Keeping the Noise Down in ES Cells

A new report (Szutorisz et al., 2006) suggests that embryonic stem (ES) cells adopt an unusual strategy to remain perpetually poised for differentiation. Apparently, ES cells use the proteasome to target transcriptional preinitiation complexes, thus minimizing transcriptional noise at genes that promote differentiation

Pluripotency Network in Embryonic Stem Cells: Maybe Leibniz Was Right All Along

The transcription factors Tcf3 and Nanog regulate many genes in embryonic stem cells, but according to two reports in this issue of Cell Stem Cell (Festuccia et al., 2012, Martello et al., 2012), only one, Esrrb, encoding an orphan nuclear hormone receptor, truly matters in the maintenance of self-renewal

Development: Sketch for a Theory of Oct4

SummaryHow is it that Oct4, a transcription factor that controls pluripotency in stem cells, also controls lineage specification? A recent study investigating common Oct4 targets in vertebrate species indicates an evolutionarily conserved role in mediating cell adhesion. This finding may help decipher Oct4’s versatility in governing stem cell behaviors

Blimp1 Expression Predicts Embryonic Stem Cell Development In Vitro

SummaryDespite recent critical insights into the pluripotent state of embryonic stem cells (ESCs), there is little agreement over the inaugural and subsequent steps leading to its generation [1–4]. Here we show that inner cell mass (ICM)-generated cells expressing Blimp1, a key transcriptional repressor of the somatic program during germ cell specification [5, 6], emerge on day 2 of blastocyst culture. Single-cell gene expression profiling indicated that many of these Blimp1-positive cells coexpress other genes typically associated with early germ cell specification. When genetically traced in vitro, these cells acquired properties normally associated with primordial germ cells. Importantly, fate-mapping experiments revealed that ESCs commonly arise from Blimp1-positive precursors; indeed, prospective sorting of such cells from ICM outgrowths increased the rate of ESC derivation more than 9-fold. Finally, using genetic ablation or distinct small molecules [7, 8], we show that epiblast cells can become ESCs without first acquiring Blimp1 positivity. Our findings suggest that the germ cell-like state is facultative for the stabilization of pluripotency in vitro. Thus, the association of Blimp1 expression with ESC development furthers understanding of how the pluripotent state of these cells is established in vitro and suggests a means to enhance the generation of new stem cell lines from blastocysts

Does FXIII Deficiency Impair Wound Healing after Myocardial Infarction?

Inadequate healing of myocardial infarction may contribute to local expansion of the infarct, frequently leading to chamber dilation, heart failure, or myocardial rupture. Experimental evidence in mouse models suggests that Factor XIII might play a key role in wound healing, and low persistent values lead to increased incidence of cardiac rupture following myocardial infarction. Here we would like to share our initial clinical experiences with strikingly similar observations in patients with this grave disease, and compare these observations to experimental findings

Human Coronary Artery Remodeling, Beginning and End of the Atherosclerotic Process

BACKGROUND, AIMS OF THE STUDY: The objective of the study was to relate the progress of coronary artery remodeling to the earliest stages of the atherosclerotic process. For this purpose, a mathematical model for description of dimensional change of the coronary artery wall and its constituent components was developed and applied. MATERIALS AND METHODS: The study used coronary artery samples randomly taken from each of 83 consecutive, unselected postmortems. All samples were routinely fixed and processed to paraffin for the preparation of right-angled, 5-micron sections, routinely stained and mounted for subsequent analysis. Computer assisted image analysis, using 32 systematic random, radial sampling lines, was used for interactive measurements of distance from centre of lumen to points defining intima, media and adventitia thickness along the radial intercept, which were subsequently tabled for analysis of variance, calculations of (group –vessel) means, and related to stage of pathology. RESULTS: Pre-atherosclerotic changes, before any localised changes in especially intima dimensions, are found, consisting of a process of gradual vascular widening, associated with temporally at least partly dissociated increases in width, which as a fraction of total vessel radius show a phased process. In these, the intima first increases, subsequently remains stable, and finally reduces in width proportionally to the increasing diameter. The media shows a similar initial increase, on average stabilising in the third phase after reaching a plateau value in the second. The adventitia, already increasing in phase 1, continues to increase in phase 2, accelerating in phase 3. The complex process, as found, occurs systematically in all vessels, is distributed circumferentially, and precedes the development of localised lesions of the intima. CONCLUSIONS: The findings suggest the existence of a diffuse complex of changes, consisting of a gradual vascular widening followed by narrowing, with associated mural changes reflecting the atherosclerotic process

RONIN Is an Essential Transcriptional Regulator of Genes Required for Mitochondrial Function in the Developing Retina

SummaryA fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression

A Novel Motif Identified in Dependence Receptors

Programmed cell death signaling is a critical feature of development, cellular turnover, oncogenesis, and neurodegeneration, among other processes. Such signaling may be transduced via specific receptors, either following ligand binding—to death receptors—or following the withdrawal of trophic ligands—from dependence receptors. Although dependence receptors display functional similarities, no common structural domains have been identified. Therefore, we employed the Multiple Expectation Maximization for Motif Elicitation and the Motif Alignment and Search Tool software programs to identify a novel transmembrane motif, dubbed dependence-associated receptor transmembrane (DART) motif, that is common to all described dependence receptors. Of 3,465 human transmembrane proteins, 25 (0.7%) display the DART motif. The predicted secondary structure features an alpha helical structure, with an unusually high percentage of valine residues. At least four of the proteins undergo regulated intramembrane proteolysis. To date, we have not identified a function for this putative domain. We speculate that the DART motif may be involved in protein processing, interaction with other proteins or lipids, or homomultimerization

Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries

Random peptide libraries displayed on the ribosome are becoming a new tool for the in vitro selection of biologically relevant macromolecules, including epitopes, antagonists, enzymes, and cell-surface receptors. Ribosome display is a cell-free system of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes) by the formation of stable protein-ribosome-mRNA complexes and permitting the selection of a functional nascent protein by iterative cycles of panning and reverse transcription-polymerase chain reaction (RT-PCR) amplification in vitro. The complexity of the random peptide library is critical for the success of a panning experiment; greater the diversity of sequences within the library, the more likely it is that the library comprises sequences that can bind a given target with specific affinity. Here, we have used the cell-free system Escherichia coli S30 lysate to construct high-complexity random peptide libraries (>1014 independent members) by introducing strategies that are different from the methods described by Mattheakis et al. and Lamla et al. The key step in our method is to produce nanomole (nmol) amounts of DNA elements that are necessary for in vitro transcription/translation by using PCR but not plasmid DNA. Library design strategies and protocols that facilitate rapid identification are also presented

The Effect of Oxidant and the Non-Oxidant Alteration of Cellular Thiol Concentration on the Formation of Protein Mixed-Disulfides in HEK 293 Cells

Cellular molecules possess various mechanisms in responding to oxidant stress. In terms of protein responses, protein S-glutathionylation is a unique post-translational modification of protein reactive cysteines forming disulfides with glutathione molecules. This modification has been proposed to play roles in antioxidant, regulatory and signaling in cells under oxidant stress. Recently, the increased level of protein S-glutathionylation has been linked with the development of diseases. In this report, specific S-glutathionylated proteins were demonstrated in human embryonic kidney 293 cells treated with two different oxidative reagents: diamide and hydrogen peroxide. Diamide is a chemical oxidizing agent whereas hydrogen peroxide is a physiological oxidant. Under the experimental conditions, these two oxidants decreased glutathione concentration without toxicity. S-glutathionylated proteins were detected by immunoblotting and glutathione concentrations were determined by high performance liquid chromatography. We further show the effect of alteration of the cellular thiol pool on the amount of protein S-glutathionylation in oxidant-treated cells. Cellular thiol concentrations were altered either by a specific way using buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis or by a non-specific way, incubating cells in cystine-methionine deficient media. Cells only treated with either buthionine sulfoximine or cystine-methionine deficient media did not induce protein S-glutathionylation, even though both conditions decreased 65% of cellular glutathione. Moreover, the amount of protein S-glutathionylation under both conditions in the presence of oxidants was not altered when compared to the amount observed in regular media with oxidants present. Protein S-glutathionylation is a dynamic reaction which depends on the rate of adding and removing glutathione. Phenylarsine oxide, which specifically forms a covalent adduct with vicinal thiols, was used to determine the possible role of vicinal thiols in the amount of glutathionylation. Our data shows phenylarsine oxide did not change glutathione concentrations, but it did enhance the amount of glutathionylation in oxidant-treated cells