[Post-treatment assessment of Helicobacter pylori eradication, by polymerase chain reaction in gastric biopsies]. / Evaluación de la erradicación postratamiento del Helicobacter pylori por reaccion en cadena de la polimerasa en biopsias gástricas.

BACKGROUND: The pre-treatment detection of H.p. in the stomach of patients is easily achieved with routine methods. Conversely, with conventional methods, it is difficult to detect the presence of H.p. after treatment. OBJECTIVE: To estimate the actual percentage of successfully treated patients by using a more sensitive and specific technique (PCR) in the same biopsies where standard methods were negative for H.p. MATERIALS AND METHODS: We selected 97 treated patients (31 Gastric Ulcers/66 Duodenal Ulcers, 62 male/35 female, age: 49 +/- 14 years), in whom success of treatment was defined by histological means and CLO Test. In the same gastric biopsies H.p-DNA PCR was performed. Different therapeutic schemes were utilized, but all included Proton Pump Inhibitors + ATB. Eight weeks after the end of the treatment, without medication, the patients were controlled as follows: 5 biopsies per patient, 2 of antrum, 2 of corpus (in different zones) and 1 for CLO Test. H.p. eradication was defined on histological grounds (gastric biopsy histology: 10% formaldehide buffer fixation, paraffin inclusion, Giemsa, HE staining and inmunohistochemistry), CLO Test (Delta West Pty. Ltd. Bentley, Australia) and by the absence of H.p.-DNA by PCR (amplification of a 296 bp of the species-specific antigen of H.p. and visualization of the amplified product in agarose gel with Ethidium Bromide and UV light). RESULTS: [table: see text] CONCLUSIONS: The higher sensitivity of PCR (10(3) fold more than conventional methods) allowed us in this group of patients to detect 13% of false eradication. It would be necessary to follow up this group of patients in order to know whether they develop or not clinical symptoms and/or histological evidence of disease. If such a case PCR could become an important tool for treatment evaluation.

[Detection of Helicobacter pylori by polymerase reaction in bile samples from gallbladder and bile stones]. / Deteccion del Helicobacter pylori por reacción en cadena de la polimerasa en muestras de bilis de pacientes con litiasis vesicular y coledociana.

Some papers report helicobacter pylori existence in bile from surgical specimens obtained during gallbladder or bile ducts surgery. The aim of this work was search by PCR, H. Pylori presence in bile specimens from patients suffering of gallbladder stones or by bile ducts stones. Bile samples were obtained by gallbladder punction during cholecystectomy in 26 patients, 19 of them with gallbladder stones and 7 also with gallbladder stones and bile duct stones. Age ranged from 22-69 years old, median 49.6 years old. Samples were sent to specialized biomolecular laboratory to perform PCR techniques. Two of 26 patients (7.6%) had positive reaction for the presence of DNA of H. Pylori in bile samples. Our research suggest that DNA of H. Pylori can be founded in bile samples patients with gallbladders and duct stones in Argentina.

A stationary-phase acyl-coenzyme A synthetase of Streptomyces coelicolor A3(2) is necessary for the normal onset of antibiotic production.

The fadD1 and macs1 genes of Streptomyces coelicolor are part of a two-gene operon. Both genes encode putative acyl coenzyme A synthetases (ACSs). The amino acid sequence of FadD1 has high homology with those of several ACSs, while MACS1 has the closest homology with medium-chain ACSs, broadly known as SA proteins. Like FadD of Escherichia coli, FadD1 also has a broad substrate specificity, although saturated long-chain fatty acids appears to be the preferred substrate. fadD1 is a growth-phase-regulated gene, and its mRNA is detected only during the stationary phase of growth. Interestingly, a mutation in fadD1 alters the levels of another ACS or ACSs, both at the stationary phase and at the exponential phase of growth, at least when glucose is used as a main carbon source. The mutant also shows a severe deficiency in antibiotic production, and at least for Act biosynthesis, this deficiency seems to be related to delayed expression of the Act biosynthetic genes. Antibiotic production is restored by the introduction of a wt fadD1 allele into the cell, demonstrating a strict link between ACS activity and the biosynthesis of secondary metabolites. The results of this study indicate that the ACSs may be useful targets for the design of rational approaches to improving antibiotic production.

Role of an essential acyl coenzyme A carboxylase in the primary and secondary metabolism of Streptomyces coelicolor A3(2).

Two genes, accB and accE, that form part of the same operon, were cloned from Streptomyces coelicolor A3(2). AccB is homologous to the carboxyl transferase domain of several propionyl coezyme A (CoA) carboxylases and acyl-CoA carboxylases (ACCases) of actinomycete origin, while AccE shows no significant homology to any known protein. Expression of accB and accE in Escherichia coli and subsequent in vitro reconstitution of enzyme activity in the presence of the biotinylated protein AccA1 or AccA2 confirmed that AccB was the carboxyl transferase subunit of an ACCase. The additional presence of AccE considerably enhanced the activity of the enzyme complex, suggesting that this small polypeptide is a functional component of the ACCase. The impossibility of obtaining an accB null mutant and the thiostrepton growth dependency of a tipAp accB conditional mutant confirmed that AccB is essential for S. coelicolor viability. Normal growth phenotype in the absence of the inducer was restored in the conditional mutant by the addition of exogenous long-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. Thus, AccB, together with AccA2, which is also an essential protein (E. Rodriguez and H. Gramajo, Microbiology 143:3109-3119, 1999), are the most likely components of an ACCase whose main physiological role is the synthesis of malonyl-CoA, the first committed step of fatty acid synthesis. Although normal growth of the conditional mutant was restored by fatty acids, the cultures did not produce actinorhodin or undecylprodigiosin, suggesting a direct participation of this enzyme complex in the supply of malonyl-CoA for the synthesis of these secondary metabolites.

Evaluacion de la erradicacion postratamiento del Helicobacter pyloripor reaccion en cadena de la polimerasa en biopsias gastricas / Post-treatment control of Helicobacter pylori erradication, by polymerase chain reaction in gastric biopsies

Introducción: La detección del H.p. en el estómago de pacientes vírgenes de tratamiento ha sido solucionada; un aspecto no resuelto, al menos en nuestro medio, es la pesquisa del H.p. luego de la terapéutica, para asegurar su erradicación. Objetivos: Utilizar una metodología sensible y específica para evaluar la presencia del H.p. en biopsias gástricas postratamiento, comparándola con otras técnicas similares clásicas. De esa manera, aumentando la precisión en el diagóstico, se podrá diferenciar la recrudescencia (falsa erradicación) de la reinfección y conocer el exacto porcentaje de curación. Materiales y Métodos: La PCR para H.p. fue realizada en 97 pacientes (62 hombres y 35 mujeres) con edad propedio de 49 años (+/- 14), en los cuales ya se había confirmado la erradicación por histología-Giemsa más CLO TEST. La metodología fue la siguiente: se realizaron 5 biopsias gástricas endoscópicas por paciente, 2 de antro, 2 de cuerpo, y 1 para CLO; todos los pacientes fueron estudiados luego de 6 ou 8 semanas de terminado el tratamiento y durante ese período no recibieron ninguna medicación. La metodología fue: las biopsias gástricas fueron fijadas en formol buffer al 10 por ciento, incluídas en parafina, y se colorearon con Giemsa y hematoxilina-eosina. El CLO TEST usado fue de Delta West Pty. Ltd. Bentley. La PCR se efectuó amplificando un fragmento de 296 pares de bases correspondiente al gen codificante del antígeno especie especifico de Helicobacter pylori, y la visualización del producto amplificado se realizó por electroforesis en gel de agarosa teñido con bromuro de etidio y U.V. Resultados: ninguno de estos pacientes presentaba úlcera gástrica o duodenal en el momento del control.Conclusiones: La PCR puede detectar secuencias de ADN especificas del H.p., incluso en su forma cocoide de resistencia, como se observan postratamiento. Las tinciones más utilizadas en biopsia (Giemsa/W-S) detectan aproximadamente 100000 microorganismos por ml; la PCR puede detectar 100, vale decir que es tres órdenes más sensible que la metodología clásica. En nuestra experiencia detectamos un 13 por ciento de falsos erradicados, los cuales clínicamente deberán ser considerados como recrudescencia y no como reinfección.

Detection del Helicobacter pylori por reacción en cadena de la polimerasa en muestras de bilis de pacientes con litiasis vesicular y coledociana / Detection of Helicobacter pylori by polimerase reaction in bile samples from gallblader and bile stones

Algunos trabajos describen la presencia de H. Pylori en muestras de bilis obtenidas durante la cirurgía por litiasis en vesícula y vías biliares. El objetivo de este trabajo, ha sido detectar la presencia del ADN del H. Pylori por medio de la Reacción en cadena de la Polimerasa (PCR) en muestras de bilis de pacientes con litasis vesicular y/o de vías biliares. Las muestras de bilis fueron obtenidas de 26 pacientes, 19 con litiasis vesicular y 7 con litiasis vesicular y coledociana, con edades comprendidas entre 22 a 69 años, media de 49,6 años, por punción de vesicular durante la colecistectomia. Las muestras fueron tratadas adecuadamente y preparadas para su investigación por PCR. 2 de 26 casos (7,6 por ciento) fueron positivos para la presencia en bilis del DNA del H. Pylori. Nuestro trabajo sugiere que el DNA del H P puede ser encontrado en muestras de bilis de pacientes portadores de litiasis biliar en la Argentina.