Quantification of histone H2AX phosphorylation in white blood cells induced by ex vivo gamma irradiation of whole blood by both flow cytometry and foci counting as a dose estimation in rapid triage.

A quick, reliable, and reproducible biological assay to distinguish individuals with possible life-threatening risk following radiological or nuclear incidents remains a quest in biodosimetry. In this paper, we examined the use of a γ-H2AX assay as an early dose estimation for rapid triage based on both flow cytometry and image analyses. In the experiment, whole blood from 11 donors was irradiated ex vivo inside a water phantom by gamma rays from Co-60 at 0.51 Gy/min. After the lysis of red blood cells, the white blood cells were collected for immunofluorescence labeling of γ-H2AX, CD45, and nuclear stained for signal collection and visualization. Analysis by flow cytometry showed that the relative γ-H2AX intensities of lymphocytes and granulocytes increased linearly with absorbed doses from 0 to 6 Gy with a large variation among individuals observed above 2 Gy. The relative γ-H2AX intensities of lymphocytes assessed by two different laboratories were highly correlated (ICC = 0.979). Using confocal microscopic images, γ-H2AX foci were observed to be discretely distributed inside the nuclei and to increase proportionally with doses from 0 to 2 Gy, whereas large plagues of merged foci appeared at 4 and 6 Gy, resulting in the saturation of foci counts above 4 Gy. The number of total foci per cell as well as the number of foci per plane were significantly different at 0 vs 1 and 2 vs 4 Gy doses (p < 0.01). Blind tests at 0.5 Gy and 1 Gy doses showed that dose estimation by flow cytometry had a mean absolute difference of less than 0.5 Gy from the actual value. In conclusion, while flow cytometry can provide a dose estimation with an uncertainty of 0.5 Gy at doses ≤ 1 Gy, foci counting can identify merged foci that are prominent at doses ≥ 4 Gy.

Polygonum odoratum leaf extract attenuates oxidative stress and cell death of Raw 264.7 cells exposed to low dose ionizing radiation.

This study aims to investigate the effect of Polygonum odoratum leaf extract (POE) on oxidative stress markers and cell death induced by low dose ionizing radiation (LDIR) in Raw 264.7 cells. The biological activities, chromatographic fingerprint, and cytotoxicity of POE were investigated. To determine the radioprotective effect of POE, Raw 264.7 cells were incubated with POE for 1 hr prior to 100 mGy x-irradiation. The cell viability, oxidative stress damage marker (malondialdehyde level; MDA), and endogenous antioxidant markers (superoxide dismutase: SOD, catalase: CAT, and glutathione peroxidase: GSH-Px) were also determined. The results showed that POE contained 8 essential substances and exhibited a potent antioxidant without any cytotoxicity. It was found that POE significantly decreased the MDA level and activated cell viability, SOD, CAT, and GSH-Px activities. The results from this study indicate that POE is a potent antioxidant, which can be developed as a radioprotector for diagnostic procedures. PRACTICAL APPLICATIONS: Polygonum odoratum leaf extract (POE) is a potent antioxidant that attenuates oxidative stress and cell death induced by low dose ionizing radiation (LDIR). POE might protect against cell damage from LDIR, particularly in diagnostic radiology procedures. Therefore, the development of functional food containing POE might be beneficial for patients who plan to undergo the diagnostic radiology procedure. The functional food containing POE might prevent stochastic and deterministic effects for these patients.

Radical intermediate generation and cell cycle arrest by an aqueous extract of Thunbergia Laurifolia Linn. In human breast cancer cells.

Thunbergia Laurifolia Linn. (TL) is one of the most familiar plants in Thai traditional medicine that is used to treat various conditions, including cancer. However, the antitumor activity of TL or its constituents has never been reported at the molecular level to support the folklore claim. The present study was designed to investigate the antitumor effect of an aqueous extract of TL in human breast cancer cells and the possible mechanism(s) of action. An aqueous crude extract was prepared from dried leaves of TL. Folin-Ciocalteu colorimetric assays were used to determine the total phenolic content. Antiproliferative and cell cycle effects were evaluated in human breast adenocarcinoma MCF-7 cells by MTT reduction assay, cell growth inhibition, clonogenic cell survival, and flow cytometric analysis. Free radical generation by the extracts was detected using electron paramagnetic resonance spectroscopy. The exposure of human breast adenocarcinoma MCF-7 cells to a TL aqueous extract resulted in decreases in cell growth, clonogenic cell survival, and cell viability in a concentration-dependent manner with an IC50 value of 843 µg/ml. Treatments with extract for 24 h at 250 µg/ml or higher induced cell cycle arrest as indicated by a significant increase of cell population in the G1 phase and a significant decrease in the S phase of the cell cycle. The capability of the aqueous extract to generate radical intermediates was observed at both high pH and near-neutral pH conditions. The findings suggest the antitumor bioactivities of TL against selected breast cancer cells may be due to induction of a G1 cell cycle arrest. Cytotoxicity and cell cycle perturbation that are associated with a high concentration of the extract could be in part explained by the total phenolic contents in the extract and the capacity to generate radical intermediates to modulate cellular proliferative signals.

A role for the TOC complex in Arabidopsis root gravitropism.

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes.

Complex physiological and molecular processes underlying root gravitropism.

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway.

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

ALTERED RESPONSE TO GRAVITY is a peripheral membrane protein that modulates gravity-induced cytoplasmic alkalinization and lateral auxin transport in plant statocytes.

ARG1 (ALTERED RESPONSE TO GRAVITY) is required for normal root and hypocotyl gravitropism. Here, we show that targeting ARG1 to the gravity-perceiving cells of roots or hypocotyls is sufficient to rescue the gravitropic defects in the corresponding organs of arg1-2 null mutants. The cytosolic alkalinization of root cap columella cells that normally occurs very rapidly upon gravistimulation is lacking in arg1-2 mutants. Additionally, vertically grown arg1-2 roots appear to accumulate a greater amount of auxin in an expanded domain of the root cap compared with the wild type, and no detectable lateral auxin gradient develops across mutant root caps in response to gravistimulation. We also demonstrate that ARG1 is a peripheral membrane protein that may share some subcellular compartments in the vesicular trafficking pathway with PIN auxin efflux carriers. These data support our hypothesis that ARG1 is involved early in gravitropic signal transduction within the gravity-perceiving cells, where it influences pH changes and auxin distribution. We propose that ARG1 affects the localization and/or activity of PIN or other proteins involved in lateral auxin transport.

Arabidopsis thaliana: A Model for the Study of Root and Shoot Gravitropism.

UNLABELLED: For most plants, shoots grow upward and roots grow downward. These growth patterns illustrate the ability for plant organs to guide their growth at a specified angle from the gravity vector (gravitropism). They allow shoots to grow upward toward light, where they can photosynthesize, and roots to grow downward into the soil, where they can anchor the plant as well as take up water and mineral ions.Gravitropism involves several steps organized in a specific response pathway. These include the perception of a gravistimulus (reorientation within the gravity field), the transduction of this mechanical stimulus into a physiological signal, the transmission of this signal from the site of sensing to the site of response, and a curvature-response which allows the organ tip to resume growth at a predefined set angle from the gravity vector.The primary sites for gravity sensing are located in the cap for roots, and in the endodermis for shoots. The curvature response occurs in the elongation zones for each organ. Upon gravistimulation, a gradient of auxin appears to be generated across the stimulated organ, and be transmitted to the site of response where it promotes a differential growth response. Therefore, while the gravity-induced auxin gradient has to be transmitted from the cap to the elongation zones in roots, there is no need for a longitudinal transport in shoots, as sites for gravity sensing and response overlap in this organ.A combination of molecular genetics, physiology, biochemistry and cell biology, coupled with the utilization of Arabidopsis thaliana as a model system, have recently allowed the identification of a number of molecules involved in the regulation of each phase of gravitropism in shoots and roots of higher plants. In this review, we attempt to summarize the results of these experiments, and we conclude by comparing the molecular and physiological mechanisms that underlie gravitropism in these organs. ABBREVIATIONS: GSPA: gravitational set point angle; IAA: indole-3-acetic acid; NAA: 1-naphthalene acetic acid; NPA: 1-N-naphthylphthalamic acid; 2,4-D: 2,4-dichlorphenoxy acetic acid; TIBA: 2,3,5-triiodobenzoic acid.