Novel glycylated sugar alcohols protect ESC-specific microRNAs from degradation in iPS cells.

Excessive accumulation of embryonic stem cell (ESC)-specific microRNAs occurs in both ESCs and induced pluripotent stem cells (iPSC); yet, the mechanism involved is unknown. In iPSCs, we for the first time found that novel glycylated sugar alcohols, particularly glycylglycerins, are tightly bound with ESC-specific microRNA precursors (pre-miRNA), such as pre-miR-302. Among these isolated glycylglycerins, we further identified that 1,3-diglycylglycerin and 1,2,3-triglycylglycerin are two major compounds bonded with negatively charged nucleic acids via electro-affinity and subsequently forming sugar-like coats in the hairpin-like double helix structures of pre-miRNAs. As a result, such glycylglycerin-formed coating serves as a protection layer against miRNA degradation. Moreover, we found that the pH value of iPSC cytosol determines the charges of these glycylglycerins. During iPSC differentiation, the cytosol pH is increased and hence neutralizes the charges of glycylglycerins, consequently leading to fast miRNA degradation. Therefore, the current findings not only explain how ESC-specific miRNAs are preserved and accumulated in iPSCs and ESCs but also demonstrate an important function of glycylglycerins in protecting the structural integrity of highly degradable miRNAs, providing a useful means for maintaining miRNA/siRNA function as well as developing the related RNA interference (RNAi) applications.

p32, a novel binding partner of Mcl-1, positively regulates mitochondrial Ca(2+) uptake and apoptosis.

Mcl-1 is a major anti-apoptotic Bcl-2 family protein. It is well known that Mcl-1 can interact with certain pro-apoptotic Bcl-2 family proteins in normal cells to neutralize their pro-apoptotic functions, thus prevent apoptosis. In addition, it was recently found that Mcl-1 can also inhibit mitochondrial calcium uptake. The detailed mechanism, however, is still not clear. Based on Yeast Two-Hybrid screening and co-immunoprecipitation, we identified a mitochondrial protein p32 (C1qbp) as a novel binding partner of Mcl-1. We found that p32 had a number of interesting properties: (1) p32 can positively regulate UV-induced apoptosis in HeLa cells. (2) Over-expressing p32 could significantly promote mitochondrial calcium uptake, while silencing p32 by siRNA suppressed it. (3) In p32 knockdown cells, Ruthenium Red treatment (an inhibitor of mitochondrial calcium uniporter) showed no further suppressive effect on mitochondrial calcium uptake. In addition, in Ruthenium Red treated cells, Mcl-1 also failed to suppress mitochondrial calcium uptake. Taken together, our findings suggest that p32 is part of the putative mitochondrial uniporter that facilitates mitochondrial calcium uptake. By binding to p32, Mcl-1 can interfere with the uniporter function, thus inhibit the mitochondrial Ca(2+) uploading. This may provide a novel mechanism to explain the anti-apoptotic function of Mcl-1.

CDK1 switches mitotic arrest to apoptosis by phosphorylating Bcl-2/Bax family proteins during treatment with microtubule interfering agents.

Microtubule interfering agents (MIAs), that can stabilise or depolymerise microtubules, are an important class of cancer chemotherapeutic drugs. They can lead to mitotic arrest and subsequent apoptosis. We demonstrate that cell cycle-dependent kinase 1 (CDK1) is important in switching cells from mitotic arrest to apoptosis during MIAs treatment. Overexpression of non-degradable cyclin B1 sustained CDK1 activation and mitotic arrest, followed by caspase-3 dependent apoptosis. CDK1 is responsible for the phosphorylation of several pro- and anti-apoptotic Bcl-2 family proteins during MIAs treatment. CDK1-mediated Bcl-2 serine 70 phosphorylation enhances its pro-apoptotic function, whereas CDK1-mediated Bad serine 128 phosphorylation promotes apoptosis. Blockage of CDK1 activity with a specific pharmacological inhibitor suppresses Mcl-1 phosphorylation, degradation and its anti-apoptotic function. Therefore, the death of cancer cells under MIAs treatment was caused by imbalance between CDK1-induced alterations in the pro-apoptotic and anti-apoptotic functions of phosphorylated Bcl-2 family proteins.

Regulation of somatic cell reprogramming through inducible mir-302 expression.

Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation.

Toxoplasma gondii infection confers resistance against BimS-induced apoptosis by preventing the activation and mitochondrial targeting of pro-apoptotic Bax.

In order to accomplish their life style, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the mitochondrial apoptotic pathway remain unknown. Here, we used the conditional expression of the BH3-only protein Bim(S) to pinpoint the interaction of T. gondii with the intrinsic pathway of apoptosis. Infection of epithelial cells with T. gondii dose-dependently abrogated Bim(S)-triggered release of cytochrome c from host-cell mitochondria into the cytosol, induction of activity of caspases 3, 7 and 9, and chromatin condensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infected host cells. Although total cellular levels and mitochondrial targeting of Bim(S) was not altered by the infection, the activation of pro-apoptotic effector proteins Bax and Bak was strongly impaired. Inhibition of Bax and Bak activation by T. gondii was seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax but not their cellular protein levels. Blockade of Bax and Bak activation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, the BH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of Bim(S)-triggered apoptosis. These results indicate that T. gondii targets activation of pro-apoptotic Bax and Bak to inhibit the apoptogenic function of mitochondria and to increase host-cell viability.

Single-cell electroendocytosis on a micro chip using in situ fluorescence microscopy.

Electroendocytosis (EED), i.e. electric field-induced endocytosis, is a technique for bio-molecule and drug delivery to cells using a pulsed electric field lower than that applied in electroporation (EP). Different from EP in which nanometer-sized electropores appear on the plasma membrane lipid bilayer, EED induces cell membrane internalization and fission via endocytotic vesicles. In this study, we conduct comprehensive experimental study on the EED of HeLa cells using a micro chip and the corresponding endocytotic vesicles were visualized and investigated by using FM4-64 fluorescent dye and in situ fluorescence microscopy. The uptake of molecules by the EED of cells was characterized by average intracellular fluorescent intensity from a large number (>2,000) of single cells. The EED efficiency was determined as a function of three electric parameters (electric field strength, pulse duration, total electric treatment time). The EED efficiency as a function of electric field strength clearly shows biphasic characteristics at different experimental conditions. The EED experiments using cytoskeleton inhibitors illustrate unique mechanisms distinct from EP. This study provides a foundation for further on-chip study of the time-dependent mechanism of EED at the single-cell level.

Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state.

Renewal of stem cells differs from cancer cell growth in self-controlled cell division. The mir-302 microRNA (miRNA) family (mir-302s) is expressed most abundantly in slow-growing human embryonic stem (ES) cells, and quickly decreases after cell differentiation and proliferation. Therefore, mir-302s was investigated as one of the key factors essential for maintenance of ES cell renewal and pluripotency in this study. The Pol-II-based intronic miRNA expression system was used to transgenically transfect the mir-302s into several human cancer cell lines. The mir-302-transfected cells, namely, miRNA-induced pluripotent stem (mirPS) cells, not only expressed many key ES cell markers, such as Oct3/4, SSEA-3, SSEA-4 ,Sox2, and Nanog, but also had a highly demethylated genome similar to a reprogrammed zygotic genome. Microarray analyses further revealed that genome-wide gene expression patterns between the mirPS and human ES H1 and H9 cells shared over 86% similarity. Using molecular guidance in vitro, these mirPS cells could differentiate into distinct tissue cell types, such as neuron-, chondrocyte-, fibroblast-, and spermatogonia-like primordial cells. Based on these findings, we conclude that mir-302s not only function to reprogram cancer cells into an ES-like pluripotent state but also to maintain this state under a feeder-free cultural condition, which may offer a great opportunity for therapeutic intervention.

Bioassay guided discovery of apoptosis inducers from gamboge by high-speed counter-current chromatography and high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry.

A screening system, composed of high-speed counter-current chromatography and high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry, was established to find bioactive lead compound. This system succeeded in discovering apoptosis inducers from gamboge, the resin of Garcinia hanburyi. High-speed counter-current chromatography was used to provide well-separated fractions for bioassay and the resulted active fractions were rapidly identified using high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry. The solvent system of n-hexane/ethyl acetate/methanol/water was optimized to the ratio of 7:3:7:3 (v/v/v/v) by a K value analysis. As a result, two active fractions were obtained. They showed apoptosis inducing effects as potent as that of taxol (500 nM) at the concentration of 1 microg/ml. Gambogenic acid (72.1%) and epimeric isogambogic acids (25.3%) were identified in one of the fractions. The other active fraction mainly contained two epimeric mixtures, gambogic acids (68.7%) and gambogoic acids (26.9%). Among them, gambogenic acid, without epimerization, has priority to be lead compound.

Bioassay-guided isolation of xanthones and polycyclic prenylated acylphloroglucinols from Garcinia oblongifolia.

Bioassay-guided fractionation of the acetone extract of the bark of Garcinia oblongifolia has resulted in the isolation of three new xanthones, oblongixanthones A-C (1-3), three new polyprenylated benzoylphloroglucinols, oblongifolins E-G (4-6), and 12 known compounds. Oblongifolins I (5) and J (6) are the first natural products that have similar structural features to those of two known oxidation products of garcinol. The structures of the new compounds 1-6 were characterized by spectroscopic data interpretation. All isolates were assayed for their apoptosis-inducing effects against HeLa-C3 cells. Oblongifolin C (16) was found to be the most potent apoptotic inducer of the compounds evaluated.

Calmodulin bound to stress fibers but not microtubules involves regulation of cell morphology and motility.

Calmodulin (CaM) is a major cytoplasmic calcium receptor that performs multiple functions including cell motility. To investigate the mechanism of the regulation of CaM on cell morphology and motility, first we checked the distribution of CaM in the living cells using GFP-CaM as an indicator. We found that GFP-CaM showed a fiber-like distribution pattern in the cytosol of living Potorous tridactylis kidney (PtK2) cells but not in living HeLa cells. The endogenous CaM in heavily permeabilized HeLa was also found to display a fiber-like distribution pattern. Further examination showed that the distribution pattern of GFP-CaM was same as that of stress fibers, but not microtubules. Co-immunoprecipitation also showed that CaM can interact with actin directly or indirectly. The microinjection of trp peptide, a specific inhibitor of CaM, attenuated the polymerization of stress fibers and induced the alteration of cell morphology. A wound-healing assay and a single cell tracking experiment showed that CaM in PtK2 cells could increase cell motility. The data we have got from living cells suggested that CaM affect cell morphology and motility through binding to stress fibers and regulate f-actin polymerization.

Mobilization of Ca2+ from endoplasmic reticulum to mitochondria plays a positive role in the early stage of UV- or TNFalpha-induced apoptosis.

Previously it was known that cytosolic Ca(2+) elevation was involved in regulating UV- or TNFalpha-induced apoptosis. Here, we reported new evidence that mitochondrial Ca(2+) signal is also involved in the apoptotic process. First, using living cell imaging techniques, we observed multiple mitochondrial Ca(2+) spikes during the early stage of UV- or TNFalpha-induced apoptosis. Second, the mitochondrial Ca(2+) spikes were synchronous with cytosolic Ca(2+) spikes observed in apoptosis, which preceded cytochrome c (cyt-c) release. Third, blocking the mitochondrial Ca(2+) elevation by applying a mitochondrial uniporter inhibitor could suppress UV-induced apoptosis in HeLa cells. Finally, overexpressing an anti-apoptotic protein, Bcl-2, could suppress the mitochondrial Ca(2+) elevation. Furthermore, it appeared that the elevation of mitochondrial Ca(2+) during apoptosis was caused by a direct coupling between endoplasmic reticulum (ER) and mitochondria through IP(3) receptors. Taken together, these findings suggest that Ca(2+) mobilization from ER to mitochondria can play a significant role in the apoptotic signaling pathway.

Bis(7)-tacrine prevents glutamate-induced excitotoxicity more potently than memantine by selectively inhibiting NMDA receptors.

We have recently reported that bis(7)-tacrine could prevent glutamate-induced neuronal apoptosis through NMDA receptors. In this study, we demonstrated that in cultured rat cortical neurons, bis(7)-tacrine (IC(50), 0.02 microM) prevented glutamate-induced excitotoxicity more substantially than memantine (IC(50), 0.7 microM). In addition, bis(7)-tacrine was more efficient than memantine in buffering the intracellular Ca(2+) triggered by glutamate. In cultured rat hippocampal neurons, bis(7)-tacrine inhibited 50 microM NMDA-activated current in a concentration-dependent manner with an IC(50) of 0.68+/-0.07 microM, which is five times more potent than that produced by memantine (IC(50), 3.41+/-0.36 microM; p<0.05). By contrast, bis(7)-tacrine, up to 5 microM, did not significantly affect the current activated by 50 microM AMPA or 50 microM kainate. These results suggest that bis(7)-tacrine is more potent than memantine against glutamate-induced neurotoxicity by selectively inhibiting NMDA-activated current.

The microRNA (miRNA): overview of the RNA genes that modulate gene function.

MicroRNAs (miRNAs), widely distributed, small regulatory RNA genes, target both messenger RNA (mRNA) degradation and suppression of protein translation based on sequence complementarity between the miRNA and its targeted mRNA. Different names have been used to describe various types of miRNA. During evolution, RNA retroviruses or transgenes invaded the eukaryotic genome and inserted in the non-coding regions of DNA, conceivably acting as transposon-like jumping genes, providing defense from viral invasion and fine-tuning of gene expression as a secondary level of gene modulation in eukaryotes. When a transposon is inserted in the intron, it becomes an intronic miRNA, taking advantage of the protein synthesis machinery, i.e., mRNA transcription and splicing, as a means for processing and maturation. Recently, miRNAs have been found to play an important, but not life-threatening, role in embryonic development. They might play a pivotal role in diverse biological systems in various organisms, facilitating a quick response and accurate plotting of body physiology and structures. Based on these unique properties, man-made intronic miRNAs have been developed for in vitro evaluation of gene function, in vivo gene therapy and generation of transgenic animal models. The biogenesis and identification of miRNAs, potential applications, and future directions for research are presented, hopefully providing a guideline for further miRNA and gene function studies.

Xanthones with growth inhibition against HeLa cells from Garcinia xipshuanbannaensis.

Eight prenylated xanthones, bannaxanthones A-H (1-8), together with seven known compounds, were isolated from the acetone extract of the twigs of Garcinia xipshuanbannaensis. Their structures were elucidated by spectroscopic data interpretation. The cytotoxic activities of these compounds were evaluated using the MTT method. The results showed that xanthones with an unsaturated prenyl group had stronger cytotoxic activity against cancer cells, whereas those with hydroxylated prenyl groups had none.

Tanshinone IIA, an isolated compound from Salvia miltiorrhiza Bunge, induces apoptosis in HeLa cells through mitotic arrest.

AIMS: Tanshinone IIA (Tan IIA) is a compound isolated from Salvia miltiorrhiza Bunge (Danshen). The aim of this study is to investigate the mechanisms of its anti-cancer effect. MAIN METHODS: To clearly delineate the cell cycle-dependent effects of Tan IIA, we used either synchronized cells or single living cell analysis to conduct our studies. Subcellular fractionation, Western blot analysis, immuno-fluorescence staining and FACS analysis were also employed in our study. KEY FINDINGS: We found that Tan IIA could arrest cancer cells in mitosis by disrupting the mitotic spindle and subsequently triggered cells to enter apoptosis through the mitochondria-dependent apoptotic pathway. Thus, Tan IIA could selectively kill mitotic cells over interphase cells. In comparison with other existing anti-cancer drugs that cause mitotic arrest by interfering with the microtubule structure (such as vincristine or taxol), Tan IIA destroyed only the mitotic spindle during the M phase but not the microtubule structure in interphase cells. Furthermore, Tan IIA could trigger the mitotic arrested cells to enter apoptosis faster than vincristine or taxol. SIGNIFICANCE: Since Tan IIA can selectively induce cancer cells to enter apoptosis through mitotic arrest, it has the potential to be developed into an anti-cancer drug.

Nonlinear current response of micro electroporation and resealing dynamics for human cancer cells.

This paper presents a novel method to measure the dynamic process of membrane permeability during electroporation (EP) on microchips for human cancer cells. Micro EP chips with three-dimensional gold electrodes accommodating a single cell in between were fabricated with a modified electroplating process. Electrochemical impedance spectroscopy (EIS) was carried out with an electrochemistry analyzer on micro EP chips and a nonlinear equivalent circuit model was proposed to describe the dynamic response of the whole system. Using such a method, micro EP current was isolated from undesired leakage current to study the corresponding electroporation dynamics under different input voltages. In addition, cell membrane recovery dynamics after electroporation was also studied and the resealing time constants were determined for different pulse treatments.

Polyprenylated xanthones from Garcinia lancilimba showing apoptotic effects against HeLa-C3 cells.

Three new prenylated xanthones, 1-3, along with ten known compounds, were isolated from the stem bark of Garcinia lancilimba. Their structures were elucidated by extensive spectroscopic analysis, including 1D- and 2D-NMR spectra, as well as HR-MS experiments. Some of these compounds showed apoptotic effects or growth-inhibition effects against HeLa cells expressing a caspase sensor protein.

The MicroRNA.

MicroRNAs (miRNAs), widely distributed, small regulatory RNA genes, target both messenger RNA (mRNA) degradation and suppression of protein translation based on sequence complementarity between the miRNA and its targeted mRNA. Different names have been used to describe various types of miRNA. During evolution, RNA retroviruses or transgenes invaded the eukaryotic genome and were inserted itself in the noncoding regions of DNA, conceivably acting as transposon-like jumping genes, providing defense from viral invasion and fine-tuning of gene expression as a secondary level of gene modulation in eukaryotes. When a transposon is inserted in the intron, it becomes an intronic miRNA, taking advantage of the protein synthesis machinery, i.e., mRNA transcription and splicing, as a means for processing and maturation. MiRNAs have been found to play an important, but not life-threatening, role in embryonic development. They might play a pivotal role in diverse biological systems in various organisms, facilitating a quick response and accurate plotting of body physiology and structures. Based on these unique properties, manufactured intronic miRNAs have been developed for in vitro evaluation of gene function, in vivo gene therapy, and generation of transgenic animal models. The biogenesis of miRNAs, circulating miRNAs, miRNAs and cancer, iPSCs, and heart disease are presented in this chapter, highlighting some recent studies on these topics.

Isolation and Identification of Gene-Specific MicroRNAs.

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducing RNA interference (RNAi)-related gene silencing, we have developed an intronic miRNA expression system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hence successfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanism has been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA so obtained is transcribed by type-II RNA polymerases, coexpressed within a primary gene transcript, and then excised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII) endonucleases further process the spliced introns into mature miRNAs. Using this intronic miRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitro but also in zebrafishes, chicken embryos, and adult mice in vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design various intronic pre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

Using a micro electroporation chip to determine the optimal physical parameters in the uptake of biomolecules in HeLa cells.

In this study, a new micro electroporation (EP) cell chip with three-dimensional (3D) electrodes was fabricated by means of MEMS technology, and tested on cervical cancer (HeLa) cells. Extensive statistical data of the threshold electric field and pulse duration were determined to construct an EP "phase diagram", which delineates the boundaries for 1) effective EP of five different size molecules and 2) electric cell lysis at the single-cell level. In addition, these boundary curves (i.e., electric field versus pulse duration) were fitted successfully with an exponential function with three constants. We found that, when the molecular size increases, the corresponding electroporation boundary becomes closer to the electric cell lysis boundary. Based on more than 2000 single-cell measurements on five different size molecules, the critical size of molecule was found to be approximately 40 kDa. Comparing to the traditional instrument, MEMS-based micro electroporation chip can greatly shorten the experimental time.