Michel Darmon

Abnormal maturation pathway of keratinocytes in psoriatic skin

We compared the maturation pathway of normal and psoriatic epidermis using three different markers: (I) Involucrin, which is normally detected in the stratum granulosum in normal skin, was detected in all but the basal layer of involved psoriatic skin; (2) an antigen, recognized by the murine monoclonal antibody Ψ3, was present in all but the basal layer of involved psoriatic skin but was absent from uninvolved and normal skin; (3) fibronectin, which normally localizes in the dermis and the epidermal‐dermal junction, was also detected intra‐ and extracellularly in the psoriatic epidermis. These results indicate that the alterations in keratinocyte maturation found in psoriasis do not arise from a truncation of the normal maturation pathway but rather reflect the onset of an abnormal pathway of differentiation characterized by the expression of Ψ3 antigen and fibronectin and the premature appearance of involucrin.

Effects of cyclic AMP pulses on adenylate cyclase and the phosphodiesterase inhibitor of D. discoideum

STARVATION triggers the differentiation of Dictyostelium discoideum amoebae to aggregation competence. The differentiated cells orientate and migrate towards attracting centres, presumably consisting of amoebae which autonomously and rhythmically emit cyclic AMP as a chemotactic signal. Signals, emitted with a periodicity of 5–8 min (refs 1–3, 5), are presumably generated by rhythmic changes in adenylate cyclase activity6, levels of which dramatically increase during the first few hours before the expression of aggregation competence7,8. Cellular responses to external cyclic AMP are probably mediated by plasma membrane receptors9. When stimulated by a pulse of cyclic AMP, amoebae amplify10 and relay1,5,10,11 the chemotactic signal, which results in a coordinated aggregation. The decay of the chemotactic signal is determined by the activity of the extracellular cyclic AMP phosphodiesterase12,13 whose activity is modulated by an inhibitor excreted by cells during the early hours of starvation14. Cyclic AMP pulses have recently also been shown to regulate the differentiation of cells to aggregation competence15. To determine their mechanism of action, we examined the influence of applied cyclic AMP pulses on the two earliest biochemical events implicated in the control of cyclic AMP levels, the rise in adenylate cyclase activity and the appearance of the extracellular phosphodiesterase inhibitor. The results show that adenylate cyclase may exist in active or inactive forms. Applied cyclic AMP pulses seem to induce cell differentiation by activating the enzyme. They also are shown to repress the production of the phosphodiesterase inhibitor.

Periodic changes in adenylate cyclase and cAMP receptors in Dictyostelium discoideum

Information basic to the construction of a multicellular organism is transmitted between starved Dictyostelium discoideum amebae as CAMP pulses [l-3] . In response to these signals, cells differentiate into aggregation-competent amebae [4] and chemotact toward centers.[l] consisting, presumably, of cells \irhich autonomously and rhythmically release CAMP [5] . The effects of CAMP pulses are probably mediated by plasma membrane receptors, as evidenced by cell-surface CAMP-binding sites [6] . Within the first few minutes after stimulation by a pulse of CAMP, cells transitorily increase their cellular CAMP levels [3] and excrete the cyclic nucleotide [2,3] . This relay of the CAMP pulses results in the co-ordinated aggregation of a large cell-population [ 1,5] . The unidirectional movement of amebae is explained by a refractory period, of 9 few minutes’ duration, during which time cells are incapable of relaying the signal [7]. On the basis of computer analyses, Golbeter and Segel have advanced a model for autonomous oscillations and relay of CAMP in which extracellular CAMP, via its receptor, activates adenylate cyclase [8]. Recent support for this model has been obtained by Roos and Gerisch who have shown that a transient stimulation of adenylate cyclase activity can be evoked by an applied pulse of CAMP [9]. In the absence of applied CAMP pulses, differentiating amebae rhythmically synthesize and excrete CAMP [2,10]. In this communication we present evidence for spontaneous oscillations in adenylate cyclase. We also demonstrate periodic changes in binding of

Dietary fish oil up-regulates cholesterol 7α-hydroxylase mRNA in mouse liver leading to an increase in bile acid and cholesterol excretion

To investigate the molecular events controlling reverse cholesterol transport, we compared gene expression of normal mouse liver to that of mice fed a long chain (LC) ω‐3 fatty acid‐enriched diet. Using cDNA microarrays, we assessed expression levels of 1176 genes, and we found that D‐site binding protein (DBP) was three‐fold increased in mice on a LC ω‐3 fatty acid‐rich diet compared to controls. DBP is known to increase transcriptional level of cholesterol 7α‐hydroxylase (C7α), the rate‐limiting enzyme for bile acid production and cholesterol excretion, and we found that C7α mRNA was also up‐regulated by LC ω‐3 fatty acids. Moreover, liver X receptor‐α, another transcription factor up‐regulating C7α, was three‐ to four‐fold increased in liver of treated mice. On the other hand, we demonstrated that bile acid and cholesterol excretion were two‐fold increased. These results show that LC ω‐3 fatty acids control cholesterol metabolism in mice at a new endpoint.

Effects of amino acids and glucose on adenylate cyclase and cell differentiation of Dictyostelium discoideum

Abstract Starvation triggers the differentiation of Dictyostelium discoideum amoebas to aggregation competence. To determine more precisely the nature of the starvation signal, the ability of various components of the growth medium to inhibit differentiation was examined. Changes in adenylate cyclase (the enzyme which generates the cAMP pulses basic to the differentiation process), various physiological and biochemical markers of developing cells, and the ability of amoebas to form specific intercellular contacts were monitored. We show that amino acid mixtures inhibit cell differentiation by preventing the increase of adenylate cyclase activity which normally occurs during the early hours of starvation. High concentrations of glucose also inhibit the differentiation process but at a later stage: The rise in adenylate cyclase still occurs when cells are starved in the presence of sugar, but the enzyme does not appear to function in vivo. Exogenously generated cAMP pulses are not able to bypass the block exerted by amino acids but can bypass the block exerted by glucose. Results support the hypothesis that the presence of amino acids inhibits adenylate cyclase synthesis, while the presence of 3% glucose blocks endogenous activation of adenylate cyclase, perhaps as a consequence of high osmotic pressure.

Phenotypic suppression of morphogenetic mutants of Dictyostelium discoideum

Abstract The effects of cAMP pulses on the capacity of 15 aggregateless mutants to differentiate and construct fruiting bodies are compared to those obtained when mutant cells are starved with wild-type amoebae. Mutant strains are classified into three main groups depending upon the degree to which their phenotypic defects can be corrected. These data extend studies published earlier [Darmon, M., Brachet, P., and Pereira da Silva, L. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 72, 3163–3166; Pereira da Silva, L., Darmon, M., Brachet, P., Klein, C., and Barrand, P. (1975). Induction of cell differentiation by the chemotactic signal in Dictyostelium discoideum. In “Proceedings of the Tenth FEBS Meeting,” pp. 269–276]. (1) Only one mutant was unresponsive both to cAMP pulses and to the presence of wild-type amoebae and did not display any of the properties of differentiated cells. (2) Following treatment with cAMP pulses, 11 mutants developed certain properties of aggregation-competent amoebae. They increased their levels of cellular phosphodiesterase, showed an enhanced chemotactic sensitivity to cAMP, and established specific cell contacts. None of these amoebae could differentiate further. They did co-aggregate to some extent with wild-type cells, but failed to differentiate into spores. Rather, mutant cells were excluded from the pseudoplasmodium during the process of morphogenesis of the fruiting body. (3) In contrast, the aggregateless phenotype of three mutants was fully corrected by both cAMP pulses and the presence of wild-type cells. These findings are discussed on the basis of a relationship between the chemotactic signal and cell differentiation.

LAMININ PROVIDES A BETTER SUBSTRATE THAN FIBRONECTIN FOR ATTACHMENT, GROWTH, AND DIFFERENTIATION OF 1003 EMBRYONAL CARCINOMA CELLS'

SummaryCulture of cells in hormonally defined media has allowed (a) the demonstration of physiological responses from cells usually unable to express them in vitro and (b) the study of the effects on growth and differentiation of diffusible factors and attachment factors. The embryonal carcinoma line 1003 forms multidifferentiated tumors in vivo but is unable to differentiate in vitro when grown in serum-containing medium. In a defined medium containing insulin, transferrin, selenium, and fibronectin as attachment factors, 1003 cells grow for several generations and differentiate into neurons and embryonic mesenchyme (Darmon et al., 1981, Dev. Biol. 85: 463–473). In the present work the effects of fibronectin and laminin were compared. In the presence of laminin the cells attached and spread better, grew faster, and could be plated at lower densities. Neurite extension was also better under these conditions and most importantly, it was found that laminin induced an important formation of muscular tissue when the cells had been seeded at low densities. Multinucleated myotubes could be stained with antibodies directed against embryonic muscular myosin. Coating the dishes with polylysine or adding FGF or serum-spreading factor to the medium allowed growth of low-density cultures with fibronectin instead of laminin but muscular differentiation was not detected under these conditions. Addition of fibronectin to laminin-containing medium did not inhibit muscular differentiation.

A PHOSPHODIESTERASE-DEFECTIVE MUTANT OF DICTYOSTELIUM DISCOIDEUM

Publisher Summary This chapter presents a data concerning an aggregate-less mutant of Dictyostelium discoideum, which appears defective in phosphodiesterase (PD) production. Its aggregation-deficient phenotype may be corrected upon addition to the starvation media of rat brain PD. Mutant amoebae also respond to supernatant media in which Dictyostelium discoideum amoebae were starved. The active factor present in these media is thought to be a peculiar fraction of the extracellular PD activity that is resistant to the inhibitor. Genetic analysis of the mutant strain suggests that the mutation responsible for the aggregate-less character is located on linkage group 4. The chapter also presents existence of an extracellular inhibitor-resistant PD activity.

Neural Differentiation of Pluripotent Embryonal Carcinoma Cells

The use of the teratocarcinoma of the mouse (Pierce, 1967; Stevens, 1967) for the study of neural differentiation offers two major advantages. First, it allows one to obtain large amounts of cells corresponding to the very early stages of neural differentiation, and second, since teratocarcinoma stem cells [embryonal carcinoma (EC) cells] can differentiate into other cell types, it offers the opportunity to study the segregation of the neuronal lineage and to evaluate which changes are specific for the neural pathway.

Human serum reacting specifically with a subset of β‐tubulin isoforms

The serum of a patient suffering from myeloma was found to decorate microtubules and mitotic spindles of cultured cells. Immunoblots performed after one‐ and two‐dimensional electrophoresis showed a reaction with a certain subset of β‐tubulin isoforms, but not with β′‐ and α‐tubulins. The tubulin subset contained both ubiquitous (β‐3) and neurospecific (β‐4,5,6) isoforms.