Ehrlichia canis TRP36 diversity in naturally infected-dogs from an urban area of Colombia.

Ehrlichia canis is the etiologic agent of a highly prevalent tick-borne disease, canine monocytic ehrlichiosis (CME). Four defined E. canis genotypes based on the trp36 gene sequences have been reported, three of them identified in North or South America. The diversity of E. canis has been investigated using genetic and serologic approaches based on distinct 36 kDa tandem repeat protein (trp36) gene sequences that have been reported. The main objectives of this study were to determine the prevalence of E. canis infection in dogs from Medellín, Colombia by PCR and determine the E. canis diversity using molecular and serologic approaches. Blood was collected from dogs (n = 300) with clinical signs of CME for PCR detection of E. canis 16S rRNA, dsb and trp36 DNA. Phylogenetic analysis of trp36 gene sequences was performed using MEGA. A serological evaluation was performed using immunofluorescence microscopy and ELISA with species-specific peptides from E. canis TRP19 and TRP36 (3 genotypes) and E. chaffeensis (TRP32). E. canis DNA (16S rRNA and/or dsb) was detected in 18 % (53/300) of dogs by PCR amplification. The trp36 gene was amplified and sequenced from 35/53 16S rRNA/dsb PCR positive samples revealing three genotypes: United States (US; n = 21), Costa Rica (CR; n = 11), and Brazil (BR; n = 3). Most dogs (33/35) with detectable trp36 DNA had anti-E. canis TRP19 and TRP36 peptide antibodies that corresponded to the genotype detected by PCR. Dogs that had antibodies to the TRP19 peptide (82/300; 38 %), also had antibodies to one or more genotype-specific TRP36 peptides. Based on TRP36 serology, the dogs exhibited highest frequency of infection with the US genogroup (US = 26), followed by the CR genogroup (CR = 19) and the BR genogroup (BR = 11). Notably, 26/53 trp36 PCR positive dogs had detectable antibodies to multiple E. canis genotypes (US/BR/CR = 8, BR/CR = 7, US/CR = 6 and US/BR = 5) suggesting coinfection or multiple sequential infections with different genotypes. Colombian dogs did not have antibodies to E. chaffeensis as determined by a TRP32 species-specific ELISA. Our results demonstrate the presence of three previously defined genotypes in North and South America in Colombian dogs (US, BR, CR). These results also demonstrate that TRP19 and TRP36 serology can provide valuable information regarding E. canis exposure and the potential genotype(s) involved in infection.

The effect of surface coverage on N2, NO and N2O formation over Pt(111).

The effect of surface coverage of species, θ, on the kinetic parameters of N2, NO and N2O formation in a system simulating ammonia oxidation over Pt(111) has been studied by using periodic density functional theory (DFT). The energy barriers for product formation decrease as θ increases, with the effect being more significant above 0.25 ML. The heat of surface reaction decreases as θ increases, making the dissociation of the products less favourable due to a weaker interaction of the intermediates with the surface. The effect of θ on the binding energy is stronger for N* than for either O* or NO*, but it is more apparent in the co-adsorption with O* and NO*. Similarly, the coverage of N* more strongly affects the activation energy of N2 and N2O desorption than does the coverage of O* for NO* and N2O formation.

Chitosan-Mediated shRNA Knockdown of Cytosolic Alanine Aminotransferase Improves Hepatic Carbohydrate Metabolism.

Alanine aminotransferase (ALT) catalyses a transamination reaction that links carbohydrate and amino acid metabolism. In this study, we examined the effect of silencing cytosolic ALT (cALT) expression on the hepatic metabolism in Sparus aurata. A number of siRNA and shRNA designed to down-regulate cALT expression were validated in HEK-293 cells transfected with plasmids expressing S. aurata cALT or mitochondrial ALT (mALT) isoforms: ALT silencing significantly decreased the expression levels of S. aurata mRNA cALT1 to 62% (siRNA) and 48% (shRNA) of the values observed in control cells. The effect of cALT silencing was analysed in the liver of S. aurata 72 h after intraperitoneal injection of chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a shRNA to down-regulate cALT expression (pCpG-si1sh1). In fish fed diets with different ratio of protein to carbohydrate and treated with chitosan-TPP-pCpG-si1sh1, cALT1 and cALT2 mRNA levels significantly decreased irrespective of the diet. Consistently, ALT activity decreased in liver of treated animals. In the liver of S. aurata treated with chitosan-TPP-pCpG-si1sh1 nanoparticles, down-regulation of cALT expression increased the activity of key enzymes in glycolysis (6-phosphofructo-1-kinase and pyruvate kinase) and protein metabolism (glutamate dehydrogenase). Besides showing for the first time that administration of chitosan-TPP-pCpG-si1sh1 nanoparticles silences hepatic cALT expression in vivo, our data support that down-regulation of cALT could improve the use of dietary carbohydrates to obtain energy and spare protein catabolism.

Restoration of direct pathway glycogen synthesis flux in the STZ-diabetes rat model by insulin administration.

Type 1 diabetes subjects are characterized by impaired direct pathway synthesis of hepatic glycogen that is unresponsive to insulin therapy. Since it is not known whether this is an irreversible defect of insulin-dependent diabetes, direct and indirect pathway glycogen fluxes were quantified in streptozotocin (STZ)-induced diabetic rats and compared with STZ rats that received subcutaneous or intraperitoneal insulin (I-SC or I-IP). Three groups of STZ rats were studied at 18 days post-STZ treatment. One group was administered I-SC and another I-IP as two daily injections of short-acting insulin at the start of each light and dark period for days 9-18. A third group did not receive any insulin, and a fourth group of nondiabetic rats was used as control. Glycogen synthesis via direct and indirect pathways, de novo lipogenesis, and gluconeogenesis were determined over the nocturnal feeding period using deuterated water. Direct pathway was residual in STZ rats, and glucokinase activity was also reduced significantly from control levels. Insulin administration restored both net glycogen synthesis via the direct pathway and glucokinase activity to nondiabetic control levels and improved the lipogenic pathway despite an inefficient normalization of the gluconeogenic pathway. We conclude that the reduced direct pathway flux is not an irreversible defect of insulin-dependent diabetes.

Effects of alanine aminotransferase inhibition on the intermediary metabolism in Sparus aurata through dietary amino-oxyacetate supplementation.

In liver, through the reaction catalysed by alanine aminotransferase (ALT), alanine becomes an effective precursor for gluconeogenesis. In the present study amino-oxyacetate (AOA) was used to evaluate its effect on liver ALT activity of the carnivorous fish Sparus aurata. Moreover, the derived metabolic effects on metabolites and other key enzymes of glycolysis, gluconeogenesis and the pentose phosphate pathway were also studied. A dose-effect-dependent inhibition of AOA on hepatic cytosolic and mitochondrial ALT activity was observed in vitro. In vivo, AOA behaved as an inhibitor of hepatic cytosolic ALT activity. A long-term exposure to AOA increased pyruvate kinase activity in the liver irrespective of the composition of the diet supplied to fish. 1H NMR studies showed that inclusion of AOA to the diet decreased the hepatic levels of alanine, glutamate and glycogen. Moreover, 2H NMR analysis indicated a higher renewal rate for alanine in the liver of fish fed with a high-carbohydrate/low-protein diet, while AOA decreased alanine 2H-enrichment irrespective of the diet. The present study indicates that AOA-dependent inhibition of the cytosolic ALT activity could help to increase the use of dietary carbohydrate nutrients.

[Evaluation of the utility and efficacy of a day hospital in a gastroenterology department]. / Valoración de la utilidad y eficacia de un hospital de día en un servicio de patología digestiva.

Because public health funds are limited, full advantage should be taken of the resources available. Consequently, hospital stay should be reduced with no loss of health care quality. In our Gastroenterology Department, investigations or treatment requiring a short hospital stay are carried out in a day hospital. Between March 1998 and March 2000, 2,169 patients were treated: 1,862 outpatients and 307 patients referred from another hospital. A total of 95.2% of the outpatients were discharged. Hospital stay was prolonged in 4.8% (91/1,862): 13 due to incomplete investigations, 29 because of worsening of their underlying disease before the procedure and 49 due to complications. The overall complication rate was 2.8% (62/2,169). Six complications developed after discharge; of these two appeared within 24 hours. After the opening of the day hospital, the mean hospital stay in the Gastroenterology Department was reduced from 9.05 days to 6.07 days (p = 0.001). In conclusion, the use of a day hospital in a gastroenterology department is useful, effective, safe and profitable.

[Aplasia after azathioprine administration: role of the thiopurine methyltransferase genetic polymorphism]. / Aplasia medular tras administración de azatioprina: papel de polimorfismo genético de la tiopurina metiltransferasa.

BACKGROUND: Azathioprine is an immunosuppressor drug widely used in the treatment of autoimmune diseases. Adverse effects during treatment are related to the activity of thiopurine methyltransferase (TPMT), an enzyme which plays a role in azathioprine metabolism. The presence of the allelic variants of the TPMT gene allows us to classify patients into three different groups: high, moderate and low risk of myelosuppression after receiving standard doses of azathioprine. PATIENTS AND METHODS: Study of the allelic variants of the TPMT gene in the positions 460 and 719 with PCR methods in a patient with Crohn's disease, who developed aplasia after receiving azathioprine. The study was extended to his relatives. RESULTS: The patient under study carried the most frequent variant allele of the TPMT gene associated with low enzymatic activity. The mother and one sister of the patient were also carriers of this allelic variant. CONCLUSIONS: Genotyping the allelic variants of the TPMT gene is a useful method to identify patients at moderate or high risk of myelosuppression after administration of azathioprine.

[Morphology of chronic gastritis associated with Campylobacter pylori infection]. / Morfología de la gastritis crónica asociada a infección por Campylobacter pylori.

Recently, a close relation has been found between infection of the gastric mucosa by Campylobacter pylori and chronic gastritis. To establish the possible existence of characteristic morphologic changes in this disease, which can be differentiated from other unrelated forms of gastritis, we analyzed the antral biopsies obtained from 75 patients, 35 with duodenal peptic ulcer and 40 with nonulcerous dyspepsia. The diagnosis of C. pylori infection is based on positive biopsy culture or, if not, when following three requirements are met: positive urease test before 24 hours, identification of the germ by Gram stain and visualization in the tissue of microorganisms with morphology similar to that of C. pylori. We found that 85.5% of the 55 patients with C. pylori infection present active chronic gastritis with lymphoid nodes (GCA + NL), while this morphology is only found in 5 of the 20 uninfected patients. The association of GCA + NL with C. pylori infection is highly significant (p less than 0.0001). We think that it could be a local immunologic response to the stimulus of the bacterial antigen, and that it has sufficient morphologic entity to differentiate it from other inflammatory processes of the gastric mucosa of still unknown etiology.