Regulation of c-fos expression by RNA polymerase elongation competence.

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.

Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway.

Apoptosis, differentiation, and proliferation are cellular responses which play a pivotal role in wound healing. During this process PPARbeta translates inflammatory signals into prompt keratinocyte responses. We show herein that PPARbeta modulates Akt1 activation via transcriptional upregulation of ILK and PDK1, revealing a mechanism for the control of Akt1 signaling. The resulting higher Akt1 activity leads to increased keratinocyte survival following growth factor deprivation or anoikis. PPARbeta also potentiates NF-kappaB activity and MMP-9 production, which can regulate keratinocyte migration. Together, these results provide a molecular mechanism by which PPARbeta protects keratinocytes against apoptosis and may contribute to the process of skin wound closure.

Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa.

In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.

Transferencia en la adquisición de segundas lenguas. Un estudio con alumnado rumano en un contexto bilingüe = Transfer in second language acquisition. A study with Romanian schoolchildren in a bilingual context

Situados en el contexto catalán, el artículo estudia la influencia de la L1 (rumano) en algunos aspectos morfosintácticos de la adquisición de las L2s. Para ello se analizan las competencias lingüísticas en catalán y castellano de un grupo de escolares cuya L1 es el rumano y que cursan 2.º y 4.º de ESO. Los datos muestran que los alumnos cuya L1 es el rumano, a pesar de dominar una lengua románica cercana a las lenguas de aprendizaje (L2/L3), presentan dificultades comparables a otros colectivos con otras lenguasde origen. Por otra parte, nuestra investigación confirma que la L1 de este alumnado juega un importante papel en la adquisición de ambas lenguas, concluyendo que parte de los errores hallados son aquellos que se basan en estructuras de la lengua propia

Localized in vivo hyperpolarization transfer sequences.

In vivo localized and fully adiabatic homonuclear and heteronuclear polarization transfer experiments were designed and performed in the rat brain at 9.4 T after infusion of hyperpolarized sodium [1,2-(13)C(2)] and sodium [1-(13)C] acetate. The method presented herein leads to highly enhanced in vivo detection of short-T(1) (13)C as well as attached protons. This indirect detection scheme allows for probing additional molecular sites in hyperpolarized substrates and their metabolites and can thus lead to improved spectral resolution such as in the case of (13)C-acetate metabolism.

Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

In vivo imaging of T cell delivery to tumors after adoptive transfer therapy.

Adoptive transfer therapy of in vitro-expanded tumor-specific cytolytic T lymphocytes (CTLs) can mediate objective cancer regression in patients. Yet, technical limitations hamper precise monitoring of posttherapy T cell responses. Here we show in a mouse model that fused single photon emission computed tomography and x-ray computed tomography allows quantitative whole-body imaging of (111)In-oxine-labeled CTLs at tumor sites. Assessment of CTL localization is rapid, noninvasive, three-dimensional, and can be repeated for longitudinal analyses. We compared the effects of lymphodepletion before adoptive transfer on CTL recruitment and report that combined treatment increased intratumoral delivery of CTLs and improved antitumor efficacy. Because (111)In-oxine is a Food and Drug Administration-approved clinical agent, and human SPECT-CT systems are available, this approach should be clinically translatable, insofar as it may assess the efficacy of immunization procedures in individual patients and lead to development of more effective therapies.

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

Leishmania RNA virus: when the host pays the toll.

The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic "metastatic factors" or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically for the maintenance of LRV infection in Leishmania parasites, which is often at the expense of its human host.

A comparison of methods for differential expression analysis of RNA-seq data.

BACKGROUND: Finding genes that are differentially expressed between conditions is an integral part of understanding the molecular basis of phenotypic variation. In the past decades, DNA microarrays have been used extensively to quantify the abundance of mRNA corresponding to different genes, and more recently high-throughput sequencing of cDNA (RNA-seq) has emerged as a powerful competitor. As the cost of sequencing decreases, it is conceivable that the use of RNA-seq for differential expression analysis will increase rapidly. To exploit the possibilities and address the challenges posed by this relatively new type of data, a number of software packages have been developed especially for differential expression analysis of RNA-seq data. RESULTS: We conducted an extensive comparison of eleven methods for differential expression analysis of RNA-seq data. All methods are freely available within the R framework and take as input a matrix of counts, i.e. the number of reads mapping to each genomic feature of interest in each of a number of samples. We evaluate the methods based on both simulated data and real RNA-seq data. CONCLUSIONS: Very small sample sizes, which are still common in RNA-seq experiments, impose problems for all evaluated methods and any results obtained under such conditions should be interpreted with caution. For larger sample sizes, the methods combining a variance-stabilizing transformation with the 'limma' method for differential expression analysis perform well under many different conditions, as does the nonparametric SAMseq method.

Curcumin, quercetin, and tBHQ modulate glutathione levels in astrocytes and neurons: importance of the glutamate cysteine ligase modifier subunit.

A decrease in GSH levels, the main redox regulator, can be observed in neurodegenerative diseases as well as in schizophrenia. In search for substances able to increase GSH, we evaluated the ability of curcumin (polyphenol), quercetin (flavonoid), and tert-butylhydroquinone (tBHQ) to up-regulate GSH-synthesizing enzymes. The gene expression, activity, and product levels of these enzymes were measured in cultured neurons and astrocytes. In astrocytes, all substances increased GSH levels and the activity of the rate-limiting synthesizing enzyme, glutamate cysteine ligase (GCL). In neurons, curcumin and to a lesser extent tBHQ increased GCL activity and GSH levels, while quercetin decreased GSH and led to cell death. In the two cell types, the gene that showed the greatest increase in its expression was the one coding for the modifier subunit of GCL (GCLM). The increase in mRNA levels of GCLM was 3 to 7-fold higher than that of the catalytic subunit. In astrocytes from GCLM-knock-out mice showing low GSH (-80%) and low GCL activity (-50%), none of the substances succeeded in increasing GSH synthesis. Our results indicate that GCLM is essential for the up-regulation of GCL activity induced by curcumin, quercetin and tBHQ.

Non-surgical transfer of 4',6'-diamidino-2-phenylindole-stained equine embryos

Selostus: Hevosen alkioiden värjääminen DAPI:lla ennen vastaanottajaan siirtoa