Cardioprotective effects of phosphoramidon on myocardial structure and function in murine Chagas' disease

Chagas' disease is an important cause of cardiomyopathy. Endothelin-1, a vasoactive peptide has been implicated in the pathogenesis of chagasic cardiomyopathy. C57BL/6 x 129sv and CD1 mice were thus, infected with trypomastigotes of Trypanosoma cruzi (Brazil strain) and these infected mice were compared with infected mice treated with phosphoramidon. This compound inhibits endothelin-converting enzyme and neutral endopeptidases and does not affect the growth of the parasite in culture. Phosphoramidon was given in a dose of 10mg/kg for the initial 15 days post-infection None of the C57Bl/6 x 129sv mice died as a result of infection. However, there was marked myocardial inflammation and fibrosis in infected, untreated mice. The hearts of the infected, phosphoramidon-treated mice showed significantly less pathology. Cardiac magnetic resonance imaging of infected mice revealed right ventricular dilation that was less severe in those treated with phosphoramidon. Phosphoramidon-treated CD1 mice survived the acute infection. Transthoracic echocardiography demonstrated left ventricular dilation and reduced percent fractional shortening and relative wall thickness. These alterations were also attenuated as a result of phosphoramidon treatment. These data suggest that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy and interventions that inhibit the synthesis of endothelin-1 and/or neutral endopeptidase might have a protective effect on myocardial structure and function in murine Chagas' disease.     

Oligogalacturonides inhibit the induction of late but not of early auxin-responsive genes in tobacco

Oligogalacturonides (OGs) released from the plant cell wall regulate several defense responses, as well as various aspects of plant growth and development. In these latter effects, OGs exhibit auxin-antagonist activity. To shed light on the mechanism by which OGs antagonise auxin, we analysed the ability of these oligosaccharides to inhibit the activity of four auxin-up-regulated promoters [pGm-GH3 of soybean (Glycine max L. Merr.), pNt114 of tobacco (Nicotiana tabacum L.), and prolB and prolD of Agrobacterium rhizogenes] driving the expression of the beta-glucuronidase reporter gene (GUS) in transgenic tobacco seedlings. Our results indicate that OGs at submicromolar concentrations inhibit the activation by auxin of pNt114, prolB and prolD, but not that of pGm-GH3. Comparative analysis of the kinetics of activation of the four promoters in response to the hormone shows that, while pGm-GH3 is rapidly activated, the other three promoters exhibit a delayed activation, with a lag of at least 4 h before the appearance of GUS activity. The lack of effect of the OGs on early auxin-responsive genes was confirmed by RNA gel blot analysis of the tobacco genes Nt-GH3 and Nt-iaa2.3/2.5. Our results suggest that the auxin-antagonist action of OGs affects the expression of late but not of early auxin-responsive genes.     

Reduction of alpha-Gal expression by relocalizing alpha-galactosidase to the trans-Golgi network and cell surface

Historically, the most effective means of modifying cell surface carbohydrates has required the intracellular overexpression of glycosyltransferases or glycosidases and is dependent on the enzymes occupying a cellular localization close to the carbohydrate structures they modify. We report on relocalizing the lysosomal resident glycosidase human alpha-galactosidase to other regions of the cell, Golgi and cell surface, where it is in closer proximity for cleaving the carbohydrate structure Galalpha(1,3)Gal. Relocalization of alpha-galactosidase was achieved by using the transmembrane and cytoplasmic domains from the human protein furin, which is known to localize in the trans-Golgi network (TGN) and cell surface. Two chimeric forms of alpha-galactosidase were generated, one directing it to the TGN of the cell and the other to the cell surface, as shown by confocal microscopy. The relocalized enzymes have the ability to cleave terminal alpha-galactose as detected by expression on the cell surface. Furthermore, when expressed as a transgene in mice, the TGN form of alpha-galactosidase was more effective at decreasing cell surface terminal alpha-galactose than was the native lysosomal form. When expressed in conjunction with the alpha1,2fucosyltransferase that also decreases Galalpha(1,3)Gal, the reduction was additive. The ability to relocalize enzymes that modify cell surface carbohydrate structures has far-reaching implications in biology and may be useful in such fields as xenotransplantation and treatment of glycosidase disorders.     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

Development of Genus/Species-Specific PCR Analysis for Identification of <Emphasis Type="Italic">Carnobacterium</Emphasis> Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes. Received: 25 July 2001 / Accepted: 19 October 2001     

Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line

This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l. At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.