Normal red blood cells partially decrease diepoxybutane-induced chromosome breakage in cultured lymphocytes from Fanconi anaemia patients

Objectives: Fanconi anaemia (FA) is a cancer‐prone chromosome instability syndrome characterized by hypersensitivity to DNA cross‐linking agents, such as diepoxybutane (DEB). Previous studies have shown that normal red blood cells (RBC) can protect cultured lymphocytes against chromosomal breaks induced by DEB. The present study was designed to analyse influence of RBCs from normal individuals on frequency of DEB‐induced chromosome breaks in lymphocyte cultures from FA patients. Materials and methods: A comparative study was performed between DEB‐induced chromosome breaks in cultures of FA lymphocytes with either autologous or heterologous RBCs. A further comparative study was carried out between whole blood cultures from FA patients performed on two occasions, before and 1 week after transfusion of RBCs. Results: It was observed that normal RBCs compared to FA RBCs, partially reduced chromosome breaks in cultured FA lymphocytes. A significant reduction in DEB‐induced breaks was also observed in FA cultured lymphocytes obtained 1 week after transfusion of RBCs, in comparison to those observed in the same patients before RBC transfusion. Conclusions: This study shows that DEB‐induced chromosome instability in FA lymphocytes is partially reduced by normal RBCs. This effect may have some clinical relevance in vivo, whenever FA patients receive a RBC transfusion.     

Chromone-2- and -3-carboxylic acids inhibit differently monoamine oxidases A and B

Chromone carboxylic acids were evaluated as human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitors. The biological data indicated that only chromone-3-carboxylic acid is a potent hMAO-B inhibitor, with a high degree of selectivity for hMAO-B compared to hMAO-A. Conversely the chromone-2-carboxylic acid resulted almost inactive against both MAO isoforms. Docking experiments were performed to elucidate the reasons of the different MAO IC₅₀ data and to explain the absence of activity versus selectivity, respectively.     

n-3 PUFA and lipotoxicity

Excess lipid accumulation in nonadipose tissues may occur in the setting of high levels of plasma free fatty acids or triglycerides (TGs) in a process called “lipotoxicity”. Evidence from human studies and animal models suggests that lipid accumulation in the heart, skeletal muscle, pancreas, and liver play an important role in the pathogenesis of heart failure, obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM). During the past few years, several studies have shown that n-3 polyunsaturated fatty acids (PUFA) have potentially cardioprotective effects, especially in high-risk patients with dyslipidemia, and might therefore be expected to be of benefit in T2DM. Moreover, new information has demonstrated the beneficial effects of consuming n-3 PUFA in preventing the complications of lipotoxicity. n-3 PUFA dietary intake thus had positive effects on fatty liver in patients with non-alcoholic fatty liver disease (NAFLD), with an improvement in liver echotexture and a significant regression of hepatic brightness, associated with improved liver hemodynamics. The n-3 PUFA also had beneficial effects on ectopic fat accumulation inside the heart, with stabilization of cardiac myocytes and antiarrhythmic effects. On the other hand, recent data from animal models suggest that oral dosing of eicosapentaenoic acid (EPA) could contribute to protect against β-cell lipotoxicity. This review discusses the latest hypotheses regarding lipotoxicity, concentrating on the impact of the n-3 PUFA that contribute to ectopic lipid storage, affecting organ function. Further human studies are needed to test the evidence and elucidate the mechanisms involved in this process.     

Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH

TASK-2 (KCNK5 or K_2P5.1) is a background K⁺ channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH_o) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH_i) is independent from that under the command of pH_o. It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH_o had normal pH_i-dependent gating. Increasing extracellular K⁺ concentration acid shifts pH_o activity curve of TASK-2 yet did not affect pH_i gating of TASK-2. pH_o modulation of TASK-2 is voltage-dependent, whereas pH_i gating was not altered by membrane potential. These results suggest that pH_o, which controls a selectivity filter external gate, and pH_i act at different gating processes to open and close TASK-2 channels. We speculate that pH_i regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys²⁴⁵) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH_i. We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH_i-dependence curve of TASK-2 as expected from its lower pK_a. We conclude that intracellular pH, together with pH_o, is a critical determinant of TASK-2 activity and therefore of its physiological function.     

Semiautomatic algorithm for lymph node analysis corrected for partial volume effects in combined positron emission tomography-computed tomography

Positron emission tomography-computed tomography (PET-CT) is superior compared to stand-alone PET in evaluation of malignancies. Few studies have employed high-resolution structural information to correct PET. We designed a semiautomatic algorithm using CT and PET to obtain a partial volume corrected (PVC) standardized uptake value (SUV) and a combined morphologic and functional parameter (multimodal SUV) for lymph node assessment. Lesions were segmented by a semiautomatic algorithm in CT images. Lesion volume was used for PVC and for calculating the multimodal SUV. The method was applied to 47 lymph nodes (30 patients) characterized as suspicious in 18F-fluorodeoxyglucose-PET-CT. In phantoms, PVC improved significantly the measured uptake of the lesion. In patients, 36 lymph nodes could be segmented without problems; in 11 lesions, a manual interaction was necessary. SUVs before PVC (mean 1.29) increased significantly (p < .0005) after PVC (mean 2.8). If SUV 2.5 was used as a threshold value to distinguish between benign and malignant lesions, 11 of the 47 lesions changed from benign to malignant after the PVC. The mean multimodal SUV was 0.39 mL for the benign lesions and 4.47 mL for the malignant lesions. In this work we presented a method for quantitative analysis of lymph nodes in PET-CT. PVC leads to significant differences in SUV.     

Structure of interphase chromosomes in the nuclei of Drosophila cells

Fluorescent images of interphase chromatin structures and chromosome structures isolated from reversibly permeable Drosophila cells were analyzed. Decondensed chromatin in early S phase (2.0-2.5 C-value) consisted of a veil-like fibrillary network. Fibrillar chromatin formed rodlets later in the early S phase (2.5-2.75 C). Drosophila chromosomes contain several smaller subunits called rodlets. Fibrillar chromatin turned to chromatin ribbon and the early mid-S-phase globular chromosomes (2.75-3.0 C), then to opened fibrous globular forms later in the mid-S-phase (3.0-3.25 C), to late-S-phase supercoiled ribbons (3.25-3.5 C), end-S-phase elongated prechromosomes (3.5-3.75 C), bent and linear chromosomes (3.75-4.0 C). Early-S phase chromatin fibrils in the nuclei of Drosophila cells are thinner than the veil-like structures in mammalian cells. The connectivity of chromosomes shows linear arrangement (3, 1, 2, 4), with larger chromosomes (1 and 2) inside and smaller chromosomes (3, 4) at the two ends in the chromosomal chain.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

The deletions of 22q11--the Portuguese experience

The patients with a chromosome 22q11 deletion have a variable phenotype which includes DiGeorge (DG) and Velocardiofacial (VCF) syndromes. The aim of the present study is to characterize the phenotype of DG and VCF using facial biometry in 12 portuguese patients. We found 4/12 patients with the DG phenotype: 3/4 had telecanthus, small mouth and retrognathia; 1/4 had telecanthus, short nose with bulbous tip and a normal mouth. These patients had major cardiac defects associated with hypoplastic or absent thymus and monosomy 22q11. We did not find velopharyngeal insufficiency in patients with the so called DG phenotype 8/12 patients had the VCF phenotype: typical facies with variable features. Four of these had velopharyngeal insufficiency and learning disabilities. Four patients had cardiac defects and 5/8 had monosomy 22q11. Probably this clinical variability is due to mutations in critical genes involved in embryonic development.     

Successful Transfection of Lymphocytes by Ternary Lipoplexes

Transgene expression in lymphoid cells may be useful for modulating immune responses in, and gene therapy of, cancer and AIDS. Although cationic liposome-DNA complexes (lipoplexes) present advantages over viral vectors, they have low transfection efficiency, unfavorable features for intravenous administration, and lack of target cell specificity. The use of a targeting ligand (transferrin), or an endosome-disrupting peptide, in ternary complexes with liposomes and a luciferase plasmid, significantly promoted transgene expression in several T- and B-lymphocytic cell lines. The highest levels of luciferase activity were obtained at a lipid/DNA (±) charge ratio of 1/1, where the ternary complexes were net negatively charged. The use of such negatively charged ternary complexes may alleviate some of the drawbacks of highly positively charged plain lipoplexes for gene delivery.