Alternative amendment for vineyards from by-products of pyro-bituminous shale: Effect on wine amino acids and biogenic amines.

With the aim of looking for a model of agroecological production, the use of by-products from pyro-bituminous shale as amendment, and its effect on wine amino acids and biogenic amines has been evaluated. Field trials aimed to compare the effect of different doses of conventional and limestone shale from by-products of pyro-bituminous. Four replicates for six different fertilization treatments were arranged in a split plot design during 2009/2010 and 2010/2011 vintage. A chromatographic analysis was carried out to evaluate the impact of fertilization treatments on the amino acid and biogenic amine content of wines produced. Results showed few significant differences among fertilization treatments tested according to the amino acids composition of wines, although it seemed that a combination of conventional and pyro-bituminous shale could be the best option. By-products of pyro-bituminous shale seem to be a good partial substitutive amendment for Brazilian vineyards. This research seems to be a new approach for sustainable revalorization of domestic fertilizers to enable minor environmental impacts and lower production costs without detriment to quality.

Mitochondrial swelling and incipient outer membrane rupture in preapoptotic and apoptotic cells.

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix.

gamma-Linolenic acid and eicosapentaenoic acid induce modifications in mitochondrial metabolism, reactive oxygen species generation, lipid peroxidation and apoptosis in Walker 256 rat carcinosarcoma cells.

The polyunsaturated fatty acids gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA) are cytotoxic to tumour cells. GLA inhibits Walker 256 tumour growth in vivo, causing alterations in mitochondrial ultrastructure and cellular metabolism. The objective of the present study was to investigate the mechanisms behind fatty acid inhibition of Walker 256 tumour growth under controlled in vitro conditions. At a concentration of 150 microM, both GLA and EPA caused a decrease in cell proliferation and an increase in apoptotic index. Increases in reactive oxygen species (ROS) and lipid peroxide production were identified, as well as alterations in energy metabolism and the deposition of large amounts of triacylglycerol in the form of lipid droplets. Mitochondrial respiratory chain complexes I+III and IV had significantly decreased activity and mitochondrial membrane potential was greatly diminished. Intracellular ATP concentrations were maintained at 70-80% of control values despite the decreased mitochondrial function, which may be in part due to increased utilisation of glucose for ATP generation. Cytochrome c release from mitochondria was found, as was caspase-3-like activation. DNA fragmentation in situ revealed many apoptotic events within the cell population. The mechanism(s) by which ROS and lipid peroxides induce apoptosis remains unclear, but the effects of GLA and EPA appear to involve the mitochondrial pathway of apoptosis induction leading to cytochrome c release, caspase activation, loss of mitochondrial membrane potential and DNA fragmentation.

Modifications in mitochondrial metabolism and ultrastructure and their relationship to tumour growth inhibition by gamma-linolenic acid.

Walker 256 tumour-bearing rats were fed pelleted chow containing low-gamma-linolenic acid (GLA) (2.98%) or high-GLA (5.55%) during the twelve-day period after subcutaneous implantation of the tumour. The presence of n-6, polyunsaturated GLA in the diet caused a concentration-dependent decrease in tumour growth, reaching an almost 50% reduction in final tumour weight in the high-GLA group. The eicosatrienoic acid content of the whole tumour homogenate and of the Percoll-purified mitochondrial fraction was increased by the GLA-rich diets. Changes in the fatty acid composition of the cytoplasmic acyl CoA pool were also found, with increases in GLA content in both the low- and high-GLA groups. Additionally, increases in eicosatrienoic acid and arachidonic acid were found in the high-GLA group. Both the cytoplasmic acyl CoA content and the mitochondrial acyl CoA synthetase activity were increased by GLA in the diet and lipid peroxidation was also increased as determined by an increase in TBARS content. Changes in mitochondrial fatty acid composition were accompanied by a decrease in the mitochondrial membrane potential in the high-GLA group. Tumours from the control and GLA groups were examined by transmission electron microscopy. This revealed an increase in mitochondrial area and volume in the high-GLA group, in comparison with the control group, as well as a change in general cell ultrastructure, with many cells found in an apoptotic state or in a necrotic state, possibly secondary to apoptosis. The data presented show that the addition of GLA to the diet of Walker 256 tumour-bearing rats can greatly decrease the rate of development of the tumour burden. This may be, in part, due to the accumulation of poorly metabolised acyl CoA's within the tumour cell cytoplasm which, when coupled with altered mitochondrial composition, membrane potential and ultrastructure, may be a signal for cell death.

Eicosapentaenoic acid and docosahexaenoic acid effects on tumour mitochondrial metabolism, acyl CoA metabolism and cell proliferation.

In order to investigate the effects of high-fat diets rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Wistar rats bearing subcutaneous implants of the Walker 256 tumour were fed pelleted chow containing low DHA/EPA or high DHA/EPA. The presence of n-3 polyunsaturated fatty acids (PUFAs) led to a marked suppression (35-46%) of tumour growth over a 12 day period. Both the whole tumour homogenate and the Percoll-purified mitochondrial fraction presented significant changes in fatty acid composition. The levels of EPA increased in both n-3 dietary groups while the levels of DHA increased only in the high DHA/EPA group, in comparison with the control chow-fed group. The presence of n-3 PUFAs led to an increase in mitochondrial acyl CoA synthetase activity, but neither the cytoplasmic acyl CoA content nor the n-3 fatty acid composition of the cytoplasmic acyl CoAs was altered by the diet. The content of thiobarbituric acid-reactive substances (TBARS) was increased in the low DHA/EPA group but was unchanged in the high DHA/EPA group. In vitro studies with the Walker 256 cell line showed a 46% decrease in cell growth in the presence of either EPA or DHA which was accompanied by a large decrease in the measured mitochondrial membrane potential. The TBARS content was increased only in the EPA-exposed cells. Cell cycle analysis identified a decrease in G0-G1 phase cells and an increase in G2-M phase cells and apoptotic cells, for both EPA and DHA-exposed cells. The data show that the presence of n-3 PUFAs in the diet is able to significantly after the growth rate of the Walker 256 tumour. The involvement of changes in mitochondrial membrane composition and membrane potential have been indicated for both EPA and DHA, while changes in lipid peroxidation have been identified in the presence of EPA but not of DHA.

Infection by Trypanosoma cruzi. Identification of a parasite ligand and its host cell receptor.

The infective trypomastigote stage of Trypanosoma cruzi expresses a set of surface glycoproteins that are known collectively as Tc85 and belong to the gp85/trans-sialidase supergene family. A member of this family, Tc85-11, with adhesive properties to laminin and cell surfaces was recently cloned. In this report, the Tc85-11 domain for cell binding and its corresponding receptor on epithelial cell LLC-MK(2) are described. Using synthetic peptides corresponding to the Tc85-11 carboxyl-terminal segment, we show that the mammalian cell-binding domain colocalizes to the most conserved motif of the trypanosome gp85/trans-sialidase supergene family (VTVXNVFLYNR). Even though Tc85-11 binds to laminin, the 19-residue cell-binding peptide (peptide J) does not contain the laminin-binding site, because it does not bind to laminin or inhibit cell binding to this glycoprotein. The host cell receptor for the peptide was characterized as cytokeratin 18. Addition of anti-cytokeratin antibodies to the culture medium significantly inhibited the infection of epithelial cells by T. cruzi. Tc85-11 is a multiadhesive glycoprotein, encoding at least two different binding sites, one for laminin and one for cytokeratin 18, that allow the parasite to overcome the barriers imposed by cell membranes, extracellular matrices, and basal laminae to reach the definitive host cell. This is the first description of a direct interaction between cytokeratin and a protozoan parasite.

Psoralen activity and binding sites in melanotic and amelanotic human melanoma cells.

The biological activity and specific binding sites of 8-methoxypsoralen (8-MOP) are assayed using two human melanoma cell lines, melanotic SK-Mel 28 and amelanotic C32TG. Long-term (72 hr) treatment with 8-MOP at a concentration of 10(-4)M results in an increase in melanogenesis and a decrease in proliferation, similar in both cell lines. Daily exposure of these cells to ultraviolet A (UVA) irradiation (1.28 mJ/cm(2)) does not enhance the response to the compound. Daily pulse application (30 min daily) of 8-MOP does not promote any response. However, in combination with UVA, 8-MOP pulse treatment becomes as effective as the long-term treatment. A decrease in cell proliferation in the constant presence of 8-MOP is not coupled with apoptosis, since no increase in the number of apoptotic nuclei was observed after the treatment. The flow cytometry indicates that 8-MOP arrests the cells at the G0/G1 phase, irrespective of the presence or absence of UVA light. In view of the lack of epidermal growth factor (EGF) receptors in both cell lines, it is not likely that such an arrest is associated with the down-regulation of EGF receptors by 8-MOP. It is noted that this compound elicits a biphasic cell response, since cell proliferation increases after the first 24-hr treatment, whereas it decreases in the subsequent 48 hr and thereafter. Competition binding assays using 3H-8-MOP disclosed: 1) the specific binding of the compound in both cell lines occurs in the presence or absence of UVA light, and 2) a higher binding rate at low concentrations of the compound is in SK-Mel 28 (72%) rather than C32TG (58%) cells. The competition assays in the presence of UVA suggest a possible occurrence of covalent bindings between psoralen and receptor, as DNA covalent binding accounted to only 3-5% of the total binding in both cell lines.

Structural elements common to mitosis and apoptosis.

Both mitotic and apoptotic cells display hypercondensation of the chromatin and loss of the nuclear envelope (Lazebnik et al., 1993). Herein, we describe a third similarity between the two processes. We have observed, initially in apoptotic cells of the PC-12 lineage clusters of 40-60 (approximately 50) nm vesicles adjoined by a minor contingent of tubule vesicular elements of 100-200 nm which are indistinguishable from their vesicular counterparts in mitotic PC-12 cells. The clusters of approximately 50 nm vesicles were subsequently observed in all studied rat tissue cells in apoptosis (plasma cells and macrophages, secretory epithelial cells from pancreatic acini, ventral lobe of prostate and mammary gland). Clusters of approximately 50 nm vesicles comparable to those of the PC-12 cells were found in HeLa cells treated with human alfa TNF, in WEHI-3 cells exposed to VM 26 (a teneposide) (Sesso et al., 1997) and in HL-60 cells treated with thapsigargin. PC-12 and HeLa cells affixed to coverslips were double labelled and examined with the fluorescence microscope to reveal simultaneously the disposition of the chromatin with Hoechst stain and the distribution of the fluorescence of Golgi or of Golgi-associated proteins. A common pattern of fluorescence was observed in a minor proportion of apoptotic cells using three different antibodies used. The label frequently appeared as finely dispersed granules in the cytoplasm. In some apoptotic cells, relatively coarse granules were observed. This pattern of label distribution is compatible with the disposition of vesicular clusters we have encountered in apoptotic PC-12 cells sectioned serially or semi serially. In such sections of both mitotic and apoptotic PC-12 cells, we noticed that the conglomerates of 50 nm vesicles were frequently associated with cisternae of the rough ER. Vesicles of similar size were also noted pinching off from the extremities of Golgi cisternae reduced in size. These cisternae diminish in length and width when they are in the process of disassembling at the very beginning of mitosis and in apoptosis.

Lower intracellular hydrogen peroxide levels in cells overexpressing CuZn-superoxide dismutase.

Transfection of V79 Chinese hamster cells produced clones in which CuZn-superoxide dismutase (CuZn-SOD) activities were 2.2- to 3. 5-fold higher than in the parental cells. An overall reduction of antioxidant enzyme activities and both total and oxidized glutathione levels had been found in these clones. Aconitase activities in these cells were determined to indirectly measure the O2- steady-state levels. As expected, in cells overexpressing CuZn-SOD, both total and cytosolic aconitase activities have increased. Because these clones showed reduced oxidized glutathione contents, it is unlikely that they present higher H2O2 steady-state levels as a consequence of the higher SOD levels. This was confirmed by measuring H2O2 steady-state levels in cells by flow cytometric analysis of 2',7'-dichlorofluorescein diacetate-treated cells. Despite the decreased antioxidant defenses, three of the clones overexpressing CuZn-SOD showed reduced H2O2 steady-state levels. These reduced H2O2 steady-state levels were found even when the cells were treated with the O2- generator 2,3-dimethoxy-1, 4-naphthoquinone. These data provide in vivo support for the hypothesis proposed by Liochev and Fridovich [Liochev, S. I. & Fridovich, I. (1994) Free Radical Biol. Med. 16, 29-33] that O2- dismutation prevents the formation of higher H2O2 levels by other reactions.

A region of the cellobiohydrolase I promoter from the filamentous fungus Trichoderma reesei mediates glucose repression in Saccharomyces cerevisiae, dependent on mitochondrial activity.

The upstream activating region that controls cellulose-induced expression of the glucose-repressible cellobiohydrolase I gene (UARcb1) of the filamentous fungus Trichoderma reesei is shown to mediate transcription and glucose repression of a reporter gene in Saccharomyces cerevisiae, a unicellular microorganism that lacks the genes required for the utilization of cellulose. Glucose-controlled transcription mediated by UARcb1 requires the products of the genes SNF1 and SSN6, a protein kinase and a repressor, respectively, that regulate glucose-repressible yeast genes. Previously, it has been shown that mitochondrial function is implicated in cellobiohydrolase I gene expression in T. reesei and this sensitivity to the metabolic state of the mitochondria was shown to be transcriptionally controlled by the 5'-flanking sequence of the cbh1 gene [Abrahão-Neto et al. (1995) Biochemistry 34, 10456-10462]. Remarkably, transcription of the reporter gene controlled by UARcb1 in S. cerevisiae also showed a requirement for active mitochondria, suggesting that a common mechanism involving mitochondrial activity controls glucose-repressible genes in both microorganisms.

One-year follow-up of 66 premature infants weighing 500 to 699 grams treated with a single dose of synthetic surfactant or air placebo at birth: results of a double-blind trial. American Exosurf Neonatal Study Group I.

In a multicenter, double-blind, placebo-controlled trial that enrolled 215 premature infants with birth weights of 500 to 699 gm, 106 infants were treated prophylactically with a single 5 ml/kg dose of synthetic surfactant and 109 infants were given an equivalent dose of air placebo shortly after birth. In each group, 40 children survived infancy: 36 children in the air placebo group and 30 children in the synthetic surfactant group were available for follow-up. Weight, height, and head circumference measurements were similar for both groups at 1-year adjusted age. Infants who received synthetic surfactant at birth had statistically similar Bayley Scales of Infant Development scores (mental developmental index, 92 vs 83; psychomotor developmental index, 87 vs 82) compared with controls. Mild to moderate impairments in the synthetic surfactant group were 7% versus 29% in the control group; these differences were not statistically significant. The incidence of retinopathy of prematurity, the number of hospital readmissions, the need for surgery after day 28, evidence of chronic lung disease, the need for respiratory support at 1-year adjusted age, and the incidence of allergies were also not different between the two groups. However, the proportion of infants surviving without impairment was modestly higher in the synthetic surfactant group (8%, air placebo group; 23%, synthetic surfactant group). The findings from this small study indicate that infants weighing 500 to 699 gm who receive a single prophylactic dose of synthetic surfactant at birth have neurodevelopmental outcomes at least equivalent to those of infants given air placebo at 1-year follow-up.

Double-blind developmental evaluation at 1-year corrected age of 597 premature infants with birth weights from 500 to 1350 grams enrolled in three placebo-controlled trials of prophylactic synthetic surfactant. American Exosurf Neonatal Study Group I.

Infants enrolled in three clinical trials of prophylactic treatment for respiratory distress syndrome with a single dose of synthetic surfactant (Exosurf Neonatal) or air placebo were monitored through 1-year adjusted age. A total of 1046 infants with birth weights from 500 to 1350 gm were enrolled in the three trials; of the 735 infants who survived to 1 year of age, follow-up evaluations were completed for 597 (80%, air placebo group; 82%, synthetic surfactant group). Infants in the air placebo and synthetic surfactant treatment groups had no differences in general health, growth, or nerodevelopmental outcomes or impairments. These follow-up results at 1-year adjusted age for infants who received a single dose of synthetic surfactant indicate that a single dose of synthetic surfactant reduces mortality without increasing the absolute number or proportion of infants with impairments at 1 year of age.

Molecular and phenotypic variability in the congenital alveolar proteinosis syndrome associated with inherited surfactant protein B deficiency.

Congenital alveolar proteinosis (CAP) is an often fatal cause of respiratory failure in term newborn infants, which has been associated with a genetic deficiency of surfactant protein B (SP-B) as a result of a frameshift mutation (121ins2) in a family with three affected siblings. In the index cases the deficiency of SP-B was associated with qualitative and quantitative abnormalities of the surfactant proteins A and C. Immunostaining for lung surfactant proteins and a search for the 121ins2 mutation by restriction enzyme analysis of DNA extracted from paraffin-embedded lung tissue was performed for 7 additional affected infants from 6 families, bringing to 10 the total number of patients with CAP who have been studied. In six infants, the surfactant protein immunostaining pattern was similar to that of the index cases. Of these, three patients were homozygous for the 121ins2 mutation; one was a compound heterozygote with the 121ins2 in one allele and a different mutation in the other; and three patients lacked the mutation in both alleles. One infant had an abundance of SP-B, suggesting phenotypic heterogeneity in CAP. Lung ultrastructural abnormalities, such as a reduced number of lamellar bodies, absent tubular myelin, and basal secretion of surfactant lipids and proteins, suggest a significant derangement of surfactant metabolism. The phenotypic heterogeneity in infants with CAP raises the possibility that variable degrees of SP-B deficiency may be more common than previously suspected.

Effects of two rescue doses of a synthetic surfactant on mortality rate and survival without bronchopulmonary dysplasia in 700- to 1350-gram infants with respiratory distress syndrome. The American Exosurf Neonatal Study Group I.

In a multicenter, double-blind, placebo-controlled rescue trial conducted at 21 American hospitals, two 5 ml/kg doses of a synthetic surfactant (Exosurf Neonatal) or air were administered to 419 infants weighing 700 to 1350 gm who had respiratory distress syndrome and an arterial/alveolar oxygen pressure ratio less than 0.22. The first dose was given between 2 and 24 hours of age; the second dose was given 12 hours later to those infants remaining on ventilatory support. Infants were stratified at entry by birth weight and gender. Among infants receiving synthetic surfactant, improvements in alveolar-arterial oxygen pressure gradient, arterial/alveolar oxygen pressure ratio, and oxygen and ventilator needs through 7 days of age were apparent. Death from respiratory distress syndrome was reduced by two thirds (21 vs 7; p = 0.007), and the overall neonatal mortality rate was reduced by half (50 vs 23; p = 0.001). Although there was no significant reduction in the incidence of bronchopulmonary dysplasia (39 vs 31; p = 0.107), the hypothesis that survival through 28 days without bronchopulmonary dysplasia would be enhanced by two rescue doses of synthetic surfactant was proved true (21% improvement, from 132 to 156 patients; p = 0.001). In addition, the incidence of pneumothorax was reduced by one third (62 vs 40; p = 0.022), and the incidence of pulmonary interstitial emphysema was reduced by half (102 vs 51; p = 0.001). The only side effect identified was an increase in the incidence of apnea (102 vs 134; p = 0.001). These findings indicate that rescue use of a synthetic surfactant can improve the morbidity and mortality rates for premature infants with respiratory distress syndrome.

Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells.

In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. This is a complex phenomenon whose biological significance has not been fully assessed. In Chinese hamster V79 cells this recovery seems to be directly coupled to an enhanced rate of double-stranded DNA elongation. The presence of the DNA polymerase alpha inhibitor, aphidicolin, after u.v. irradiation produces two different responses. At low concentration, sufficient to inhibit 95% of DNA replication but having no effect on excision repair, the drug has no effect on the recovery. This shows that ongoing replicative DNA synthesis is not required for recovery. At higher concentrations of aphidicolin, sufficient to block excision repair, the recovery phenomenon was prevented. The recovery was also prevented by actinomycin D at a concentration that inhibits 60% of RNA synthesis. In quantitative autoradiography experiments in which previously irradiated cells were fused with unirradiated cells the nuclei of the latter exhibited a higher resistance to inhibition by u.v. than nuclei from non-fused cells. These results indicate that: (1) even the low repair rate exhibited by V79 cells (relative to human cells) is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. After irradiation these cells replicate DNA faster than untreated cells. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of smaller replicons. This may also be the mechanism whereby recovery from inhibition occurs after u.v. irradiation.

[Comparative study of methods for the determination of urea in biological fluids]. / Estudo comparativo de métodos para a determinação da uréia em fluidos biológicos

The authors have studied comparatively two methods to determine urea in relationship to the reference technic that utilizes urease. The authors observed that the diacetylmonoxime with thiosemicarbazide method showed the most similar results in comparison with the reference technic.