p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31^comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31^comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31^comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31^comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31^comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31^comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31^comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31^comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31^comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31^comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.     

Proteomic analysis of microvesicles derived from human mesenchymal stem cells

Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC's self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.     

Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling

Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.     

Allithiamine Exerts Therapeutic Effects on Sepsis by Modulating Metabolic Flux during Dendritic Cell Activation

Recent studies have highlighted that early enhancement of the glycolytic pathway is a mode of maintaining the pro-inflammatory status of immune cells. Thiamine, a well-known co-activator of pyruvate dehydrogenase complex, a gatekeeping enzyme, shifts energy utilization of glucose from glycolysis to oxidative phosphorylation. Thus, we hypothesized that thiamine may modulate inflammation by alleviating metabolic shifts during immune cell activation. First, using allithiamine, which showed the most potent anti-inflammatory capacity among thiamine derivatives, we confirmed the inhibitory effects of allithiamine on the lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and maturation process in dendritic cells. We applied the LPS-induced sepsis model to examine whether allithiamine has a protective role in hyper-inflammatory status. We observed that allithiamine attenuated tissue damage and organ dysfunction during endotoxemia, even when the treatment was given after the early cytokine release. We assessed the changes in glucose metabolites during LPS-induced dendritic cell activation and found that allithiamine significantly inhibited glucose-driven citrate accumulation. We then examined the clinical implication of regulating metabolites during sepsis by performing a tail bleeding assay upon allithiamine treatment, which expands its capacity to hamper the coagulation process. Finally, we confirmed that the role of allithiamine in metabolic regulation is critical in exerting anti-inflammatory action by demonstrating its inhibitory effect upon mitochondrial citrate transporter activity. In conclusion, thiamine could be used as an alternative approach for controlling the immune response in patients with sepsis.     

Plant regeneration via direct somatic embryogenesis in Panax japonicus

Panax japonicus is one of the important medicinal plants. Here, we established the protocol for plant regeneration of P. japonicus via direct somatic embryogenesis. Somatic embryos were directly obtained from the segments of zygotic embryos on MS medium with 4.4 μM 2,4-D. Thereafter, somatic embryos were produced by repetitive secondary somatic embryogenesis. The secondary somatic embryo formation was enhanced by plasmolyzing pretreatment (1.0 M mannitol for 10 h). Frequency of secondary somatic embryo formation from cotyledon segments was lowered by plasmolyzing pretreatment, but the number of somatic embryos per explants was greatly increased. Plasmolyzing pretreatment resulted in retardation of embryo growth and required subculture to fresh medium for further growth of embryos into cotyledonary stage. Without plasmolyzing pretreatment, cotyledonary embryos were obtained after 8 weeks of culture. All the cotyledonary somatic embryos germinated by 5 μM GA₃ treatment, but only 15.3% were germinated on hormone-free medium. After 2 months of culture on 1/2 strength WPM medium, plantlets produced flowers spontaneously. In the anthers of in vitro flowers, microsporogenesis occurred normally with low number of pollen grains.     

Effect of cigarette smoke on fibroblast-mediated gel contraction is dependent on cell density

Cigarette smoke exposure has been associated with a variety of diseases, including emphysema. The current study evaluated the interaction of cell density and cigarette smoke extract (CSE) on fibroblast contraction of collagen gels. Protein levels of transforming growth factor (TGF)-beta1, fibronectin, PGE(2), and TGF-beta1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 x 10(5) cell/ml), only 5% CSE augmented contraction in higher-density cultures (3-5 x 10(5) cells/ml). CSE also inhibited fibronectin and TGF-beta1 production in low-density cultures but stimulated fibronectin production in high-density cultures. Active TGF-beta1 was readily detectable only in higher-density cultures and was markedly augmented by 5% CSE. In contrast, although TGF-beta1 mRNA expression was inhibited in high-density cultures by 10% CSE, expression was increased in the presence of 5% CSE. These results suggest that CSE-induced inhibition of low-density fibroblast contraction is due to inhibition of fibronectin production, whereas CSE's stimulatory effect on high-density cells is the result of increased release of TGF-beta1. These effects may help explain the varied pathologies associated with exposure to cigarette smoke.     

Up-regulation of skeletal muscle LIM protein 1 gene by 25-hydroxycholesterol may mediate morphological changes of rat aortic smooth muscle cells

Changes in the expression level of the skeletal muscle LIM protein 1 (SLIM1) in cultured A10 cells were monitored in response to 25-hydroxycholesterol (25-HC), an oxidized form of cholesterol present in the oxidized low-density lipoproteins. The level of SLIM1 mRNA was elevated in a time- and concentration-dependent manner by treatment of 25-HC. Expressions of smooth muscle (SM) alpha-actin and calponin-1 (CNN-1), early markers for SMC differentiation, were also increased by the 25-HC treatments. Expressions of all three genes (SLIM1, SM alpha-actin and CNN-1) were simultaneously elevated in the cells treated with 9-cis retinoic acid (RA). On the other hand, the SLIM1 expression induced by the 25-HC or 9-cis RA (as well as SM alpha-actin and CNN-1) was decreased by the treatment of 15d-PGJ2. Since the 25-HC, 9-cis RA and 15d-PGJ2 were ligands for the LXR, RXRalpha and PPARgamma respectively, there might be a functional positive cross-talk between LXR and RXRalpha pathways and a negative cross-talk between PPARgamma and LXR and/or RXRalpha pathways in the regulation of SLIM1 expression. The cells stably transfected with the expressional vector for SLIM1 also showed an elevation in the levels of SM alpha-actin and CNN-1. In addition, an over-production of SLIM1 in the cells resulted in a change in the cell-shape into a spindle-like form, which is identical to that observed after a prolonged treatment of the cells with cholesterol.