Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts

Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in developed and developing countries. The main purpose of this study was to investigate whether transplantation of bone marrow stromal cells (BMSC) directly into the myocardium could improve the performance of healed infarcted rat hearts. Cell culture medium with or without BMSC was injected into borders of cardiac scar tissue 4 wk after experimental infarction. Cardiac performance was evaluated 2 wk after cellular (n = 10) or medium (n = 10) injection by electro- and echocardiography. Histological study was performed 3 wk after treatment. Electrocardiography of BMSC-treated infarcted rats showed electrical and mechanical parameters more similar to those in control than in medium-treated animals: a normal frontal QRS axis in 6 of 10 BMSC-treated and all control rats and a rightward deviation of the QRS axis in all 10 medium-treated animals. BMSC treatment, assessed by echocardiography, improved fractional shortening (39.00 +/- 4.03%) compared with medium-treated hearts (18.20 +/- 0.74%) and prevented additional changes in cardiac geometry. Immunofluorescence microscopy revealed colocalization of 4',6-diamidino-2-phenylindole-labeled nuclei of transplanted cells with cytoskeletal markers for cardiomyocytes and smooth muscle cells, indicating regeneration of damaged myocardium and angiogenesis. These data provide strong evidence that BMSC implantation can improve cardiac performance in healed infarctions and open new promising therapeutic opportunities for patients with postinfarction heart failure.     

Immobilization of Chacoan peccaries (Catagonus wagneri) using medetomidine, Telazol, and ketamine

A combination of medetomidine, Telazol, and ketamine hydrochloride was used to immobilize captive Chacoan peccaries (Catagonus wagneri) for translocation within Paraguay during August-October 2002. Animals were darted in enclosed areas of varying size. The average dose used was 32.5+/-7.2 microg/kg of medetomidine, 0.63+/-0.2 mg/kg of Telazol, and 3.9+/-0.65 mg/kg of ketamine. First effects were noted at 4.3+/-2.1 min, and ability to handle the animals was achieved by 12.6+/-3.7 min. Heart and respiratory rates declined and oxygen saturation increased during anesthesia. Muscle relaxation was good. Atipamezole was used to antagonize the medetomidine, although recoveries were still slow. This drug combination provided adequate immobilization of Chacoan peccaries; however, this protocol would not be considered to be reversible, and confinement during recovery is recommended.     

Thermoperiodic effect on flowering and endogenous hormonal status in Dendrobium (Orchidaceae)

Two year-old cloned plants of Dendrobium Second Love were submitted to 25 °C (light) and 10 °C (dark) under a 12 h photoperiod (60 μmol m⁻¹ s⁻¹) for 30 days. The endogenous levels of IAA, ABA, and the cytokinins Z, [9R]Z, iP, and [9R]iP were measured 15, 22, and 30 days after the start of the thermoperiodic treatment in lateral buds and leaves. The endogenous levels of IAA and cytokinins, especially the zeatin-derived forms, increased significantly in buds after 15 days of treatment. On the other hand, the amount of ABA decreased progressively and significantly throughout the treatment. The treatment conspicuously accelerated flower-bud development. The found correlation suggests that hormones are involved in the signal transduction pathway of thermoperiodic flowering control.     

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments

AIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS AND RESULTS: The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. CONCLUSIONS: The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries.     

In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.     

Caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties

Caffeic acid and some of its derivatives such as caffeic acid phenetyl ester (CAPE) and octyl caffeate are potent antioxidants which present important anti-inflammatory actions. The present study assessed the in vitro and in vivo effects of five caffeic acid derivatives (caffeic acid methyl, ethyl, butyl, octyl and benzyl esters) and compared their actions to those of CAPE. In the model of LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages, the pre-incubation of all derivatives inhibited nitrite accumulation on the supernatant of stimulated cells, with mean IC₅₀ (μM) values of 21.0, 12.0, 8.4, 2.4, 10.7 and 4.80 for methyl, ethyl, butyl, octyl, benzyl and CAPE, respectively. The effects of caffeic acid derivatives seem to be related to the scavenging of NO, as the compounds prevented SNAP-derived nitrite accumulation and decreased iNOS expression. In addition, butyl, octyl and CAPE derivatives significantly inhibited LPS-induced iNOS expression in RAW 264.7 macrophages. Extending the in vitro results, we showed that the pre-treatment of mice with butyl, octyl and CAPE derivatives inhibited carrageenan-induced paw edema and prevented the increase in IL-1β levels in the mouse paw by 30, 24 and 36%, respectively. Butyl, octyl and CAPE derivatives also prevented carrageenan-induced neutrophil influx in the mouse paw by 28, 49 and 31%, respectively. Present results confirm and extend literature data, showing that caffeic acid derivatives exert in vitro and in vivo anti-inflammatory actions, being their actions mediated, at least in part by the scavenging of NO and their ability to modulate iNOS expression and probably that of other inflammatory mediators.     

Selective involvement of the PI3K/PKB/bad pathway in retinal cell death

The phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB)/Bad signal transduction pathway is engaged in the control of apoptosis in many different cell types, particularly through phosphorylation of the Bcl-2 family protein Bad. We examined the involvement of this pathway in the control of programmed cell death in the retina of developing rats. PKB is constitutively phosphorylated in retinal tissue in vitro, whereas Bad was dephosphorylated both in Ser112 and Ser136. Cell death induced by either the PI3K inhibitor LY294002, or the general kinase inhibitor 2-aminopurine, were followed by PKB dephosphorylation, but PKB was not modulated during cell death induced by the protein synthesis inhibitor anisomycin. Treatment of retinal tissue cultures with forskolin, which increases intracellular levels of cAMP, partially blocked apoptosis induced by both anisomycin and 2-aminopurine, but not by LY294002, whereas forskolin invariably induced phosphorylation of Bad on both Ser112 and Ser136. The data suggest that Bad may be engaged in survival pathways in the immature retina, but pathways other than PI3K/PKB/Bad, and phosphorylation sites other than Ser112 and Ser136 in the Bad protein control cell survival in retinal tissue.     

Maize C4 NADP-malic enzyme. Expression in Escherichia coli and characterization of site-directed mutants at the putative nucleoside-binding sites

Malic enzymes catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO(2), and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C(4)-specific NADP-ME has evolved from C(3)-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C(4)-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties and K(m) for the substrates as those of the naturally occurring NADP-ME previously characterized. However, the k(cat) was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C(4) NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.     

In vitro evaluation of antibacterial substances produced by bacteria isolated from different marine organisms

Bacteria from several groups of marine organisms were isolated and, using direct antibiograms, identified those that produce antibacterial substances, using a human pathogenic strain of Staphylococcus aureus ATCC6538 as revealing microorganism. Bacteria which produce substances that inhibited S. aureus growth were identified through morphological, physiological and biochemical tests. Out of 290 bacteria, 54 (18.6%) inhibited the growth of S. aureus, but only 27 survived for identification. Bivalves, sponges and corals were the most represented from which 41.2, 33.3 and 29.7%, respectively, produced antibacterial substances of the isolated bacteria in each group. The marine species with highest proportions of these bacteria were the hard coral Madracis decactis (62.5%), the sponges Cliona sp. (57.1%) and the octocoral Plexaura flexuosa (50.0%). Out of the 27 strains that produced antibacterial substances, 51.8% were Aeromonas spp. and 14.8% Vibrio spp. Marine bacteria that produce antibacterial substances are abundant, most belong in the Vibrionacea group and were isolated mainly from corals and bivalve mollusks.     

Effects of maturational stage, cumulus cells and coincubation of mature and immature cumulus-oocyte complexes on in vitro penetrability of porcine oocytes

The in vitro penetrability of porcine oocytes is conditioned by several factors, some of which remain unclear. Knowledge of the different effects of the cellular components involved in penetrability would no doubt serve to simplify laboratory IVF methods. This study was designed to evaluate the effects of the following factors on penetrability: oocyte maturational stage, the presence of isolated or oocyte-attached cumulus cells, and coincubation of in vitro-matured and immature oocytes. Immature oocytes and oocytes matured in Waymouth medium were obtained from non atretic follicles and fertilized in TCM 199 medium. Sperm-rich fractions were collected by the gloved hand method and semen was used for IVF at a final concentration of 1 x 10(6) cells/mL in all experiments. Under the same conditions of IVF, the penetrability of the immature cumulus-oocyte complexes (COCs) was significantly lower than that of mature COCs, in terms of penetration rate and mean number of sperm per penetrated oocyte. This difference was abolished when the oocytes were denuded, leading to similar penetration rates. Coincubation of mature and immature COCs reduced the penetrability of immature COCs compared with that observed when these were incubated in isolation. However, neither the addition of isolated cumulus cells from decumulated mature oocytes nor the addition of denuded mature oocytes to immature COCs modified the penetration rate. These findings suggest that the presence of surrounding cumulus cells is mainly responsible for the differences observed in penetrability, regardless of the maturational stage of the oocyte. Moreover, when mature and immature COCs are coincubated, penetrability of immature COCs is diminished by the effects of the mature COC and not by the independent actions of the cellular components.