Daniel Tsun-Yee Chiu

Impaired production of nitric oxide, superoxide, and hydrogen peroxide in glucose 6‐phosphate‐dehydrogenase‐deficient granulocytes

Since the generation of superoxide and hydrogen peroxide by NADPH oxidase and nitric oxide (NO) by NO synthase (NOS) in granulocytes is NADPH‐dependent, we investigated the production of NO, superoxide and H2O2 in glucose 6‐phosphate dehydrogenase (G6PD)‐deficient human granulocytes. Our results showed that upon stimulation with either 5 μg/ml of lipopolysaccharide (LPS) or 10 μM of phorbol 12‐myristate 13‐acetate (PMA), the production of nitrite in normal granulocytes was elevated, 252±135% and 239±72%, respectively, compared to the resting stage. In contrast, G6PD‐deficient granulocytes did not produce more nitrite upon stimulation with either LPS or PMA compared to the resting stage. Western blot analysis indicated a normal expression pattern of inducible NOS in G6PD‐deficient granulocytes. In addition, the production of H2O2 and superoxide was also significantly impaired in G6PD‐deficient granulocytes compared to control cells. These data demonstrate that G6PD deficiency causes an impairment in the production of NO, superoxide and H2O2.

Disabled-2 small interfering RNA modulates cellular adhesive function and MAPK activity during megakaryocytic differentiation of K562 cells.

Previous studies have shown that Disabled‐2 (DAB2) is up‐regulated during megakaryocytic differentiation of human K562 cells. To delineate the consequences of DAB2 induction, a DNA vector‐based small interfering RNA (siRNA) was designed to intervene in DAB2 expression. We found that DAB2 siRNA specifically inhibited DAB2 induction, resulting in the modulation of cell–cell adhesion and mitogen‐activated protein kinase (MAPK) phosphorylation. The morphological changes and β3 integrin expression associated with megakaryocytic differentiation were not affected. Since the MAPK pathway has been shown to involve DAB2 induction [Tseng et al., Biochem. Biophys. Res. Commun. 285 (2001) 129–135], our results suggest a reciprocal regulation between DAB2 and MAPK in the differentiation of K562 cells. In addition, we have demonstrated for the first time that DAB2 siRNA is a valuable tool for unveiling the biological consequences of DAB2 expression.

Broad-Range Ribosomal RNA Real-Time PCR after Removal of DNA from Reagents: Melting Profiles for Clinically Important Bacteria

With the ability to exponentially amplify regions of DNA, two different PCR-based strategies have been developed for nonculture diagnosis of bacteremia. The first approach targets the species-specific genes for amplification (1), whereas the second approach involves universal PCR amplification of conserved bacterial DNA sequences, such as the 16S rRNA, the 23S rRNA, and the 16S-23S rRNA interspace regions (2)(3)(4). Although universal PCR is not able to distinguish bacteria to the species level, numerous studies have shown that this method provides valuable information complementary to the results of time-consuming and subjective phenotypic tests used in detection of bacterial infection (5) and can be used to differentiate bacterial from viral or other infections (6)(7). In clinical applications, real-time PCR for broad-range amplification of bacterial DNA sequences could offer additional benefits: it is less labor-intensive, less time-consuming, and reduces the risk of PCR carryover contamination.nnA major obstacle for broad-range PCR amplification is the presence of bacterial DNA in the Taq DNA polymerase and real-time PCR master mixture (8)(9)(10). The contaminating DNA is effectively amplified, giving rise to false-positive results. Efforts have been taken to eliminate contaminating DNA, including the use of Sau 3AI restriction endonuclease (11), DNase I (12)(13), and ultraviolet irradiation (7)(8). Although these strategies may be effective for conventional PCR, the study by Corless et al. (14) indicated that the contamination issue could not be avoided without affecting sensitivity when TaqMan probe-based real-time PCR is performed. Therefore, an appropriate method for removal of contaminating bacterial DNA and subsequent real-time amplification is required. In this study, we address this issue and report optimized conditions for broad-range amplification of the bacterial 16S rRNA gene by real-time PCR using SYBR Green I dye (15) as the fluorescent signal.nnBacterial …

ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These resultsxa0identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.

Glucose-6-phosphate dehydrogenase – beyond the realm of red cell biology

Abstract Glucose-6-phosphate dehydrogenase (G6PD) is critical to the maintenance of NADPH pool and redox homeostasis. Conventionally, G6PD deficiency has been associated with hemolytic disorders. Most biochemical variants were identified and characterized at molecular level. Recently, a number of studies have shone light on the roles of G6PD in aspects of physiology other than erythrocytic pathophysiology. G6PD deficiency alters the redox homeostasis, and affects dysfunctional cell growth and signaling, anomalous embryonic development, and altered susceptibility to infection. The present article gives a brief review of basic science and clinical findings about G6PD, and covers the latest development in the field. Moreover, how G6PD status alters the susceptibility of the affected individuals to certain degenerative diseases is also discussed.

Diminished COX-2/PGE2-Mediated Antiviral Response Due to Impaired NOX/MAPK Signaling in G6PD-Knockdown Lung Epithelial Cells

Glucose-6-phosphate dehydrogenase (G6PD) provides the reducing agent NADPH to meet the cellular needs for reductive biosynthesis and the maintenance of redox homeostasis. G6PD-deficient cells experience a high level of oxidative stress and an increased susceptibility to viral infections. Cyclooxygenase-2 (COX-2) is a key mediator in the regulation of viral replication and inflammatory response. In the current study, the role of G6PD on the inflammatory response was determined in both scramble control and G6PD-knockdown (G6PD-kd) A549 cells upon tumor necrosis factor-α (TNF-α) stimulation. A decreased expression pattern of induced COX-2 and reduced production of downstream PGE2 occurred upon TNF-α stimulation in G6PD-kd A549 cells compared with scramble control A549 cells. TNF-α-induced antiviral activity revealed that decreased COX-2 expression enhanced the susceptibility to coronavirus 229E infection in G6PD-kd A549 cells and was a result of the decreased phosphorylation levels of MAPK (p38 and ERK1/2) and NF-κB. The impaired inflammatory response in G6PD-kd A549 cells was found to be mediated through NADPH oxidase (NOX) signaling as elucidated by cell pretreatment with a NOX2-siRNA or NOX inhibitor, diphenyleneiodonium chloride (DPI). In addition, NOX activity with TNF-α treatment in G6PD-kd A549 cells was not up-regulated and was coupled with a decrease in NOX subunit expression at the transcriptional level, implying that TNF-α-mediated NOX signaling requires the participation of G6PD. Together, these data suggest that G6PD deficiency affects the cellular inflammatory response and the decreased TNF-α-mediated antiviral response in G6PD-kd A549 cells is a result of dysregulated NOX/MAPK/NF-κB/COX-2 signaling.

Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling.

Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α) and GTPase myxovirus resistance 1 (MX1)—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E) and enterovirus 71 (EV71) infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH) sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

Tryptophan as a surrogate prognostic marker for diabetic nephropathy

Diabetic nephropathy is one of the leading causes of end‐stage renal disease. Unfortunately, reliable surrogate markers for predicting the prognostic outcome of diabetic nephropathy are as yet absent. In order to find new markers in predicting the progression of diabetic nephropathy, we carried out a prospective study by investigating the correlation between serum metabolites and the annual change of estimated glomerular filtration rate (eGFR).

Glucose-6-phosphate dehydrogenase is indispensable in embryonic development by modulation of epithelial-mesenchymal transition via the NOX/Smad3/miR-200b axis

Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme involved in the pentose phosphate shunt for producing nicotinamide adenine dinucleotide phosphate (NADPH). Severe G6PD deficiency leads to embryonic lethality, but the underlying mechanism is unclear. In the current study, the effects of G6PD on epithelial–mesenchymal transition (EMT), especially during embryonic development, were investigated. The knockdown of G6PD induced morphological changes, accompanied by the suppression of epithelial markers, E-cadherin and β-catenin, in A549 and MDCK cells. Such modulation of EMT was corroborated by the enhancement of migration ability in G6PD-knockdown A549 cells. Zebrafish embryos with g6pd knockdown exhibited downregulation of the E-cadherin/β-catenin adhesion molecules and impaired embryonic development through reduction in epiboly rate and increase in cell shedding at the embryo surface. The dysregulation in zebrafish embryonic development caused by g6pd knockdown could be rescued through human G6PD or CDH1 (E-cadherin gene) cRNA coinjection. The Smad3/miR-200b axis was dysregulated upon G6PD knockdown, and the reconstitution of SMAD3 in G6PD-knockdown A549 cells restored the expression of E-cadherin/β-catenin. The inhibition of NADPH oxidase (NOX) activation through the loss of p22phox signaling was involved in the dysregulation of the Smad3/miR-200b axis upon G6PD knockdown. The reconstitution of G6PD led to the recovery of the regulation of NOX/Smad3/miR-200b signaling and increased the expression of E-cadherin/β-catenin in G6PD-knockdown cells. Thus, these results suggest that in the EMT process, G6PD plays an important regulatory role as an integral component of the NOX/Smad3/miR-200b axis.

Oxidative stress in biology and medicine

In this issue of Biomedical Journal, we have organized a special section related to “oxidative stress in biology and medicine” with four invited review articles. These four articles are written by invited speakers of the “Biennial Meeting of Society for Free Radical Research‐Asia” held in Chang Gung University of Taiwan from October 16 to 19, 2013. The first two review articles in this special section are written by Professor Barry Halliwell of Singapore and Professor Etsuo Niki of Japan, respectively, and are part of their Plenary Lecture during the aforementioned meeting. The third review article is written by Professor Steven Qian and his associate of the United States. The fourth review article is written by Professor Jiangang Shen and his associates of Hong Kong. Professor Barry Halliwell’s review article is entitled “Cell culture, oxidative stress, and antioxi‐ dants: Avoiding pitfalls.” In this article, Professor Halliwell points out the potential artifacts created by reagents, particularly “antioxidants,” because these chemicals can react with the constituents of the cell culture media to produce H 2 O 2 and degradation products which can influence cell behavior. This article would be extremely helpful to the free radical/antioxidant community to have greater awareness of the potential artifacts in cell culture studies. The second article written by Professor Etsuo Niki is entitled “Antioxidants: Basic principles, emerging concepts, and problems.” It is well accepted that radical‐scavenging antioxidants play an essential role in the maintenance of health and prevention of diseases. However, Professor Niki points out in his article that there is no evidence to support the use of non‐discriminative antioxidant supple‐ ment for prevention of diseases. On the other hand, recent data show that antioxidants may be effective in the prevention and/or treatment of diseases when the right antioxidant is given to the right subject at the right time for the right duration. Recent findings in a clinical study by Professor Chandan Sen and his associates that tocotrienol provides multimodal protection against stroke (Oxygen Club of California Meeting at Davis, CA, USA, 2014) provide additional strong support to this concept. The third article written by Professor Steven Qian and his associate is entitled “Anti‐cancer activi‐ ties of ω‐6 polyunsaturated fatty acids.” Although it is well‐known that the ω‐6s are abundant in our daily diet and could be implicated in many pathological processes including cancer development, the beneficial effects of γ‐linolenic acid (GLA) and dihomo‐γ‐linolenic acid (DGLA), two of arachidonic acid upstream ω‐6s, have been just started to be recognized. In this paper, Professor Qian reviews the documented anticancer activities of ω‐6 polyunsaturated fatty acids (PUFAs), including the recent findings regarding the anticancer effects of free radical–mediated DGLA peroxidation. Such an under‐ standing of the beneficial effects of ω‐6 PUFAs would be helpful for the development of a ω‐6–based diet care strategy for cancer prevention and treatment. The fourth article written by Professor Jiangang Shen and his associates is entitled “Pros and cons of current approaches for detecting peroxynitrite and their applications.” Peroxynitrite, a representative of reactive nitrogen species, plays important roles in the physiological and pathological processes of health and diseases. However, due to its high reactivity and short half‐life, accurate measurement of peroxynitrite in biological systems is a great challenge. In this article, three major approaches for peroxynitrite detec‐ Oxidative Stress in Biology and Medicine