Angel Herráez

Biomolecules in the computer: Jmol to the rescue

Jmol is free, open source software for interactive molecular visualization. Since it is written in the Java™ programming language, it is compatible with all major operating systems and, in the applet form, with most modern web browsers. This article summarizes Jmol development and features that make it a valid and promising replacement for Rasmol and Chime in the development of educational materials, as well as in basic investigation of biomolecular structure. The description is set up by comparison with the well known abilities of Rasmol and Chime. Jmol is suitable for molecular model display and analysis in biochemistry, molecular biology, organic and inorganic chemistry, crystallography, and materials science.

Ganoderma lucidum induced apoptosis in NB4 human leukemia cells: Involvement of Akt and Erk

AIM OF THE STUDY The final goal of this work was to study the toxic and apoptosis effects induced by fractions from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] on NB4 human leukemia cells. MATERIALS AND METHODS Two aqueous extracts and a methanol-extracted column-chromatography semipurified fraction were obtained from Ganoderma lucidum fruiting body. Flow cytometry analyses were used to measure cell viability, cell cycle and DNA fragmentation and to quantify apoptosis. Western-blot analyses were used to quantify changes in apoptosis proteins and intracellular kinases. RESULTS Aqueous extracts slightly reduce cell viability and induce DNA fragmentation in NB4 cells. Methanol-extracted semipurified fraction at dilutions down to 15% or 40% of the initial fraction concentration reduced significantly the viability of these leukemia cells (treated for 19h) with induction of DNA fragmentation and induction of apoptosis. Overmore, the dilution down to 15% of the initial E3 concentration induced a reduction of p53 levels, of the Bcl2/Bax relationship as well as reduced levels of both unphosphorylated and phosphorylated Akt (Protein kinase Akt, protein kinase B) and Erk (Erk1 and 2). CONCLUSIONS Induction of apoptosis and alterations in signal transduction kinases (Akt and Erk) are produced by active fractions from Ganoderma lucidum on human leukemia cells. These data could be of important relevance from the viewpoint of antitumor actions of compounds from Ganoderma lucidum. Eventual therapy applications in leukemia cells might be developed.

In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross-linking or diamide treatment

Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red‐blood‐cell membrane surface, namely oxidant compounds, such as ascorbate/Fe+2 and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3‐cross‐linking reagents. We monitored the phagocytosis of oxidized or Band 3‐cross‐linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe3+, but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross‐linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS3) and 3,3′‐dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS3‐cross‐linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short‐term delivery to cells of the mononuclear phagocytic system.

Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism.

Acute promyelocytic leukemia (APL) is a type of cancer, in which immature cells called promyelocytes proliferate abnormally. Human NB4 cell line appears to be a suitable in vitro model to express the characteristics of APL. In this work, we have investigated the effects of esculetin, a coumarin derivative with antioxidant properties, on the viability, the induction of apoptosis and the expression of apoptotic factors in NB4 cells. Cells treated with esculetin at several concentrations (20-500 μM) and for different times (5-24 h) showed a concentration- and time-dependent viability decrease with increased subdiploid DNA production. Esculetin inhibited cell cycle progression and induced DNA fragmentation. Moreover, annexin-V-FITC cytometry assays suggested that increased toxicity is due to both early and late apoptosis. This apoptosis process is be mediated by activation of caspase-3 and caspase-9. Treatments with progressively increasing concentrations (from 100 μM to 500 μM) of esculetin produced a reduction of Bcl2/Bax ratio in NB4 cells at 19 h, without affecting p53 levels. Proapoptotic action of esculetin involves the ERK MAP kinase cascade since increased levels of phosphorylated ERK were observed after those treatments. Increments in the levels of phosphorylated-Akt were also observed. Additionally, esculetin induced the loss of mitochondrial membrane potential with a release of cytochrome c into the cytosol which starts at 6 h of treatment with esculetin and increases up to 24 h. Esculetin induced an increase in superoxide anion at long times of treatment and a reduction of peroxides at short times (1 h) with an observed increase at 2-4 h of treatment. No significant changes in NO production was observed. Esculetin reduced the GSH levels in a time-dependent manner. In summary, the present work shows the cytotoxic action of esculetin as an efficient tool to study apoptosis mechanism induction on NB4 cell line used as a relevant model of APL disease.

Analysis by partitioning in aqueous two-phase systems of the loss of transferrin-binding capacity during maturation of rat reticulocytes

A decrease in the number of binding sites for125I-transferrin, without an apparent modification of the association constant, has been observed during the maturation of reticulocytes into erythrocytes. As an experimental model, different red cell populations from phenylhydrazinic anaemic rates (95% to 12% reticulocyte-rich) have been used. The fractionation by multiple partition in two-phase systems of these red cell populations has been applied here to show the relationship between number of transferrin receptors and rate of reticulocyte maturation.

FLUORESCENCE ANALYSIS OF CARRIER RAT AND HUMAN ERYTHROCYTES LOADED WITH FITC-DEXTRAN

Rat and human erythrocytes are inherently different with respect to slow dialysis encapsulation used in preparing carrier erythrocytes. The incorporation process, commonly measured with radioactive tracers, is always larger in human erythrocytes, mainly because the rat carrier cells are more fragile. When FITC-Dextran (Dx) is used in the encapsulation process, and loaded rat and human RBCs are studied by fluorescence intensity, some additional events are evident. Not all cells of each population appear with a fluorescence signal, and not all show similar fluorescence intensity. Human RBCs show a higher percentage of marked cells and a higher fluorescence intensity than rat RBCs. Two populations, of high and low fluorescence, appear in FITC-Dx loaded rat erythrocytes. The human loaded RBCs show a similar peak distribution together with another peak in the middle scale of fluorescence. Therefore, a heterogeneity in the cell population as a result of the encapsulation process is manifested for both species. The fractionation of RBCs, loaded with either FITC-Dx or 125I-CA, by centrifugation on Ficoll-Paque reveals that the low density cells have much more substance incorporation than the counterpart cell subpopulation in the pellet. Therefore, the cell modifications produced by the encapsulation process are independent of the substance being incorporated. On the other hand, FITC-Dx, but not 125I-CA, shows a certain degree of association to RBCs membranes, especially in humans.

CYTOTOXIC ACTION OF GANODERMA LUCIDUM ON INTERLEUKIN-3 DEPENDENT LYMPHOMA DA-1 CELLS: INVOLVEMENT OF APOPTOSIS PROTEINS

Aqueous extracts and a semipurified fraction obtained by methanol extraction and column chromatography were isolated from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] and their effects on interleukin 3‐dependent lymphoma cells (DA‐1) were studied. Cell viability was reduced by the action of unboiled aqueous extract and by the methanol‐extracted column‐chromatography semipurified fraction, producing DNA fragmentation in DA‐1 cells. Treatments with aqueous extracts showed increments of Bax after 13 h, increments of p53 and Mdm2 after 19 h and a reduction of these three proteins after 24 h. The methanol‐extracted semipurified fraction also induced increments of p53 and Mdm2 factors at 19 h with a reduction after 24 h. The methanol‐extracted column‐chromatography semipurified fraction from Ganoderma lucidum produced minor changes in the level of Akt after treatments for 19 h in DA‐1 cells with a slight reduction in the levels of NFkB‐p65 factor. Both the unboiled aqueous extract and the methanol‐extracted column‐chromatography semipurified fraction produced cleavage of inactive caspase 3, as a clear indication of induction of apoptosis by compounds present in Ganoderma lucidum. Copyright

Factores determinantes del índice de masa corporal en escolares españoles a partir de las Encuestas Nacionales de Salud

INTRODUCTION Spanish National Health Surveys do not establish synergistic relations between variables. The purpose of this study was to perform a deeper historical analysis of body mass index (BMI) and its relation to other parameters included in the questionnaire for children. MATERIAL AND METHODS Data from interviews conducted (between 1987 and 2006) to parents and guardians of schoolchildren aged 9-15 years were analyzed. Height and weight reported by parents were selected and used to calculate BMI. Subjects were stratified by age, gender, time spent sleeping and watching television and, finally, frequency of physical activity. The historical trend of BMI and its dependence on the above factors were analyzed using ANOVA tests. RESULTS AND CONCLUSIONS Significant weight and height increases were seen, which were more marked in boys aged 12 to 15 years. Influence of physical activity on BMI was shown, but decreased (P<.001) as exercise time increased. By contrast, BMI increased (P<.001) as time spent watching television increased. On the other hand, the role of sleep as modulator of body size was confirmed, since schoolchildren aged 9-11 years who slept over 9h had lower BMIs (P<.001). In subjects aged 12 to 15, BMI decreased (P<.001) from 6h of sleep.

ELEASY: a program for processing experimental enzymoimmunoassay data

A computer program is described that automates processing of data from enzyme immunoassays. This includes statistics, data editing, graphic display and fitting of data to both a straight line and a sigmoidal curve. The program can work either without the need for the user to enter choices, initial values, etc., or else with user-controlled choices for adaptation to special data sets.

Weight and height percentiles calculated by the LMS method in Argentinean schoolchildren. A comparative references study

Abstract Background: The Argentinean population is characterized by ethnic, cultural and socio-economic diversity. Aim: To calculate the percentiles of weight-for-age (W/A) and height-for-age (H/A) of schoolchildren from Argentina employing the LMS method; and to compare the obtained percentiles with those of the international and national references. Subjects and methods: Anthropometric data of 18 698 students (8672 girls and 10 026 boys) of 3–13 years old were collected (2003–2008) from Buenos Aires, Catamarca, Chubut, Jujuy, La Pampa and Mendoza. Percentiles of W/A and H/A were obtained with the LMS method. Statistical and graphical comparisons were established with the WHO (international reference) and with that published by the Argentinean Paediatric Society (national reference). Results: Differences in W/A and H/A, regarding the references, were negative and greater at the highest percentiles and in most of the age groups. On average, the differences were greater for boys than girls and for national than international references. Conclusion: The distribution of weight and height of schoolchildren, coming from most regions of the country, differs from those of national and international references. It should be advisable to establish a new national reference based on internationally recognized methodological criteria that adequately reflect the biological and cultural diversity of the Argentinean populations.