Serum repressing efflux pump CDR1 in Candida albicans

BACKGROUND: In the past decades, the prevalence of candidemia has increased significantly and drug resistance has also become a pressing problem. Overexpression of CDR1, an efflux pump, has been proposed as a major mechanism contributing to the drug resistance in Candida albicans. It has been demonstrated that biological fluids such as human serum can have profound effects on antifungal pharmacodynamics. The aim of this study is to understand the effects of serum in drug susceptibility via monitoring the activity of CDR1 promoter of C. albicans. RESULTS: The wild-type C. albicans cells (SC5314) but not the cdr1/cdr1 mutant cells became more susceptible to the antifungal drug when the medium contained serum. To understand the regulation of CDR1 in the presence of serum, we have constructed CDR1 promoter-Renilla luciferase (CDR1p-RLUC) reporter to monitor the activity of the CDR1 promoter in C. albicans. As expected, the expression of CDR1p-RLUC was induced by miconazole. Surprisingly, it was repressed by serum. Consistently, the level of CDR1 mRNA was also reduced in the presence of serum but not N-acetyl-D-glucosamine, a known inducer for germ tube formation. CONCLUSION: Our finding that the expression of CDR1 is repressed by serum raises the question as to how does CDR1 contribute to the drug resistance in C. albicans causing candidemia. This also suggests that it is important to re-assess the prediction of in vivo therapeutic outcome of candidemia based on the results of standard in vitro antifungal susceptibility testing, conducted in the absence of serum

TAK1 inhibition-induced RIP1-dependent apoptosis in murine macrophages relies on constitutive TNF-alpha signaling and ROS production

Background: Transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1) is a key regulator of signal cascades of TNF-alpha receptor and TLR4, and can induce NF-kappa B activation for preventing cell apoptosis and eliciting inflammation response. Results: TAK1 inhibitor (TAKI) can decrease the cell viability of murine bone marrow-derived macrophages (BMDM), RAW264.7 and BV-2 cells, but not dermal microvascular endothelial cells, normal human epidermal keratinocytes, THP-1 monocytes, human retinal pigment epithelial cells, microglia CHME3 cells, and some cancer cell lines (CL1.0, HeLa and HCT116). In BMDM, TAKI-induced caspase activation and cell apoptosis were enhanced by lipopolysaccharide (LPS). Moreover, TAKI treatment increased the cytosolic and mitochondrial reactive oxygen species (ROS) production, and ROS scavengers NAC and BHA can inhibit cell death caused by TAKI. In addition, RIP1 inhibitor (necrostatin-1) can protect cells against TAKI-induced mitochondrial ROS production and cell apoptosis. We also observed the mitochondrial membrane potential loss after TAKI treatment and deterioration of oxygen consumption upon combination with LPS. Notably TNF-alpha neutralization antibody and inhibitor enbrel can decrease the cell death caused by TAKI. Conclusions: TAKI-induced cytotoxicity is cell context specific, and apoptosis observed in macrophages is dependent on the constitutive autocrine action of TNF-alpha for RIP1 activation and ROS production

Celecoxib Induces Heme Oxygenase-1 Expression in Macrophages and Vascular Smooth Muscle Cells Via Ros-Dependent Signaling Pathway

The multiple cytoprotective mechanisms of heme oxygenase HO- 1 make it a promising therapeutic target. This study investigated whether the selective cyclooxygenase COX-2 inhibitor, celecoxib, can upregulate HO-1 expression. Murine J774 macrophages and rat aortic vascular smooth muscle cells VSMCs were used to study the effect of celecoxib on HO -1 expression . A signal transduction pathway involving reactive oxygen species ROS was also investigated. We found that celecoxib can upregulate HO-1 gene and protein expressions in J774 macrophages and VSMCs. This effect was not diminished by prostaglandin E-2 or 15dPGJ2, while it was additive to hypoxia-induced HO-1 expression, suggesting an event independent of COX-2 activity or hypoxia-inducible factor-1 alpha. Moreover, celecoxib activated ERK, p38, Akt, and Nrf2 as well as increased ROS production. All these events contributed to the increase in the expression of HO-1 caused by celecoxib. In this study, we also, for the first time, demonstrated that AMP-activated protein kinase AMPK can mediate HO-1 expression via the downstream activation of p38 and Akt. However, the HO-1-inducing actions of celecoxib and hypoxia were not associated with AMPK. This study demonstrates a COX-2-independent action of celecoxib in upregulating HO-1 in macrophages and VSMCs. This action is dependent on ROS, Akt, ERK, p38, and Nrf2 activation. These findings provide new insights into the action mechanism of celecoxib with broad implications for anti- inflammation therapy

Analysis of Embryo-Specific Genes from 10-day Embryos of Rice (Oryza sativa L. cv Tainung 67 )

本試驗以實驗室初步篩選的99個台農67號品種水稻之10天胚特有的殖系為 材料進行水稻10天胚特有基因的選殖與分析。目的為：(1)選殖水稻胚早 期發育相關 的基因，(2)分析這些基因在胚發育過程之表現特性，(3)探 討這些基因的結構 與組成。將99個殖系重複進行南方墨點差異篩選後， 選出20個10天胚特有基因。經核酸定序，比對其3''端 100- 250個核酸序 列，可將殖系分成四組。前三組的各殖系在組別內之相似性極高，可能為 同一個基因或基因族。第四組的各殖系間相似性較低，可能為不同的基因 。接著完成了12個殖系，全部 insert 片段之核酸定序。其中 pRE31D( U25968)、pRE1M(U25969)、pRE16J(U25970)，帶有疑似ORF，分別可推論 出 526，402，159個胺基酸，比對結果顯示 pRE31D與 glutelin 有34﹪ 的確定度(Identity)，pRE1M 與已發表資料之相似性不高，而pRE16J 則 與 oleosin 有71﹪的確定度，經過一些胺基酸序列的分析後，推斷pRE16 J 可能屬於 oleosin 類的蛋白質。 以四組中代表性殖系做為探針， 與5、10、15、20、25、30天、成熟胚和葉片之mRNA做北方轉漬雜交，結 果顯示這些殖系的時序性表現，在10天時最高、之後隨時間增加而遞減， 至成熟胚和葉片組織內便幾乎偵測不到。以pRE31D、pRE1M、pRE16J三個 殖系做為探針，與10、15天、成熟胚和葉片之mRNA做北方墨點雜交，顯示 選殖系中 pRE1M、pRE16J、 pRE31D 三個殖系可能為全長之cDNA clones 。以 ABA 及 NaCl 處理水稻幼苗，均會誘導 pRE31D、pRE1M、pRE16J 三 個選殖系的表現；南方墨點分析顯示 pRE31D、pRE1M、pRE16J 三個殖系 在染色體組上可能由單一或少數的基因座構成。The objectives of the current research are to isolate the embryo specific genes from 10-day embryos of rice (Oryza sativa L. cv. Tainung 67), to characterize the nucleic acid sequences of the selected genes, to examine the spatial-specific and temporal-specific expression of the selected genes, and to study the genomic orgamization of these genes. Twenty specific cDNA clones were selected by differential hybridization from 99 cDNA clones which were preliminary screened in our laboratory. According to the nucleic acid sequences at the 3''-end of the genes, the selected twenty clones could be divided into four different groups. The clones in the first three groups were highly homologous within the same group, while clones in the fourth group were the different clones. Open reading frames were found in pRE31D, pRE1M and pRE16J that was judged by nucleotide sequencing of fully length. Comparison of nucleotide and deduced amino acid sequence with computer database revealed that the identities of pRE31D, pRE1M were still unknown, while pRE16J could be belong to oleosin protein. The expression of pRE31D, pRE1M and pRE16J could be induced in the rice seedling by NaCl or ABA treatment. Highest expression of pRE31D, pRE16L, RE1M, pRE16J, pRE13G, pRE37N and pRE15N were found in 10-day embryos, and then declined thereafter. The results of the southern hybridization indicated that pRE1M, pRE16J and pRE31D were the single or a few copy genes in the chromosome

Regulation of c-Fos gene expression by NF-κB: a p65 homodimer binding site in mouse embryonic fibroblasts but not human HEK293 cells.

The immediate early gene c-Fos is reported to be regulated by Elk-1 and cAMP response element-binding protein (CREB), but whether nuclear factor (NF)-κB is also required for controlling c-Fos expression is unclear. In this study, we determined how NF-κB's coordination with Elk/serum response factor (SRF) regulates c-fos transcription. We report that PMA strongly induced c-Fos expression, but tumor necrosis factor (TNF)-α did not. In mouse embryonic fibroblasts, the PMA induction of c-Fos was suppressed by a deficiency in IKKα, IKKβ, IKKγ, or p65. By contrast, in human embryonic kidney 293 cells, PMA induced c-Fos independently of p65. In accordance with these results, we identified an NF-κB binding site in the mouse but not human c-fos promoter. Under PMA stimulation, IKKα/β mediated p65 phosphorylation and the binding of the p65 homodimer to the NF-κB site in the mouse c-fos promoter. Furthermore, our studies demonstrated independent but coordinated functions of the IKKα/β-p65 and extracellular signal-regulated kinase (ERK)-Elk-1 pathways in the PMA induction of c-Fos. Collectively, these results reveal the distinct requirement of NF-κB for mouse and human c-fos regulation. Binding of the p65 homodimer to the κB site was indispensable for mouse c-fos expression, whereas the κB binding site was not present in the human c-fos promoter. Because of an inability to evoke sufficient ERK activation and Elk-1 phosphorylation, TNF-α induces c-Fos more weakly than PMA does in both mouse and human cells