Alejandro Venegas

Gastric Cancer is Related to Early Helicobacter pylori Infection in a High-Prevalence Country

Background and Aims: Chile ranks fifth in the world among countries with the highest incidence of gastric cancer. The aim was to quantify the association between Helicobacter pylori infection and gastric cancer mortality at the county of residence. Methods: A cross-sectional household survey, a probability sample of the Chilean adult population, provided 2,615 participants in whom serum H. pylori IgG antibodies were measured (ELISA). The spatial pattern of 48,367 deaths due to gastric cancer which occurred from 1985 to 2002 was analyzed using a hierarchical Poisson regression model; 333 counties were categorized as low, medium, and high gastric cancer mortality with median gastric cancer death rates of 11.4, 19.1, and 26.0 per 100,000 inhabitants, respectively. The association between H. pylori positivity and gastric cancer mortality in the county of residence was assessed by multivariate Poisson regression for complex samples. Results: H. pylori prevalence was 73.0% [95% confidence intervals (CI), 70.0-76.0], higher in men [prevalence rate ratio (PRR), 1.1 (95% CI, 1.01-1.20)], peaked at ages 45 to 64, and dropped after age 65. It was higher among residents in counties with high gastric cancer mortality (79.7%; 95% CI, 76.4-82.6) compared to counties with low gastric cancer mortality (62.3%; 95% CI, 53.8-70.2; corresponding PRR, 1.3; 95% CI, 1.1-1.5); under age 24, H. pylori infection was 79.7% (95% CI, 72.2-85.6) versus 39.8% (95% CI, 19.6-64.2) among residents in counties with high and low gastric cancer mortalities, respectively (PRR, 2.0; 95% CI, 1.1-3.7). Conclusions: The high prevalence of H. pylori at younger ages was associated with high gastric cancer mortality in the base population. (Cancer Epidemiol Biomarkers Prev 2007;16(4):662–7)

Pex3p-dependent peroxisomal biogenesis initiates in the endoplasmic reticulum of human fibroblasts.

The mechanisms of peroxisomal biogenesis remain incompletely understood, specially regarding the role of the endoplasmic reticulum (ER) in human cells, where genetic disorders of peroxisome biogenesis lead to Zellweger syndrome (ZS). The Pex3p peroxisomal membrane protein (PMP) required for early steps of peroxisome biogenesis has been detected in the ER in yeast but not in mammalian cells. Here, we show that Pex3p‐GFP expressed in a new ZS cell line (MR), which lacks peroxisomes due to a mutation in the PEX3 gene, localizes first in the ER and subsequently in newly formed peroxisomes. Pex3p bearing an artificial N‐glycosylation site shows an electrophoretic shift indicative of ER targeting while en route to preformed peroxisomes in normal fibroblast. A signal peptide that forces its entry into the ER does not eliminate its capability to drive peroxisome biogenesis in ZS cells. Thus, Pex3p is able to drive peroxisome biogenesis from the ER and its ER pathway is not privative of ZS cells. Cross‐expression experiments of Pex3p in GM623 cells lacking Pex16p or Pex16p in MR cells lacking Pex3p, showed evidence that Pex3p requires Pex16p for ER location but is dispensable for the ER location of Pex16p. These results indicate that Pex3p follows the ER‐to‐peroxisomal route in mammalian cells and provides new clues to understand its function. J. Cell. Biochem. 107: 1083–1096, 2009.

Enhanced resistance to bacterial infection by Erwinia Carotovora subsp. Atroseptica in transgenic potato plants expressing the attacin or the cecropin SB-37 genes

Blackleg and soft rot diseases, caused by the bacteriumErwinia 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 fromHyalophora cecropia, or with the gene encoding the cecropin analog peptide SB37. These clones were evaluated for soft rot and blackleg resistance, after inoculation with the bacterial strainErwinia 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.

Downregulated Th17 responses are associated with reduced gastritis in Helicobacter pylori-infected children.

Helicobacter pylori induces less gastric inflammation in children than adults. Here we investigated whether this reduced inflammation involves dysregulated T helper type 17 (Th17) responses. H. pylori–infected children and adults in Santiago, Chile had similar levels of H. pylori colonization, proportions of bacteria containing cagA and s1/s2 vacA markers of virulence, and strain genotypes (predominantly hpEurope), but the children had significantly reduced levels of gastric inflammation and neutrophil infiltration. The reduced neutrophil accumulation in the infected children was accompanied by significantly fewer gastric Th17 cells and significantly lower levels of interleukin (IL)-17-specific mRNA and protein compared with the infected adults. The gastric mucosa of H. pylori–infected children also contained higher numbers of IL-10+ cells and increased levels of both IL-10 and Foxp3 mRNA compared with that of the infected adults. Thus, reduced gastric inflammation, including diminished neutrophil accumulation, in H. pylori–infected children compared with infected adults is likely due to downregulated gastric Th17/IL-17 responses as a consequence of enhanced mucosal regulatory T-cell activity in the children.

Active site-directed inhibition of E. coli DNA-dependent RNA polymerase by pyridoxal 5′-phosphate

Summary DNA-dependent RNA polymerase from E. coli is rapidly inhibited by preincubation with pyridoxal 5′-phosphate. The enzyme is not inhibited by pyridoxamine 5′-phosphate or pyridoxine. Pyridoxal is about 10 times less effective than pyridoxal 5′-phosphate. Nucleoside triphosphates but not DNA, protect the enzyme from inhibition. Spectral data from the reaction mixture and NaBH4 reduction products indicate the formation of a Schiff base between the aldehyde group of pyridoxal 5′-phosphate and amino group of the enzyme. The results show that the inhibitor is reacting with a critical amino group presumably at the nucleoside phosphate binding site of the active center of the enzyme.

Inactivation of E. coli RNA polymerase by pyridoxal 5′-phosphate: Identification of a low pKa lysine as the modified residue

Abstract The inactivation of E. coli RNA polymerase (3.3 × 10 −7 M) by pyridoxal 5′-phosphate (1 × 10 −4 M to 5 × 10 −4 M) is a first order process with respect to the remaining active enzyme. Studies of the variation of the first order rate constant with the concentration of pyridoxal 5′-phosphate show that the inactivation reaction follows saturation kinetics. The formation of a reversible enzyme-inhibitor intermediate is postulated. Kinetic studies at different pH values indicate that the inactivation rate constant depends on the mole fraction of one conjugate base with pK a 7.9. The apparent equilibrium constant (association) for the inactivation reaction is independent of the pH and is 1.8 × 10 4 M −1 . By electrophoretic and chromatographic analysis of enzyme hydrolyzates after pyridoxal 5′-phosphate and NaBH 4 treatment only N-e-pyridoxyllysine was found. It is postulated that a lysine e-amino group with a low pK a is critical for the activity of the enzyme.

Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi

Erwinia 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 of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. 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.

Expression of the chicken lysozyme gene in potato enhances resistance to infection by Erwinia carotovora subsp. atroseptica.

Infection of potato plants and tubers with the bacteriumErwinia 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 genechly encoding the enzyme lysozyme from chicken was introduced into potato plants (cv. Desirée) viaAgrobacterium- 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 bacteriumE. 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.ResumenLa infección de plantas y tubérculos de la papa con la bacteriaErwinia carotovora subsp.atroseptica produce las enfermedades de pie negro y pudrición blanda, las cuales causan pérdidas significativas en su cultivo y aunacenamiento. Con el propósito de obtener resistencia contra esta bacteria, se introdujo en plantas de papa (cv. Desirée) el genchly, que codifica para la enzima lisozima de pollo, via transformación mediada porAgrobacterium. La resistencia a pie negro y pudrición blanda se evaluó en ensayos de invernadero en 63 y 69 clones transgénicos, respectivamente. Los resultados reportados en este trabajo indican que entre el 21 y el 29% de los clones mostraron un aumento en la resistencia a la infección por la bacteria E. c. subsp.atroseptica T7, revelado por una reducida severidad en los sintomas de pie negro y pudrición blanda. Se realizó un análisis molecular de 9 clones seleccionados que mostraron diferentes niveles de resistencia. En estos clones se estimé el número de copias del transgén integrado al genoma de la planta, mediante análisis de “Southern blot”. El nivel de expresión del transgén en estos clones, detectado mediante análisis de “Northern blot”, se correlacionó con el nivel de resistencia bacteriana obtenido.

Serological response to Helicobacter pylori recombinant antigens in Chilean infected patients with duodenal ulcer, non-ulcer dyspepsia and gastric cancer

We 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%, 82%, 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.

A rapid procedure for purifying a restriction endonuclease from thermus thermophilus (Tth I)

Restriction enzymes have proved to be a powerful tool for mapping genomes [l] and developing molecular cloning and DNA sequencing techniques [2]. A few restriction enzymes have been isolated from thermophilic bacteria [3], showing a high thermostability and resistance to protein denaturing agents. These properties could be useful to study DNA structure at higher temperatures. Looking for a stable enzyme with a new recognition sequence, we have isolated Tth I, an enzyme from the extreme thermophile Thermus thermophilus HB8 [4]. This thermostable enzyme turned out to be an isoschizomer of Taq I, which recognizes the sequence 5’-TCGA-3’ [5]. Thermus thermophilus is a very convenient source for purifying this enzyme since this bacterium does not produce the pigmented ‘slime’ described in Thermus aquaticus [6], which interferes with the purification of Taq I [5,7] and other enzymes [8,9]. Furthermore, Tth I was readily released by osmotic shock, which allowed us to develop a simplified, rapid two-step procedure to obtain an enzyme preparation free of contaminating nucleases. We report here the purification method and some of the properties of Tth I.