Nitroblue Tetrazolium Staining Research Articles

Protein carbonylation in barley seedling roots caused by aluminum and proton toxicity is suppressed by salicylic acid

Proton (H+) and aluminum (Al3+) toxicities are major factors limiting crop production on acid soils. To study whether salicylic acid (SA) is functional in alleviating protein damage caused by H+ and Al3+ toxicities, an investigation of the antioxidant defense response regulated by SA was carried out on barley (Hordeum vulgare L.) seedlings under H+, Al3+, and combined stresses. It was found that the relative root elongation of seedlings, which grew in the solutions supplemented with SA, was significantly higher than that of seedlings without SA treatment after 24-h treatments with H+, Al3+, and combined stresses. The lesser amount of carbonylated proteins with molecular weights ranging from 14.4 to 97 kD, was accumulated in seedlings treated with SA than that in the seedlings without SA treatment. The higher activities of antioxidant enzymes and lesser content of MDA were observed in seedlings treated with SA compared with the seedlings without SA treatment. Moreover, the nitroblue tetrazolium staining of roots showed that ROS accumulation was decreased by SA treatments. This study suggested that SA could alleviate cell damage caused by H+ and Al3+ toxicities on acid soils by both activating antioxidant defense responses and reducing the contents of carbonylated proteins caused by ROS in barley seedlings.

Quantitative determination of superoxide in plant leaves using a modified NBT staining method

In plants, the ROS (reactive oxygen species) level is tightly regulated because their accumulation produces irreversible damage leading to cell death. However, ROS accumulation plays a key role in plant signaling under biotic or abiotic stress. Although various methods were reported to evaluate ROS accumulation, they are restricted to model plants or provide only qualitative information. Develop a simple method to quantify superoxide radicals produced in plant tissues, based on the selective extraction of the formazan produced after nitroblue tetrazolium (NBT) reduction in histochemical staining. Plant leaves were stained with a standard NBT method and the formazan precipitated in tissues was selectively extracted using chloroform. The organic phase was dried and formazan residue dissolved in dimethylsulfoxide-potassium hydroxide and quantified by spectrophotometry. The method was tested in strawberry plant leaves under different stressing conditions. Formazan extracted from leaves subjected to stress conditions showed similar absorption spectra to those obtained from standard solutions using pure formazan. Calibration curves showed a linear relationship between absorbance and formazan amounts, within the range 0.5-8 µg. Outcomes suggested that formazan was retained in the solid residue of leaf tissues. This protocol allowed us to quantify superoxide radicals produced under different stress conditions. Chloroform allowed a selective formazan extraction and removal of potential endogenous, exogenous or procedural artefacts that may interfere with the quantitative determination. This protocol can be used to quantify the superoxide produced in plant tissues using any traditional qualitative NBT histochemical staining method.

Inhibition of semicarbazide-sensitive amine oxidase attenuates myocardial ischemia–reperfusion injury in an in vivo rat model

Aims This study tested the hypothesis that the inhibition of semicarbazide-sensitive amine oxidase (SSAO) after ischemia could attenuate myocardial ischemia–reperfusion (I/R) injury. Main methods Anesthetized male Sprague–Dawley rats underwent myocardial I/R injury. Saline, semicarbazide (SCZ, 30 mg/kg), hydralazine (HYD, 10 mg/kg), or LJP 1207 (30 mg/kg) was administered intraperitoneally 3 min before reperfusion. After 30 min of ischemia and 180 min of reperfusion, the myocardial infarct size was determined using nitroblue tetrazolium staining. Myocardial myeloperoxidase activity was determined through biochemical assay. HE staining was used for histopathological evaluation. Myocardial SSAO activity was assayed with high performance liquid chromatography analysis. Additionally, the endothelial expression of P-selectin was evaluated using immunohistochemistry after 30 min of ischemia and 20 min of reperfusion. Key findings Myocardial SSAO activity was increased in myocardial I/R injury. Administration of SCZ, HYD, or LJP 1207 reduced the myocardial infarct size and decreased leukocyte infiltration and endothelial P-selectin expression in myocardial I/R injury in vivo. Significance These data suggest that myocardial I/R injury up-regulates myocardial SSAO activity, and the inhibition of SSAO prior to reperfusion is able to attenuate acute myocardial I/R injury.

Catestatin Improves Post-Ischemic Left Ventricular Function and Decreases Ischemia/Reperfusion Injury in Heart

The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium.

Protective effect of Xinning tablet on acute myocardial ischemia in anesthesia dogs

Abstract This work aimed to observe the protective effect of Xinning tablets on experimental myocardial ischemia. The canine model of acute myocardial infarction was established by ligating the anterior descending coronary artery of dogs. The ultrasound Doppler method was adopted to determine myocardial blood flow. Heart membrane electrical diagram coup and nitro blue tetrazolium (N-BT) staining were used to calculate the extent of myocardial ischemia and myocardial infarct size. Radioimmunoassay was used to determine plasma ET, TXB 2 and 6-Keto-PGF 1α levels. 30-180 min after administration of Xinning tablets at 1.2 and 0.6 g/kg, the dog myocardial ischemia icon measured from the epicardial electrogram was significantly reduced; the infarct area was decreased according to NBT staining. 30-120 min after the administration, cardiac blood flow was significantly increased in the myocardial ischemia area, the levels of plasma ET and TXB 2 were greatly inhibited, and the 6-Keto-PGF1α content to TXB 2 /6-Keto-PGF1α ratio was increased. Taken together, Xinning tablets have significant protective effect on dog acute myocardial ischemia and myocardial infarction, the mechanism of which may be related to ET inhibition, TXA 2 release and increased myocardial blood flow, consequently improving the impaired heart function.

Changes in susceptibility of grape berries to splitting are related to impaired osmotic water uptake associated with losses in cell vitality

Background and Aims: High rainfall events after the onset of ripening commonly cause the skin of grape berries to split. The aim was to verify the suggestion, based on vineyard observations, that the susceptibility of grapes to splitting decreases during ripening, and to establish the role of cell vitality in this process. Methods and Results: The susceptibility of detached, ripening berries, cv. Shiraz, to splitting was assessed by immersing them in sucrose solutions or deionised water. At the onset of ripening, susceptibility to splitting was highest and decreased only slightly over 30 days. Thereafter, susceptibility decreased greatly until becoming negligible about 30 days later. Cell vitality within the pericarp, as determined by nitroblue tetrazolium staining, decreased from the onset of ripening. Conclusions: The reduction in the susceptibility of grape berries to splitting is attributed to a decrease in turgor-generating capacity within the berry as an increasing proportion of pericarp cells lose vitality. The loss of cell vitality corroborates previous evidence of this phenomenon, and indicates that this is a general feature of grape ripening. Significance of the Study: This study demonstrates the proportion of vital cells within the pericarp is the driver of turgor-induced splitting, in contrast to previous models in which the whole berry, enclosed in a semi-permeable skin, has been regarded as the osmotically functional system.

Superoxide anion and hydrogen peroxide in the yeast antagonist–fruit interaction: A new role for reactive oxygen species in postharvest biocontrol?

The importance of reactive oxygen species (ROS) in plant defense responses against certain pathogens is well documented. There is some evidence that microbial biocontrol agents also induce a transient production of ROS in a host plant which triggers local and systemic defense responses to pathogens. The ability of biocontrol agents used to control postharvest diseases to induce defense-related oxidative responses in fruits, however, has not been explored. Here we show that the yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate greater levels of super oxide anion (O 2 −) on intact fruit surfaces (poor in nutrients) then those applied on a nutrient-poor agar medium. Even though yeast antagonists show a high level of O 2 − on nutrient-rich media, when applied on fruits around wounds (areas abundant in nutrients) accumulation of O 2 −, as detected by nitro blue tetrazolium staining, occurred much more rapidly on the latter. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H 2O 2 accumulation in host tissue. In citrus fruit, the level of H 2O 2 around inoculated wounds increased by 4-fold compared to controls (wounds inoculated with water) as early as 18 h after inoculation. Yeast continued to stimulate H 2O 2 production in citrus fruit up to 66 h after inoculation and H 2O 2 levels were still 3-fold above the control. Living yeast cells were detected in fruit wounds at this time point indicating the ability of M. fructicola to tolerate host ROS, which has been reported to be an intrinsic characteristic of efficient yeast antagonists. Similar increase in H 2O 2 accumulation around yeast-inoculated wounds was observed in apple fruit exocarp. The present data, together with our earlier discovery of the importance of H 2O 2 production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases.

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem Cell Transplantation

Possible cardiac repair by adult stem cell transplantation is currently hampered by poor cell viability and delivery efficiency, uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, positron emission tomography, and immunohistochemistry, we show that injection of a hyaluronan mixed ester of butyric and retinoic acid (HBR) into infarcted rat hearts afforded substantial cardiovascular repair and recovery of myocardial performance. HBR restored cardiac [18F]fluorodeoxyglucose uptake and increased capillary density and led to the recruitment of endogenous Stro-1-positive stem cells. A terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay demonstrated that HBR-treated hearts exhibited a decrease in the number of apoptotic cardiomyocytes. In isolated rat cardiomyocytes and Stro-1 stem cells, HBR enhanced the transcription of vascular endothelial growth factor, hepatocyte growth factor, kdr, akt, and pim-1. HBR also increased the secretion of vascular endothelial growth factor and hepatocyte growth factor, suggesting that the mixed ester may have recruited both myocardial and Stro-1 cells also. An increase in capillarogenesis was induced in vitro with medium obtained from HBR-exposed cells. In the infarcted myocardium, HBR injection increased histone H4 acetylation significantly. Acetyl-H4 immunoreactivity increased in rat cardiomyocytes and Stro-1 cells exposed to HBR, compared with untreated cells. In conclusion, efficient cardiac regenerative therapy can be afforded by HBR without the need of stem cell transplantation or vector-mediated gene delivery.

Postembryonic seedling lethality in the sterol-deficient Arabidopsis cyp51A2 mutant is partially mediated by the composite action of ethylene and reactive oxygen species.

Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14alpha-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.

The Effect of Vitamin E Succinate on Ischemia Reperfusion Injury

Ischemia-reperfusion (I/R) injury is, in part, related to the burst of reactive oxygen species (ROS) generated after reperfusion. Vitamin E has been shown to exert its biological effects as an antioxidant, inhibiting the ROS. In this report, the effect of Vitamin E succinate (VES) on ischemia/reperfusion injury and NF-κB expression was studied in a rat skeletal muscle model during reperfusion following a 4-h ischemic period. The study group consisted of muscle flaps infused with 150mg/kg of VES given intraperitoneally 1h post-initiation of ischemia. Muscle viability based on nitroblue tetrazolium staining, edema, and Doppler blood flow was measured in a control and a study group. Muscle samples were analyzed by standard gel shift assay. The VES experimental group showed an increase in muscle viability compared to controls (average of 44.675% versus 31.925%, respectively, p = 0.0415). Blood flow, measured by Doppler 24h after reperfusion, was increased in the VES study group compared to controls (10.3 vs. 5.1ml/g/s p = 0.00355). Additionally, the VES group showed a trend of decreasing edema compared with the control; however, not at a level that was statistically significant (p = 0.1267). The VES-treated group showed a decreased expression of NF-κB as compared to controls (p < 0.05). These results show that vitamin E succinate has a protective effect in preventing I/R injury as measured by increased muscle viability and reperfusion blood flow. Vitamin E can exert its efforts as an antioxidant as well as other biological roles, including inhibition of NF-κB.

NOVEL FHL1 MUTATIONS IN FATAL AND BENIGN REDUCING BODY MYOPATHY

Reducing body myopathy (RBM) is a rare disorder characterized pathologically by the presence of intracytoplasmic inclusions strongly stained by menadione-NBT (nitroblue tetrazolium) staining in the absence of the substrate α-glycerophosphate. The causative gene for RBM was recently identified as FHL1 on chromosome Xq27 encoding four and a half LIM domains 1.1 FHL1 is a 32 kDa protein, composed of four LIM domains preceded by a single N-terminal zinc finger. FHL1 is highly expressed in skeletal muscle and heart. Here, we searched for FHL1 mutations in three sporadic cases2-4 and one familial case5 of RBM we previously reported. ### Methods. All clinical materials used in this study were obtained for diagnostic purpose with informed consent. Patient 1 and patient 2 have fatal infantile form,2,3 and patient 3 has adult-onset form.4 Patients 4 (son) and 5 (his mother) had familial cases.5 We directly sequenced all exons and their flanking intronic regions of FHL1 in the five RBM patients and 250 Japanese controls. Frozen muscle specimens were examined by immunohistochemistry and immunoblotting using standard technique. ### Results. We identified four novel mutations in FHL1 : a heterozygous missense mutation of c.449G>A (p.C150Y) in patient 1 and c.302G>T (p.C101F) in patient 2, an in-frame 9 bp deletion at c.304-312delAAGGGGTGC (p.102-104delKFC) in patient 3, and a hemizygous mutation c.310T>C (p.C104R) in patient 4. The mother (patient 5) had the same mutation in heterozygous mode. All mutations we identified are located in the second LIM domain of FHL1 (figure e-1 on the Neurology ® Web site at www.neurology.org). Immunohistochemical analysis of …

Transcriptional Activation in Yeast in Response to Copper Deficiency Involves Copper-Zinc Superoxide Dismutase

Copper is an essential trace element, yet excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. In Saccharomyces cerevisiae, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. Here we show that the copper-dependent enzyme Sod1 (Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either CCS1 or SOD1 results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. Given the presence of both Sod1 and copper-responsive transcription factors in higher eukaryotes, these studies may yield important insights into how copper deficiency is sensed and appropriate cellular responses are coordinated.

A new nutrient polypeptide-Fe and its antioxidant ability

Polypeptide-Fe is the hemoglobin that is purified highly from animal blood. It is an active material obtained by connecting heme with a different-length peptide section after physical and chemical processing. Polypeptide-Fe is non-toxic to the human body, does not have the side effects, and namely has iron enhancement function. Nitroblue tetrazolium staining showed that it retained the superoxide radical scavenging activity of hemoglobin. Polypeptide-Fe achieves the elimination of free radical by degradations of the polypeptide connected to iron. Oxidative stress has been considered to play an important role in the pathogenesis of iron-deficiency anemia. The primary free radicals in most of biological system are the superoxide anion, hydrogen peroxide, and the hydroxyl free radical. We performed experiments to detect the clearance ability on these three free radicals and maleic dialdehyde by polypeptide-Fe. Results of the present study showed that polypeptide-Fe had strong antioxidant ability and it may provide some useful information relevant to an additional contribution to the treatment of iron-deficiency anemia in future.

Groucho/TLE Corepressors Regulate Myeloid Differentiation and Size of the Stem Cell/Progenitor Population.

The Groucho (Gro)/TLE family of transcriptional co-repressor proteins have been called master regulatory genes based on their interaction with a variety of transcription factors and their critical role in development. The TLEs have also been shown to play major roles in brain and lymphocyte differentiation. We became interested in this gene family after finding two family members, TLE1 and TLE4, localized to the commonly deleted region on chromosome 9q in acute myeloid leukemia (AML). This deletion is tightly associated with t(8;21), and we recently showed loss of TLE1 and TLE4 cooperated with AML1-ETO to affect myeloid cell proliferation and survival, implicating TLE1 and 4 as potential tumor suppressor genes in AML. Based on their known ability to inhibit the function of several signaling pathways and transcription factors including Wnt/b-catenin, NF-kB, AML1, and Pu.1 known to be important in leukemogenesis and hematopoiesis, we undertook a series of experiments to determine whether the TLEs could affect myeloid cell differentiation and proliferation. We showed that expression of either TLE1 or TLE4 was able to induce differentiation in the HL-60 myeloid cell line as demonstrated by morphological changes, increased expression of the myeloid differentiation makers CD11b, CD14 and CD15, downregulated myeloperoxidase activity, as well as increased nitroblue tetrazolium (NBT) staining. Furthermore, when HL-60 cells were induced to differentiate by exposure to all-trans-retinoic acid (ATRA) we observed a 150-fold transient increase in TLE1 message after three days. Knockdown of TLE1 with specific shRNAs partially blocked ATRA-induced differentiation as monitored by CD11b expression, suggesting this burst of TLE1 expression is critical for ATRA induced myeloid cell differentiation. To determine the role of the TLEs in primary human cells, human cord blood cells were sorted, and TLE mRNA levels were determined in progenitor cells (CD34+/CD33−/CD16−/CD15−/CD14−), early myeloid precursors (CD34−/CD33+/CD16−/CD15−CD14−) and mature granulocytes (CD34−/CD33+/CD16+/CD15−CD14−). Lowest levels of TLE1 and TLE4 were found in progenitor cells with a peak of expression in early myeloid precursors. To help determine the significance of these low TLE levels in hematopoietic stem/progenitor cells, we designed shRNAs to simultaneously knockdown both TLE1 and TLE4. Knockdown of TLE1/4 in cord blood cells resulted in a 6.3-fold expansion of CD34+CD38− cells after 4 days' coculture with M2-10B4 in the presence of hSCF, hFlt3-ligand, and hTPO, as compared with only 2.6-fold expansion of cells transfected with control shRNA. Similarly, using mouse bone marrow cells cultured in vitro in the presence of mIL-3, mIL-6 and mSCF, we found a 2.1-fold increase in mouse bone marrow Lin−/Sca-1+/c-kit+ (LSK) cells transfected with TLE1/4 shRNA as compared to cells transfected with scrambled control shRNA. Our results indicate that modulation of TLE levels is capable of influencing both myeloid differentiation, as well as expansion of the stem cell and/or progenitor population.

The Chromate-Inducible chrBACF Operon from the Transposable Element Tn OtChr Confers Resistance to Chromium(VI) and Superoxide

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.

Multidetector computed tomography (MDCT) evaluation of myocardial viability: intraindividual comparison of monomeric vs. dimeric contrast media in a rabbit model

To evaluate the influence of different types of iodinated contrast media on the assessment of myocardial viability, acute myocardial infarction (MI) was surgically induced in six rabbits. Over a period of 45 min, contrast-enhanced cardiac MDCT (64 x 0.6 mm, 80 kV, 680 mAs(eff.)) was repeatedly performed using a contrast medium dose of 600 mg iodine/kg body weight. Animals received randomized iopromide 300 and iodixanol 320, respectively. Attenuation values of healthy and infarcted myocardium were measured. The size of MI was computed and compared with nitroblue tetrazolium (NBT)-stained specimen. The highest attenuation differences between infarcted and healthy myocardium occurred during the arterial phase with 140.0+/-3.5 HU and 141.0+/-2.2 HU for iopromide and iodixanol, respectively. For iodixanol the highest attenuation difference on delayed contrast-enhanced images was achieved 3 min post injection (73.5 HU). A slightly higher attenuation difference was observed for iopromide 6 min after contrast medium injection (82.2 HU), although not statistically significant (p=0.6437). Mean infarct volume as measured by NBT staining was 33.5%+/-13.6%. There was an excellent agreement of infarct sizes among NBT-, iopromide- and iodixanol-enhanced MDCT with concordance-correlation coefficients ranging from rho(c)=0.9928-0.9982. Iopromide and iodixanol both allow a reliable assessment of MI with delayed contrast-enhanced MDCT.

Gefitinib potentiates myeloid cell differentiation by ATRA

Gefitinib potentiates myeloid cell differentiation by ATRA

Migration of Vascular Smooth Muscle Cells by High Glucose is Reactive Oxygen Dependent

Background: Oxidative stress contributes to vascular diseases in patients with diabetes. As the mechanism of development and progression of diabetic vascular complications is poorly understood, th is study was aimed to assess the potential role of hyperglycemia-induced oxidative stress and to determine whether the oxidative stress is a major factor in hyperglycemia-induced migration of vascular smooth muscle cells (VSMCs). Methods: We treated primary cu ltured rat aortic smooth muscle cells for 72 hours with medium containing 5.5 mM D-glucose (normal glucose), 30 mM D-glucose (high glucose) or 5.5 mM D-glucose plus 24.5 mM mannitol (osmotic control). We me asured the migration of VSMCs and superoxide production. Immunoblotting of PKC isozymes using phoshospecific antibodies was performed, and PKC activity was also measured. Results: Migration of VSMCs incubated under high glucose condition were markedly increased compared to normal glucose condition. Treatment with diphenyleneiodonium (DPI, 10 μmol/L) and superoxide dismutase (SOD, 500 U/mL) significantly suppressed high glucose-induced migration of VSMCs. Superoxide production was significantly increased in high glucose condition and was markedly decreased after treatment with DPI and SOD. High glucose also markedly increased activity of PKC-δ isozyme. When VSMCs were treated with rottlerin or transfected with PKC-δ siRNA, nitro blue tetrazolium (NBT) staining and NAD(P)H oxidase activity were significantly attenuated in the high glucose-treated VSMCs. Furthermore, inhibition of PKC-δ markedly decreased VSMC migration by high glucose. Conclusion: These results suggest that high glucose-induced VSMC migration is dependent upon activation of PKC-δ, which may responsible for elevated intracellular ROS producti on in VSMCs, and this is mediated by NAD(P)H oxidase. (KOREAN DIABETES J 32:185-195, 2008)

A tip-high, Ca(2+) -interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes.

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca(2+)](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca(2+)](cyt) gradient. Such modulation of intracellular [Ca(2+)](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.

Histochemical studies on the accumulation of reactive oxygen species (O 2− and H 2O 2) in the incompatible and compatible interaction of wheat— Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O 2 −) and hydrogen peroxide (H 2O 2), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O 2 − and H 2O 2 was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H 2O 2 accumulation in guard cells. In the incompatible interaction, a rapid increase of O 2 − and H 2O 2 generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H 2O 2 accumulation further increased, whereas those exhibiting O 2 − accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O 2 − and H 2O 2 generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H 2O 2 accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H 2O 2 accumulation was observed in mesophyll cells surrounding the rust lesion.