Insights into the dynamics of genome size and chromosome evolution in the early diverging angiosperm lineage Nymphaeales (water lilies).

Nymphaeales are the most species-rich lineage of the earliest diverging angiosperms known as the ANA grade (Amborellales, Nymphaeales, Austrobaileyales), and they have received considerable attention from morphological, physiological, and ecological perspectives. Although phylogenetic relationships between these three lineages of angiosperms are mainly well resolved, insights at the whole genome level are still limited because of a dearth of information. To address this, genome sizes and chromosome numbers in 34 taxa, comprising 28 species were estimated and analysed together with previously published data to provide an overview of genome size and chromosome diversity in Nymphaeales. Overall, genome sizes were shown to vary 10-fold and chromosome numbers and ploidy levels ranged from 2n = 2x = 18 to 2n = 16x = ∼224. Distinct patterns of genome diversity were apparent, reflecting the differential incidence of polyploidy, changes in repetitive DNA content, and chromosome rearrangements within and between genera. Using model-based approaches, ancestral genome size and basic chromosome numbers were reconstructed to provide insights into the dynamics of genome size and chromosome number evolution. Finally, by combining additional data from Amborellales and Austrobaileyales, a comprehensive overview of genome sizes and chromosome numbers in these early diverging angiosperms is presented.

Swarm of terminal 35S in Cheirolophus (Asteraceae, Centaureinae).

Island radiation constitutes a playground for species diversification, which has long fascinated researchers and still does today. Because only a small subset of taxa within the pool of island colonizers is concerned by this process, the question is raised on whether some factors could make a taxon prone to radiate. Cheirolophus is the only genus of Centaureinae subtribe to have experienced a radiation in the Canary Islands. Cytogenetic characterization through FISH of 5S and 35S ribosomal RNA genes in eight Cheirolophus species from continent and Canary Islands revealed an unusually high number of 35S predominantly at terminal position, together with a single interstitial 5S rDNA locus in all the studied taxa. Such an abundance of 35S rDNA signals is unique among Centaureinae and predates Cheirolophus arrival in Canary Islands. The possible link of the rDNA profile with radiation process is discussed through a comparison with two other case studies, the closely related Rhaponticum group and the genus Centaurea.

Elimination of Ehrlich tumours by ATP-induced growth inhibition, glutathione depletion and X-rays.

ATP-induced tumour growth inhibition is accompanied by a selective decrease in the content of the tripeptide glutathione (GSH) within the cancer cells in vivo. Depletion of cellular GSH sensitizes tumours to chemotherapy and radiation, but the usefulness of this depletion depends on whether the levels of GSH can be reduced in the tumour relative to normal tissues. We report here that administration of ATP in combination with diethylmaleate and X-rays leads to complete regression of 95% of Ehrlich ascites tumours in mice. This shows that an aggressive tumour can be eliminated by using a therapy based on modulation of GSH levels in cancer cells.

[Cost minimisation analysis for darbepoetin alpha vs. epoetin alpha in chronic kidney disease patients on haemodialysis]. / Análisis de minimización de costes de darbepoetina alfa frente a epoetina alfa en pacientes con insuficiencia renal crónica sometidos a hemodiálisis.

INTRODUCTION: Multiple studies have shown that epoetin alpha (r-HuEpo) and darbepoetin alpha (NESP) are similarly effective and safe for maintaining haemoglobin levels in patients with chronic kidney disease (CKD). Nevertheless, there is some debate over their cost-effectiveness. The purpose of this study is to carry out a cost-minimisation analysis including a comparison of the costs to the hospital arising from treatment with r-HuEpo vs. NESP. METHODS: Prospective observational study. We included CRF patients on haemodialysis with no iron, vitamin B12 or folate deficiencies, treated with stable doses of IV r-HuEpo. Follow-up was performed over three periods: the first during six months, maintaining prior treatment with r-HuEpo; the second for eight months, after changing to NESP, and the third, during the final eight months, following resuming r-HuEpo treatment. For converting both treatments, the conversion factor established on technical sheet 1:200 was used. RESULTS: 51 patients completed the study and were valid for analysis. Their mean age was 68.3 years, and 18 were women (35.3%). The mean weekly doses at the end of each period were 8,058.8 (SD 3,911.1) IU for the EPO1 period, 39.4 (SD 21.6) microg for NESP and 7,882.4 (SD 4,594.1) IU for EPO2. The weekly costs for each treatment showed significant differences between NESP and r-HuEpo: the cost of NESP was higher. CONCLUSION: In our study, we found that r-HuEpo and NESP were similarly effective in patients with CRF on haemodialysis, but that there was a significant cost increase associated with NESP treatment.

[Cost-effectiveness analysis of the empirical antifungal strategy in oncohaematological patients]. / Estudio coste-efectividad de la estrategia empírica antifúngica en pacientes oncohematológicos.

OBJECTIVE: Observational study performing a cost-effectiveness analysis of the empirical antifungal strategy in high-risk oncohaematological patients, from the hospital perspective and with an average time horizon of 10.8 days of treatment. METHOD: Data gathered: effectiveness, purchase costs and other costs (diagnostic tests, hospitalisation, and second-line antifungal therapy). A total of 107 patients were analysed, 115 invasive fungal infection sub-episodes and 138 empirical treatments. RESULTS: The effectiveness and average cost/treatment were: voriconazole 88% and 20,108.8 euro, caspofungin 68% and 49,067.7 euro, Amphotericin B Lipid Complex (ABLC) 58% and 30,375.2 euro, and Amphotericin B Liposome (AB-L) 50% and 38,234.5 euro. The first tree designed shows voriconazole as the dominant option, although there are few case studies. The second tree selects ABLC in comparison to AB-L and caspofungin, with an average CE of 52,371 euro, the nearest figure to the established availability to pay (50,000 euro). The sensitivity analysis evaluates the most influential parameters. The variation in the cost of purchasing do not modify the sense of the analysis, and the modification of 25% in other costs for caspofungin reverses the ratio, making this the most cost-effective option. The ICE indicates that using voriconazole instead of caspofungin saves 144,794 euro. With regard to caspofungin, ABLC increases the cost by 186,925 euro, a deceptive figure influenced by a level of effectiveness that is not very different; and AB-L increases the cost by 60,184 euro. CONCLUSIONS: The analysis provides relevant information from the perspective of clinical practice in spite of the limitations of the unconsidered costs (nephrotoxicity). This type of analysis contributes to rationalising the use of antifungal agents in the hospital setting and in high-risk patients such as oncohaematological ones.

Origin and evolution of binucleate cells in cultures of HEp-2 cells.

The origin and evolution of binucleate cells in cultures of HEp-2 cells have been studied by means of interval photography and time-lapse video-recording. Binucleate cells most frequently formed by the fusion of two sister cells born in a previous mitosis. The study of binucleate cells has shown that they are a cellular type able to successfully undergo mitosis. However, the mitosis may be bipolar, tripolar or multipolar. The daughter cells arising from these divisions do not follow a clear pattern in the number of nuclei they have, instead showing a wide range of possibilities.

Blood glutathione as an index of radiation-induced oxidative stress in mice and humans.

The effect of x-rays on GSH and GSSG levels in blood was studied in mice and humans. An HPLC method that we recently developed was applied to accurately determine GSSG levels in blood. The glutathione redox status (GSH/GSSG) decreases after irradiation. This effect is mainly due to an increase in GSSG levels. Mice received single fraction radiotherapy, at total doses of 1.0 to 7.0 Gy. Changes in GSSG in mouse blood can be detected 10 min after irradiation and last for 6 h within a range of 2.0-7.0 Gy. The highest levels of GSSG (20.1 +/- 2.9 microM), a 4.7-fold increase as compared with controls) in mouse blood are found 2 h after radiation exposure (5 Gy). Breast and lung cancer patients received fractionated radiotherapy at total doses of 50.0 or 60.0 Gy, respectively. GSH/GSSG also decreases in humans in a dose-response fashion. Two reasons may explain the radiation-induced increase in blood GSSG: (a) the reaction of GSH with radiation-induced free radicals resulting in the formation of thyl radicals that react to produce GSSG; and (b) an increase of GSSG release from different organs (e.g., the liver) into the blood. Our results indicate that the glutathione redox ratio in blood can be used as an index of radiation-induced oxidative stress.

Mitochondrial glutathione depletion by glutamine in growing tumor cells.

The effect of L-glutamine (Gln) on mitochondrial glutathione (mtGSH) levels in tumor cells was studied in vivo in Ehrlich ascites tumor (EAT)-bearing mice. Tumor growth was similar in mice fed a Gln-enriched diet (GED; where 30% of the total dietary nitrogen was from Gln) or a nutritionally complete elemental diet (SD). As compared with non-tumor-bearing mice, tumor growth caused a decrease of blood Gln levels in mice fed an SD but not in those fed a GED. Tumor cells in mice fed a GED showed higher glutaminase and lower Gln synthetase activities than did cells isolated from mice fed an SD. Cytosolic glutamate concentration was 2-fold higher in tumor cells from mice fed a GED ( approximately 4 mM) than in those fed an SD. This increase in glutamate content inhibited GSH uptake by tumor mitochondria and led to a selective depletion of mitochondrial GSH (mtGSH) content (not found in mitochondria of normal cells such as lymphocytes or hepatocytes) to approximately 57% of the level found in tumor mitochondria of mice fed an SD. In tumor cells of mice fed a GED, 6-diazo-5-norleucine- or L-glutamate-gamma-hydrazine-induced inhibition of glutaminase activity decreased cytosolic glutamate content and restored GSH uptake by mitochondria to the rate found in EAT cells of mice fed an SD. The partial loss of mtGSH elicited by Gln did not affect generation of reactive oxygen intermediates (ROIs) or mitochondrial functions (e.g., intracellular peroxide levels, O(2)(-)(*) generation, mitochondrial membrane potential, mitochondrial size, adenosine triphosphate and adenosine diphosphate contents, and oxygen consumption were found similar in tumor cells isolated from mice fed an SD or a GED); however, mitochondrial production ROIs upon TNF-alpha stimulation was increased. Our results demonstrate that glutamate derived from glutamine promotes an inhibition of GSH transport into mitochondria, which may render tumor cells more susceptible to oxidative stress-induced mediators.

Glutamine potentiates TNF-alpha-induced tumor cytotoxicity.

L-glutamine (Gln) sensitizes tumor cells to tumor necrosis factor (TNF)-alpha-induced cytotoxicity. The type and mechanism of cell death induced by TNF-alpha was studied in Ehrlich ascites tumor (EAT)-bearing mice fed a Gln-enriched diet (GED; where 30% of the total dietary nitrogen was from Gln). A high rate of Gln oxidation promotes a selective depletion of mitochondrial glutathione (mtGSH) content to approximately 58% of the level found in tumor mitochondria of mice fed a nutritionally complete elemental diet (standard diet, SD). The mechanism of mtGSH depletion involves a glutamate-induced inhibition of GSH transport from the cytosol into mitochondria. The increase in reactive oxygen intermediates (ROIs) production induced by TNF-alpha further depletes mtGSH to approximately 35% of control values, which associates with a decrease in the mitochondrial transmembrane potential (MMP), and elicits mitochondrial membrane permeabilization and release of cytochrome c. Mitochondrial membrane permeabilization was also found in intact tumor cells cultured with a Gln-enriched medium under conditions of buthionine sulfoximine (BSO)-induced selective GSH synthesis inhibition. Enforced expression of the bcl-2 gene in tumor cells could not avoid the glutamine- and TNF-alpha-induced cell death under conditions of mtGSH depletion. However, addition of GSH ester, which delivers free intracellular GSH and increases mtGSH levels, preserved cell viability. These findings show that glutamine oxidation and TNF-alpha, by causing a change in the glutathione redox status within tumor mitochondria, activates the molecular mechanism of apoptotic cell death.

Ultrastructural localization of cisplatin in Ehrlich ascites tumor cells.

The incorporation of cis-diammine Dichloro Platinum (II) (cisplatin) on the Ehrlich ascites carcinoma (EAT) cells has been studied in this paper. Ultrastructural study of cells treated 'in vivo' with cisplatin showed that a new treatment with this substance after fixation, blocks uranyl acetate staining with the consequent lack of heterochromatin contrast.

Possible mechanisms for tumour cell sensitivity to TNF-alpha and potential therapeutic applications.

TNF is a macrophage/monocyte-derived cytokine with cytostatic and cytotoxic anti-tumour activity. TNF-alpha can cause haemorrhagic necrosis and regression of experimental tumours. Nevertheless, the TNF-alpha doses required to cure tumour-bearing mice lead to injury of normal tissues and, eventually, may cause a lethal shock syndrome. This toxicity implies severe limitations for the therapeutic use of TNF-alpha. Reactive oxygen intermediates (ROls) are involved in TNF-alpha-induced cell killing. Different studies are consistent with the hypothesis that tumour cell sensitivity to TNF-alpha is related to its capacity to buffer oxidative attack. Recently, we have demonstrated that the sensitivity of Ehrlich ascites tumor (EAT) cells to TNF depends on their glutathione (GSH, the most prevalent nonprotein thiol in mammalian cells) content and their rate of proliferation. This is important because tumour cell populations under active proliferative states may show higher GSH levels, and drug- and/or radiation-resistant tumours have increased cellular levels of GSH. TNF-alpha induces a shift towards oxidation in the mitochondrial glutathione (mtGSH) status, a fact that is consistent with the hypothesis that mtGSH plays a key role in scavenging TNF-induced ROIs. GSH, which is not synthesized within mitochondria but is neccessary for their normal function, needs to be taken up from the cytosol through a high affinity multicomponent transport system. In consequence, different approaches that lead to depletion of mtGSH may improve the anticancer efficacy of TNF-alpha both in vitro and in vivo. As an example, EAT-bearing mice fed a glutamine-enriched diet (GED) show a selective increase of glutamate content witihin the tumour cells. Glutamate inhibits GSH uptake by tumour mitochondria and leads to a selective depletion of mtGSH content (not found in mitochondria of normal cells) to approx. 57% of the level found in tumour mitochondria of mice fed a standard diet (SD). Administration of rhTNF-alpha, which increases generation of mitochondrial ROIs, to EAT-bearing mice fed a SD does not affect significantly the rate of tumour growth. However, when tumour-bearing mice fed a GED where treated with rhTNF-alpha the number of viable tumour cells was decreased to approx. 38% of controls.

Thixotropy of highly viscous sodium (carboxymethyl)cellulose hydrogels.

A general method to quantify the thixotropic behavior of systems with very low thixotropy is proposed. The areas enclosed by the rheograms tau = f(gamma) must be fitted to functions with well-determined boundary conditions. From these equations the corresponding thixotropic areas are obtained, together with the theoretical area enclosed by the rheogram corresponding to the maximum rheodestruction. The proposed method is applied to high viscosity sodium (carboxymethyl)cellulose gels.

A time-dependent expression for thixotropic areas. Application to aerosil 200 hydrogels.

Experimental determination of the areas enclosed under the up curve, sigma = sigma(;gamma), corresponding to time zero, and the down curve, which would be obtained following complete rheodestruction of the system structure, is not an easy matter. In the present study a semi-empirical procedure for calculating the hypothetical values of these parameters is proposed. With these parameters it is possible to obtain a time-dependent expression for the thixotropic areas, S(T). This procedure has been applied to the study of the thixotropic behavior of Aerosil 200 hydrogels at different concentrations. The comparative analysis of these systems has been realized by studying the relative thixotropic areas and relative thixotropic rates that are obtained from S(T).

Shear stress synergism index and relative thixotropic area.

Shear stress synergism index and relative thixotropic area are introduced as quantifiers of viscous synergism and comparative thixotropy. The utility of these terms is demonstrated by their application to microcrystalline cellulose-sodium carboxymethyl cellulose (MCC-NaCMC) hydrogel, a starch of different botanical origins hydrogels and their combinations.

Possible relationship between micronucleated and binucleated cells induced by cisplatin in cultured CHO cells.

Chinese hamster ovary (CHO) cells were treated with a single dose (10 micrograms/ml) of cis-diamminodichloroplatinum (II) (cisplatin) for 1 h and the effect of the drug on the kinetics of proliferation of the cultures was studied. It was found that the drug produces a delay in the proliferation rates of the treated cultures. The induction of micronuclei and binucleated cells (BC) at different times after treatment have also been studied, and the ability of these cells to undergo DNA synthesis (measured as the ability to incorporate [3H]thymidine) is shown. It was also found that cisplatin induced a particular type of BC that contains one or more micronuclei rather than a pure population of BC. The results obtained show a possible relationship between micronuclei and BC. The possibility that some of the micronucleated cells evolve in subsequent cell divisions to BC with micronuclei is suggested.

Induction of micronucleated and binucleate cells in Chinese hamster ovary (CHO) cells by cis-diamminedichloroplatinum (II): a morphological and morphometric study.

The mutagenicity and cytotoxicity of cis-diamminedichloroplatinum (II) (cisplatin) at doses of 5, 10 and 20 micrograms/ml in Chinese hamster ovary (CHO) cells have been examined. A morphological characterization of several cell types induced by cisplatin was carried out. The frequencies of both cells with micronuclei and binucleate cells as a time-dependent parameter have also been studied. Whilst the number of cells with micronuclei was found to decrease with time, the number of binucleate cells increased. The possible kinetic mechanism for the production of binucleate cells and cells with micronuclei is discussed. A morphometric analysis was also performed. The nuclear area in both treated and control nuclei was measured with the IBAS image analysis system. The results of this analysis show that a continuous reduction in the nuclear size in the control cells is produced. However the size of the treated cells increased after treatment.

Calculation of the wetting parameter from a cluster model in the framework of nanothermodynamics.

The critical wetting parameter omega(c) determines the strength of interfacial fluctuations in critical wetting transitions. In this Brief Report, we calculate omega(c) from considerations on critical liquid clusters inside a vapor phase. The starting point is a cluster model developed by Hill and Chamberlin in the framework of nanothermodynamics [Proc. Natl. Acad. Sci. USA 95, 12779 (1998)]. Our calculations yield results for omega(c) between 0.52 and 1.00, depending on the degrees of freedom considered. The findings are in agreement with previous experimental results and give an idea of the universal dynamical behavior of the clusters when approaching criticality. We suggest that this behavior is a combination of translation and vortex rotational motion (omega(c)=0.84).

Regulation of tumour cell sensitivity to TNF-induced oxidative stress and cytotoxicity: role of glutathione.

Glutathione (GSH) and the rate of cellular proliferation determine tumour cell sensitivity to tumour necrosis factor (TNF). Buthionine sulphoximine (BSO), a selective inhibitor of GSH synthesis, inhibits tumour growth and increases recombinant human TNF (rhTNF)-alpha cytoxicity in vitro. Administration of sublethal doses of rhTNF-alpha to Ehrlich ascites-tumour (EAT)-bearing mice induces oxidative stress (as measured by increases in intracellular peroxide levels, O2.- generation and mitochondrial GSSG). ATP-induced selective GSH depletion, when combined with rhTNF-alpha administration, affords a 61% inhibition of tumour growth and results in a significant extent of host survival. Administration of N-acetylcysteine (NAC) or GSH ester abolishes the rhTNF-alpha and ATP-induced effects on tumour growth by maintaining high GSH levels in the cancer cells. TNF-induced mitochondria GSH depletion appears critical in the cascade of events that lead to cell death.