Gemma Rodríguez-Tarduchy

Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes☆

The effect of phorbol esters on the proliferation and survival of interleukin-2(IL-2)-dependent cells was studied using an IL-2-dependent T cell line (CTLL-2) and blasts of BALB/c mouse spleen cells stimulated with Concanavalin A. Addition of phorbol 12,13-dibutyrate (PDBu) to CTLL-2 or ConA blasts induces a mitogenic response which is 25-40% of that elicited by IL-2. Interleukin 2 deprivation leads to a marked decline in the number of viable cells (50% of CTLL-2 cells have died after 8-10 hours incubation in IL-2-free medium). The mechanism of cell death seems to correspond to the suicide process known as apoptosis since an early degradation of DNA into oligonucleosome-size fragments could be observed after removal of the growth factor. When present, PDBu inhibits both the activation of the endonuclease and the development of the cell death process in CTLL-2 cells and ConA-blasts deprived of IL-2. Taken together, our results suggest that the tumor promoters phorbol esters inactivate in T cells the mechanism of cell elimination triggered by IL-2 deprivation and may help to explain why transformation of T cells decreases or even abolishes their requirements of IL-2 for survival and growth.

1H HR-MAS and genomic analysis of human tumor biopsies discriminate between high and low grade astrocytomas

We investigate the profile of choline metabolites and the expression of the genes of the Kennedy pathway in biopsies of human gliomas (n = 23) using 1H High Resolution Magic Angle Spinning (HR‐MAS, 11.7 Tesla, 277 K, 4000 Hz) and individual genetic assays. 1H HR‐MAS spectra allowed the resolution and relative quantification by the LCModel of the resonances from choline (Cho), phosphocholine (PC) and glycerophosphorylcholine (GPC), the three main components of the combined tCho peak observed in gliomas by in vivo 1H NMR spectroscopy. All glioma biopsies depicted a prominent tCho peak. However, the relative contributions of Cho, PC, and GPC to tCho were different for low and high grade gliomas. Whereas GPC is the main component in low grade gliomas, the high grade gliomas show a dominant contribution of PC. This circumstance allowed the discrimination of high and low grade gliomas by 1H HR‐MAS, a result that could not be obtained using the tCho/Cr ratio commonly used by in vivo 1H NMR spectroscopy. The expression of the genes involved in choline metabolism has been investigated in the same biopsies. High grade gliomas depict an upregulation of the β gene of choline kinase and phospholipase C, as well as a downregulation of the cytidyltransferase B gene, the balance of these being consistent with the accumulation of PC. In the low grade gliomas, phospholipase A1 and lysophospholypase are upregulated and phospholipase D is downregulated, supporting the accumulation of GPC. The present findings offer a promising procedure that will potentially help to accurately grade glioma tumors using 1H HR‐MAS, providing in addition the genetic background for the alterations of choline metabolism observed in high and low grade gliomas. Copyright

Apoptosis but Not Other Activation Events Is Inhibited by a Mutation in the Transmembrane Domain of T Cell Receptor β That Impairs CD3ζ Association

The transmembrane domain of T cell receptor (TCR) β contains a conserved immunoreceptor tyrosine-based activation-like motif consisting of a duplicated YXXL sequence. The motif is also present in TCRγ, the equivalent chain to TCRβ in γδ T lymphocytes but is absent in TCRα and TCRδ. To determine the putative role of this sequence in TCR·CD3 complex assembly and signal transduction, a TCRβ chain cDNA was mutated in the C-terminal tyrosine of the motif, cloned in an expression vector, and transfected into TCRβ-negative Jurkat cells. Transfectants of the mutated chain (MUT) expressed, on average, much less TCR·CD3 complex on the membrane than wild type TCRβ transfectants. Radiolabeling experiments suggested that the mutation caused a loose association of the CD3ζ chain resulting in a defective assembly. However, stimulation of high TCR·CD3 expressing wild type and MUT clones with monoclonal antibodies and Staphylococcus aureus enterotoxin B resulted in similar levels of CD25 and CD69 expression, interleukin-2 secretion, and TCR·CD3 complex down-regulation. By contrast, MUT cells were clearly resistant to activation-induced cell death, and they did not express CD95-ligand upon activation. These results suggest a differentiated intracellular signaling pathway leading to apoptosis in which Tyr-TM11 of the immunoreceptor tyrosine-based activation motif-like motif and CD3ζ appear to be involved.

Early effects of copper accumulation on methionine metabolism

Abstract.Wilson’s disease is characterized by longterm hepatic accumulation of copper leading to liver disease with reduction of S-adenosylmethionine synthesis. However, the initial changes in this pathway remain unknown and constitute the objective of the present study. Using the Long Evans Cinnamon rat model, early alterations were detected in the mRNA and protein levels, as well as in the activities of several enzymes of the methionine cycle. Notably, the main change was a redox-mediated 80% decrease in the mRNA levels of the methionine adenosyltransferase regulatory subunit as compared to the control group. Moreover, changes in S-adenosylmethionine, S-adenosylhomocysteine, methionine and glutathione levels were also observed. In addition, in vitro experiments show that copper affects the activity and folding of methionine adenosyltransferase catalytic subunits. Taken together, these observations indicate that early copper accumulation alters methionine metabolism with a pattern distinct from that described previously for other liver diseases.

Determination of enzymatic activities and metabolites in microliter sample size

A new generation of spectrophotometers able to measure a wide range of absorbance in microliter aliquots is currently used for the determination of DNA, RNA, and proteins. The object of this article is to show that these instruments could be easily adapted for routine evaluation of enzymes and metabolites in 1-2-microl volumes of biological samples.

An AFLP based method for the detection and identification of indigenous yeast in complex must samples without a microbiological culture

Ribera de Duero Spanish wines are appreciated worldwide for their organoleptic characteristics; however, the wine market is very competitive, and the demand for high quality natural wines has been increasing in recent years. The microbiology of the process, specifically the yeasts involved in the alcoholic fermentation, constitutes an essential element directly related to the complexity and quality of the wine. Our work has focused on the development of a procedure to identify the indigenous wine yeasts present in complex samples of must and wine, without requiring colony isolation or a microbiological culture. The procedure is based on the use of AFLP molecular markers. The AFLP allele profiles obtained from complex samples are compared with the species-specific ones previously determined and included in a database using a sorting algorithm. The system allows a fast and efficient identification of yeast species and strains present in complex must and wine samples. This information can then be used by the enologists during the fermentation process in order to obtain signed wines.