IRES mediated translational regulation of p53 isoforms

p53 is a well known tumor suppressor protein that plays a critical role in cell cycle arrest and apoptosis. It has several isoforms which are produced by transcriptional and posttranscriptional regulatory mechanisms. p53 mRNA has been demonstrated to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform N-p53 by the use of alternative translation initiation sites. The mechanism of translation regulation of these two isoforms was further elucidated by the discovery of IRES elements in the p53 mRNA. These two IRESs were shown to regulate the translation of p53 and N-p53 in a distinct cell-cycle phase-dependent manner. This review focuses on the current understanding of the regulation of p53 IRES mediated translation and the role of cis and trans acting factors that influence expression of p53 isoforms. (C) 2013 John Wiley & Sons, Ltd.

Roles of the troponin isoforms during indirect flight muscle development in Drosophila

Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, a majority of the myofibrillar proteins in the indirect flight muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

Reversible induction of translational isoforms of p53 in glucose deprivation

Tumor suppressor protein p53 is a master transcription regulator, indispensable for controlling several cellular pathways. Earlier work in our laboratory led to the identification of dual internal ribosome entry site (IRES) structure of p53 mRNA that regulates translation of full-length p53 and Delta 40p53. IRES-mediated translation of both isoforms is enhanced under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum stress, oncogene-induced senescence and cancer. In this study, we addressed nutrient-mediated translational regulation of p53 mRNA using glucose depletion. In cell lines, this nutrient-depletion stress relatively induced p53 IRES activities from bicistronic reporter constructs with concomitant increase in levels of p53 isoforms. Surprisingly, we found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. Importantly under these conditions polypyrimidine-tract-binding protein, an established p53 ITAF did not show nuclear-cytoplasmic relocalization highlighting the novelty of SMAR1-mediated control in stress. In vivo studies in mice revealed starvation-induced increase in SMAR1, p53 and Delta 40p53 levels that was reversible on dietary replenishment. SMAR1 associated with p53 IRES sequences ex vivo, with an increase in interaction on glucose starvation. RNAi-mediated-transient SMAR1 knockdown decreased p53 IRES activities in normal conditions and under glucose deprivation, this being reflected in changes in mRNAs in the p53 and Delta 40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis such as p21, TIGAR and Bax. This study provides a new physiological insight into the regulation of this critical tumor suppressor in nutrient starvation, also suggesting important functions of the p53 isoforms in these conditions as evident from the downstream transcriptional target activation.

Efecto de concentraciones de aceite de algodón sobre la viabilidad, patogenicidad, estabilidad de la cepa CB-32 de Beauveria bassiana (Bals y vuils)

En Nicaragua, Plutella xylostella es la plaga que causa mayores daños al cultivo de repollo (Brassica oleracea) lo que implica una gran inversión en el control de la misma. Para contribuir a la búsqueda de alternativas para el Manejo integrado de esta plaga se realizó este estudio en las instalaciones del Centro Nacional de Protección Vegetal (CENAPROVE) y en la Universidad Nacional Agraria durante el periodo comprendido entre Febrero 1991 y Febrero 1992. Se evaluó la Cepa CB-32 del hongo entomopatógeno Beauveria bassiana (Bals & Vuils) en 6 formulaciones conteniendo 0%, 2%, 4%, 6%. 8% y 10% de aceite de algodón en agua sobre larvas del segundo instar de P. xylostella. La concentración de conidias en las formulaciones fué de 3.35x108 En un rango de tiempo de 14-30 horas después de la inoculación en PDA se distinguieron con un mayor porcentaje de germinación de conidias las formulaciones con O y 2% de aceite de algodón. Las mismas demostraron mayor sedimentación reportando 0.2 y 0.5 ml seis horas después de ser sometidas a reposo. Para la evaluación del efecto de la radiación solar sobre las conidias de B. bassiana se realizó un análisis de varianza y pruebas de Tukey con un 95% de confianza, las respaldan que las formulaciones pueden separarse en cuatro categorías estadísticas diferentes de las cuales en primer lugar tenemos las formulaciones con O y 2% de aceite que presentan el mayor porcentaje de germinación de conidias expuestas a 24 horas de radiación solar (86.61% y 80.36%) respectivamente. A la variable patogenicidad de las conidias de B. bassiana en huésped de Plutella xylostella se efectúo un análisis de varianza y prueba de Tuckey y resulto que las formulaciones pueden clasificarse en 3 categorías estadísticas, sobresaliendo la patogenicidad de las conidias de las formulaciones con O y 2% de aceite reportando 95 y 80% respectivamente. El tiempo letal para las formulaciones no varió provocando la mortalidad dos días después de la inoculación de las larvas por el método de inmersión.

GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease

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Differential activity of GSK-3 isoforms regulates NF-kappa B and TRAIL- or TNF alpha induced apoptosis in pancreatic cancer cells

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3 alpha or GSK-3 beta. In contrast, depletion of GSK-3 beta, but not GSK-3 alpha, sensitized PDA cell lines to TNF alpha-induced cell death. Further experiments demonstrated that TNF alpha-stimulated I kappa B alpha phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3 beta-deficient MEFs. Nonetheless, inhibition of GSK-3 beta function in MEFs or PDA cell lines impaired the expression of the NF-kappa B target genes Bcl-xL and cIAP2, but not I kappa B alpha. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3 beta targeted to the nucleus but not GSK-3 beta targeted to the cytoplasm, suggesting that GSK-3 beta regulates NF-kappa B function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3 beta overexpression and nuclear localization contribute to TNF alpha and TRAIL resistance via anti-apoptotic NF-kappa B genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.

SynGAP isoforms exert opposing effects on synaptic strength.

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5' rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing.

母鸡输卵管子宫部初级感觉神经元的CB-HRP法定位研究

应用CB-HRP溶液注入母鸡输卵管子宫部浆膜下,四甲基联苯胺(TMB)组织化学呈色,以探察母鸡输卵管子宫部初级感觉神经元的定位。结果表明:母鸡输卵管子宫部的初级感觉神经元位于双侧T1-LS13脊神经节、颈静脉神经节和结状神经节。标记细胞数分布不均,在体左侧多于体右侧,在脊神经节多于颈静脉神经节和结状神经节。在脊神经节内标记细胞有T5-LS1和LS8-LS11前后两个相对集中区,峰值分别在T7和LS11。说明尽管母鸡输卵管子宫部是单侧发育成熟脏器,但其感觉沿双侧脊神经和迷走神经传入中枢,以体左侧传入为主;且

用CB微核法预测癌细胞的放射敏感性

用细胞质分裂阻断微核技术（简称ＣＢ微核法）研究了癌细胞的放射敏感性，结果表明；ＣＢ 微核法可用来预测癌细胞对重离子和γ射线的放射敏感性，具有省时且成功率高的特点。细胞 受重离子作用后，每双核细胞的微核与剂量的效应关系适合方程 Y＝ c＋ ｋＤｎ或 Y＝ｃ＋ａＤ＋βＤ２，受γ射线作用后，每双核细胞的微核与剂量的效应关系适合方程 Y＝ｃ＋ ａＤ。在相同剂量下，重离子效应高于ｒ射线效应。

辐射效应对LDPE/CB复合物电性能的影响

研究了γ射线辐照交联后的低密度聚乙烯 (LDPE) /碳黑 (CB)复合物在不同温度下的导电性能。应用扫描电子显微镜研究了不同剂量对复合体系正温度系数 (PTC)效应以及负温度系数(NTC)效应的影响。结果表明 ,辐射剂量对PTC和NTC效应的影响很大。在 4 0 0kGy时 ,NTC效应基本消除 ,当辐射剂量继续增加时 ,NTC效应又重新出现。

Characterization of the human and mouse ETV1/ER81 transcription factor genes: Role of the two alternatively spliced isoforms in the human

The Ets transcription factors of the PEA3 group - E1AF/PEA3, ETV1/ER81 and ERM - are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1α) or the absence (ETV1β) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1β) and the full-length isoform (human ETV1α) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1α and human ETV1β expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

Human E2F6 is alternatively spliced to generate multiple protein isoforms

E2F6 protein belongs to the family of the E2F transcription factors. Here, we showed that the human E2F6 gene contains nine exons distributed along 20.4kbp of genomic DNA on chromosome 2 leading to the transcription of six alternatively spliced E2F6 mRNAs that encode four different E2F6 proteins. Moreover, we identified an E2F6 pseudogene localized on chromosome 22 completely spliced and devoid of exons 2, 3, and 4, and part of exons 1 and 5. Definition of the transcriptional initiation site and sequence analysis show that the gene contains a TATA less, CAAT less, GC-rich promoter with multiple transcription start sites. Regulatory elements necessary for basal transcription reside within a 134bp fragment as determined by transient transfection experiments. © 2004 Elsevier Inc. All rights reserved.