(ARF) in mouse and p14(ARF) in man) has become a central actor of the cell cycle regulation process as it participates to the ARF-MDM2-p53 pathway and the Rb-E2F-1 pathway. By use of immunoprecipitation and Western blotting (IP/WB), we now show that ARF physically associates with topoisomerase I (Topo I). ARF-Topo I immune complexes were detected in SF9 insect cells infected with recombinant baculoviruses encoding the two genes as well as in 293 cells that express endogenously these proteins. Preparations of a GST-ARF recombinant protein stimulated the DNA relaxation activity of Topo I but, in contrast, had no effect on the decatenation activity of Topo II. The Topo I stimulation was also detected in cell extracts of SF9 cells expressing both proteins. A confocal microscopy study indicated that part of ARF and Topo I colocalized in the granular component structure of the nucleolus. As a whole, our data indicate that Topo I is a new partner of ARF and suggest that ARF is involved in cell reactions that require Topo I.

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences.

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.

Regulation of expression and function of muscarinic receptors.

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

[Variations in bone calcium content according to age and sex in a group of control subjects aged 3 to 18 years (author's transl)]. / La densitométrie osseuse chez l'enfant par absorption d'un faisceau monochromatique (méthode de Norland-Cameron). I. Etude des variations de la charge calcique suivant l'âge et le sexe dans un groupe de sujets témoins de 3 à 18 ans.

More than a thousand bone calcium content measurements were made in a group of children by means of densitometry, using the Norland-Cameron apparatus. The region chosen was that dividing the middle from the lower third of the radius, and the method used ensures that only a very weak dose is delivered to a limited field of the forearm. Errors occur more frequently in children than in adults because of lack of immobility and the large amounts of fatty tissue present. Normal curves were established as a function of age and sex in 405 control children of French nationality. These were similar to those obtained in a group of children from the Cameron but French children appear to have lower mineral levels than American children. This method is reproducible and suitable for surveillance of changes in mineral levels in the same subject.

[Variations in bone calcium content as a function of various pathological or iatrogenic disorders (author's transl)]. / La densitométrie osseuse chez l'enfant par absorption d'un faisceau monochromatique (méthode de Norland-Cameron). II. Etude des variations de la charge calcique en fonction de diverses circonstances pathologiques ou iatrogènes.

Bone calcium content was studied in 200 children who had received long-term therapy with anticonvulsants or corticoids, or who presented retarded growth (more particularly, pituitary deficiency treated with somatotropin, thyroid deficiency treated with thyroid extracts, or Turner's syndrome). It was demonstrated that 25 p. cent of children on anticonvulsant therapy, 30 p. cent of those on corticoid treatment, and nearly all those with retarded growth had a mineral content much below normal levels. Bone densitometry by absorption of a monochromatic iodine-125 beam is a rapid, precise method, which is not dangerous at the dose delivered, and appears to be well adapted for surveillance of children with anomalies in calcium content.

[X-ray computed tomographic diagnosis of primary cardiac malignant lymphoma in AIDS]. / Diagnostic tomodensitométrique d'un lymphome malin cardiaque primitif au cours du SIDA.

Primary lymphomas rarely affect the heart. The myocardial disease is usually latent and the diagnosis is based on post mortem observations. The cardiac symptoms do not reveal the disease and symptomatology is not specific. Our observation shows the complementarity of non invasive techniques, for a better screening of cardiac tumoral forms. Although echocardiography is the main examination, CT scan provides a detection of infiltrative forms and of extracardiac extension. Concurrently, MRI remains the method of choice to display beginning infiltrative forms, revealed by pericardial effusion in AIDS disease.

Differential regulation of cAMP-mediated gene transcription by m1 and m4 muscarinic acetylcholine receptors. Preferential coupling of m4 receptors to Gi alpha-2.

We have used a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of the regulation by muscarinic acetylcholine receptors (mAChRs) of intracellular cAMP levels and cAMP-regulated gene expression. Treatment with the muscarinic agonist carbachol results in an increase in luciferase activity in JEG-3 cells transiently transfected with mouse m1 (8-10-fold) and chick m4 (3-5-fold) mAChRs. Control experiments indicate that these responses are not due to a calcium-mediated pathway and are dependent upon a functional protein kinase A. The m1 and m4 responses are not sensitive to pertussus toxin and the m4 response was potentiated by it. Thus, these responses do not result from direct stimulation of adenylate cyclase by beta gamma subunits released from pertussis toxin-sensitive G-proteins. Atropine treatment of cells transfected with high levels of m4 mAChR, but not m1, causes an elevation in basal levels of cAMP response element-mediated luciferase expression in the absence of agonist. This suggests that the m4 receptor is spontaneously active and can cause constitutive inhibition of adenylyl cyclase that is relieved by atropine treatment. Surprisingly, the m4 receptor exhibits little if any agonist-induced inhibition of luciferase expression at either low or high levels of receptor expression. JEG-3 cells express Gi alpha-1 and Gi alpha-3 but not Gi alpha-2. Cotransfection of Gi alpha subunits with m4 demonstrates that the m4 receptor requires Gi alpha-2 for optimal agonist-mediated inhibition. Even in the presence of Gi alpha-2, high levels of receptor increased luciferase expression at high concentrations of agonist. Thus, the m4 mAChR can undergo a switch in functional coupling from inhibition to stimulation of adenylyl cyclase. This switch is dependent on the level of receptor expression, the subtypes of G-proteins coexpressed with the receptor, and the concentration of agonist. Furthermore, we demonstrate that the Gi alpha-2 G-protein alpha subunit preferentially couples the m4 mAChR to inhibition of adenylyl cyclase in JEG-3 cells.

Regulation of cAMP-mediated gene transcription by wild type and mutated G-protein alpha subunits. Inhibition of adenylyl cyclase activity by muscarinic receptor-activated and constitutively activated G(o) alpha.

We have used a luciferase reporter gene under the transcriptional control of a cAMP response element (CRE) to monitor the effects of G-protein alpha subunits on cAMP-regulated gene expression and to examine muscarinic acetylcholine receptor (mAChR) functional coupling to G-proteins. Expression in JEG-3 cells of a mutationally activated Gi alpha-2 in which glutamine 205 is replaced with leucine (Q205L) decreased forskolin-stimulated expression from the CRE-luciferase gene by up to 75%. Similarly, mutation of glycine 43 (corresponding to glycine 12 in p21ras) to valine decreased forskolin-stimulated expression from the CRE-luciferase gene by a maximum of 50%, indicating that this mutation activates the G-protein and is potentially oncogenic. Transfection of the activated Q205L G(o) alpha subunit decreased forskolin stimulation of CRE-luciferase expression. Transfected wild type G(o) alpha was also able to couple the m4 mAChR receptor to inhibition of AC. The amino-terminal myristoylation site was removed from wild type Gi alpha-2 and Q205L Gi alpha-2 by changing glycine 2 to alanine (G2A). Gi alpha-2 with the G2A and Q205L mutations was unable to decrease forskolin stimulation of CRE-mediated luciferase activity. Furthermore, G2A Gi alpha-2 was unable to couple the m4 mAChR to inhibition of AC. Thus, myristoylation is required both for the function of constitutively active Q205L Gi alpha-2 and for receptor-mediated activation of wild type Gi alpha-2.

Differential coupling of m2 and m4 muscarinic receptors to inhibition of adenylyl cyclase by Gi alpha and G(o)alpha subunits.

We compared the G-protein requirements for coupling of human and chicken m2 and m4 muscarinic acetylcholine receptors (mAChRs) to inhibition of adenylyl cyclase, using a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of intracellular cAMP levels. Previously, we used this system to demonstrate that the chick m4 receptor preferentially coupled to Gi alpha-2 and G(o)alpha over Gi alpha-1 and Gi alpha-3. We found that both the chick and human m2 mAChRs can couple to Gi alpha-1, Gi alpha-2, Gi alpha-3, and G(o)alpha, while the human m4 mAChR preferentially couples to Gi alpha-2 and G(o)alpha. Both the G(o)1 and G(o)2 forms of the G(o)alpha subunit were effective in reconstituting coupling of the m2 and m4 mAChRs to inhibit adenylyl cyclase activity. The m2 and m4 mAChRs thus couple to inhibition of adenylyl cyclase by overlapping but different sets of G-protein alpha subunits.

Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells.

We have characterized the muscarinic ACh receptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK) strain II epithelial cells. Binding studies with the membrane-impermeable antagonist N-[(3)H]methylscopolamine demonstrated that mAChRs are approximately 2.5 times more abundant on the basolateral than on the apical surface. Apical, but not basolateral, mAChRs inhibited forskolin-stimulated adenylyl cyclase activity in response to the agonist carbachol. Neither apical nor basolateral mAChRs exhibited detectable carbachol-stimulated phospholipase C activity. Carbachol application to the apical or the basolateral membrane resulted in a threefold increase in intracellular Ca(2+) concentration, which was completely inhibited by pertussis toxin on the apical side and partially inhibited on the basolateral side. RT-PCR analysis showed that MDCK cells express the M(4) and M(5) receptor mRNAs. These data suggest that M(4) receptors reside on the apical and basolateral membranes of polarized MDCK strain II cells and that the M(5) receptor may reside in the basolateral membrane of a subset of cells.

A novel mechanism for coupling of m4 muscarinic acetylcholine receptors to calmodulin-sensitive adenylyl cyclases: crossover from G protein-coupled inhibition to stimulation.

Muscarinic m4 acetylcholine receptors are normally coupled through Gi to inhibition of adenylyl cyclases. In the olfactory bulb and some cultured cells, however, m4 receptors can couple to stimulation of adenylyl cyclase activity. In this study, m4 receptors and specific isozymes of adenylyl cyclases were coexpressed in HEK-293 cells to characterize the mechanism(s) for m4 receptor stimulation of adenylyl cyclases. The calmodulin-sensitive type I and type III adenylyl cyclases were chosen for this study because neither enzyme is stimulated by the beta/gamma complex of G coupling proteins. M4 receptors exhibited either inhibition or stimulation of type I and III adenylyl cyclases depending upon receptor density and agonist concentration. Inhibition of adenylyl cyclase was apparently due to M4 coupling through Gi. Adenylyl cyclase stimulation through m4 receptors was not due to increases in intracellular Ca2+ and stimulation of the calmodulin-sensitive enzymes since it was evident in isolated membranes in the absence of free Ca2+ and with whole cells preloaded with the Ca2+ chelator BAPTA/AM. Stimulation of adenylyl cyclase activities by m4 receptors was apparently mediated via Gs since it was GTP-dependent, was insensitive to pertussis toxin, and was not due to beta/gamma stimulation. Synthetic peptides derived from a G protein activating region of the m4 receptor mimicked the m4-mediated stimulation of adenylyl cyclase activity. These data demonstrate a novel mechanism for muscarinic regulation of adenylyl cyclases that may involve crossover from inhibitory to stimulatory G protein coupling.