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Biomedical subjects

R Pacheco

Publications and source records attributed to R Pacheco.

At least 19 recordsLinked to original sources
PubMed2006

HLA-G, -E and -F: allelism, function and evolution.

The major histocompatibility complex class I genes comprise a bunch of classical genes (A, B, C), non-classical genes (E, F and G), pseudogenes and truncated genes. MHC-E, -F and -G are now considered immune "tolerization" molecules, which not only interact with NK cells but with T lymphocyte subsets and other cells and have a role in fetus acceptation. A strong positive directional selection is acting for maintaining the observed low polymorphism on E, F and G loci in human and apes. Invariance must be important for this non-classical class I proteins function.

Alleles↗
PubMed2005

Single (B or C), dual (BC or BD) and triple (BCD) viral hepatitis in HIV-infected patients in Madrid, Spain.

INTRODUCTION: There are very limited data about the prevalence of multiple hepatitis virus infections in HIV infected individuals. In HIV uninfected individuals with triple BCD hepatitis, hepatitis D virus (HDV) appears to be the dominant virus. However, in HIV infected patients with triple hepatitis it is not known if HDV replication inhibits hepatitis B virus (HBV) and/or hepatitis C virus (HCV) replication. METHODS: We calculated the prevalence of single (B or C), dual (BC) and triple (BCD) hepatitis in 423 HIV-infected patients with positive HCV serum antibodies and/or positive serum HBsAg. In patients with multiple infections we performed an evaluation of serum markers of HBV, HCV and HDV replication. RESULTS: The prevalence of multiple hepatitis was 4.7% (95% confidence interval, 2.7-6.7%). Multiple hepatitis occurred only among patients who acquired HIV through injection drug use. The most common multiple hepatitis was triple BCD. Patients with hepatitis BC and past or chronic hepatitis D were significantly more likely to have cirrhosis and a negative serum HBeAg and HCV PCR than patients with single hepatitis B or hepatitis C. Patients with chronic hepatitis D showed uniform suppression of HBV and HCV replication markers. CONCLUSIONS: In our geographic area approximately 5% of HIV infected patients with hepatitis suffer multiple hepatitis virus infection. In patients with triple hepatitis BCD virus infection, HDV appears to be the dominant virus causing inhibition of both HBV and HCV replication.

Adult↗
PubMed2005

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase.

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.

Amidohydrolases↗
PubMed2005

CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse.

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A(1) adenosine receptors, or A(2B) adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC(50) for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADA-anchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3- to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-gamma, TNF-alpha, and IL-6.

Adenosine Deaminase↗
PubMed2003

Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy.

A method based on Fourier transform infrared spectroscopy (FT-IR) has been developed for assaying the Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain. The kinetic of acetamide hydrolysis by the enzyme, in aqueous media, was monitored by measuring the intensity of the acetamide amide I band maximum at 1635 cm(-1) as a function of time. A value of 0.5mM(-1) cm(-1) was obtained for the extinction coefficient (epsilon) of acetamide at this frequency. The rate of the hydrolysis was found to be linear with the concentration of the enzyme up to 90 microM. The Michaelis-Menten kinetics parameters V and K(m) were determined as 30.7 U/mg and 4mM, respectively. These results were similar to those obtained using high-performance liquid chromatography analysis of the same hydrolytic reaction catalyzed by amidase either in water or in buffer. This suggests that the precision of the FT-IR method is suitable for the kinetic studies of amidase with the additional advantage of being able to perform a real-time measurement of the enzymatic activity.

Acetamides↗
PubMed2001

The CIDA-QUEST large-scale survey of Orion OB1: evidence for rapid disk dissipation in a dispersed stellar population.

We are conducting a large-scale, multiepoch, optical photometric survey [Centro de Investigaciones de Astronomia-Quasar Equatorial Survey Team (CIDA-QUEST)] covering about 120 square degrees to identify the young low-mass stars in the Orion OB1 association. We present results for an area of 34 square degrees. Using photometric variability as our main selection criterion, as well as follow-up spectroscopy, we confirmed 168 previously unidentified pre-main sequence stars that are about 0.6 to 0.9 times the mass of the sun (Mo), with ages of about 1 million to 3 million years (Ori OB1b) and about 3 million to 10 million years (Ori OB1a). The low-mass stars are spatially coincident with the high-mass (at least 3 Mo) members of the associations. Indicators of disk accretion such as Halpha emission and near-infrared emission from dusty disks fall sharply from Ori OB1b to Ori OB1a, indicating that the time scale for disk dissipation and possibly the onset of planet formation is a few million years.

Journal Article↗
PubMed2001

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability.

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

Amidohydrolases↗
PubMed1999

Human and porcine Taenia solium infection in a village in the highlands of Cusco, Peru. The Cysticercosis Working Group in Peru.

A serological survey was performed using the enzyme-linked immunoelectrotransfer blot assay (EITB) in a village in the highlands of Peru where there are three distinct but close neighborhoods, to determine if there is a direct relationship between human and porcine Taenia solium infection. One hundred and eight out of 365 individuals were sampled, and 14 were seropositive (human seroprevalence 13%). Most seropositive individuals were neurologically asymptomatic. Thirty-eight out of 89 sampled pigs (43%) were seropositive. There was a clear geographical clustering of cases, and positive correlation between human and porcine seroprevalence found when comparing the three neighborhoods. Cysticercosis is an important cause of neurological morbidity in most developing countries, and control/eradication trials are now being increasingly applied. Porcine serology provides an appropriate indicator of T. solium environmental contamination and should be used to estimate the risk of infection when evaluating control measures.

Adult↗
PubMed1999

PCR-ELISA for diagnosis of mucocutaneous leishmaniasis.

In this work we demonstrate that the PCR-ELISA technique is sufficiently sensitive and specific for use as a diagnostic test in cases of mucocutaneous leishmaniasis. DNA was extracted from cultures of Leishmania braziliensis, Leishmania infantum, Leishmania tropica, Leishmania mexicana, Trypanosoma cruzi, and blood samples from individuals who presented a clinical diagnosis of leishmaniasis as well as from healthy individuals. The DNA was PCR amplified and the product obtained was hybridised with a biotin-labelled probe, the sequence of which was designed in our laboratory. The result of the hybridisation was visualised by means of an ELISA technique using antifluorescein antibody labelled with alkaline phosphatase and p-nitrophenylphosphate (pNFF) as chromogen. The optical density of the products of the pNFF hydrolysis was quantified in a spectrophotometer at a wavelength of 405 nm. Using this technique the percentage of detection was 83.3% in blood samples from patients clinically diagnosed as having mucocutaneous leishmaniasis. No false positive results were obtained.

Animals↗
PubMed1999

Inverted double-gap isolation chamber for high-resolution calcium fluorimetry in skeletal muscle fibers.

Here we describe an improved inverted double-grease-gap isolation chamber that allows the formation of grease seals of high mechanical stability and high electrical resistance. We also provide a detailed description of the procedure used to mount the muscle fibers onto the apparatus. The new chamber permits the electrophysiological study of muscle fibers using an inverted microscope and high-resolution objectives, thus complying with the requirements of modern fluorescence confocal detection methods. The simplicity and reliability of the mounting procedure make this chamber preferable over other gap isolation chambers currently used for simultaneous electrophysiological and optical studies of calcium release. Experimental results obtained from amphibian muscle fibers are presented to illustrate the performance of the chamber when using global fluorescence detection, confocal spot detection, and laser confocal scanning imaging.

Animals↗
PubMed1998

Effect of food on the bioavailability of triclabendazole in patients with fascioliasis.

AIMS: Preliminary results indicate higher absorption of triclabendazole (TCBZ) administered postprandially. Therefore, the influence of food on the pharmacokinetics of TCBZ and its active sulphoxide (TCBZ-SO) and sulphone (TCBZ-SO2) metabolites was investigated. METHODS: Two single doses (10 mg kg(-1)) of TCBZ were administered to 20 patients with fascioliasis. Ten patients were first given the drug after a high energy breakfast and then, 48 h later, after an overnight fast. The other 10 patients first received the drug in fasting state and then, 48 h later, after breakfast. A low energy breakfast was served 2 h after drug administration for fasting state. RESULTS: Compared with the fasting state, an increased AUC and Cmax after food intake (significant, P < 0.0001) was shown from the values of TCBZ, TCBZ-SO and TCBZ-SO2. The mean AUC for TCBZ (fasting: 1.55, fed: 5.72 micromol l(-1) h), TCBZ-SO (fasting: 177, fed: 386 micromol l(-1) h) and TCBZ-SO2 (fasting: 13.9, fed: 30.5 micromol l(-1) h) indicated a large availability increase with food and the strong systemic predominance of the active sulphoxide metabolite over the unchanged drug. (All patients were cured at the end of the trial except one who required a second course of two postprandial doses of triclabendazole (10 mg kg(-1) each). Tolerability to the treatment among the patients was good. CONCLUSIONS: The administration of triclabendazole with food is recommended for improved systemic availability in patients with fascioliasis or paragonimiasis.

Adolescent↗
PubMed1996

New trends in the drug therapy of localized and disseminated Mycobacterium avium complex infection.

Recent advances in the drug therapy of localized and disseminated infection with Mycobacterium avium complex (MAC) are reviewed. MAC infection is the most commonly reported bacterial infection in patients with AIDS, and the frequency of this infection in patients negative for the human immunodeficiency virus (HIV) is increasing. The main portals of entry for MAC are the gastrointestinal and respiratory tracts. Localized MAC infection is more common in HIV-negative than HIV-infected patients. The symptoms of disseminated MAC disease are those typical of advanced HIV disease. The most reliable diagnosis is provided by blood cultures; radiometric culturing techniques are favored. The overall treatment of MAC infection has improved greatly with the introduction of new agents during the past 15 years; survival time has been extended. Clarithromycin and azithromycin have proven effective against both localized and disseminated MAC infection. Clarithromycin is the cornerstone of therapy for disseminated infection. Ciprofloxacin has been successfully used to treat disseminated infection as part of a four-drug regimen including rifampin, ethambutol, and clofazimine. Rifabutin has substantial efficacy when combined with other agents. Liposomal aminoglycosides, such as amikacin, and interferon gamma have shown some initial promise. Rifabutin is currently recommended for the prevention of MAC disease in HIV-infected patients. Clarithromycin and azithromycin have also shown efficacy for prophylaxis, and fluoroquinolones may play a preventive role as well. New drug therapies are improving the outlook for persons infected with MAC.

AIDS-Related Opportunistic Infections↗
PubMed1994

Distribution of hydatidosis and cysticercosis in different Peruvian populations as demonstrated by an enzyme-linked immunoelectrotransfer blot (EITB) assay. The Cysticercosis Working Group in Peru (CWG).

A serosurvey for human hydatidosis and cysticercosis was performed in different regions of Peru. Those regions included a known endemic area for cystic hydatid disease, a cooperative in the central Peruvian Andes near the city of Tarma, Department of Junin; three areas endemic for cysticercosis in the Departments of Ancash, Cuzco, and San Martin, where the status of hydatid disease is not well defined; and an urban shantytown near Lima, where neither zoonosis is known to be present. A seroprevalence for hydatidosis 1.9% (6 of 309) was found with both the enzyme-linked immunoelectrotransfer blot (EITB) and double diffusion assays in the area endemic for hydatidosis. Seroprevalence in the other zones tested was zero using only the EITB assay. Cysticercosis seroprevalence was high in pig-raising zones but low in the high-altitude, sheep-raising areas and in the seaport of Callao. No cross-reactions between Echinococcus granulosus and cysticercosis were noted in any of the regions studied. Hydatid infection remains a major health problem in the central Peruvian Andes where sheep raising is widely practiced; however, in those regions where mainly swine are raised, human hydatid infection is not a problem.

Abattoirs↗
PubMed1994

Medicolegal problems associated with do not resuscitate orders.

The use and potential abuse of the do not resuscitate (DNR) order has wide-ranging ethical, legal and economic implications. A DNR order is essential for clear communication between health care professionals, but the order itself may affect the care that patients subsequently receive. In addition, the DNR order may be a crucial therapeutic decision point in the care of the critically ill. Although general guidelines exist for withholding resuscitative measures from the terminally ill and general principles have been established, Spanish legislation is ambiguous on a number of points, forcing medical professionals to make difficult choices. This has led to the systematic, indiscriminate application of resuscitative measures, motivated in many cases by the desire to avoid a possible future lawsuit. We believe clearer and more precise legislation should be developed to bring Spanish criteria into line with those of international organizations that provide a common set of guidelines on important issues relating to the right to life and the right to refuse treatment.

Beneficence↗
PubMed1991

[Changes in autopsies in cancer in Navarra from 1980 to 1988].

BACKGROUND: Autopsies may be used to know the causes of death in population. In Navarra, the number of patients who died by cancer and were autopsied has increased. A similar pattern appeared in other communities. METHODS: We reviewed 2,643 autopsies performed in the main hospitals of Navarra. In each autopsy, age, sex, existence of primary malignant tumor, and its histologic type and localization were analyzed. The results were compared to the statistics of mortality of the Tumoral Register of Navarra. The modifications from 1980 to 1988 were studied. RESULTS: There is a significant increase of autopsy and mortality by cancer in Navarra. In autopsy cases, there is a high incidence of colon carcinoma and hepato-biliary carcinoma. The incidence in all cancer dead patients is similar being greater in hepato-biliary carcinoma and pancreas carcinoma. The percentage of autopsies in patients over 60 years of age is decreased, but it has increased in all cancer dead patients of the same group of age in the community of Navarra. In the autopsies of patients over 60 years of age there has been a rise in breast carcinoma, while there has been a general increase in all dead patients. In all deaths of people under 60 years of age, there has been a decrease, but in autopsy cases there has been an increase of colon carcinoma. CONCLUSIONS: In autopsy series from 1980 to 1988, cancer deaths increased significantly. There is not relationship between cancer dead patients and autopsy cases in the studied data. In hepato-biliary carcinoma, there is an increase in autopsy cases and cancer dead patients. Discrepancies between the number of deaths of cancer patients and autopsy patients revealed that the performance of autopsy depends of multiple causes.

Age Factors↗