Browsing by Author "Venegas, A"
Now showing 1 - 9 of 9
Results Per Page
Sort Options
- ItemChromogenic plate assay distinguishing bacteriolytic from bacteriostatic activity of an antibiotic agent(2000) Mardones, G; Venegas, AA solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. The assay uses a bacterial culture harboring beta-galactosidase enzyme as reporter of cellular lysis. When a drop of bacteriolytic compound is placed on the agar, beta-galactosidase is released from the bacteria to the external solid medium where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic compounds consistently did not display blue halo at the inhibition zone. (C) 2000 Elsevier Science B.V. All rights reserved.
- ItemCloning and comparison of ten gene sequences of a Chilean H-pylori strain with other H-pylori strains revealed higher variability for VacA and CagA virulence factors(2002) Müller, I; Medina-Selby, A; Palacios, JL; Martinez, P; Opazo, P; Bruce, E; Mancilla, M; Valenzuela, P; Yudelevich, A; Venegas, AWe have cloned and sequenced ten Helicobacter pylori genes from a Chilean strain (CH-CTX1) including: a cytotoxin VacA fragment, a CagA fragment (A 17), a species-specific protein (TsaA), urease subunits (UreA, UreB), a flagellin subunit (FlaB), heat shock proteins (HspA and HspB), adhesin (HpaA) and a lipoprotein (Lpp20). We compared their deduced amino acid sequences with the corresponding sequences from three unrelated H. pylori strains, including fully sequenced strains 26695(UK) and J99(USA), and found that eight of them (UreA, UreB, FlaB, HspA, HspB, Lpp20, TsaA and HpaA) presented more than 97.3% identity. In contrast, VacA partial sequence showed lower identity values (93.2-94.9%). Moreover, we found major differences in the A] 7 region respect to the number and arrangement of the internal repeated elements when sequences from different strains were aligned. The A17 regions from strains CH-CTX1 and 26695 are very similar (91.8% identity) but lacked 6 repeated elements when compared to the Australian strains ATCC 43526 and NCTC 11637. The CCUG 17874 A17 region showed the largest deletion involving 9 repeats. A 17 size differences between strains CCUG 17874 and CH-CTX1 were verified by PCR and polypeptide size. Such differences may explain variations in virulence among H. pylori strains as well as diversity in serum immunoreactivity.
- ItemCongenital lipoid adrenal hyperplasia caused by a novel splicing mutation in the gene for the steroidogenic acute regulatory protein(ENDOCRINE SOC, 2004) Gonzalez, AA; Reyes, ML; Carvajal, CA; Tobar, JA; Mosso, LM; Baquedano, P; Solar, A; Venegas, A; Fardella, CESteroidogenic acute regulatory protein (StAR) plays a crucial role in the transport of cholesterol from the cytoplasm to the inner mitochondrial membrane, facilitating its conversion to pregnenolone by cytochrome P450scc. Its essential role in steroidogenesis was demonstrated after observing that StAR gene mutations gave rise to a potentially lethal disease named congenital lipoid adrenal hyperplasia, in which virtually no steroids are produced. We report here a 2-month-old female patient, karyotype 46XY, who presented with growth failure, convulsions, dehydration, hypoglycemia, hyponatremia, hypotension, and severe hyperpigmentation suggestive of adrenal insufficiency. Serum cortisol, 17OH-progesterone, dehydroepiandrosterone sulfate, testosterone, 17OH-pregnenolone, and aldosterone levels were undetectable in the presence of high ACTH and plasma renin activity levels. Immunohistochemical analysis of testis tissues revealed the absence of StAR protein. Molecular analysis of StAR gene demonstrated a homozygous G to T mutation within the splice donor site of exon 1 (IVS1 + 1G>T). Her parents and one brother were heterozygous for this mutation. In vitro analysis of the mutation was performed in COS cells transfected with minigenes coding regions spanning exon-intron 1 to 3 carrying the mutant and the wild-type sequences. RT-PCR analyses of the mutant gene showed an abnormal mRNA transcript of 2430 bp (normal size 433 bp). Sequence analysis of the mutant mRNA demonstrated the retention of intron 1. Immunolocalization of the StAR minigene product detected the peptide in the mitochondria of COS cells transfected with the wild-type minigene but not in those transfected with the mutant minigene. We conclude that this mutation gives rise to a truncated StAR protein, which lacks an important N-terminal region and the entire lipid transfer domain.
- ItemConstruction of a gene encoding the insect bactericidal protein attacin. Studies on its expression in Escherichia coli.(1997) Santibanez, E; Gomez, I; Martinez, MT; Bruce, E; Venegas, AAttacin, a bactericidal small protein is produced by the giant silk moth Hyalophora cecropia. This paper deals with our efforts to clone the attacin cDNA in a bacterial vector to express it in Escherichia coli and produce the protein in sufficient amount for further studies. We chose two inducible expression vector/bacterial cell systems: pPL-lambda/N99cI(+) cells which is able to be induced by nalidixic acid, and pET3d/BL21(DE3) cells carrying a T7 RNA polymerase gene which is IPTG-inducible. After cloning in the pPL-lambda system and under no addition of the inducer, isolated transformants carried this plasmid with at least 2 concurrent deletions that drastically affected attacin expression, even though attacin gene seems to be intact as deduced by its PCR amplification. It was concluded that basal attacin expression occurred in this system and bacterial growth was limited. Plasmid deletions may have emerged by selection pressure as a way to avoid bactericidal expression and allow bacteria survival. The second cloning attempt was done in pET3d vector/BL21 cells, that should not express the cloned sequence (they lack T7 RNA polymerase gene). Transformed BL21 cells gave 3 recombinant plasmids, 2 of them presented a C deletion that generated an early stop signal in the attacin coding region. The third clone, pET-ATT18, carrying an intact gene, was transferred to BL21(DE3)-IPTG inducible cells in order to be expressed. Attacin was undetectable in stained gels or by Western blot analysis. However, expression was visualized in grown cells after 30 min of IPTG induction and 5 min of [S-35]-methionine labeling, as a 22.5 kDa protein bond by using gel electrophoresis and fluorography. This low level of expression drastically affected bacterial growth. Considering that attacin has no lytic activity, these results suggest that this molecule should block bacterial growth directly at the cytoplasm by an unknown mechanism, since no signal peptide coding sequence was incorporated in this gene construction, precluding periplasmic or external destination of this protein.
- ItemEnhanced resistance to bacterial infection by Erwinia carotovora subsp atroseptica in transgenic potato plants expressing the attacin or the cecropin SB-37 genes(1999) Arce, P; Moreno, M; Gutierrez, M; Gebauer, M; Dell'Orto, P; Torres, H; Acuña, I; Oliger, P; Venegas, A; Jordana, X; Kalazich, J; Holuigue, LBlackleg and soft rot diseases, caused by the bacterium Erwinia carotovora, are among the diseases that cause important losses in culture and storage of potato. In this paper, we introduced bacterial resistance into potato, via genes encoding for proteins with antibacterial activity. For this purpose, potato clones were transformed either with the gene encoding the acidic attacin protein from Hyalophora cecropia, or with the gene encoding the cecropin analog peptide SB-37. These clones were evaluated for soft rot and blackleg resistance, after inoculation with the bacterial strain Erwinia carotovora subsp. atroseptica T7. Results reported in this paper indicate that a considerable percentage of the potato clones (15-22%) showed increased resistance to bacterial infection, revealed by reduced severity of blackleg or soft rot symptoms. Expression of the transgenes was demonstrated in some of the clones by Northern blot analysis. This is the first report indicating that. expression of the gene encoding for an attacin protein and for the cecropin SB-37 peptide in transgenic potato confers increased resistance to bacterial infection.
- ItemExpression of the chicken lysozyme gene in potato enhances resistance to infection by Erwinia carotovora subsp, Atroseptica(2000) Serrano, C; Arce-Johnson, P; Torres, H; Gebauer, M; Gutierrez, M; Moreno, M; Jordana, X; Venegas, A; Kalazich, J; Holuigue, LInfection of potato plants and tubers with the bacterium Erwinia carotovora subsp, atroseptica produces blackleg and soft rot diseases, which cause significant losses to crops and stored potatoes. In order to obtain resistance against this bacterium, the gene chly encoding the enzyme lysozyme from chicken was introduced into potato plants (cv. Desiree) via Agrobacterium-mediated transformation. Sixty-three and 69 transgenic potato clones were evaluated in the greenhouse for resistance to blackleg and soft rot diseases, respectively. Results reported in this paper indicate that 21%-29% of the potato clones showed increased resistance to infection by the bacterium E. c, subsp, atroseptica T7, as revealed by a reduced severity of blackleg or soft rot symptoms. Nine clones showing different levels of resistance were selected for further molecular analysis. The number of copies of the transgene integrated in the plant genome of these clones was estimated by Southern blot analysis. The level of transgene expression, detected by Northern blot analysis, correlated with the level of resistance detected in these clones.
- ItemIn vivo expression of β-galactosidase by rat oviduct exposed to naked DNA or messenger RNA(2002) Rios, M; Venegas, A; Croxatto, HBIntra-oviductal administration of RNA obtained from oviducts of estradiol-treated rats resulted in accelerated egg transport (Rios et al., 1997). It is probable that estradiol-induced messenger RNA (mRNA) entered oviductal cells and was translated into the proteins involved in accelerated egg transport. In order to test this interpretation we deposited in vivo 50 mug of pure beta-galactosidase (beta-gal) mRNA, 50 mug of pure DNA from the reporter gene beta-gal under SV40 promoter or the vehicle (control oviducts) into the oviductal lumen of rats. Twenty four hours later the beta-gal activity was assayed in oviductal tissue homogenates using o-nitrophenyl-beta-D-galactopyranoside as a substrate. The administration of beta-gal mRNA and pSVBgal plasmid increased beta-gal activity by 71% and 142%, respectively, over the control oviducts. These results indicate that naked DNA and mRNA coding for beta-gal can enter oviductal cells and be translated into an active enzyme. They are consistent with the interpretation that embryo transport acceleration caused by the injection of estradiol-induced RNA in the oviduct involves translation of the injected mRNA.
- ItemSerological response to Helicobacter pylori recombinant antigens in Chilean infected patients with duodenal ulcer, non-ulcer dyspepsia and gastric cancer(1999) Opazo, P; Müller, I; Rollán, A; Valenzuela, P; Yudelevich, A; García-de la Guarda, R; Urra, S; Venegas, AWe have previously cloned 10 Helicobacter pylori antigen genes from a Chilean strain including: cytotoxin VacA, a truncated region of CagA (called A17), a species-specific protein (Ag26), urease subunits (UreA, UreB), a flagellin, (FlaB), heat shock proteins (HspA and HspB), an adhesin (HpaA) and a lipoprotein (Lpp20). Immunogenicity of these antigens was tested by immunoblot with sera of Chilean infected patients, revealing that HpaA, A17, HspB and VacA were more frequently recognized (86%, 820/o, 68% and 68%, respectively). According to the clinical condition, it was determined that Lpp20 was preferentially recognized by sera from non-ulcer dyspepsia patients (80%), A17 and VacA by patients with duodenal ulcer (92% and 83% respectively), and HspB by patients with duodenal ulcer (83%) and gastric cancer (90%). An ELISA was developed with a purified mixture of A17 and VacA antigens to test the different groups of patients. It was found that sera from duodenal ulcer patients showed higher values than those from non-ulcer dyspepsia patients, but this difference was not significant (p<0.2). Moreover, sera from gastric cancer patients showed values lower than those from non-ulcer dyspepsia patients (p<0.019). These results indicate that, in the Chilean population, antibodies raised against VacA and A17 are not markers either for duodenal ulcer or for gastric cancer.
- ItemSubset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi(2001) Palacios, JL; Zaror, I; Martínez, P; Uribe, F; Opazo, P; Socías, T; Gidekel, M; Venegas, AErwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments off. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DW. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs, Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.