Boeun Kim

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A2 and Δ12-PGJ2

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and Δ12-PGJ2 induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca2+ ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA2 and Δ12-PGJ2 in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.

Activation of caspase-8 in 3-deazaadenosine-induced apoptosis of U-937 cells occurs downstream of caspase-3 and caspase-9 without Fas receptor-ligand interaction

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.

Cytochrome C-dependent Fas-independent apoptotic pathway in HeLa cells induced by delta12-prostaglandin J2.

Cyclopentenone prostaglandins (PGs) have antiproliferative activity on various tumor cell growth in vitro. Particularly, 9-deoxy-(9,12)-13,14-dihydro PGD2( Δ12-PGJ2) was reported for its antineoplastic and apoptotic effects on various cancer cells, but its mechanism inducing apoptosis is still not clear. In this study, we have characterized apoptosis induced by Δ12-PGJ2in HeLa cells. Treatment of Δ12-PGJ2induced apoptosis as indicated by DNA fragmentation, chromatin condensation, and formation of apoptotic body. We also observed release of cytochrome c from mitochondria and activation of caspase cascade including caspase-3, -8, and -9. And the pan-caspase inhibitor z-Val-Ala-Asp (OMe) fluoromethyl-ketone (z-VAD-fmk) and Q-Val-Asp (OMe)-CH2-OPH (Q-VD (OMe)-OPH) prevented cell death induced by Δ12-PGJ2 showing participation of caspases in this process. However, protein expression level of Bcl-2 family was not altered by Δ12-PGJ2, seems to have no effect on HeLa cell apoptosis. And ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase 8 indicating that Fas receptor-ligand interaction was not involved in this pathway. Treatment of Δ12-PGJ2 also leads to suppression of nuclear factor κB (NF-κB) as indicated by nuclear translocation of p65/RelA and c-Rel and its DNA binding ability analyzed by EMSA. Taken together, our results suggest that Δ12-PGJ2-induced apoptosis in HeLa cell utilized caspase cascade without Fas receptor-ligand interaction and accompanied with NF-κB inactivation.

FEASIBILITY OF PERCUTANEOUS CONTRAST ULTRASOUND-GUIDED CHOLECYSTOGRAPHY IN DOGS

Differentiating hepatocellular disease versus biliary obstruction can be challenging in dogs presented for icterus. The purpose of this prospective study was to determine the feasibility of percutaneous contrast ultrasound-guided cholecystography in dogs. Ten normal dogs weighing 7.6-13.0 kg (median 9.8 kg) were recruited. All dogs were considered normal based on complete blood count, serum chemistry profile, ultrasound examination, and percutaneous radiographic cholecystography. Percutaneous contrast ultrasound-guided cholecystography was performed using 0.5 ml of commercially available contrast agent and two conventional ultrasound machines for simultaneous scanning at two different locations. Two observers independently evaluated the time to initial detection of contrast in the proximal duodenum and duration of contrast enhancement via visual monitoring. Dynamic contrast enhancement was calculated using time-intensity curves. Mean (± SD) and median (range) of time to initial detection were 8.60 s (± 3.35) and 8.0 s (2.0-11.0), respectively, and mean and median duration were 50.45 s (± 23.24) and 53.0 s (20.0 - 70.0), respectively. Mean, median, and range of peak intensity were 114.1 mean pixel value (MPV) (SD ± 30.7), 109.2 MPV, and 79.7-166.7, respectively, and mean, median, and range of time to peak intensity were 26.1 s (SD ± 7.1 s), 24.0 s, and 19.0-41.0 s, respectively. Findings indicated that percutaneous contrast ultrasound-guided cholecystography is a feasible technique for detecting and quantifying patency of the bile duct in normal dogs. Future studies are needed to assess the diagnostic utility of this technique for dogs with biliary obstruction.

Clinical applications and characteristics of apparent diffusion coefficient maps for the brain of two dogs

Diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) mapping are functional magnetic resonance imaging techniques for detecting water diffusion. DWI and the ADC map were performed for intracranial lesions in two dogs. In necrotizing leukoencephalitis, cavitated lesions contained a hypointense center with a hyperintense periphery on DWI, and hyperintense signals on the ADC maps. In metastatic sarcoma, masses including a necrotic region were hypointense with DWI, and hyperintense on the ADC map with hyperintense perilesional edema on DWI and ADC map. Since DWI and ADC data reflect the altered water diffusion, they can provide additional information at the molecular level.

Donor-dependent variation of human umbilical cord blood mesenchymal stem cells in response to hypoxic preconditioning and amelioration of limb ischemia

With the rapidly growing demand for mesenchymal stem cell (MSC) therapy, numerous strategies using MSCs for different diseases have been studied and reported. Because of their immunosuppressive properties, MSCs are commonly used as an allogeneic treatment. However, for the many donors who could potentially be used, it is important to understand the capacity for therapeutic usage with donor-to-donor heterogeneity. In this study, we aimed to investigate MSCs as a promising therapeutic strategy for critical limb ischemia. We evaluated MSCs from two donors (#55 and #64) and analyzed the capacity for angiogenesis through in vivo and in vitro assays to compare the therapeutic effect between different donors. We emphasized the importance of intra-population heterogeneity of MSCs on therapeutic usage by evaluating the effects of hypoxia on activating cellular angiogenesis in MSCs. The precondition of hypoxia in MSCs is known to enhance therapeutic efficacy. Our study suggests that sensitivity to hypoxic conditions is different between cells originating from different donors, and this difference affects the contribution to angiogenesis. The bioinformatics analysis of different donors under hypoxic culture conditions identified intrinsic variability in gene expression patterns and suggests alternative potential genetic factors ANGPTL4, ADM, SLC2A3, and CDON as guaranteed general indicators for further stem cell therapy.Stem cell therapy: Finding the best cellsStem cells from some donors have a stronger therapeutic effect than others, and can be identified by genetic markers. Adult or mesenchymal stem cells (MSCs), which can differentiate into many other cell types, show promise to treat many diseases. Kyung-Sun Kang at Seoul National University and co-workers investigated whether MSCs from some donors show better therapeutic efficacy than others. They tested MSCs from several donors in a mouse model simulating a stroke in a hindlimb, and measured regrowth of blood vessels. MSCs from one donor showed higher regenerative efficacy, triggering regrowth of many blood vessels and allowing the limb to be saved. Kang and co-workers used genetic analysis to identify genes that showed higher expression in these MSCs. This gene expression profile could be used to select MSCs with a higher regenerative capacity.

Retrospective study of degenerative mitral valve disease in small-breed dogs: survival and prognostic variables

Small-breed dogs (n = 168; weight < 15 kg) diagnosed with myxomatous mitral valve degeneration based on a routine clinical examination, radiology, electrocardiography, and echocardiography at the Seoul National University Veterinary Medical Teaching Hospital were included in this study. Survival periods were determined, and there were significant differences in survival rates among the three International Small Animal Cardiac Health Council classes. The mean follow-up period was 14.3 ± 12.1 months. Univariate analysis revealed that dyspnea, pulmonary edema, and vertebral heart score were significantly associated with survival time (p < 0.05). Additionally, age, left atrial-to-aortic root ratio, ejection fraction, and left ventricular end diastolic volume were associated with an increased risk of death (p < 0.1), while body weight, body condition score, systolic blood pressure, arrhythmia, syncope, fractional shortening, and end systolic volume were not associated with an increased risk of death. These results suggest that among the assessed variables dyspnea, pulmonary edema, and vertebral heart score could be useful prognostic factors for providing patient information to owners.

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA:

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.