DNA binding affinity and sequence permutation preference of the telomere protein from Euplotes crassus

Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single-strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins, we have characterized the DNA binding properties of the telomere end binding protein from Euplotes crassus (EcTEBP). EcTEBP and its predicted amino-terminal DNA-binding domain, EcTEBP-N, were expressed in Escherichia coli and purified. Each protein formed stoichiometric (1:1) complexes with single-strand DNA oligos derived from the precisely defined d(TTTTGGGGTTTTGG) sequence found at DNA termini in Euplotes. Dissociation constants for DNA x EcTEBP and DNA x EcTEBP-N complexes were comparable: K(D-DNA) = 38 +/- 2 nM for the full-length protein and K(D-DNA) = 60 +/- 4 nM for the N-terminal domain, indicating that the N-terminal domain retains a high affinity for DNA even in the absence of potentially stabilizing moieties located in the C-terminal domain. Rate constants for DNA association and DNA dissociation corroborated a slightly improved DNA binding performance for the full-length protein (ka = 45 +/- 4 microM(-1) s(-1), kd = 0.10 +/- 0.02 s(-1)) relative to that of the N-terminal domain (ka = 18 +/- 1 microM(-1) s(-1), kd = 0.15 +/- 0.01 s(-1)). Equilibrium dissociation constants measured for sequence permutations of the telomere repeat spanned the range of 55-1400 nM, with EcTEBP and EcTEBP-N binding most tightly to d(TTGGGGTTTTGG), the sequence corresponding to that of mature DNA termini. Additionally, competition experiments showed that EcTEBP recognizes and binds the telomere-derived 14-nucleotide DNA in preference to shorter 5'-truncation variants. Compared with the results for multisubunit complexes assembled with telomere single-strand DNA from Oxytricha nova, our results highlight the relative simplicity of the E. crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single-strand DNA.     

Archaeology, land use, pollen and restoration in the Yazoo Basin (Mississippi, USA)

Increasingly, modern ecologists are realizing that the history of ecological systems is crucially important for understanding the landscape and that human land use has a great impact on the trajectory of ecosystems. The Yazoo Basin of Mississippi (USA) is one area in which palaeoecological and archaeological research has been done, but at a time when interpretations of the results relied on paradigms that gave credence only to climate change as a causal factor in explaining vegetation histories of plant communities. This paper uses knowledge of ecological processes and patterns of plant colonization and succession to make testable expectations for vegetation composition and change related to human action. An existing pollen record from the area is then examined in light of these expectations and reveals evidence that humans were an integral part of the ecosystem in this area, influencing the trajectory of vegetation history over thousands of years.     

Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.     

Evaluation of cDNA Libraries from Different Developmental Stages of Schistosoma mansoni for Production of Expressed Sequence Tags (ESTs)

A comparative study of the gene expression profile in differentdevelopmental stages of Schistosoma mansoni has been initiatedbased on the expressed sequence tag(EST) approach. A total of1401 ESTs were generated from seven different cDNA librariesconstructed from four distinct stages of the parasite life cycle.The libraries were first evaluated for their quality for a large-scalecDNA sequencing program. Most of them were shown to have lessthan 20% useless clones and more than 50% new genes. The redundancyof each library was also analyzed, showing that one adult wormcDNA library was composed of a small number of highly frequentgenes. When comparing ESTs from distinct libraries, we coulddetect that most genes were present only in a single library,but others were expressed in more than one developmental stageand may represent housekeeping genes in the parasite. When consideringonly once the genes present in more than one library, a totalof 466 unique genes were obtained, corresponding to 427 newS. mansoni genes. From the total of unique genes, 20.2% wereidentified based on homology with genes from other organisms,8.3% matched S. mansoni characterized genes and 71.5% representunknown genes.     

Leptin receptor expression and signaling in lymphocytes: kinetics during lymphocyte activation, role in lymphocyte survival, and response to high fat diet in mice

Leptin has direct effects not only on neuroendocrine function and metabolism, but also on T cell-mediated immunity. We report in this study that leptin receptor (ObR) is expressed on resting normal mouse CD4(+), CD8(+), B cells, and monocyte/macrophages. ObR expression is up-regulated following cell activation, but with different kinetics, in different lymphocyte subsets. Leptin binding to ObR results in increased STAT-3 activation in T cells, with a different activation pattern in resting vs anti-CD3 Ab stimulated T cells. Leptin also promotes lymphocyte survival in vitro by suppressing Fas-mediated apoptosis. B lymphocytes appear to be more susceptible to the antiapoptotic effects of leptin, and they show higher surface expression of ObR, compared with T cells. Moreover, CD4(+) T cells isolated from ObR-deficient mice displayed a reduced proliferative response, compared with normal controls. Furthermore, ObR/STAT-3-mediated signaling in T lymphocytes is decreased in the diet-induced obese mouse model of obesity and leptin resistance. In summary, our findings show that the ObR is expressed on normal mouse lymphocyte subsets, that leptin plays a role in lymphocyte survival, and that leptin alters the ObR/STAT-3-mediated signaling in T cells. Taken together, our data further support the notion that nutritional status acting via leptin-dependent mechanisms may alter the nature and vigor of the immune response.