Acute Immune Response in Venoarterial and Venovenous Extracorporeal Membrane Oxygenation Models of Rats.

Although experimental extracorporeal membrane oxygenation (ECMO) animal models have been reported, there are few studies on the immune response to ECMO. We developed the venoarterial (VA) and venovenous (VV) model in rats and serially investigated the changes in the distribution of immune cells. Forty rats underwent both VA and VV modes of ECMO, and blood samples were collected at 1 day before ECMO (D-1), at the end of ECMO run (D+0), and 3 days after the ECMO (D+3). Flow cytometry was used to characterize surface marker expression (CD3, CD4, CD8, CD43, CD45, CD45R, CD161, and His48) on immune cells. Granulocytes were initially activated in both ECMO types and were further reduced but not normalized until 3 days of decannulation. Monocyte and natural killer cells were decreased initially in VA mode. B lymphocytes, helper T lymphocytes, and cytotoxic T lymphocytes also significantly decreased in VA modes after ECMO, but this phenomenon was not prominent in the VV modes. Overall immune cells proportion changed after ECMO run in both modes, and the immunologic balance altered significantly in the VA than in VV mode. Our ECMO model is feasible for the hemodynamic and immunologic research, and further long-term evaluation is needed.

BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production.

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.

The microRNA miR-132 targets Lrrfip1 to block vascular smooth muscle cell proliferation and neointimal hyperplasia.

OBJECTIVE: The proliferation and remodeling of vascular smooth muscle cells (VSMCs) is an important pathological event in atherosclerosis and restenosis. Here we report that microRNA-132 (miR-132) blocks vascular smooth muscle cells (VSMC) proliferation by inhibiting the expression of LRRFIP1 [leucine-rich repeat (in Flightless 1) interacting protein-1]. METHODS AND RESULTS: MicroRNA microarray revealed that miR-132 was upregulated in the rat carotid artery after catheter injury, which was further confirmed by quantitative real-time RT-PCR. Transfection of a miR-132 mimic significantly inhibited the proliferation of VSMCs, whereas transfection of a miR-132 antagomir increased it. miR-132 mimic inhibited VSMC migration and induced apoptosis. miR-132 mimic increased the protein amounts of both p27 and smooth muscle (SM) α-actin, whereas it decreased SM α-actin and Bcl2. Bioinformatics showed that LRRFIP1 is a target candidate of miR-132. miR-132 down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of Lrrfip1 in a sequence-specific manner. LRRFIP1 induced VSMC proliferation and increased phosphorylation of ERK. Immunohistochemical analysis revealed that Lrrfip1 was clearly expressed along with the basal laminar area of smooth muscle, and its expression pattern was disrupted 7 days after arterial injury. LRRFIP1 mRNA was decreased 14 days after injury. Delivery of miR-132 to rat carotid artery reduced LRRFIP1 expression and attenuated neointimal proliferation in carotid artery injury models. CONCLUSIONS: Our results suggest that miR-132 is a novel regulator of VSMC proliferation that represses neointimal formation by inhibiting LRRFIP1 expression.

Exceptional production of both prodigiosin and cycloprodigiosin as major metabolic constituents by a novel marine bacterium, Zooshikella rubidus S1-1.

A Gram-negative, red-pigment-producing marine bacterial strain, designated S1-1, was isolated from the tidal flat sediment of the Yellow Sea, Korea. On the basis of phenotypic, phylogenetic, and genetic data, strain S1-1 (KCTC 11448BP) represented a new species of the genus Zooshikella. Thus, we propose the name Zooshikella rubidus sp. nov. Liquid chromatography and mass spectrometry of the red pigments produced by strain S1-1 revealed that the major metabolic compounds were prodigiosin and cycloprodigiosin. In addition, this organism produced six minor prodigiosin analogues, including two new structures that were previously unknown. To our knowledge, this is the first description of a microorganism that simultaneously produces prodigiosin and cycloprodigiosin as two major metabolites. Both prodigiosin and cycloprodigiosin showed antimicrobial activity against several microbial species. These bacteria were approximately 1.5-fold more sensitive to cycloprodigiosin than to prodigiosin. The metabolites also showed anticancer activity against human melanoma cells, which showed significantly more sensitivity to prodigiosin than to cycloprodigiosin. The secondary metabolite profiles of strain S1-1 and two reference bacterial strains were compared by liquid chromatography-mass spectrometry. Multivariate statistical analyses based on secondary metabolite profiles by liquid chromatography-mass spectrometry indicated that the metabolite profile of strain S1-1 could clearly be distinguished from those of two phylogenetically related, prodigiosin-producing bacterial strains.

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line.

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn(2+)) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn(2+) with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl(2) markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn(2+) has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) blunted the increase in Zn(2+) levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn(2+) contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.

Studies on the Dermal and Ocular Irritation of Prodigiosin Isolated from Zooshikella rubidus.

This study was carried out to investigate the irritation of the prodigiosin isolated from Zooshikella rubidus on the skin and eyes in New Zealand white rabbits. The tests were performed on the basis of Korea Food and Drug Administration (KFDA) guidelines. Prodigiosin induced severe eye irritation at high concentration (0.5 g/site/ml) but there was no eye irritation at low concentration (0.3 mg/site/ ml). The primary irritation index was calculated from higher concentration (0.5 g/site/ml) to lower concentration (0.3 mg/site/ml). There were found non-irritation or induced mild irritation at lower concentration of prodigiosin application. On the basis of this study, it could be concluded that the prodigiosin may be non-irritant to mild irritant of usual application at lower concentration (0.3 mg/site) resulting it is safe and useful in dyeing technology of fabrics.

Altered patterns of gene expression in response to chronic atrial fibrillation.

To obtain greater insight into atrial remodeling at the molecular level we analyzed the changes in gene expression in human atrial tissue between patients with chronic atrial fibrillation (AF) and those with normal sinus rhythm (NSR). cDNA microarray analysis was used to identify genes differentially expressed during sustained AF of more than 6 months (n = 9, mean age, 45 +/- 12, 6 males and 3 females) as compared to those with NSR (n = 9, mean age, 47 +/- 13, 6 males and 3 females). Western blot analysis was performed to confirm the altered gene expression and to establish the changes in protein expression. DNA gel electrophoresis to establish DNA ladder formation, which was associated with apoptosis in response to chronic AF, was performed. Microscopic findings were observed via electron microscopy. In the microarray analysis, out of 8,167 candidate genes, 66 genes showed a significant change in the expression level in the patients with chronic AF, which was in contrast to those with NSR. Among those, 31 genes were consistently down-regulated and 35 up-regulated more than 2-fold. The relative amounts of the Bcl-2 and p27 in the atrial tissue were decreased and angiotensin II type 2 (AT2) receptor and p21 were increased in the patients with chronic AF as compared to those with NSR. The atrial cardiomyocytes in chronic AF showed a prominent DNA ladder, which is a biochemical hallmark of apoptosis. The expression of Bcl-2, AT2 receptor, p21, and p27 were consistent with a significant role in the apoptosis of cardiac myocytes in the patients with chronic AF.

Involvement of ROS and JNK1 in selenite-induced apoptosis in Chang liver cells.

Selenium is a dietary essential trace nutrient with important biological roles. Selenocompounds were reported to induce apoptosis in many types of tumor cells. In this study, we investigated the signaling pathway involved in the selenite-induced apoptosis using Chang liver cells as a non-malignant cell model. The Chang liver cell apoptosis induced by selenite (10 microM) was confirmed by DNA fragmentation and typical apoptotic nuclear changes. Treatment of selenite increased intracellular reactive oxygen species (ROS) level and c-Jun N-terminal kinase1 (JNK1) phosphorylation. The selenite-induced cell death was attenuated by SP600125, a specific inhibitor of JNK, and by dominant negative JNK1 (DN-JNK1). Antioxidants such as glutathione (GSH), N-acetyl cysteine (NAC), curcumin, epigallocatechin gallate (EGCG) and epicatechin (EC) inhibited selenite-induced intracellular ROS elevation and JNK1 phosphorylation. Our results suggest that selenite-induced apoptosis in Chang liver cells was preceded by the ROS generation and JNK1 activation.

Nosocomial infection of malnourished patients in an intensive care unit.

Malnutrition is one of the most important factors for the development of nosocomial infection (NI). We performed a study of the correlation between abnormal nutritional factors and NI risk by investigating the patients who stayed longer than 3 days in the intensive care unit (ICU) of our university hospital. The patients were classified into three groups based on serum albumin levels and total lymphocyte counts (TLC). The criteria of Group I (well nourished group) were serum albumin level of 3.5 g/dl or higher and TLC of 1,400/mm3 or higher. The criteria of Group III (severely malnourished group) were serum albumin of less than 2.8 g/dl and TLC of less than 1,000/mm3. The other patients were classified as Group II (moderately malnourished group). The occurrences of NI were monitored during the study period and the APACHE III Score was calculated. The probability of first NI infection in Group III was 2.4 times higher than that in Groups I and II. The mortality rate of 20.5% was more significantly correlated with APACHE III Score than nutritional status. Nineteen (53%) of the total 36 NI patients were infected within 10 days after ICU admission and they all belonged to Group III. When we compared the gap period between infections, the time to first infection was significant.