On the evolution of erythrocyte programmed cell death: apoptosis of Rana esculenta nucleated red blood cells involves cysteine proteinase activation and mitochondrion permeabilization

Batracian Rana esculenta erythrocytes cell death induced by either calcium influx, or staurosporine, involves typical apoptotic phenotype. Our data reveal: (i) a drastic modification of the cell morphology with loss of the ellipsoidal form as assessed by phase contrast microscopy and scanning electron microscopy; (ii) an exposure of the phosphatidylserine residues in the outer leaflet of the cell membrane; (iii) a caspase-3-like activity; (iv) a mitochondrial membrane potential (Delta Psi m) loss; and (v) a chromatin condensation and fragmentation. Erythrocyte chromatin condensation and fragmentation are prevented by caspase and calpain peptide inhibitors. These inhibitors also prevent Delta Psi m loss supporting the idea that mitochondria is a central sensor for Rana erythrocytes cell death. Our observations highlight the conservation of the programmed cell death machinery in erythrocytes across kingdom.     

Molecular enantiorecognition of l‐glucose and d‐glucose in whole blood samples

In the past years, enantioanalysis became very important for clinical analysis; biomarkers/substances of biomedical importance with chiral structure should be analyzed and their presence correlated with the specific disorder. Therefore, we developed a method for the assay of l ‐ and d ‐glucose, based on molecular recognition of l ‐ and d ‐glucose. While for d ‐glucose there are many methods to assess its quantity, the l ‐enantiomer is not routinely detected by standard methods. Two stochastic microsensors based on the immobilization of Copper(II)phthalocyanine and Ni(II)phthalocyanine, in natural diamond powder, were proposed for the enantioanalysis of glucose. The proposed methods proved to have high sensitivities and were able to be used for determination of concentrations as low as 2.5 pg mL^?1 for d ‐glucose and as low as 2.5 fg mL^?1 for l ‐glucose. The enatioanalysis was performed with good results in whole blood samples collected from diabetic patients.     

Lysosomal-Mitochondrial Axis in Zoledronic Acid-induced Apoptosis in Human Follicular Lymphoma Cells

Bisphosphonates (BPs) are potent inhibitors of osteoclast function, widely used to treat excessive bone resorption associated with bone metastases, that also have anti-tumor activity. Zoledronic acid (ZOL) represents a potential chemotherapeutic agent for the treatment of cancer. ZOL is the most potent nitrogen-containing BPs, and it inhibits cell growth and induces apoptosis in a variety of cancer cells. Recently we demonstrated that accumulation of isopentenyl pyrophosphate and the consequent formation of a new type of ATP analog (ApppI) after mevalonate pathway inhibition by nitrogen-containing BPs strongly correlates with ZOL-induced cell death in cancer cells in vitro. In this study we show that ZOL-induced apoptosis in HF28RA human follicular lymphoma cells occurs exclusively via the mitochondrial pathway, involves lysosomes, and is dependent on mevalonate pathway inhibition. To define the exact signaling pathway connecting them, we used modified HF28RA cell lines overexpressing either BclXL or dominant-negative caspase-9. In both mutant cells, mitochondrial and lysosomal membrane permeabilization (MMP and LMP) were totally prevented, indicating signaling between lysosomes and mitochondria and, additionally, an amplification loop for MMP and/or LMP regulated by caspase-9 in association with farnesyl pyrophosphate synthetase inhibition. Additionally, the lysosomal pathway in ZOL-induced apoptosis plays an additional/amplification role of the intrinsic pathway independently of caspase-3 activation. Moreover, we show a potential regulation by Bcl-XL and caspase-9 on cell cycle regulators of S-phase. Our findings provide a molecular basis for new strategies concomitantly targeting cell death pathways from multiple sites.     

Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging.

BACKGROUND: A highly sensitive, fast, and simple flow cytometric assay to assess human red blood cell (RBCs) viability and aging is reported. METHODS: The assay described in this report is based on the use of acetoxymethyl ester of calcein (calcein-AM), a fluorescein derivative and nonfluorescent vital dye that passively crosses the cell membrane of viable cells and is converted by cytosolic esterases into green fluorescent calcein, which is retained by cells with intact membranes and inactive multidrug resistance protein. The loss of calcein can be easily determined by flow cytometry, and the cytosolic localization of esterases was demonstrated by spectrofluorometric analyses. RESULTS: We found that RBCs incubated with Ca(2+), which induces a rapid and modulated self-death that shares several features with apoptosis (Bratosin et al., Cell Death Differ 2001;8:1143-1156), externalized phosphatidylserine and lost calcein staining and cytosolic adenosine triphosphate content. Double labeling using phycoerythrin-labeled annexin-V and calcein-AM showed that the decrease of esterase activity is an early event that precedes the externalization of phosphatidylserine residues. In addition, this assay allowed us to distinguish young and aged RBCs isolated by ultracentrifugation in a self-forming Percoll gradient and can be considered as a reliable marker of RBC aging. CONCLUSIONS: Calcein-AM assay may represent a wide application for assessing RBC viability, particularly in blood banks.     

Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L‐T3, L‐T4, and D‐T4

A fast screening method of whole blood was proposed for enantiorecognition of free L‐T₃ , L‐T₄, and D‐T₄. Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L‐T₃ , L‐T₄, and D‐T₄. For the enantiorecognition of free L‐T₄ and D‐T₄ in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L‐T₃ (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L‐T4, and D‐T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP‐based microsensor. For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). Free L‐T₃ , L‐T₄, and D‐T₄ were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). Chirality 27:973–978, 2015. © 2015 Wiley Periodicals, Inc.