The BTB (bric-a-brac, tramtrak and broad complex) ⁄ POZ (poxvirus zinc finger) domain is a proteinprotein interaction domain first described in several proteins of Drosophila melanogaster and poxvirus [1,2]. BTB ⁄ POZ domain-containing proteins constitute a diverse group of proteins involved in transcriptional repression, cytoskeletal regulation, and ion channel function [3]. More recently, some BTB proteins have been characterized as substrate-specific adaptors for cullin(CUL)3-based E3 ligases [4][5][6][7]. The BTB domain of these substrate-specific adaptors binds to CUL3, whereas additional domains in these polypeptides, such as zinc fingers, meprin and traf homology (MATH) domain, and Kelch repeats, work as substrate recognition domains. The first protein shown to be regulated by a CUL3 ligase was MEI-1 in Caenorhaditis elegans. This protein is part of the katanin-like microtubule severing complex [5,6] Potassium channel tetramerization domain (KCTD) proteins contain a bric-a-brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage-gated potassium channels. Some BTB-domain-containing proteins have been shown recently to participate as substrate-specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty-two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post-transcriptionally in peripheral blood lymphocytes stimulated through the T-cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N-terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate-specific adaptor for cullin3-based E3 ligases.Abbreviations AU, arbitrary unit; BTB, bric-a-brac, tramtrak and broad complex; CT, cycle threshold; CUL, cullin; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; IL-2, interleukin-2; KCTD, potassium channel tetramerization domain; MATH, meprin and traf homology; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin; PMA, 4b-phorbol 12-myristate 13-acetate; POZ, poxvirus zinc finger; Ub, ubiquitin.

The p21 is a downstream effector of p53/ p73 and belongs to the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs). It is, therefore, a potential tumor suppressor gene and probably plays an important role in tumor development. Moreover, reduced expression of p21 has been reported to have prognostic value in several human malignancies. In contrast with other CDKIs, mutational inactivation of p21 is infrequent, but gene inactivation by an alternative mechanism seems to be the general pathway. In this study, we analyzed the methylation status of the p21 promoter region using semiquantitative polymerase chain reaction in 124 patients with acute lymphoblastic leukemia (ALL). We observed p21 hypermethylation in bone marrow cells from 41% (51 of 124) of ALL patients. Hypermethylation within promoter strongly correlated with decreased p21 messenger RNA expression in tumoral cells. Clinical, molecular, and laboratory features and complete remission rate did not differ significantly between hypermethylated and normally methylated patients. Estimated diseasefree survival (DFS) and overall survival at 7 and 9 years, respectively, were 59% and 65% for healthy patients and 6% and 8% for hypermethylated patients (P ‫؍‬ .00001 and P ‫؍‬ .006). Multivariate analysis of potential prognostic factors demonstrated that p21 methylation status was an independent prognostic factor in predicting DFS (P ‫؍‬ .0001). Our results indicate that the p21 gene is subject to methylation regulation at the transcription level in ALL and seems to be an important factor in predicting the clinical outcome of these patients. (Blood. 2002;99:2291-2296

We address the specific role of cytoplasmic Ca 2؉ overload as a cell death trigger by expressing a receptoroperated specific Ca 2؉ channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca 2؉ uptake through the VR1 channel, but not capacitative Ca 2؉ influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca 2؉ ] rises, exposure of phosphatidylserine, and cell death. Ca 2؉ influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca 2؉ -induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca 2؉ influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.Release of mitochondrial intermembrane proteins to the cytosol is a fundamental step in the cell death machinery during apoptosis and necrosis (1). Among other triggers, massive calcium uptake into isolated mitochondria induces the collapse of the mitochondrial membrane potential (⌬⌿ m ) 1 (1), the opening of the mitochondrial permeability transition pore or megachannel (PTP) and the release of proapoptotic factors such as cytochrome c (2) and/or apoptosis inducer factor (AIF) (3). It has been shown that high [Ca 2ϩ ] (100 -500 M) is necessary for PTP opening in isolated mitochondria (1) ] c ) (6), its extent critically depends on both the magnitude and duration of the [Ca 2ϩ ] c rise (7). In the ischemic/excitotoxic stress model, the precise role of Ca 2ϩ overload in neuronal cell death is too complex to trace (5). Stress stimuli often produce, in addition to Ca 2ϩ entry through the plasma membrane, other effects, including (i) changes in membrane potential that can activate a variety of ion channels, (ii) metabolic changes that may lead to free radical production, (iii) activation of kinases, and (iv) release of Ca 2ϩ from the intracellular Ca 2ϩ stores. Release of Ca 2ϩ from the stores may be particularly relevant, as Ca 2ϩ depletion of the endoplasmic reticulum (ER) blocks protein synthesis and is proapoptotic per se (8, 9).Apoptosis is induced through Ca 2ϩ -dependent pathways in several cell types. This raises the question of whether Ca 2ϩ influx is sufficient for cell death. An apoptotic pathway involving up-regulation of the FasL gene has been extensively studied in T cells (10 -12). This activation-induced cell death (AICD) pathway can be triggered by receptors that stimulate phosphoinositide turnover, such as the T cell receptor or the human muscarinic...

One of the most important determinants of meat quality is the intramuscular fat (IMF) content. The development of high-throughput techniques as RNA-seq allows identifying gene pathways and networks with a differential expression (DE) between groups of animals divergent for a particular trait. The Iberian pig is characterized by having an excellent meat quality and a high content of intramuscular fat. The objectives of the present study were to analyze the longissimus dorsi transcriptome of purebred Iberian pigs divergent for their IMF breeding value to identify differential expressed genes and regulatory factors affecting gene expression. RNA-seq allowed identifying ∼10,000 of the 25,878 annotated genes in the analyzed samples. In addition to this, 42.46% of the identified transcripts corresponded to newly predicted isoforms. Differential expression analyses revealed a total of 221 DE annotated genes and 116 DE new isoforms. Functional analyses identified an enrichment of overexpressed genes involved in lipid metabolism (FASN, SCD, ELOVL6, DGAT2, PLIN1, CIDEC, and ADIPOQ) in animals with a higher content of IMF and an enrichment of overexpressed genes related with myogenesis and adipogenesis (EGR1, EGR2, EGR3, JUNB, FOSB, and SEMA4D) in the animals with a lower content of IMF. In addition to this, potential regulatory elements of these DE genes were identified. Co-expression networks analyses revealed six long non-coding RNAs (lncRNAs) (ALDBSSCG0000002079, ALDBSSCG0000002093, ALDBSSCG0000003455, ALDBSSCG0000004244, ALDBSSCG0000005525, and ALDBSSCG0000006849) co-expressed with SEMA4D and FOSB genes and one (ALDBSSCG0000004790) with SCD, ELOVL6, DGAT2, PLIN1, and CIDEC. Analyses of the regulatory impact factors (RIFs) revealed 301 transcriptionally regulatory factors involved in expression differences, with five of them involved in adipogenesis (ARID5B, CREB1, VDR, ATF6, and SP1) and other three taking part of myogenesis and development of skeletal muscle (ATF3, KLF11, and MYF6). The results obtained provide relevant insights about the genetic mechanisms underlying IMF content in purebred Iberian pigs and a set of candidate genes and regulatory factors for further identification of polymorphisms susceptible of being incorporated in a selection program.

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