Phospho‐dependent signaling during the general stress response by the atypical response regulator and ClpXP adaptor RssB

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two‐domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here, we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF₃ ⁻. Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a “meta‐active” state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y–T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins.     

Production,characterization, and assessment of a stable analog of the response regulator CheY‐phosphate from Thermotoga maritima

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY‐phosphate have been challenging due to the rapid hydrolysis of the aspartyl‐phosphate in vitro. An inert analog of Tm CheY‐phosphate, phosphono‐CheY, was synthesized by chemical modification and purified by cation‐exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono‐CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono‐CheY bound a peptide derived from FliM, a native partner of CheY‐phosphate, with a dissociation constant of ～29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono‐CheY is a useful and unique analog of Tm CheY‐phosphate.     

The crosstalk between trace elements with DNA damage response,repair, and oxidative stress in cancer

DNA damage response (DDR) is a regulatory system responsible for maintaining genome integrity and stability, which can sense and transduce DNA damage signals. The severity of damage appears to determine DDRs, which can include damage repair, cell-cycle arrest, and apoptosis. Furthermore, defective components in DNA damage and repair machinery are an underlying cause for the development and progression of various types of cancers. Increasing evidence indicates that there is an association between trace elements and DDR/repair mechanisms. In fact, trace elements seem to affect mediators of DDR. Besides, it has been revealed that oxidative stress (OS) and trace elements are associated with cancer development. In this review, we discuss the role of some critical trace elements in the risk of cancer. In addition, we provide a brief introduction on DDR and OS in cancer. Finally, we will further review the interactions between some important trace elements including selenium, zinc, chromium, cadmium, and arsenic, and DDR, and OS in cancer.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

The effect of menadione-induced oxidative stress on the in vivo reactive oxygen species and antioxidant response system of Phanerochaete chrysosporium

The antioxidant response system of Phanerochaete chrysosporium against menadione-induced oxidative stress was investigated in this study. The superoxide anion radical levels in tested menadione-supplemented conditions generally decreased over the incubation period. The level of hydrogen peroxide and the activities of NAD(P)H oxidase, superoxide dismutase (SOD) and catalase (CAT) were higher than those in the controls at all incubation times. The highest NADH and NADPH oxidase activities were determined to be 4.9- and 5.0-fold higher than those in the control, respectively in cells exposed to 0.75 mM menadione. The SOD and CAT activities increased with increasing menadione, and their highest activities were 5.4- and 5.1-fold higher than those in the control, respectively. In 0.1–0.5 mM menadione exposed cells, the lipid peroxidation levels did not change significantly when compared to each other, except 8th hour of incubation (p > 0.01). Our result shows that although menadione induces the formation of reactive oxygen species, the antioxidant response system of P. Chrysosporium is able to negate menadione-induced oxidative stress up to relatively high menadione concentrations, as 0.75 mM. These results are important to determine the effects of menadione, as a medicine, on the antioxidant response system of eukaryotic models and the resulting level of damage.     

Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans virulence properties

AIM: To assess potential function of each two-component signal transduction system in the expression of Streptococcus mutans virulence properties. METHODS AND RESULTS: For each two-component system (TCS), the histidine kinase-encoding gene was inactivated by a polymerase chain reaction (PCR)-based deletion strategy and the effects of gene disruption on the cell's ability to form biofilms, become competent, and tolerate acid, osmotic, and oxidative stress conditions were tested. Our results demonstrated that none of the mutations were lethal for S. mutans. The TCS-2 (CiaRH) is involved in biofilm formation and tolerance to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal competence development. CONCLUSIONS: Our results confirmed the physiological role of the TCS in S. mutans cellular function, in particular the SncRK-like TCS and TCS-9 as they may represent new regulatory systems than can be involved in S. mutans pathogenesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple TCS govern important biological parameters of S. mutans enabling its survival and persistence in the biofilm community.     

氧化应激在高原重体力劳动过程中急性高原反应发生中的作用

目的：观察氧化应激在高原重体力劳动过程中急性高原反应（AHAR）发生中的作用。方法：由低海拔（1500m）快速进入高原（3700m）并从事重体力劳动的男性官兵96名,年龄18～35岁。根据AHAR症状评分,分为重度AHAR组（A组,n=24）、轻中度AHAR组（B组,n=47）和无AHAR组（C组,n=25）,在该高度逗留50d后下撤前及返回低海拔（1500m）后12h、15d分别测定血清8．异前列腺素F2a（8-iso-PGF2a）、超氧化物歧化酶（SOD）、丙二醛（MDA）,并与低海拔（1500m）50名健康官兵（D组）比较。结果：A组血清8-iso-PGF2a、MDA[分别为（9．53±0．47）μg／L、（8．91±0．39）μmol／L]水平显著高于B组[分别为（8．34±O．42）μg／L、（7．31±0．32）μmol／L]、C组[分别为（7．02±0．48）μg／L、（6．41±0．23）μmol／L和D组[分别为（5．13±0．56）μg／L、（5．48±0．33）μmol／L]（均P〈0．01）,SOD（52．08±3．44）μ／mL水平显著低于B组（62．27±2．54）μ/mL、C组（71．99±3．35）μ/mL和D组（80．78±3．44）μ/mL,（均P〈0．01）,B组与c组之间和C组与D组之间亦有显著性差异（均P〈0．01）。海拔3700mAHAR总计分与血清8-iso-PGF2α、ⅣⅡ）A呈显著正相关（均P〈0．01）,与血清SOD显著负相关（P〈0．01）;8-iso-PGF2α、MDA与SOD显著负相关（均P〈0．01）。海拔3700m50d,血清8-iso-PGF2α、MDA水平显著高于,SOD水平显著低于海拔1500m12h、15d和D组（均P〈0．01）,海拔1500m12h与15d之间有显著性差异（均P〈0．01）,海拔1500m 15d与D组之间无显著性差异。结论：人体在高原低氧并重体力时氧化应激和氧化．抗氧化失衡与AHAR的发病和程度有密切关系,氧化应激和氧化．抗氧化失衡越严重,AHAR越重。返回低海拔后12h有显著改善,15d恢复到正常水平。     

Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea

In this study, we compare some antioxidative responses of leaves and roots associated to growth reduction in cowpea plants (Vigna unguiculata) during short-term salt stress and recovery. The salt treatment was imposed (200 mM NaCl) for six consecutive days and the salt withdrawal after 3 d. The salt treatment caused an almost complete cessation in the relative growth rate of both leaves and roots. Although NaCl withdrawal has induced an intense reduction in the Na(+) content from the leaves and roots, the growth recovery was slight, after 3 d. The leaf lipid peroxidation was increased in salt-stressed plants and slightly reduced in recovered plants after 3 d. Surprisingly, in the salt-stressed roots it decreased markedly after 3 d treatment and in the pre-stressed/recovered roots it was restored to levels near to the control. In leaves, catalase (CAT) activity showed a rapid and prominent decrease after 1 d of NaCl treatment and salt withdrawal had no effect on its recovery. In contrast, the root CAT activity was not changed by effects of both NaCl and salt withdrawal, over time interval. Leaf superoxide dismutase (SOD) activity did not change in all treatments, whereas in roots it significantly decreased after 3 d of salt treatment and recovered after NaCl withdrawal. Contrasting to the other enzymes, the guaiacol-peroxidase activity increased in leaves and roots, reaching almost 200% of control values and it significantly decreased in both organs from the pre-stressed/recovered plants. In conclusion, cowpea roots and leaves present distinct mechanisms of response to lipid peroxidation and CAT and SOD activities during salt stress and recovery. However, these responses and/or the oxidative damages caused by reactive oxygen species were not related with the growth reduction.     

Salt stress and hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp. PCC 6803.

Acclimation of microorganisms to environmental stress is closely related to the expression of various genes. We report here that salt stress and hyperosmotic stress have different effects on the cytoplasmic volume and gene expression in Synechocystis sp. PCC 6803. DNA microarray analysis indicated that salt stress strongly induced the genes for some ribosomal proteins. Hyperosmotic stress strongly induced the genes for 3-ketoacyl-acyl carrier protein reductase and rare lipoprotein A. Genes whose expression was induced both by salt stress and by hyperosmotic stress included those for heat-shock proteins and the enzymes for the synthesis of glucosylglycerol. We also found that each kind of stress induced a number of genes for proteins of unknown function. Our findings suggest that Synechocystis recognizes salt stress and hyperosmotic stress as different stimuli, although mechanisms common to the responses to each form of stress might also contribute to gene expression.     

Effect of Triphala on oxidative stress and on cell-mediated immune response against noise stress in rats

Stress is one of the basic factors in the etiology of number of diseases. The present study was aimed to investigate the effect of Triphala (Terminalia chebula, Terminalia belerica and Emblica officinalis) on noise-stress induced alterations in the antioxidant status and on the cell-mediated immune response in Wistar strain male albino rats. Noise-stress employed in this study was 100 dB for 4 h/d/15 days and Triphala was used at a dose of 1 g/kg/b.w/48 days. Eight different groups of rats namely, non-immunized: control, Triphala, noise-stress, Triphala with noise-stress, and corresponding immunized groups were used. Sheep red blood cells (5×10⁹ cells/ml) were used to immunize the animals. Biochemical indicators of oxidative stress namely lipid peroxidation, antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), ascorbic acid in plasma and tissues (thymus and spleen) and SOD, GPx and corticosterone level in plasma were estimated. Cell-mediated immune response namely foot pad thickness (FPT) and leukocyte migration inhibition (LMI) test were performed only in immunized groups. Results showed that noise-stress significantly increased the lipid peroxidation and corticosterone level with concomitant depletion of antioxidants in plasma and tissues of both non-immunized and immunized rats. Noise-stress significantly suppressed the cell-mediated immune response by decreased FPT with an enhanced LMI test. The supplementation with Triphala prevents the noise-stress induced changes in the antioxidant as well as cell-mediated immune response in rats. This study concludes that Triphala restores the noise-stress induced changes may be due to its antioxidant properties.     

Organismal regulation of XBP-1-mediated unfolded protein response during development and immune activation

The increased demand on protein folding in the endoplasmic reticulum (ER) during bacterial infection activates the unfolded protein response (UPR). OCTR-1-a G protein-coupled catecholamine receptor expressed in neurons-suppresses innate immunity by downregulating a non-canonical UPR pathway and the p38 MAPK pathway. Here, we show that OCTR-1 also regulates the canonical UPR pathway, which is controlled by XBP-1, at the organismal level. Importantly, XBP-1 is not under OCTR-1 control during development, only at the adult stage. Our results indicate that the nervous system temporally controls the UPR pathway to maintain ER homeostasis during development and immune activation.     

DNA endonuclease- and active oxygen-associated degradation of chloroplast DNA in response to paraquat-induced oxidative stress

Activity of chloroplast-localized DNA endonuclease was observed in detached tobacco leaves that had been treated with paraquat and light The DNA endonuclease was able to cleave the chloroplast, plasmid, and single-stranded DNA, as estimated on an agarose gel. Activity was sensitive to two endonuclease inhibitors: aurintricarboxylic acid and ZnSO₄. The time course for activity showed a peak 4 h after the stress treatment These results suggest that this enzyme plays a specific physiological role during oxidative stress. Probable roles for this enzyme are also discussed.     

MAPK转导通路在白念珠菌菌体抗氧化应激中的作用

目的探讨MAPK通路在念珠菌抗氧化应激中的作用。方法采用不同浓度过氧化氢刺激白念珠菌,通过流式细胞仪检测念珠菌的凋亡率,并计算其增殖指数;通过实时荧光定量PCR检测MAPK通路中8种基因的表达水平。结果随着过氧化氢的刺激浓度增高,白念珠菌的凋亡率逐渐升高,而其增殖指数下降。在不同的过氧化氢浓度刺激下,MAPK通路中各基因表达水平基本一致,即在较低的过氧化氢浓度刺激下,各基因表达水平均有一定的上升,而随着浓度增高,在高浓度的过氧化氢刺激下,各基因表达水平趋于稳定。结论在低浓度的过氧化氢刺激下,白念珠菌的凋亡率虽有所上升,但其相应的增殖指数也有所上升,即生长加快。这可能与其MAPK通路中各基因表达增强有一定的关系。