Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury

<p>Abstract</p> <p>Background</p> <p>We hypothesized that gp91^phox(NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phoxand reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phoxknockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phoxgeneration.</p> <p>Methods</p> <p>Unilateral TBI was induced in gp91^phox-/-and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91^phoxafter TBI were investigated using immunoblotting and staining techniques. Levels of O₂^-and peroxynitrite were determined <it>in situ </it>in the mouse brain. The activated phenotype in microglia that expressed gp91^phoxwas determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.</p> <p>Results</p> <p>Gp91^phoxexpression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O₂^-and peroxynitrite metabolites produced were less in gp91^phox-/-mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91^phox.</p> <p>Conclusions</p> <p>Classical activated microglia promote ROS formation through gp91^phoxand have an important role in brain damage following TBI. Modulating gp91^phoxand gp91^phox-derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.</p

Improved Adsorption of an Enterococcus faecalis Bacteriophage ΦEF24C with a Spontaneous Point Mutation

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component

The Usefulness of ^99mTc-Technegas Scintigraphy for Diagnosing Pulmonary Impairment Caused by Pulmonary Emphysema

X-ray computed tomography (CT) has been used for diagnosis of pulmonary emphysema because it can reveal the morphology of low attenuation areas. Recently, 99mTc-Technegas imaging, one of several types of scintigraphic techniques, has been used for ventilation scintigraphy. Technegas scintigraphy was performed on 15 patients with pulmonary emphysema, and we compared the extent and degree of abnormal findings on Technegas scintigraphy with the extent of low attenuation areas shown by CT. We classified the findings of Technegas imaging into three grades, from mild to severe, according to the extent of peripheral irregularity and central hot spot formation. We also classified the findings of CT as centrilobular emphysema into three grades from mild to severe according to the extent of low attention areas in the peripheral lung fields. In 5 cases, CT and Technegas assessment resulted in equivalent diagnoses. In eight cases, Technegas images showed more detailed findings than CT images. In the two remaining cases, which were diagnosed as panlobular emphysema on CT, Technegas images showed the severe stage. Technegas scintigraphy was useful for diagnostic assessment of pulmonary emphysema, especially for panlobular emphysema, which is difficult to distinguish from the normal lung condition by CT assessment.</p

Inhibition of Chlorogenic Acid-induced Cytotoxicity by CoCl_2

Chlorogenic acid (CGA) induced apoptotic cell death in human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines. CGA exhibited oxidation Potential in the culture medium, as demonstrated by NO monitor. Both cytotoxic activity and oxidatoin potential were significantly reduced by addition of CoCl_2. ESR spectrascopy showed that CGA produced seven peaks of radicals under alkaline condition, while addition of CoCL_2 altered the spectral pattern and diminished the radical intensity of CCA. CoCl_2 accelerated the CGA-induced coloration of the culture medium and modified the difference spectrum at around 325 nm, an absorption maximum characteristic of CGA. These data suggest that CoCl_2 induced conformational changes in the CGA molecule. Chlorogenic acid (CGA) has shown diverse biological activities, including anti-HIV activity (1), antioxidant activity (2-4), anticarcinogenic activity (5-8), modulating activity of cytochrome P450-linked enzyme (9, 10) and antiallergic activity (11). We have recently reported that CGA induced cytotoxicity against human oral tumor cells (human oral squamous cell carcinoma HSC-2, human salivary gland tumor HSG) (12). The cytotoxic activity of CGA was significantly reduced by various antioxidants (catalase, sodium ascorbate, N-acetyl-L-cysteine) (13), suggesting that CGA induced cytotoxicity by its pro-oxidant action. However, the mechanism of cytotoxicity induction by CGA has not yet been elucidated. We have recently found that the cytotoxic activity of CGA was significantly reduced by COCI_2 (12). At present, there are at least two possibilities: one is that COCI_2 may directly interact with CGA and transform it into an inactive form, while a second one is that COCI_2 may induce transcription factors (4) which stimulate the gene expression of glycolytic enzymes and various growth factors necessary for cell survival. This study was undertaken to test the first possibility

Interaction between Chlorogenic Acid and Antioxidants

The interaction between chlorogenic acid (CGA) and antioxidants was investigated by two different parameters: radical intensity and cytotoxicity induction. ESR spectroscopy shows that CGA produced radicals under alkaline condition. The CGA radical was scavenged by 100-300-fold lower concentrations of sodium ascorbate or N-acetyl-l-cysteine (NAC), whereas the ascorbate radical was not completely scavenged by CGA. The cytotoxic activity of CGA ageinst human oral tumor cells (HSC-2, HSG) wa completely eliminated by lower concentrations of sodium ascorbate or NAC, whereas that of sodium ascorbate or NAC was only slightly reduced by CGA. The present study demonstrated that CGA ubdyces cytotoxicity by its radical-mediated oxidation mechanism and suggests the applicability ESR spectroscopy for the screening of drug to drug interaction

フキノール酸のラジカル消去能について

今まで報告例のないフキノール酸(FA)のラジカル消去能について、クロロゲン酸(CGA)、エピガロカテキンガレート(EGCG)、没食子酸(GA)と比較して検討した。スーパーオキシドアニオン(HX-XOD反応で生成)、ヒドロキシルラジカル(フェントン反応で生成)、一酸化窒素(NOC-7より生成)およびDPPHラジカル消去能はESR法を用いて測定した。またDPPHラジカル消去能は吸光度法でも測定した。その結果、スーパーオキシドアニオン消去能はEGCG>GA>FA>CGAの順、ヒドロキシルラジカル消去能はEGCG>CGA>FA>GAの順、一酸化窒素消去能はEGCG>FA>CGAの順、ESR法で測定したDPPHラジカル消去能はEGCG>GA>FA>CGAの順、そして吸光度法で測定したDPPHラジカル消去能はEGCG>FA>CGAの順となった。本研究によりFAは抗酸化剤としての利用が期待できると考えられる。Radical scavenging activity of fukinolic acid (FA), chlorogenic acid (CGA), epigallocatechin gallate (EGCG) and gallic acid (GA) against superoxide anion (produced by HX-XOD reaction), hydroxyl radical (produced by Fenton reaction), nitric oxide (NO) (produced by NOC-7 with carboxy-PTIO) and DPPH radical was measured by ESR spectroscopy and OD method. The radical scavenging activity of these compounds was in the following order : (1) superoxide anion, EGCG>GA>FA>CGA, (2) hydroxyl radical, EGCG>CGA>FA>GA, (3) NO, EGCG>FA>CGA, and (4) DPPH radical, EGCG>GA>FA>CGA (ESR method), EGCG>FA>CGA (OD method). These data suggest that FA should be considered as a candidate of applicable antioxidant as well as other polyphenols