Circulating exosomes deliver free fatty acids from the bloodstream to cardiac cells: Possible role of CD36.

Regulation of circulating free fatty acid (FFA) levels and delivery is crucial to maintain tissue homeostasis. Exosomes are nanomembranous vesicles that are released from diverse cell types and mediate intercellular communication by delivering bioactive molecules. Here, we sought to investigate the uptake of FFAs by circulating exosomes, the delivery of FFA-loaded exosomes to cardiac cells and the possible role of the FFA transporter CD36 in these processes. Circulating exosomes were purified from the serum of healthy donors after an overnight fast (F) or 20 minutes after a high caloric breakfast (postprandial, PP). Western blotting, Immunogold Electron Microscopy and FACS analysis of circulating exosomes showed that CD36 was expressed under both states, but was higher in postprandial-derived exosomes. Flow cytometry analysis showed that circulating exosomes were able to take-up FFA directly from serum. Importantly, preincubation of exosomes with a blocking CD36 antibody significantly impeded uptake of the FFA analogue BODIPY, pointing to the role of CD36 in FFA exosomal uptake. Finally, we found that circulating exosomes could delivery FFA analogue BODIPY into cardiac cells ex vivo and in vivo in a mice model. Overall, our results suggest a novel mechanism in which circulating exosomes can delivery FFAs from the bloodstream to cardiac tissue. Further studies will be necessary to understand this mechanism and, in particular, its potential involvement in metabolic pathologies such as obesity, diabetes and atherosclerosis.

Flow cytometry diagnosis in myelodysplastic syndrome: Current practice in Latin America and comparison with other regions of the world.

BACKGROUND: Flow cytometry (FC) is a valuable tool for the diagnosis of myelodysplastic syndromes (MDS). We present results of a survey carried out to evaluate FC current practice for MDS diagnosis in Latin America (LA), focusing on markers used and characteristics of the clinical diagnostic report. Compliance to IMDSflow recommendations was also evaluated. These practices were then compared with those used in other countries. METHODS: An online survey was sent through the Grupo Latino-Americano de Mielodisplasia to LA cytometrists and other international scientific societies. RESULTS: 91 responses from 15 LA countries were received. The median of the number of markers used was 20 ± 4.5, but only 8.1% of participants adopted the complete panel proposed by the International/European LeukemiaNet Working Group (IMDSflow). We received 140 eligible answers from regions other than LA (66 Europe, 59 USA-Canada, 8 Oceania, 6 Asia and 1 Africa). LA utilized more markers for MDS diagnosis than USA/Canada (p = 0.006), but similar to Europe. The use of MDS scoring systems differed among regions: 10.3% in LA, 0% USA/Canada and 25.7% Europe reported the "Ogata score". Finally, 52.0% of all participants included a general interpretation statement in the final report about the consistency of the FC results with MDS diagnosis, with no statistical differences between regions. CONCLUSIONS: This survey shows a low compliance with the IMDSflow recommendations and a scarce use of the scoring systems proposed in the literature. However, the number of surface markers used is high. We will work to develop a FC consensus for MDS diagnosis adapted to the clinical practice requirements in LA.

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

Comparative analysis of eight cytotoxicity assays evaluated within the ACuteTox Project.

A comparative analysis of eight cytotoxicity assays [the 3T3 and normal human keratinocytes Neutral Red Uptake (NRU) assay, the primary rat hepatocytes, human HepG2 and 3T3 MTT assay, and the human A.704, SH-SY5Y and HepG2 cells propidium iodide (PI) assay] included in several work packages of the EU Integrated Project ACuteTox, has been carried out. The aim was to evaluate whether cells originating from liver, kidney and brain provided different in vitro acute toxicity results, and the influence of primary liver cells versus cell lines originated from the same tissue. Spearman rank correlation analysis and Hierarchical Cluster Analysis were performed based on the IC50 (50% inhibitory concentrations for the endpoint measured) values generated for 57 chemicals. A relatively large number of neurotoxicants and hepatotoxicants were included which allowed to examine the impact of chemicals with specific tissue toxicity on the results. Our analyses confirmed the similarity between the NRU assays and between the two hepatic cell systems related MTT assays. The type of assay appears to have the greatest influence upon the clustering result regardless of the origin of the cells used. The information provided by the NRU and MTT assays differed from that provided by the PI assay. This approach did not allow to show tissue specific toxicity but it does reveal the effectiveness of the clustering methodology for choosing assays for a testing program for predicting e.g. acute oral toxicity in humans.

The training-induced changes on automatism, conduction and myocardial refractoriness are not mediated by parasympathetic postganglionic neurons activity.

The purpose of this study is to test the role that parasympathetic postganglionic neurons could play on the adaptive electrophysiological changes produced by physical training on intrinsic myocardial automatism, conduction and refractoriness. Trained rabbits were submitted to a physical training protocol on treadmill during 6 weeks. The electrophysiological study was performed in an isolated heart preparation. The investigated myocardial properties were: (a) sinus automatism, (b) atrioventricular and ventriculoatrial conduction, (c) atrial, conduction system and ventricular refractoriness. The parameters to study the refractoriness were obtained by means of extrastimulus test at four different pacing cycle lengths (10% shorter than spontaneous sinus cycle length, 250, 200 and 150 ms) and (d) mean dominant frequency (DF) of the induced ventricular fibrillation (VF), using a spectral method. The electrophysiological protocol was performed before and during continuous atropine administration (1 µM), in order to block cholinergic receptors. Cholinergic receptor blockade did not modify either the increase in sinus cycle length, atrioventricular conduction and refractoriness (left ventricular and atrioventricular conduction system functional refractory periods) or the decrease of DF of VF. These findings reveal that the myocardial electrophysiological modifications produced by physical training are not mediated by intrinsic cardiac parasympathetic activity.

Clinical predictors of immunotolerance in heart transplantation.

BACKGROUND AND AIM: The drugs routinely administered to prevent rejection often cause lethal side effects. Tolerant patients, therefore, should be identified to minimize these problems. The aim of this analysis was to identify clinical variables that may be associated with tolerance. METHODS: We recruited 522 heart transplants (HT), excluding combined procedures, retransplantations, pediatric recipients, and subjects who died in the first year to obtain a cohort of 375 patients. Two groups were distinguished by the presence of echocardiographic, clinical, or pathological evidence of rejection in the first year (15 echocardiograms and 10 protocol biopsies per patient); 99 tolerant patients were compared with 276 nontolerant patients. We analyzed clinical variables related to morbidity and mortality. RESULTS: The univariate analysis showed few differences between the groups. The multivariate analysis showed that only major histocompatibility complex (MHC)-A and MHC-DR matched recipients were significantly associated with tolerance. Thus, the likelihood of tolerance was increased by 1.7- and 2.8-fold if 1 or 2 MHC-I matches were present and by 3.4- and 3.7-fold if 1 or 2 MHC-DR matches were present, respectively survival curves showed significant differences (P=.034). Most deaths in both groups were related to immunosuppressive drugs; among tolerant subjects, deaths were due to infection and neoplasms and among nontolerant patients, deaths were due to chronic rejection, neoplasms, and infection. CONCLUSIONS: The only clinical parameter that can determined whether a HT recipient was tolerant was MHC-A and MHC-DR matching. If there is matching, a reduced immunosuppressive load should be prescribed to prevent drug toxicity.

Nitric oxide, derived from inducible nitric oxide synthase, decreases hypoxia inducible factor-1alpha in macrophages during aspirin-induced mesenteric inflammation.

BACKGROUND AND PURPOSE: Nitric oxide (NO) modulates expression of hypoxia inducible factor-1 (HIF-1), a transcription factor regulating function of myeloid cells. Here, we have assessed the role played by NO, formed by inducible NOS (iNOS), in the inflammation induced by aspirin in the gut, by modulating HIF-1 activity. EXPERIMENTAL APPROACH: The role of iNOS-derived NO on leucocyte-endothelial interactions induced by aspirin was evaluated by intravital microscopy in mesenteric venules of rats pretreated with selective iNOS inhibitors, 1400W or l-N6-(1-iminoethyl)-lysine. NO was localized by fluorescence microscopy, using DAF-FM. iNOS, HIF-1alpha and CD36 were localized by immunohistochemistry. KEY RESULTS: Leucocyte-endothelial interactions increased at 6 h and returned to normal levels 24 h after aspirin administration. Numbers of migrated leucocytes were similar between 6 and 24 h after aspirin. iNOS expression and iNOS-derived NO synthesis were observed in leucocytes of the mesentery of aspirin-treated rats. Blockade of iNOS activity in aspirin-treated rats: (i) did not modify leucocyte infiltration at 6 h, but reduced the number of polymorphonuclear leucocyte and increased that of macrophages at 24 h; (ii) increased HIF-1alpha immunostaining in macrophages of the mesentery; and (iii) prevented the decrease in CD36 immunostaining induced by aspirin in these cells. CONCLUSIONS AND IMPLICATIONS: NO, associated with acute gut inflammation induced by aspirin, diminished HIF-1alpha stabilization in macrophages. Early inhibition of iNOS-derived NO synthesis, by increasing the activity of HIF-1 in these cells, may accelerate the clearance of leucocytes.

Immunophenotypical characterization in Andalusian horse: variations with age and gender.

Assessment of lymphocyte subsets is an effective method for characterizing disorders such as leukemia, lymphomas, autoimmune and infectious diseases. In order to clinically interpret these parameters, normal reference values should be set, estimating age- and gender-related variations. This research aimed to: (1) characterize lymphocyte subpopulations in Andalusian horse, and (2) evaluate age and gender-related variations of lymphocyte subsets. Jugular blood samples were obtained from 159 animals, 77 males and 82 females, belonging to four age groups-1: 1-2 years (N=39; 21 males and 18 females), 2: 2-3 years (N=38; 16 males and 22 females), 3: 3-4 years (N=41; 19 males and 22 females) and 4: 4-7 years (N=41; 21 males and 20 females). T lymphocytes subsets were quantified by flow cytometry with monoclonal antibodies specific for CD2, CD4 and CD8 cell markers. B and NK cell counts were estimated by using a mathematical formula. No variations were found in T, B lymphocytes and NK cells between males and females. Animals of group 1 and 2 had a higher number of CD2, T, CD4+, CD8+, B lymphocytes and NK cells than animals of groups 3 and 4. The percentage of CD2 in group 1 was significantly lower than in group 4. The percentage of T and CD4+ lymphocytes in the group 1 were significantly higher than groups 2 and 3, respectively. Whereas the percentage of B cells calculated by flow cytometry was significantly lower in group 2 compared to group 4, the percentage of B cells calculated by a mathematical formula was higher in group 1. NK cells percentage was significantly lower in group 3 and 4 than in younger animals. In conclusion, in Andalusian horse, gender does not influence absolute numbers and percentages of T, B and NK. There is an age-related decline in absolute number of CD2, T, CD4+ and CD8+ lymphocytes, B lymphocytes and NK cells, with increasing percentage of CD2, T, CD4+ and B lymphocytes, and a decrease in NK with no differences in CD4/CD8 ratio. The decline of lymphocyte population numbers with age is a natural process in many animal species, and could be the origin for immune dysfunction observed in geriatric individuals.

Cigarette smoking affects specific sperm oxidative defenses but does not cause oxidative DNA damage in infertile men.

OBJECTIVE: To evaluate the effects of tobacco consumption on the oxidative defenses of sperm, the glutathione system (GS), and sperm DNA oxidation. DESIGN: Double-blind experimental study. SETTING: Andrology laboratory in a university-affiliated private setting. PATIENT(S): One hundred seventeen semen samples from infertile males. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): (a) sperm GS enzymatic activity with respect to glutathione peroxidase isoforms GPx-1 and GPx-4, glutathione reductase (GR), and cellular glutathione (GSH) content (n = 29); (b) GPx-1, GPx-4, and GR mRNA expression analysis (n = 33); (c) oxidative DNA damage quantification using OXIDNA assay kit (n = 55). Two groups were established: nonsmoking and smoking males. The t tests were employed to detect significant differences between groups. RESULT(S): We identified a significant decrease in sperm GPx-4 activity but not in GPx-1 and GSH activity in smokers compared with nonsmokers. A significant decrease was also observed in GPx-1, GPx-4, and GR mRNA expression in the former group. Interestingly, we did not observe any significant variation in the percentage of cells with oxidative damage of the DNA or in the average level of oxidation of affected cells with respect to the smoking condition of the male. CONCLUSION(S): We demonstrate that smoking has a negative impact on intracellular antioxidant enzymes but that effect does not increase oxidative DNA damage. Thus, the effects of reduced oxidative defenses in sperm as a result of cigarette smoking are yet to be elucidated.

A method to establish the relationship between chronological age and stage of union from radiographic assessment of epiphyseal fusion at the knee: an Irish population study.

Characteristic changes during epiphyseal union provide a skeletal age, which when compared with age-based standards provides an estimation of chronological age. Currently there are no data on epiphyseal union for the purposes of age estimation specific to an Irish population. This cross-sectional study aims to investigate the relationship between stage of epiphyseal union at the knee joint and chronological age in a modern Irish population. A novel radiographic method that sub-divides the continuum of development into five specific stages of union is presented. Anteroposterior and lateral knee radiographs of 148 males and 86 females, aged 9-19 years, were examined. Fusion was scored as Stage 0, non-union; Stage 1, beginning union; Stage 2, active union; Stage 3, recent union; or Stage 4, complete union. Stage of epiphyseal union is correlated with chronological age in both males and females. Mean age gradually increases with each stage of union and also varies between male and female subjects. A statistically significant difference in mean age was recorded between stages when compared to the previous stage, for the three epiphyses. Irish children are comparable to those from previously published studies with epiphyseal union in females occurring earlier than males. A significant difference was noted between the mean age of union for males and females for each of Stages 1 and 2 for the femur and Stages 0, 1, 2 and 3 for the tibia and the fibula. The results also suggest that the stages of union occur at earlier ages in this Irish population. Implementation of standardized methodology is necessary to investigate if this is due to a secular or population variation in maturation or to a methodology which clearly identifies five stages of union.

Inhibitory effects of N-acetylcysteine on the functional responses of human eosinophils in vitro.

BACKGROUND: Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. OBJECTIVE: The aim of this work was to study the effects of the clinically used antioxidant N-acetyl-L-cysteine (NAC) on the functional responses of human-isolated eosinophils. METHODS: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca(2+) signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47(phox)-p67(phox) translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay). RESULTS: NAC (0.1-1 mm) inhibited the extracellular generation of oxygen species induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with -logIC(50) values of 3.61+/-0.03 and 3.36+/-0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5-1 mm). NAC (1 mm) did not alter the fMLP-induced Ca(2+) signal but augmented the eosinophil content of reduced GSH and inhibited p47(phox)-p67(phox) translocation. NAC inhibited the release of ECP ( approximately 90% inhibition at 1 mm) from fMLP-activated eosinophils. CONCLUSION: Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation.

A halocin acting on Na+/H+ exchanger of Haloarchaea as a new type of inhibitor in NHE of mammals

The capability of halocin H6 (a bacteriocin-like protein produced by haloarchaeaHaloferax gibbonsii) to inhibit Na+/H+ exchange (NHE) in mammalian cells and its cardio-protective efficacy on the ischemic and reperfused myocardium were evaluated in the present study. H6 inhibits NHE activity (measured by a flow cytometry method) in a dose-dependent form of cell lines of mammalian origin (HEK293, NIH3T3, Jurkat and HL-1) as well as in primary cell culture from human skeletal muscle (myocytes and fibroblasts).In vivo, an ischemia-reperfusion model in dogs by coronary arterial occlusion was used (two hours of regional ischemia and three hours of reperfusion). In animals treated with halocin H6 there was a significant reduction of premature ventricular ectopic beats and infarct size, whereas blood pressure and heart rate remained unchanged. Up to date, halocin H6 is the only described biological molecule that exerts a, specific inhibitory activity in NHE of eukaryotic cells (AU)

En el presente trabajo se evalúa la capacidad de la halocina H6 (una proteína tipo bacteriocina producida por la haloarchaeaHaloferax gibbonsii) para inhibir el intercambiador Na+/H+ (NHE) de céludas de mamífero y su posible eficacia cardioprotectora frente a los daños causados por isquemia-reperfusión del miocardio. En experimentosin vitro H6 inhibe la actividad de NHE (determinada por citometría de flujo) de forma dosis-dependiente tanto en líneas celulares de mamíferos (HEK293, NIH3T3, Jurkat y HL-1) como en cultivos primarios de miocitos y fibroblastos aislados de músculo esquelético humano. En experimentosin vivo se utilizó un modelo de isquemia-reperfusión en perros por oclusión de la arteria coronaria (dos horas de isquemia y tres de reperfusión). En animales tratados con halocina H6 se produjo una disminución significativa a nivel estadístico, tanto del número de latidos ectópicos ventriculares como del tamaño del infarto, mientras que no se produjeron cambios tanto en la presión sanguínea como en el ritmo cardíaco. Hasta la fecha la halocina H6 es la única molécula biológica descrita que ejerce una actividad inhibidora específica sobre el NHE de células eucariotas (AU)

Eosinophil-selective mediators in human strongyloidiasis.

Strongyloides stercoralis infection is characterized by the production of IgE and eosinophils in peripheral blood. Experimental studies have demonstrated that eosinophils play an important role in protection against Strongyloides stercoralis, but the mechanisms regulating eosinophils are not known. In this study we have focused on analysing the molecules that selectively regulate eosinophil migration, namely eotaxin and interleukin-5 (IL-5), using an enzyme-linked immunosorbent assay in patients with strongyloidiasis. Serum expression of eotaxin and IL-5 were significantly increased in patients compared with the control group. This rise suggests that selective mediators of the eosinophil can have a role in immunity against S. stercoralis in human infection.

Chromosomal variation and genome size support existence of cryptic species of Triatoma dimidiata with different epidemiological importance as Chagas disease vectors.

The wide geographical distribution of Triatoma dimidiata, one of the three major vectors of Chagas disease, ranges from Mexico to northern Peru. Since this species occupies a great diversity of artificial and natural ecotopes, its eradication is extremely difficult. In order to assist control efforts, we used chromosome analyses and DNA amount as taxonomic markers to study genetic variability in populations of T. dimidiata from Mexico, Guatemala, El Salvador and Colombia. We differentiated three groups or cytotypes defined by characteristic chromosome C-banding patterns and genome size measured by flow cytometry. The three cytotypes are restricted to different geographic locations. Cytotype 1 occurs in Mexico (excluding Yucatán), Guatemala (excluding Petén), El Salvador and Colombia. Cytotype 2 occurs in Yucatán and cytotype 3 occurs in Petén. Cytotype 1, commonly associated with domestic and peridomestic environments but also inhabiting sylvatic ecotopes, is the most widespread and with major epidemiological significance. In contrast, the Yucatán cytotype inhabits wild ecotopes but increasingly enters houses, while the Petén cytotype appears exclusively sylvatic. We suggest that these cytotypes represent cryptic species of T. dimidiata with different epidemiological relevance as Chagas disease vectors. Poor ability to colonize human dwellings, together with their restricted geographic distribution, indicate that the Yucatán and Petén putative species probably have much less epidemiological significance than cytotype 1. Thus, the genetic markers we describe are powerful tools to differentiate cryptic species in T. dimidiata with different epidemiological significance, contributing to planning the most effective control measures.

Toll-like receptor 4 defective mice carrying point or null mutations do not show increased susceptibility to Candida albicans in a model of hematogenously disseminated infection.

We have studied the role of TLR4 in murine defenses against Candida albicans in two TLR4-defective mouse strains: C3H/HeJ mice which have defective TLR4 signaling, and TLR4-/- knockout mice. Both TLR4-defective mice strains experimentally infected with virulent C. albicans cells showed no significant difference in survival as compared with their respective controls. Recruitment of neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR4-/-animals, but significantly enhanced in C3H/HeJ mice, compared with their control mice. In vitro production of TNF-alpha by macrophages from both types of TLR4-defective mice, in response to yeasts and hyphae of C. albicans, was not diminished as compared with production by macrophages from wild-type mice. In vitro production of TNF-alpha by yeast-stimulated splenocytes from mice intravenously infected with the low-virulence C. albicans PCA2 strain was not affected in TLR4-defective mice, but the TNF-alpha production in response to hyphae was higher in TLR4-defective than in control animals; the production of IFN-gamma by these splenocytes was similar to controls, as well as the frequency of IFN-gamma-producing CD4+T lymphocytes, indicating that TLR4-defective mice are capable of mounting a Th1 adaptive immune response. Our data indicate that TLR4 is dispensable for murine immune resistance to C. albicans.

Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size.

For about half of all Chagas disease cases T. infestans has been the responsible vector. Contributing to its genetic knowledge will increase our understanding of the capacity of geographic expansion and domiciliation of triatomines. Populations of all infestans subcomplex species, T. infestans, T. delpontei, T. platensis and T. melanosoma and the so-called T. infestans "dark morph", from many South American countries were studied. A total of 10 and 7 different ITS-2 and ITS-1 haplotypes, respectively, were found. The total intraspecific ITS-2 nucleotide variability detected in T. infestans is the highest hitherto known in triatomines. ITS-1 minisatellites, detected for the first time in triatomines, proved to be homologous and thus become useful markers. Calculations show that ITS-1 evolves 1.12-2.60 times faster than ITS-2. Despite all species analyzed presenting the same n=22 chromosome number, a large variation of the haploid DNA content was found, including a strikingly high DNA content difference between Andean and non-Andean specimens of T. infestans (mean reduction of 30%, with a maximum of up to 40%) and a correlation between presence/absence of minisatellites and larger/smaller genome size. Population genetics analysis of the eight composite haplotypes of T. infestans and net differences corroborate that there are clear differences between western and eastern populations (60%), and little genetic variation among populations (1.3%) and within populations (40%) within these two groups with migration rates larger than one individual per generation corresponding only to pairs of populations one from each of these groups. These values are indicative either of a large enough gene flow to prevent population differentiation by drift within each geographic area or a very recent spread, the latter hypothesis fitting available data better. Phylogenetic trees support a common ancestor for T. infestans and T. platensis, an origin of T. infestans in Bolivian highlands and two different dispersal lines, one throughout Andean regions of Bolivia and Peru and another in non-Andean lowlands of Chile, Paraguay, Argentina, Uruguay and Brazil.

A halocin acting on Na+/H+ exchanger of haloarchaea as a new type of inhibitor in NHE of mammals.

The capability of halocin H6 (a bacteriocin-like protein produced by haloarchaea Haloferax gibbonsii) to inhibit Na+/H+ exchanger (NHE) in mammalian cells and its cardio-protective efficacy on the ischemic and reperfused myocardium were evaluated in the present study. H6 inhibits NHE activity (measured by a flow cytometry method) in a dose-dependent form of cell lines of mammalian origin (HEK293, NIH3T3, Jurkat and HL-1) as well as in primary cell culture from human skeletal muscle (myocytes and fibroblasts). In vivo, an ischemia-reperfusion model in dogs by coronary arterial occlusion was used (two hours of regional ischemia and three hours of reperfusion). In animals treated with halocin H6 there was a significant reduction of premature ventricular ectopic beats and infarct size, whereas blood pressure and heart rate remained unchanged. Up to date, halocin H6 is the only described biological molecule that exerts a specific inhibitory activity in NHE of eukaryotic cells.

Effects of the cryopreservation procedures on recovered rice cell populations.

Cryogenic storage of plant cells allows the long-term maintenance of valuable genotypes. Cryopreservation of calli and cell suspensions is often performed using cryoprotectants and slow cooling rates. Rice calli (Oryza sativa L.) were cryopreserved by this procedure as well as by direct immersion in liquid nitrogen without cryoprotection. Subsequently, the characteristics of the recovered cells as well as the effects of putative cryoselection were investigated by microscopic observations and flow cytometric analyses. For this purpose, protoplasts were prepared from calli that had been cryopreserved by direct plunging into liquid nitrogen and from their unfrozen controls. Results show that direct immersion in liquid nitrogen of calli pre-treated with abscisic acid is a fast and highly efficient freezing procedure that maintains the main characteristics of the cell populations and appears to increase their metabolic activity

The relevance of flow cytometry for biochemical analysis.

Flow cytometry (FCM) allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cells or microscopic particles in suspension, as they flow rapidly through a sensing area. In some systems, individual cells or particles may be sorted according to the properties exhibited. By using appropriate fluorescent markers, FCM is unique in that multiple structural and functional parameters can be quantified simultaneously on a single-particle basis, whereas up to thousands of biological particles per second may be examined. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. In this critical review, we summarize the main advantages and limitations of FCM for biochemical studies and discuss briefly the most relevant parameters and analytical strategies. Graphical examples of the biological information provided by multiparametric FCM are presented. Also, this review contains specific sections on flow cytoenzymology, FCM analysis of isolated subcellular organelles, and cell-free FCM.