Esther Castellano

Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras‐like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3‐kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D‐type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.

Transcriptional networks of knockout cell lines identify functional specificities of H-Ras and N-Ras: significant involvement of N-Ras in biotic and defense responses

We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras−/− and H-ras−/− fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras−/− cells was significantly closer to that of wild-type fibroblasts than to that of N-ras−/− cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras−/− cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras−/− cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras−/− cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses

H-ras and N-ras are dispensable for T-cell development and activation but critical for protective Th1 immunity

The small guanine nucleotide binding proteins of the Ras family, including in mammals the highly homologous H-ras, N-ras, and K-ras isoforms, are rapidly activated on ligation of the T-cell antigen receptor (TCR), but whether each isoform plays specific roles in T cells is largely unknown. Here, we show, with the use of mice specifically lacking H-ras or N-ras, that these isoforms are dispensable for thymocyte development and mature T-cell activation. By contrast, CD4⁺ T cells from Ras-deficient mice exhibited markedly decreased production of the Th1 signature cytokine IFN-γ early after TCR stimulation, concomitantly with impaired induction of the Th1-specific transcription factor T-bet. Accordingly, Ras-deficient mice failed to mount a protective Th1 response in vivo against the intracellular parasite Leishmania major, although they could be rendered resistant to infection if a Th1-biased milieu was provided during parasite challenge. Collectively, our data indicate that the TCR recruits distinct Ras isoforms for signal transduction in developing and mature T cells, thus providing a mechanism for differential signaling from the same surface receptor. Furthermore, we demonstrate for the first time that H-ras and N-ras act as critical controllers of Th1 responses, mostly by transmitting TCR signals for Th1 priming of CD4⁺ T cells.

Synthesis, crystal structure and spectroscopic properties of diarganothallium (III) derivatives of 2,6-diacetyl-pyridine-monothiosemicarbazone

Abstract Dimethyl(2,6-diacetyl-pyridine-monothiosemicarbazonato)thallium(III), [TlMe2(L)], and Diphenyl-(2,6-diacetyl-pyridine-monothiosemicarbazonato)thallium(III) chloroform solvate, [TlPh2(L)],CHCl3 were prepared by reacting the corresponding diorganothallium(III) hydroxide with 2,6-diacetyl-pyridinemonothiosemicarbazone (HL). [TlMe2(L)] crystallizes in the triclinic space group P l (no. 2) with a = 7.064(3), b = 9.149(2), c = 12.377(2) A , α = 79.378(11), β = 74.92(2), γ = 82.75(2)°, Z = 2, R 1 = 0.0305, Rw 2 = 0.0673 . [TlPh2(L)].CHCl3 crystallizes in the monoclinic space group C2/c with a = 23.559(3), b = 13.582(2), c = 16.808(3) A , β = 106.04(1)°, Z = 8, R 1 = 0.0620, Rw 2 = 0.1155 . Both complexes consist of molecules in which the deprotonated ligand is S,N(3),N(4),O-bonded to the thallium atom [TlMe2(L)], Tl  S = 2.872(2), Tl  N (3) = 2.590(6), Tl  N (4) = 2.686(2), Tl  O = 2.883(6) A ; [TlPh2(L).CHC]3, Tl  S = 2.791(6), Tl  N (4) = 2.579(14), Tl  O = 2.86(1) A ]. A weak bond Tl…O′ [TlMe2(L)], 3.050(2) A; [TlPh2(L)].CHCl3, 3.42(1) A] between neighbouring molecules give rise to weakly bonded dimers. The 1H, 13C and 205Tl NMR spectra of both compounds in DMSO indicate that the bonding scheme observed in solid state is only partially retained in solution, where at least two isomers are observed.

Interactions of diorganolead(IV) with 3-(2-thienyl)-2-sulfanylpropenoic acid and/or thiamine: chemical and in vitro and in vivo toxicological results.

The reactions of PbR(2)(OAc)(2) (R = Me, Ph) with 3-(2-thienyl)-2-sulfanylpropenoic acid (H(2)tspa) in methanol or ethanol afforded complexes [PbR(2)(tspa)] that electrospray ionization-mass spectrometry (ESI-MS) and IR data suggest are polymeric. X-ray studies showed that [PbPh(2)(tspa)(dmso)] x dmso, crystallized from a solution of [PbPh(2)(tspa)] in dmso, is dimeric, and that [HQ](2)[PbPh(2)(tspa)(2)] (Q = diisopropylamine), obtained after removal of [PbPh(2)(tspa)] from a reaction including Q, contains the monomeric anion [PbPh(2)(tspa)(2)](2-). In the solid state the lead atoms are O,S-chelated by the tspa(2-) ligands in all these products, and in the latter two have distorted octahedral coordination environments. NMR data suggest that tspa(2-) remains coordinated to PbR(2)(2+) in solution in dmso. Neither thiamine nor thiamine diphosphate reacted with PbMe(2)(NO(3))(2) in D(2)O. Prior addition of H(2)tspa protected LLC-PK1 renal proximal tubule cells against PbMe(2)(NO(3))(2); thiamine had no statistically significant effect by itself, but greatly potentiated the action of H(2)tspa. Administration of either H(2)tspa or thiamine to male albino Sprague-Dawley rats dosed 30 min previously with PbMe(2)(NO(3))(2) was associated with reduced inhibition of delta-ALAD by the organolead compound, and with lower lead levels in kidney and brain, but joint administration of both H(2)tspa and thiamine only lowered lead concentration in the kidney.