Pedro S. Lazo

Signal transduction by tumor necrosis factor receptors.

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.

Melatonin blocks the activation of estrogen receptor for DNA binding

The present study shows that melatonin prevents, within the first cell cycle, the estradiol‐induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol–estrogen receptor complexes to estrogen‐responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol–estrogen receptor complex to the estrogen responsive element.—Rato, A. G., Pedrero, J. G., Martínez, M. A., del Rio, B., Lazo, P. S., Ramos, S. Melatonin blocks the activation of estrogen receptor for DNA binding. FASEB J. 13, 857–868 (1999)

Amplification of ERBB oncogenes in squamous cell carcinomas of the head and neck

The activation of ERBB oncogenes has been described in various human tumours, including squamous cell carcinomas of the head and neck (SCCHN), and, in some of them, it has been correlated with a poor prognosis. Tissue samples from 59 patients with SCCHN were studied. After DNA extraction, the ERBB1, ERBB2 and ERBB3 copy number in tumour samples was estimated with the polymerase chain reaction (PCR) method. The PCR products were analysed by agarose gel electrophoresis and quantified by image analysis techniques. 9 (15%) cases presented with ERBB1 amplification, which was correlated with lymph node involvement (P = 0.04), poorly differentiated tumours (P = 0.03) and a hypopharyngeal primary site (P = 0.035). No correlation among amplification status, recurrence, metastases and survival was observed, although this may be due to the small number of patients in the amplified group. None of the 59 cases presented amplification of ERBB2 and ERBB3 oncogenes.

NF-κB Signal Triggering and Termination by Tumor Necrosis Factor Receptor 2

Tumor necrosis factor receptor 2 (TNFR2) activates transcription factor κB (NF-κB) and c-Jun N-terminal kinase (JNK). The mechanisms mediating these activations are dependent on the recruitment of TNF receptor-associated factor 2 (TRAF2) to the intracellular region of the receptor. TNFR2 also induces TRAF2 degradation. We show that in addition to the well characterized TRAF2 binding motif 402-SKEE-405, the human receptor contains another sequence located at the C-terminal end (amino acids 425–439), which also recruits TRAF2 and activates NF-κB. In addition to that, human TNFR2 contains a conserved region (amino acids 338–379) which is responsible for TRAF2 degradation and therefore of terminating NF-κB signaling. TRAF2 degradation and the lack of NF-κB activation when both TNFR1 and TNFR2 are co-expressed results in an enhanced ability of TNFR1 to induce cell death, showing that the cross-talk between both receptors is of a great biological relevance. Induction of TRAF2 degradation appears to be independent of TRAF2 binding to the receptor. Amino acids 343-TGSSDSS-349 are essential for inducing TRAF2 degradation because deletion mutants of this region or point mutations at serine residues 345 and 346 or 348 and 349 obliterate the ability of TNFR2 to induce TRAF2 degradation.

Variability of Genetic Alterations in Different Sites of Head and Neck Cancer

Objective Tumors arising from different sites of the head and neck area have different clinical behavior. However, most of the studies on genetic alterations in head and neck squamous cell carcinomas do not make a distinction between the sites within this area. The objective of this study is to compare the genetic alterations in three different sites of the head and neck (larynx, oropharynx, and hypopharynx).

α-galactosidase (melibiase) from Saccharomyces carlsbergensis: Structural and kinetic properties

Abstract This report describes structural and kinetic properties of the purified α-galactosidase from Saccharomyces carlsbergensis . This galactosidase has many similar properties to other exocellular enzymes in yeast which have been reported. Its molecular weight of 300,000 is comparable; it has similar carbohydrate content (57%) and amino acid and carbohydrate composition. That is, 35% of its amino acid residues can be accounted for by threonine, serine, and aspartic acid. Its carbohydrate composition is primarily mannose (90–95%) with approximately 7% glucose and 1% glucosamine. The enzyme is very stable to both acidic and alkaline conditions as well as to heating to 50 °C. α-Galactosidase remains active after incubation with as much as 1% sodium dodecyl sulfate at 30 °C. However, the enzyme is denatured with urea and guanidine hydrochloride. The loss of activity is proportional to the urea concentration, the nondenatured enzyme being responsible for the remaining activity. Inactivation by urea is partially reversible. With urea or 60 °C heat denaturation, the enzyme dissociates into two types of subunits as revealed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Thus, α-galactosidase is the first external enzyme from yeast in which an oligomeric structure is reported. The enzyme catalyzes the hydrolysis of p -nitrophenyl-α- d -galactoside, melibiose, and raffinose with similar pH optima and V max . However, the affinity is 20-fold lower for raffinose than for the other two substrates. Sugars having the same configuration in carbons 2, 3, and 4 as galactose competitively inhibit the enzyme.

Primary effects of yeast killer toxin

Abstract Killer toxin from Saccharomyces cerevisiae binds to sensitive cells immediately after addition to the cells. However, 50% mortality was obtained only after 40 minutes. Although it is thought that a lag phase is required for the killer to exert its action, we report experiments showing that the killer starts affecting the cell immediately after binding. Thus, shortly after addition the toxin was able to inhibit the transport of L-|;3H|; leucine as well as that of protons which are cotransported with this amino-acid or with histidine. Moreover, killer toxin inhibited the pumping of protons to the medium by cells which were actively metabolizing glucose. These effects were a function of the concentration of toxin used. The results suggest that killer toxin acts by affecting the electrochemical proton gradient across the plasma membrane of yeast.

Molecular cloning of a mouse homologue for the Drosophila splicing regulator Tra2.

We report the identification of a mouse cDNA, SIG41, encoding a protein of 288 amino acids that is 45% identical (58% similar) to the Drosophila splicing regulator Tra2. SIG41 cDNA contains four polyadenylation signals whose alternative use gives rise to four types of transcripts (2.1, 2.0, 1.5, and 1.4 kb) in mouse cells. Northern analysis and RT‐PCR assays showed that SIG41 mRNA is present in virtually all the cell lines and tissues studied, with remarkable levels of expression in uterus and brain tissues. Differential stability of the SIG41 mRNAs was detected in mouse macrophage cells.

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.

Polyinosinic acid induces TNF and NO production as well as NF-κB and AP-1 transcriptional activation in the monocytemacrophage cell line RAW 264.7

Abstract.Objective: This study evaluates the poly inosinic acid (poly I)-induced activation in the murine monocytemacrophage cell line RAW 264.7, which led to an inflammatory phenotype.Material: RAW 264.7, and WEHI 164 cell lines were used.Results: The activation process is characterized by the acquisition of a mature macrophage morphology and the production of inflammatory mediators tumor necrosis factor (TNF) and nitric oxide (NO). The activation by poly I has distinctive features. Thus, poly I induced an increase in nuclear factor κB (NF-κB) transcriptional activity due to a long-term degradation of inhibitory NF-κB (IκB) β while lipopolysaccharide (LPS) induced the degradation of both IκBα and IκBβ. Poly I also induced an increase in activator protein 1 (AP-1) transcriptional activity, possibly due to the activation of the mitogen activated protein kinases (MAPKs) ERK, Jun N terminal kinase (JNK) and p38. Dextran sulphate (DS) efficiently inhibited the activation induced by poly I including the production of the inflammatory mediators. Dextran sulphate also inhibited AP-1 and NF-κB transcriptional activities in poly I-stimulated cells. RAW 264.7 cells express macrophage scavenger receptor 1 (Msr1) type I and Msr1 type II that are differently up-regulated upon treatment with poly I.Conclusions: The results presented demonstrate that the well-known blocker of scavenger receptors poly I activates macrophages to produce TNF and NO, triggering specific signal transduction pathways.