Springer Science and Business Media LLC

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues.

Human adipose-derived stem cells (huADSC) were generated from fat tissue of a 65-year-old male donor. Flow cytometry and reverse transcription polymerase chain reaction (RT-PCR) analyses indicated that the huADSC express neural cell proteins (MAP2, GFAP, nestin and β-III tubulin), neurotrophic growth factors (BDNF and GDNF), and the chemotactic factor CXCR4 and its corresponding ligand CXCL12. In addition, huADSC expressed the characteristic mesenchymal stem cell (MSC) markers CD29, CD44, CD73, CD90, CD105 and HLA class I. The huADSC were employed, via a right femoral vein injection, to treat rats inflicted with experimental intracerebral hemorrhage (ICH). Behavioral measurement on the experimental animals, seven days after the huADSC therapy, showed a significant functional improvement in the rats with stem cell therapy in comparison with rats of the control group without the stem cell therapy. The injected huADSC were detectable in the brains of the huADSC treated rats as determined by histochemistry analysis, suggesting a role of the infused huADSC in facilitating functional recovery of the experimental animals with ICH induced stroke.

Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), is a surface type I transmembrane core proteoglycan that is crucially involved in cell survival, migration and angiogenesis. NG2 is frequently used as a marker for the identification and characterization of certain cell types, but little is known about the mechanisms regulating its expression. In this review, we provide evidence that the regulation of NG2 expression underlies inflammation and hypoxia and is mediated by methyltransferases, transcription factors, including Sp1, paired box (Pax) 3 and Egr-1, and the microRNA miR129-2. These regulatory factors crucially determine NG2-mediated cellular processes such as glial scar formation in the central nervous system (CNS) or tumor growth and metastasis. Therefore, they are potential targets for the establishment of novel NG2-based therapeutic strategies in the treatment of CNS injuries, cancer and other conditions of these types.

Belonging to Class II of transposable elements, En/Spm transposons are widespread in a variety of distantly related plant species. Here, we report on the sequence conservation of the transposase region from sequence analyses of En/Spm-like transposons from Poaceae species, namely Zingeria biebersteiniana, Zingeria trichopoda, Triticum monococcum, Triticum urartu, Hordeum spontaneum, and Aegilops speltoides. The transposase region of En/Spm-like transposons was cloned, sequenced, and compared with equivalent regions of Oryza and Arabidopsis from the gene bank database. Southern blot analysis indicated that the En/Spm transposon was present in low (Hordeum spontaneum, Triticum monococcum, Triticum urartu) through medium (Zingeria bieberstiana, Zingeria trichopoda) to relatively high (Aegilops speltoides) copy numbers in Poaceae species. A cytogenetic analysis of the chromosomal distribution of En/Spm transposons revealed the concurence of the chromosomal localization of the En/Spm clusters with mobile clusters of rDNA. An analysis of En/Spm-like transposase amino acid sequences was carried out to investigate sequence divergence between 5 genera — Triticum, Aegilops, Zingeria, Oryza and Arabidopsis. A distance matrix was generated; apparently, En/Spm-like transposase sequences shared the highest sequence homology intra-generically and, as expected, these sequences were significantly diverged from those of O. sativa and A. thaliana. A sequence comparison of En/Spm-like transposase coding regions defined that the intra-genomic complex of En/Spm-like transposons could be viewed as relatively independent, vertically transmitted, and permanently active systems inside higher plant genomes. The sequence data from this article was deposited in the EMBL/GenBank Data Libraries under the accession nos. AY707995-AY707996-AY707997-AY707998-AY707999-AY708000-AY708001-AY708002-AY708003-AY708004-AY708005-AY708005-AY265312.

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

A novel approach to DNase I-sensitivity analysis was applied to examining genes of the spermatogenic pathway, reflective of the substantial morphological and genomic changes that occur during this program of differentiation. A new real-time PCR-based strategy that considers the nuances of response to nuclease treatment was used to assess the nuclease susceptibility through differentiation. Data analysis was automated with the K-Lab PCR algorithm, facilitating the rapid analysis of multiple samples while eliminating the subjectivity usually associated with Ct analyses. The utility of this assay and analytical paradigm as applied to nuclease-sensitivity mapping is presented.

The variant cell line U937V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937V cells was the lack of cellular disintegration, which contrasts to the formation of apoptotic bodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937V cells, the basic molecular events of apoptosis, such as internucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.

Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization of full-length human MLF1 is 14-3-3-independent, and is probably regulated by other as-yet-unknown proteins.