Fusicoccin Counteracts the Toxic Effect of Cadmium on the Growth of Maize Coleoptile Segments

The effects of cadmium (Cd; 0.1–1000 μM) and fusicoccin (FC) on growth, Cd2+ content, and membrane potential (Em) in maize coleoptile segments were studied. In addition, the Em changes and accumulation of Cd and calcium (Ca) in coleoptile segments treated with Cd2+ combined with 1 μM FC or 30 mM tetraethylammonium (TEA) chloride (K+-channel blocker) were also determined. In this study, the effects of Ca2+-channel blockers [lanthanum (La) and verapamil (Ver)] on growth and content of Cd2+ and Ca2+ in coleoptile segments were also investigated. It was found that Cd at high concentrations (100 and 1000 μM) significantly inhibited endogenous growth of coleoptile segments and simultaneously measured proton extrusion. FC combined with Cd2+ counteracted the toxic effect of Cd2+ on endogenous growth and significantly decreased Cd2+ content (not the case for Cd2+ at the highest concentration) in coleoptile segments. Addition of Cd to the control medium caused depolarization of Em, the extent of which was dependent on Cd concentration and time of treatment with Cd2+. Hyperpolarization of Em induced by FC was suppressed in the presence of Cd2+ at 1000 μM but not Cd2+ at 100 μM. It was also found that treatment of maize coleoptile segments with 30 mM TEA chloride caused hyperpolarization of Em and decreased Cd2+ content in coleoptile segments, suggesting that, in the same way as for FC, accumulation of Cd2+ was dependent on plasma membrane (PM) hyperpolarization. Similar to FC, TEA chloride also decreased Ca2+ content in coleoptile segments. La and Ver combined with Cd2+ (100 μM) significantly decreased Cd content in maize coleoptile segments, but only La completely abolished the toxic effect of Cd2+ on endogenous growth and growth in the presence of FC. Taken together, these results suggest that the mechanism by which FC counteracts the toxic effect of Cd2+ (except at 1000 μM Cd2+) on the growth of maize coleoptile segments involves both stimulation of PM H+-ATPase activity by FC as well as Cd2+-permeable, voltage-dependent Ca channels, which are blocked by FC and TEA chloride-induced PM hyperpolarization

Targeting Lysophosphatidic Acid Signaling Retards Culture-Associated Senescence of Human Marrow Stromal Cells

Marrow stromal cells (MSCs) isolated from mesenchymal tissues can propagate in vitro to some extent and differentiate into various tissue lineages to be used for cell-based therapies. Cellular senescence, which occurs readily in continual MSC culture, leads to loss of these characteristic properties, representing one of the major limitations to achieving the potential of MSCs. In this study, we investigated the effect of lysophosphatidic acid (LPA), a ubiquitous metabolite in membrane phospholipid synthesis, on the senescence program of human MSCs. We show that MSCs preferentially express the LPA receptor subtype 1, and an abrogation of the receptor engagement with the antagonistic compound Ki16425 attenuates senescence induction in continually propagated human MSCs. This anti-aging effect of Ki16425 results in extended rounds of cellular proliferation, increased clonogenic potential, and retained plasticity for osteogenic and adipogenic differentiation. Expressions of p16Ink4a, Rb, p53, and p21Cip1, which have been associated with cellular senescence, were all reduced in human MSCs by the pharmacological inhibition of LPA signaling. Disruption of this signaling pathway was accompanied by morphological changes such as cell thinning and elongation as well as actin filament deformation through decreased phosphorylation of focal adhesion kinase. Prevention of LPA receptor engagement also promoted ubiquitination-mediated c-Myc elimination in MSCs, and consequently the entry into a quiescent state, G0 phase, of the cell cycle. Collectively, these results highlight the potential of pharmacological intervention against LPA signaling for blunting senescence-associated loss of function characteristic of human MSCs

Physiological response of the retinal pigmented epithelium to 3-ns pulse laser application, in vitro and in vivo

BACKGROUND: To treat healthy retinal pigmented epithelium (RPE) with the 3-ns retinal rejuvenation therapy (2RT) laser and to investigate the subsequent wound-healing response of these cells. METHODS: Primary rat RPE cells were treated with the 2RT laser at a range of energy settings. Treated cells were fixed up to 7 days post-irradiation and assessed for expression of proteins associated with wound-healing. For in vivo treatments, eyes of Dark Agouti rats were exposed to laser and tissues collected up to 7 days post-irradiation. Isolated wholemount RPE preparations were examined for structural and protein expression changes. RESULTS: Cultured RPE cells were ablated by 2RT laser in an energy-dependent manner. In all cases, the RPE cell layer repopulated completely within 7 days. Replenishment of RPE cells was associated with expression of the heat shock protein, Hsp27, the intermediate filament proteins, vimentin and nestin, and the cell cycle-associated protein, cyclin D1. Cellular tight junctions were lost in lased regions but re-expressed when cell replenishment was complete. In vivo, 2RT treatment gave rise to both an energy-dependent localised denudation of the RPE and the subsequent repopulation of lesion sites. Cell replenishment was associated with the increased expression of cyclin D1, vimentin and the heat shock proteins Hsp27 and αB-crystallin. CONCLUSIONS: The 2RT laser was able to target the RPE both in vitro and in vivo, causing debridement of the cells and the consequent stimulation of a wound-healing response leading to layer reformation.John P. M. Wood, Marzieh Tahmasebi, Robert J. Casson, Malcolm Plunkett, Glyn Chidlo

Preoperative aqueous humour flare values do not predict proliferative vitreoretinopathy in patients with rhegmatogenous retinal detachment

Background/aims Patients with rhegmatogenous retinal detachment (RRD) who develop postoperative proliferative vitreoretinopathy (PVR) have been found to have higher preoperative laser flare values than patients with RRD who do not develop this complication. Measurement of laser flare has therefore been proposed as an objective, rapid and non-invasive method for identifying high-risk patients. The purpose of our study was to validate the use of preoperative flare values as a predictor of PVR risk in two additional patient cohorts, and to confirm the sensitivity and specificity of this method for identifying high-risk patients. Methods We combined data from two independent prospective studies: centre 1 (120 patients) and centre 2 (194 patients). Preoperative aqueous humour flare was measured with a Kowa FM-500 Laser Flare Meter. PVR was defined as redetachment due to the formation of traction membranes that required reoperation within 6 months of initial surgery. Logistic regression and receiver operating characteristic analysis determined whether higher preoperative flare values were associated with an increased risk of postoperative PVR. Results PVR redetachment developed in 21/314 patients (6.7%). Median flare values differed significantly between centres, therefore analyses were done separately. Logistic regression showed a small but statistically significant increase in odds with increasing flare only for centre 2 (OR 1.014; p=0.005). Areas under the receiver operating characteristic showed low sensitivity and specificity: centre 1, 0.634 (95% CI 0.440 to 0.829) and centre 2, 0.731 (95% CI 0.598 to 0.865). Conclusions Preoperative laser flare measurements are inaccurate in discriminating between those patients with RRD at high and low risk of developing PVR

Supplementary Material for: Thinning of Inner Retinal Layers after Vitrectomy with Silicone Oil versus Gas Endotamponade in Eyes with Macula-Off Retinal Detachment

<p><b><i>Purpose:</i></b> To evaluate retinal layer thickness with optical coherence tomography (OCT) in eyes with macula-off retinal detachment after silicone oil (SiO) or gas endotamponade. <b><i>Procedures:</i></b> Cross-sectional study of 40 eyes with macula-off rhegmatogenous retinal detachment that underwent vitrectomy. 20 eyes received SiO tamponade and 20 matched eyes received gas. 33 healthy fellow eyes served as controls. Macular spectral domain OCT was performed with automated layer detection in the 5 inner subfields of the Early Treatment Diabetic Retinopathy Study (ETDRS) map. <b><i>Results:</i></b> Comparing the SiO group with the gas group, the ganglion cell layer showed a significant thinning in all fields of the inner ring of the ETDRS map, the inner plexiform layer in the nasal, superior and temporal quadrants, and the outer plexiform layer in the nasal quadrant. <b><i>Conclusions:</i></b> Inner retinal layers in the fovea/parafovea were significantly thinner in the SiO group. Prospective studies are warranted to further elucidate possible retinal adverse effects of SiO tamponade.</p