Caspase-independent death of human osteosarcoma cells by flavonoids is driven by p53-mediated mitochondrial stress and nuclear translocation of AIF and endonuclease G

Flavonoids have antioxidant and antitumor promoting effects. Rhus verniciflua Stokes (RVS) is a flavonoid-rich herbal medicine that has long been used in Korea as both a food additive and antitumor agent. It was previous reported that a purified flavonoid fraction prepared from RVS, herein named RCMF (the RVS chloroform-methanol fraction), inhibited the proliferation and induced apoptosis in human osteosarcoma (HOS) cells. This study examined the mechanisms involved in the RCMF-mediated apoptosis in HOS cells. RCMF was shown to be capable of inducing apoptosis in HOS cells by inducing p53 in the cells resulting in the decrease in Bcl-2 level, activation of Bax, and cytoplasmic release of cytochrome c, which led to the translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus. However, the RCMF-induced apoptosis was suppressed by transfecting the cells with antisense p53 oligonucleotides but not by treating them with a MAPK or caspase inhibitor. This suppression occurred through the regulation of Bcl-2 members as well as by preventing the nuclear translocation of the mitochondrial apoptogenic factors. Overall, it appears that p53-mediated mitochondrial stress and the nuclear translocation of AIF and EndoG are mainly required for the apoptosis induced by RCMF.     

Acetate-mediated pH-stat fed-batch cultivation of transconjugant <Emphasis Type="Italic">Enterobacter</Emphasis> sp. BL-2S over-expressing <Emphasis Type="Italic">glmS</Emphasis> gene for excretive production of microbial polyglucosamine PGB-1

A unique cationic polyglucosamine biopolymer PGB-1 comprising more than 95% D-glucosamine was excretively produced from a new bacterial strain Enterobacter sp. BL-2 under acetate-mediated culture conditions. Since the biopolymer PGB-1 could be synthesized from the UDP-N-acetylglucosamine monomer derived from the hexosamine pathway, three glmS, glmM, and glmU genes in the hexosamine pathway were cloned from Enterobacter sp. BL-2, and their molecular structures were elucidated. The cloned glmS, glmM, and glmU genes were reintroduced into the parent strain Enterobacter sp. BL-2 through a conjugative transformation for the overproduction of the biopolymer PGB-1. The biopolymer production increased 1.5-fold in the transconjugant Enterobacter sp. BL-2S over-expressing the first-step glmS gene encoding glucosamine-6-phosphate synthase. The transconjugant Enterobacter sp. BL-2S was cultivated pH-stat fed-batch widely, while intermittently feeding an acetate solution to maintain a constant pH level of 8.0 for 72 h, resulting in 1.15 g/L of the extracellular polyglucosamine biopolymer PGB-1.     

Characteristics of primary and immortalized fibroblast cells derived from the miniature and domestic pigs

Background

The pig, Sus scrofa domestica includes both the miniature and commercial domestic breed. These animals have influenced the human life and economies and have been studied throughout history. Although the miniature breeds are more recent and have increasingly been used in a variety of biomedical studies, their cell lines have rarely been established. Therefore, we sought to establish primary and immortal cell lines derived from both the miniature and domestic pig to better enable insight into possible in vivo growth differences.

Results

The in vitro lifespan of primary domestic pig fibroblast (PF) and miniature pig fibroblast (MPF) cells using a standard 3T3 protocol was determined. Both of the primary PF and MPF cells were shown to have a two-step replicative senescence barrier. Primary MPF cells exhibited a relatively shorter lifespan and slower proliferation rate compared to those of primary PF cells. Beyond senescence barriers, lifespan-extended PF and MPF cells were eventually established and indicated spontaneous cellular immortalization. In contrast to the immortalized PF cells, immortal MPF cells showed a transformed phenotype and possessed more frequent chromosomal abnormalities and loss of p53 regulatory function. The lifespan of primary MPF and PF cells was extended by inactivation of the p53 function using transduction by SV40LT without any detectable senescent phenotype.

Conclusion

These results suggest that p53 signaling might be a major determinant for the replicative senescence in the MPF cells that have the shorter lifespan and slower growth rate compared to PF cells in vitro.     

Monoclonal antibody K312-based depletion of pluripotent cells from differentiated stem cell progeny prevents teratoma formation

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.     

Developing portable widefield fundus camera for teleophthalmology: Technical challenges and potential solutions

A portable, low cost, widefield fundus camera is essential for developing affordable teleophthalmology. However, conventional trans-pupillary illumination used in traditional fundus cameras limits the field of view (FOV) in a snapshot image, and frequently requires pharmacologically pupillary dilation for reliable examination of eye conditions. This minireview summarizes recent developments in alternative illumination approaches for widefield fundus photography. Miniaturized indirect illumination has been used to enable compact design for developing low cost, portable, widefield fundus camera. Contact mode trans-pars-planar illumination has been validated for ultra-widefield fundus imaging of infant eyes. Contact-free trans-pars-planar illumination has been explored for widefield imaging of adult eyes. Trans-palpebral illumination has been also demonstrated in a smartphone-based widefield fundus imager to foster affordable teleophthalmology.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor

 A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of Taxus cuspidata (Japanese yew) on a pilot-scale. Rapidly growing cell lines were selected from callus cultures derived from immature embryos of yew. The cells were inoculated in 20-l capacity bioreactors of different types to test the growth performance. The models of small-scale bioreactors incorporated in this study included a balloon-type bubble bioreactor (BTBB), a bubble-column bioreactor (BCB), a BCB with a split-plate internal loop, a BCB with a concentric draught-tube internal loop, a BCB with a fluidized bed bioreactor, and two different models of stirred tank reactors. Among the reactors, BTBB appeared to be the most efficient in promoting cell growth. The doubling time of cell growth in BTBB was 12 days with a 30% inoculation cell density. The optimum time for medium replacement or feeding was 12–15 days after inoculation as determined by monitoring both the levels of sugars and medium conductivity. When yew tree cells were grown in different sizes (100–500-l) of BTBBs, more than 70% cell viability was recorded at the time of harvest. The growth pattern of the cells in the pilot-scale BTBB appeared to be the same as that of cells in the 20-l bioreactors. Approximately 3 mg/l of taxol and 74 mg/l total taxanes were obtained after 27 days of culture. Received: 6 April 1999 / Revision received: 23 August 1999 / Accepted: 31 August 1999     

Possible biogeochemical response to the passage of Hurricane Fabian observed by satellites

Physical and biogeochemical changes induced by the HurricaneFabian in the Northwest Atlantic in early September 2003 wereobserved using composite satellite images. After the passageof the hurricane, the mean sea surface temperature (SST) alongthe track decreased on average by about 1.3°C with maximumdecrease of 10°C. At the same time, the mean Chl a concentrationincreased by about 42%. Entrainment of cold, nutrient-rich watersby vertical mixing induced by the hurricane seems to have enhancedthe phytoplankton production. Asymmetric distribution of changesin SST and Chl a (strong intensity on the right side of thestorm track) was observed from the satellite data. The storm-inducednitrate increase estimated from the satellite SST, using a localrelationship between nitrate and temperature measurements wasabout 40% on average along the track of the storm. A numericalmodel study and climatological nutrient profile showed an increasein mixed-layer depth of 26 m and nitrate increase of about 0.2µmol L^–1 after the storm passage. In addition toaltering the physicochemical conditions of the water column,physical forcing by the hurricane also changed the taxonomiccomposition of phytoplankton. It is inferred that the dominanceof diatoms after the storm is a result of the increase in nutrientconcentration within the mixed layer due to the wind forcingof the storm.