Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver

Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F1-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodamine-123 revealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.     

Genetic evidence for NAD(P)H:quinone oxidoreductase 1-catalyzed quinone reduction on passage through the mouse pulmonary circulation

The quinones duroquinone (DQ) and coenzyme Q(1) (CoQ(1)) and quinone reductase inhibitors have been used to identify reductases involved in quinone reduction on passage through the pulmonary circulation. In perfused rat lung, NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as the predominant DQ reductase and NQO1 and mitochondrial complex I as the CoQ(1) reductases. Since inhibitors have nonspecific effects, the goal was to use Nqo1-null (NQO1(-)/(-)) mice to evaluate DQ as an NQO1 probe in the lung. Lung homogenate cytosol NQO1 activities were 97 ± 11, 54 ± 6, and 5 ± 1 (SE) nmol dichlorophenolindophenol reduced·min(-1)·mg protein(-1) for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs, respectively. Intact lung quinone reduction was evaluated by infusion of DQ (50 μM) or CoQ(1) (60 μM) into the pulmonary arterial inflow of the isolated perfused lung and measurement of pulmonary venous effluent hydroquinone (DQH(2) or CoQ(1)H(2)). DQH(2) efflux rates for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs were 0.65 ± 0.08, 0.45 ± 0.04, and 0.13 ± 0.05 (SE) μmol·min(-1)·g dry lung(-1), respectively. DQ reduction in NQO1(+/+) lungs was inhibited by 90 ± 4% with dicumarol; there was no inhibition in NQO1(-/-) lungs. There was no significant difference in CoQ(1)H(2) efflux rates for NQO1(+/+) and NQO1(-/-) lungs. Differences in DQ reduction were not due to differences in lung dry weights, wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total DQ recoveries. The data provide genetic evidence implicating DQ as a specific NQO1 probe in the perfused rodent lung.     

Cyclic di-GMP activation of polynucleotide phosphorylase signal-dependent RNA processing

The second messenger cyclic diguanylic acid (c-di-GMP) is implicated in key lifestyle decisions of bacteria, including biofilm formation and changes in motility and virulence. Some challenges in deciphering the physiological roles of c-di-GMP are the limited knowledge about the cellular targets of c-di-GMP, the signals that control its levels, and the proportion of free cellular c-di-GMP, if any. Here, we identify the target and the regulatory signal for a c-di-GMP-responsive Escherichia coli ribonucleoprotein complex. We show that a direct c-di-GMP target in E. coli is polynucleotide phosphorylase (PNPase), an important enzyme in RNA metabolism that serves as a 3′ polyribonucleotide polymerase or a 3′-to-5′ exoribonuclease. We further show that a complex of polynucleotide phosphorylase with the direct oxygen sensors DosC and DosP can perform oxygen-dependent RNA processing. We conclude that c-di-GMP can mediate signal-dependent RNA processing and that macromolecular complexes can compartmentalize c-di-GMP signaling.     

Geminin escapes degradation in G1 of mouse pluripotent cells and mediates the expression of Oct4, Sox2, and Nanog

Geminin is an essential cell-cycle protein that is only present from S phase to early mitosis in metazoan somatic cells. Genetic ablation of geminin in the mouse results in preimplantation embryonic lethality because pluripotent cells fail to form and all cells differentiate to trophoblast. Here we show that geminin is present in G1 phase of mouse pluripotent cells in contrast to somatic cells, where anaphase-promoting complex/cyclosome (APC/C)-mediated proteasomal destruction removes geminin in G1. Silencing geminin directly or by depleting the APC/C inhibitor Emi1 causes loss of stem cell identity and trophoblast differentiation of mouse embryonal carcinoma and embryonic stem cells. Depletion of cyclins A2 or B1 does not induce this effect, even though both of these APC/C substrates are also present during G1 of pluripotent cells. Crucially, geminin antagonizes the chromatin-remodeling protein Brg1 to maintain expression of Oct4, Sox2, and Nanog. Our results define a pluripotency pathway by which suppressed APC/C activity protects geminin from degradation in G1, allowing sustained expression of core pluripotency factors. Collectively, these findings link the cell cycle to the pluripotent state but also raise an unexplained paradox: How is cell-cycle progression possible in pluripotent cells when oscillations of key regulatory proteins are lost?     

In Vivo Quantitative Imaging Provides Insights into Trunk Neural Crest Migration

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Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line‐derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up‐regulated in primary RMG after glial cell line‐derived neurotrophic factor stimulation, provides neuroprotective and pro‐survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b^rd1 retinas. To identify stimulated pathways in the retina, we treated Pde6b^rd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation‐site‐specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro‐survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.

     

Toxicity outcome in patients treated with modulated arc radiotherapy for localized prostate cancer

Aim

This study evaluates the acute toxicity outcome in patients treated with RapidArc for localized prostate cancer.

Background

Modern technologies allow the delivery of high doses to the prostate while lowering the dose to the neighbouring organs at risk. Whether this dosimetric advantage translates into clinical benefit is not well known.

Materials and methods

Between December 2009 and May 2012, 45 patients with primary prostate adenocarcinoma were treated using RapidArc. All patients received 1.8 Gy per fraction, the median dose to the prostate gland, seminal vesicles, pelvic lymph nodes and surgical bed was 80 Gy (range, 77.4–81 Gy), 50.4 Gy, 50.4 Gy and 77.4 Gy (range, 75.6–79.2 Gy), respectively.

Results

The time between the last session and the last treatment follow up was a median of 10 months (range, 3–24 months). The incidence of grade 3 acute gastrointestinal (GI) and genitourinary (GU) toxicity was 2.2% and 15.5%, respectively. Grade 2 acute GI and GU toxicity occurred in 30% and 27% of patients, respectively. No grade 4 acute GI and GU toxicity were observed. Older patients (>median) or patients with V60 higher than 35% had significantly higher rates of grade ≥2 acute GI toxicity compared with the younger ones.

Conclusions

RapidArc in the treatment of localized prostate cancer is tolerated well with no Grade >3 GI and GU toxicities. Older patients or patients with higher V60 had significantly higher rates of grade ≥2 acute GI toxicity. Further research is necessary to assess definitive late toxicity and tumour control outcome.