Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.     

The ATM-BID pathway regulates quiescence and survival of haematopoietic stem cells

BID, a BH3-only BCL2 family member, functions in apoptosis as well as the DNA-damage response. Our previous data demonstrated that BID is an ATM effector acting to induce cell-cycle arrest and inhibition of apoptosis following DNA damage. Here we show that ATM-mediated BID phosphorylation plays an unexpected role in maintaining the quiescence of haematopoietic stem cells (HSCs). Loss of BID phosphorylation leads to escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. We also demonstrate that BID phosphorylation plays a role in protecting HSCs from irradiation, and that regulating both quiescence and survival of HSCs depends on BID's ability to regulate oxidative stress. Moreover, loss of BID phosphorylation, ATM knockout or exposing mice to irradiation leads to an increase in mitochondrial BID, which correlates with an increase in mitochondrial oxidative stress. These results show that the ATM-BID pathway serves as a critical checkpoint for coupling HSC homeostasis and the DNA-damage stress response to enable long-term regenerative capacity.     

Dihydropyridines and atypical MDR: a novel perspective of designing general reversal agents for both typical and atypical MDR

Multidrug resistance (MDR) is defined as resistance of tumor cells to the cytotoxic action of multiple structurally dissimilar and functionally divergent drugs commonly used in chemotherapy. 1,4-Dihydropyridines (DHP(s)) are one of the major classes of Ca2+ channel blockers, and it has been shown that these agents are a new class of drug resistance reversers in cancer treatment. Analysis of various investigations on MDR reversing effects of DHPs shows that they can be a potential class for designing compounds simultaneously effective on both typical and atypical MDR. Also, it is possible to include some considerations on essential structural features for MDR reversing and further decreasing of Ca2+ channel blocking activity as an adverse effect.     

Bacterial RNase P RNA is a drug target for aminoglycoside-arginine conjugates

The ribonuclease P (RNase P) holoenzymes are RNPs composed of RNase P RNA (PRNA) and a variable number of P protein subunits. Primary differences in structure and function between bacterial and eukaryotic RNase P and its indispensability for cell viability make the bacterial enzyme an attractive drug target. On the basis of our previous studies, aminoglycoside-arginine conjugates (AACs) bind to HIV-1 TAR and Rev responsive element (RRE) RNAs significantly more efficiently than neomycin B. Their specific inhibition of bacterial rRNA as well as the findings that the hexa-arginine neomycin derivative (NeoR6) is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, led us to explore the structure-function relationships of AACs in comparison to a new set of aminoglycoside-polyarginine conjugates (APACs). We here present predicted binding modes of AACs and APACs to PRNA. We used a multistep docking approach comprising rigid docking full scans and final refinement of the obtained complexes. Our docking results suggest three possible mechanisms of RNase P inhibition by AACs and APACs: competition with the P protein and pre-tRNA on binding to P1-P4 multihelix junction and to J19/4 region (probably including displacement of Mg2+ ions from the P4 helix) of PRNA; competition with Mg2+ ions near the P15 loop; and competition with the P protein and/or pre-tRNA near the P15 helix and interfering with interactions between the P protein and pre-tRNA at this region. The APACs revealed about 10-fold lower intermolecular energy than AACs, indicating stronger interactions of APACs than AACs with PRNA.     

Insight into the mechanisms of aminoglycoside derivatives interaction with HIV-1 entry steps and viral gene transcription

In recent years, based on peptide models of HIV-1 RNA binding, NMR structures of Tat-responsive element-ligand complexes and aminoglycoside-RNA interactions, and HIV-1 Tat structure, we have designed and synthesized aminoglycoside-arginine conjugates (AACs) and aminoglycoside poly-arginine conjugates (APACs), to serve as Tat mimetics. These novel molecules inhibit HIV-1 infectivity with 50% effective concentration values in the low micromolar range, the most potent compounds being the hexa-arginine-neomycin B and nona-D-arginine-neomycin conjugates. Importantly, these compounds, in addition to acting as Tat antagonists, inhibit HIV-1 infectivity by blocking several steps in HIV-1 cell entry. The AACs and APACs inhibit HIV-1 cell entry by interacting with gp120 at the CD4-binding site, by interacting with CXCR4 at the binding site of the CXCR4 mAb 12G5, and apparently by interacting with transient structures of the ectodomain of gp41. In the current review, we discuss the mechanisms of anti-HIV-1 activities of these AACs, APACs and other aminoglycoside derivatives in detail. Targeting several key processes in the viral life cycle by the same compound not only may increase its antiviral efficacy, but more importantly, may reduce the capacity of the virus to develop resistance to the compound. AACs and APACs may thus serve as leading compounds for the development of multitargeting novel HIV-1 inhibitors.     

LIGR, a protease-activated receptor-2-derived peptide, enhances skin pigmentation without inducing inflammatory processes

The protease-activated receptor-2 (PAR-2) is a seven transmembrane G-protein-coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR-2 with Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), a known PAR-2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR-2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu-Ile-Gly-Arg-NH(2) (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno-deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho-GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR-2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR-2 signaling pathways, including [Ca(2+)] mobilization, cAMP induction, the induction of cyclooxgenase-2 (COX-2) expression and the secretion of prostaglandin E2, interleukin-6 and -8. PAR-2 siRNA inhibited LIGR-induced phagocytosis, indicating that LIGR signals via PAR-2. Our data suggest that LIGR is a more specific regulator of PAR-2-induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned-like skin color, without enhancing inflammatory processes, and without the need of UV exposure.     

Domains of the TMV movement protein involved in subcellular localization

To identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C-terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild-type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9–11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49–51 may be involved in co-alignment of the protein with microtubules. The region around residues 88–101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.