Philippe Brachet

1,25-Dihydroxyvitamin D3 regulates the synthesis of nerve growth factor in primary cultures of glial cells

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) on nerve growth factor (NGF) synthesis was investigated in primary cultures of astrocytes prepared from brain of neonatal rats. 1,25-(OH)2 D3 elicited a dose-dependent increase of NGF mRNA with a maximal effect at 10(-7) M, which persisted for at least 48 h. Northern blot analysis revealed an expression of the vitamin D3 receptor (VDR) gene in primary glial cells. Treatment of cells with 1,25-(OH)2 D3 led to an increase in the VDR mRNA levels. Similar results were obtained in C6 glioma cells. Exposure of primary glial cells to 10(-8) M 1,25-(OH)2 D3 caused only a 2-fold increase of the levels of cell-secreted NGF after 3 days of treatment. However, a 5-fold increase was observed three days after a second addition of vitamin D3. Likewise, a pretreatment with lower doses of hormone such as 10(-10) M or 10(-9) M enhanced the responsiveness of the cells to a 24 h treatment with 10(-8) M hormone. It appears, therefore, that the duration of the treatment influences the level of synthesis of NGF, possibly as a consequence of the increase of the VDR gene expression. The specificity of 1,25-(OH)2 D3 is supported by the fact that a concentration of 10(-7) M of an another vitamin D3 metabolite, 24,25-(OH)2 D3, had no effect on NGF synthesis. Several lines of evidence indicate that astrocytes constitute the major cell type responsive to 1,25-(OH)2 D3 in primary cultures of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25-dihydroxyvitamin D3, an inducer of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) has significant therapeutic potentials, in particular for neurodegenerative disorders. To determine factors that would enhance GDNF expression, we analysed the effect of 1,25-(OH)2 D3 in C6 glioma cells. Treatment of C6 cells with 10−7 M, 1,25-(OH)2 D3 for 48 h elicited an 18.5-fold increase in the level of GDNF mRNA. In addition, our results indicate that 1,25-(OH)2 D3 is effective at concentrations as low as 10−10 M and that retinoic acid has additive effects. These data indicate that 1,25-(OH)2 D3 is a potent inducer of GDNF expression and suggest that 1,25-(OH)2 D3 may contribute to the regulation of GDNF in vivo.

Isolation and characterization of deletions in bacteriophage λ residing as prophage in E. coli K12

SummarySeveral new deletions of the λ prophage have been mapped, permitting the localization of sites in the λ genome for initiation of replication, for initiation of gene c I function and for the response to regulation by gene cro.

Relations physiologiques entre les phages tempérés λ et Φ80

SummaryThis work deals with the ability of phage Φ80 to provide defective mutants of λ with their missing functions. Functions Involved in Recombination. As shown by others, the Int mechanism of Φ80 cannot excise prophage λ. However, Φ80 efficiently excises recombinants from tandem dilysogens, using its Ter mechanism. Likewise, the nonspecific mechanism Red is interchangeable between Φ80 and λ. Maturation of λ DNA by Φ80. The “Ter recombinants” excised by Φ80 from tandem dilysogens are packaged into a Φ80 protein coat. This contrasts with the fact, already mentionned by Dove, that Φ80 is extremely inefficient for packaging phage λ superinfecting a λ-lysogen. The latter result is also found when the helper phage is a hybrid with the left arm of λ (Φ80hy4 or Φ80hy41 — see Fig. 1). However, the maturation of the superinfecting λ is much more efficient if the Φ80hy used as a helper has the att-N region of λ (like Φ80hy1). Conversely a λ with the att-N region of Φ80 (λhy6 — see Fig. 1) is packaged more efficiently by Φ80 or Φ80hy4 than by Φ80hy1. It is suggested that the maturation of λ chromosome superinfecting an immune cell requires a recombination with the helper phage. Vegetative Functions. Among the replicative functoons O and P, the latter only can be supplied by Φ80. That λN mutants are efficiently helped by Φ80 does not tell that Φ80 provides the defective λ with an active N product; the λ chromosomes are simply packaged into a Φ80 coat. This shows that Φ80 is unable to switch on the late genes of λ. That neither Φ80 nor any of the Φ80hy tested can provide an active N product is shown in a more direct way by their complete failure to help λN-r14; this phage carries a polar mutation which makes the expression of genes O and P entirely N-dependant. The maturation of a λN- by Φ80 contrasts with the fact that mutants affected in late genes (A, F or H) are not efficiently helped by Φ80. This suggests that the products coded by these genes are not interchangeable between Φ80 and λ, and that packaging of λ DNA into Φ80 coats is possible but inhibited when λ late proteins are present in the cell. Activation of the Late Genes. Among the imϕ80hλ+hybrids tested, only Φ80hy41 is able to switch on the late genes of a λN defective mutant. This hybrid differs from the other hybrids studied here, by the fact that it has the Q-S-R region of λ (see Fig. 1). The results are consistant with the view that the product of Q gene is sufficient for activating the late genes of a λ DNA. N would thus control the expression of late genes only indirectly by controlling the expression of gene Q (Couturier & Dambly have independantly reached the same conclusion, 1970). Furthermore the failure of Φ80 and of the Φ80hy1 and Φ80hy4 to activate the late genes of λ would imply that these phages are unable to provide an Q product active on the λ chromosome Reciprocally, λ switches on the late genes of prophage Φ80hy41, but not of prophages Φ80hy1 and Φ80hy4. This suggests that the initiation of late genes expression takes place at a main specific site located in the Q-S-R region of the λ chromosome. The expression of the late genes would thus be sequential, and proceed through the left arm only when steaky ends cohere. Similar conclusions were reached independantly by Toussaint (1969) and by Herskowitz and Signer (1970).

Isolation and characterization of new sus (amber) mutants of bacteriophage λ

Abstract New amber (sus) mutants of bacteriophage λ have been isolated. Some of them were obtained by a new technique which yields preferentially N and R mutants. The sensitivity of these and other sus mutants towards various suppressors has been determined and is discussed. Conclusions are drawn about which λ functions are “catalytic” or “stoichiometric”.

Serum and thyroid hormones T3 and T4 regulate nerve growth factor mRNA levels in mouse L cells

Mouse L cells synthesize and secrete a neurotrophic factor related to the β subunit of the submaxillary gland nerve growth factor (NGF) of male mice. Use of a cDNA probe which encodes the β‐NGF mRNA demonstrated that L cells produce a transcript identical in size to that of the submaxillary gland. Moreover, target sites of restriction enzymes EcoRI, PstI and BamHI were not significantly rearranged in the β‐NGF gene locus of these cells. The abundance of the β‐NGF transcript was found to depend on culture conditions. Removal of serum depressed the cellular content of polyadenylated RNA by a factor of 1.7, and decreased specifically the pool of β‐NGF transcript by an additional factor of 4. The presence of 10−7 M testosterone in the serum‐free medium did not modify the level of β‐NGF mRNA, while addition of 10−7 M T3 (or T4) increased this level by a factor of 1.5. These data provide the first evidence that the β‐NGF mRNA of L cells is subjected to regulation, but in a way apparently different from that described for the submaxillary gland.

Differentially expressed genes in C6.9 glioma cells during vitamin D-induced cell death program

C6.9 rat glioma cells undergo a cell death program when exposed to 1,25-dihydroxyvitamin D3 (1,25-D3). As a global analytical approach, we have investigated gene expression in C6.9 engaged in this cell death program using differential screening of a rat brain cDNA library with probes derived from control and 1,25-D3-treated cells. Using this methodology we report the isolation of 61 differentially expressed cDNAs. Forty-seven cDNAs correspond to genes already characterized in rat cells or tissues. Seven cDNAs are homologous to yeast, mouse or human genes and seven are not related to known genes. Some of the characterized genes have been reported to be differentially expressed following induction of programmed cell death. These include PMP22/gas3, MGP and β-tubulin. For the first time, we also show a cell death program induced up-regulation of the c-myc associated primary response gene CRP, and of the proteasome RN3 subunit and TCTP/mortalin genes. Another interesting feature of this 1,25-D3 induced-cell death program is the down-regulated expression of transcripts for the microtubule motor dynein heavy chain/MAP 1C and of the calcium-binding S100β protein. Finally 15 upregulated cDNAs encode ribosomal proteins suggesting a possible involvement of the translational apparatus in this cell program. Alternatively, these ribosomal protein genes could be up-regulated in response to altered rates of cellular metabolism, as has been demonstrated for most of the other isolated genes which encode proteins involved in metabolic pathways. Thus, this study presents to our knowledge the first characterization of genes which are differentially expressed during a cell death program induced by 1,25-D3. Therefore, this data provides new information on the fundamental mechanisms which participate in the antineoplastic effects of 1,25-D3 and on the machinery of a cell death program in a glioma cell line.

Regulation of Peripherin in Mouse Neuroblastoma and Rat PC 12 Pheochromocytoma Cell Lines

Peripherin (Formerly the Y protein) is found in the peripheral nervous system. This Triton-insoluble protein is characterized by its isoelectric point (5.6), its apparent molecular weight (56,000 daltons) and its peptide map. Peripherin was also observed in a mouse neuroblastoma cell line, NIE 115, where its expression appeared regulated by the presence of an inducer of morphological differentiation. In order to analyze more precisely this control, the presence of peripherin was investigated in several neuroblastoma cell lines which exhibit different morphological patterns of differentiation and in the rat pheochromocytoma PC 12 cell line. Differentiation of these cells was induced with 1-methylcyclohexane carboxylic acid (CCA) and nerve growth factor (NGF), respectively. Peripherin was found in these different cell lines. Moreover, the cellular amount of peripherin appraised by [35S]-methionine incorporation was significatively increased in differentiated cells. In contrast, other cytoskeletal components did not undergo a similar raise. The level at which the control of the peripherin content takes place was studied in a cell-free translation system. Poly(A)-rich RNAs extracted from growing or differentiated NIE 115 cells directed the synthesis of similar amounts of peripherin in a reticulocyte lysate. In contrast, polysomes prepared from differentiated cells and the corresponding polysomal RNA programmed in vitro the synthesis of twice more peripherin than polysomes or polysomal RNA from growth-phase cells. Since peripherin synthesis is enhanced 5 times in living cells, it seems probable that the cellular amount of peripherin is controlled partly at the translational level and partly at the turn-over level.

Dexamethasone rapidly reduces the expression of the β-NGF gene in mouse L-929 cells☆

Mouse L-929 cells were treated with dexamethasone, and the cellular levels of beta-NGF mRNA were estimated by hybridization of the RNAs with a beta-NGF cDNA probe. The results revealed that the glucocorticoid decreased specifically, in a dose-dependent manner, the pool of beta-NGF transcripts. After 4 h, L-929 cells cultured with 10(-7) M dexamethasone contained one-fifth as much beta-NGF mRNA as untreated control cells, and as little as one-tenth as much when the glucocorticoid concentration was 10(-6) M. The effect of the hormone became maximal after 8 h of treatment. Amounts of beta-NGF secreted by the cells during 24 h were measured with a two-site enzyme immunoassay. They also appeared reduced in cultures exposed to the glucocorticoid. These data indicate that dexamethasone controls negatively the expression of the beta-NGF gene in L-929 cells at some pre-translational level.

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