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Biomedical subjects

R Palacios

Publications and source records attributed to R Palacios.

At least 55 records · Page 3Linked to original sources
PubMed1999

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.

Blotting, Southern↗
PubMed1998

Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus.

The cDNA encoding an allergen from the dust mite Dermatophagoides pteronyssinus has been cloned and sequenced. The allergen (Der p 10) is a tropomyosin that shared more than 65% identical residues with other invertebrate tropomyosins. The final recovery of recombinant Der p 10 from the culture media after a single purification step was as much as 26 mg/l. The recombinant allergen is reactive to shrimp antitropomyosin IgG antibodies and has a 5.6% frequency of IgE reactivity in sera from mite-allergic patients.

Allergens↗
PubMed1998

Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin).

BACKGROUND: Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua. OBJECTIVES: We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy. METHODS: Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen. RESULTS: Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin. CONCLUSION: This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

Allergens↗
PubMed1998

Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen.

Sunflower (Helianthus annuus) sensitization is not always related with occupational allergy. We have isolated the allergen profilin (Hel a 2) from this Compositae plant, cloned and sequenced five cDNAs encoding for full-length or partial Hel a 2. Natural sunflower profilin reacted with specific IgE in the 121 sera tested, at a frequency of 30.5%. Expression of the cDNA encoding Hel a 2 in Escherichia coli and a simple purification procedure by poly-L-proline chromatography allowed immunological characterization of the recombinant allergen. Binding of monoclonal antibodies against sunflower profilin revealed that some epitopes responsible for antigen-specific IgG production were not present in the recombinant allergen. High cross-reactivity has been found between recombinant Hel a 2 and profilins from other Compositae plants and also from botanically distant plants.

Allergens↗
PubMed1998

Anaphylactic reaction to ingestion of Quercus ilex acorn nut.

BACKGROUND: A patient experienced an anaphylactic reaction after eating acorn nuts, fruit of the holm oak (Quercus ilex), one of the most abundant trees in Spain. Several urticaria episodes upon ingestion of peanuts were also referred. OBJECTIVE: To assess the hypersensitivity reaction to acorn and to characterize the allergenic proteins involved. METHODS: Cutaneous tests were performed using the skin-prick technique, using a large variety of grass, tree and weed pollens as well as fresh nuts and nut extracts. Specific IgE determination was assessed by RAST. IgE binding bands were determined by SDS-PAGE immunoblotting. RESULTS: Skin-prick tests were strongly positive with acorn and peanut. Olea europaea, Quercus alba, Quercus ilex and grass pollens also elicited a weal higher than negative control. Patient serum had measurable levels of IgE antibodies especially to acorn, peanut and grass pollens. Only one protein band, of 17.9 kDa molecular mass, showed IgE-binding properties in the acorn extract. The possible homology of this strong allergenic protein with the group 1 tree pollen allergens was evidenced by the partial inhibition of the western blot with Bet v 1. CONCLUSIONS: We present a case of anaphylaxis to acorn ingestion as demonstrated by in vivo and in vitro results. A 17.9 kDa IgE-binding band, showing some homology to group 1 pollen tree allergens, was recognized by patient serum.

Anaphylaxis↗
PubMed1998

Study of a case of hypersensitivity to lettuce (Lactuca sativa).

BACKGROUND: Allergic reactions to lettuce (Lactuca sativa) are not too frequent and few cases of systemic adverse reactions after its ingestion have been described. OBJECTIVE: We report a case of clinical sensitization to lettuce on a patient who presented mucocutaneous manifestations after its ingestion, with positive skin tests, histamine release test and serum specific-IgE to lettuce. The allergens responsible for this sensitization were also characterized by means of SDS-PAGE immunoblotting. MATERIALS AND METHODS: We performed skin tests, histamine release test, serum specific IgE determination and CAP inhibition with lettuce and mugwort (Artemisia vulgaris) extracts. An aqueous and enriched lettuce (from loose leaf type) extract was subjected to SDS-PAGE immunoblotting for determination of its IgE-binding components. RESULTS AND CONCLUSIONS: CAP inhibition showed antigenic community between lettuce and mugwort. Four protein bands from the lettuce extracts with molecular weights of 50, 43, 39 and 16 kDa exhibited IgE-binding properties.

Adult↗
PubMed1998

Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici.

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.

Agrobacterium tumefaciens↗
PubMed1998

Three replicons of Rhizobium sp. Strain NGR234 harbor symbiotic gene sequences.

Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b.

DNA, Bacterial↗
PubMed1998

[Fungemia in patients with HIV infection].

OBJECTIVE: To make an analysis of fungemia in HIV-infected patients in our hospital. PATIENTS AND METHODS: We retrospectively (1989-1997) studied all HIV-infected patients with positive blood cultures for Candida sp., Cryptococcus neoformans or any other fungal infection. RESULTS: C. neoformans was isolated in 11 patients (10 men and 1 woman): Six were treated with amphotericin B and 5 with fluconazole. 2 patients died during the acute phase and the infection relapsed in 3. Blood culture for Candida sp. were positive in 9 (8 men and 1 woman): only a case was nosocomial. Seven patients were intravenous drug users and the presenting manifestations were autolimited candidemia in 3, aortic and tricuspid endocarditis in 1 and 2 cases respectively and pneumonia in another one. Six C. albicans, 2 C. krusei and 1 C. glabrata were isolated. 3 patients received amphotericin B and 3 received fluconazole. 2 patients suffering from endocarditis died and so did the patient with C. glabrata infection. A patient, who denied having travelled to endemic areas, developed histoplasmosis; blood culture was positive for H. capsulatum. He initially had a good response to amphotericin B and itraconazole. CONCLUSIONS: Fungemia is not frequent in HIV-infected patients. Cryptococcosis and histoplasmosis occur in advanced HIV-patients and candidemia is fundamentally associated with intravenous drug use.

Adult↗
PubMed1997

Sequence polymorphism and structural analysis of timothy grass pollen profilin allergen (Phl p 11).

Three cDNA clones encoding timothy grass pollen profilin (Phl p 11) were newly isolated. Comparison of the sequences of four cDNA clones, including a previously isolated clone, showed a low level of polymorphism. Isoelectrofocusing of highly purified timothy grass profilin indicated the existence of at least five isoforms. One recombinant profilin showed similar immunological properties to natural timothy grass profilin. Tertiary structure of Phleum pratense profilin was obtained by homology-based molecular modeling.

Allergens↗
PubMed1997

Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen.

BACKGROUND: Olive tree (Olea europaea) pollen allergy is one of the main causes of allergy in Mediterranean countries and some areas of North America. OBJECTIVE: To clone olive allergens and to characterize immunologically the purified recombinant allergens. METHODS: Full-length complementary deoxyribonucleic acid (cDNA) strands encoding olive allergens (Ole e 1) were cloned by polymerase chain reaction amplification and sequenced. Recombinant proteins were produced in Escherichia coli by the use of two different expression systems. Immunoreactivity of the recombinant proteins was tested by ELISA and Western blot with serum from patients with allergy to olive. RESULTS: Significant sequence polymorphism was found in both allergens. The panallergen profilin was expressed as a nonfusion protein and was purified to homogeneity after a single step of affinity chromatography with a poly-L-proline Sepharose column. One cDNA encoding an Ole e 1 isoform was expressed as a fusion protein consisting of the glutathione S-transferase of Schistosoma japonicum and Ole e 1. The fusion protein was purified to homogeneity by gel filtration chromatography and affinity chromatography with a glutathione-Sepharose column, and digested with thrombin. Both recombinant allergens shared B cell epitopes with the corresponding natural allergens. CONCLUSION: IgE-reactive Ole e 1 and olive profilin expressed in bacteria were purified after simple chromatographic procedures and may be useful for diagnostic purposes.

Allergens↗
PubMed1997

Treatment of childhood Hodgkin's disease with COPP or COPP-ABV (hybrid) without radiotherapy in Nicaragua.

BACKGROUND: Childhood Hodgkin's disease (HD) in low-income countries has been reported to have distinct presenting features, including a high prevalence of the mixed cellularity subtype, which also seems to be associated with poorer prognosis. Further investigations are needed to evaluate these issues. Another controversial aspect of childhood HD is the use of radiotherapy (RT) in its treatment and the growing concern about its serious adverse side effects. In this paper, data on the diagnosis and outcome of children treated without RT in a low-income country (Nicaragua) are reported. PATIENTS AND METHODS: Forty-eight consecutive children aged 0-15 years, diagnosed at 'La Mascota' Hospital of Managua (Nicaragua) from January 1990 to October 1995. entered this study. Follow-up was updated in May 1996. Clinical and histopathological staging was performed according to Ann Arbor and Rye criteria, respectively. Treatment consisted of COPP (six cycles) for stages I or IIA, or COPP-ABV hybrid): eight cycles for stages IIB or III, and ID cycles for stage IV. Total cumulative doses of adriamycin and bleomycin in this protocol are, respectively, 200 and 80 mg:sqm for stages II B or III and 250 and 100 mg/sqm for stage IV. RESULTS: The median age of the 48 patients at diagnosis was seven years, and the mean age was 7.9 years (range 3-15 years). Clinical stages were IA in 5, IIA in 9, IIB in 6, IIIA in 5, IIIB in 14, and IVB in 9. Histopathologically, 25 cases presented with mixed cellularity, 15 with nodular sclerosis, 5 with lymphocytic predominance and 3 with lymphocytic depletion. Four patients did not proceed with treatment and were lost to follow-up. Two patients (stages IIIB and IVB), who never achieved complete remission (CR) during treatment, presented progressive disease at the end of the scheduled chemotherapy. The remaining 42 patients were in complete remission at the end of chemotherapy. Following discontinuation of therapy, one patient (stage IA) was lost to follow-up and two patients with stage IIIB, who were in CR after the second chemotherapy cycle, relapsed 20 and 9 months following the diagnosis. EFS at three years is 100% for the 25 patients with stages I, II, IIIA and 74.9% for the 23 patients with stages IIIB or IV. CONCLUSION: The presenting features found in these patients are similar to those reported from other low-income countries. In our experience, however, the high prevalence of the mixed cellularity subtype was not associated with poorer prognosis. Satisfactory results have been achieved in patients with stages I, II or IIIA HD using COPP or COPP-ABV (hybrid) regimens without RT. The treatment was also well tolerated and can thus be recommended for these patients in low-income countries, where RT facilities may be scarce or unavailable. The use of more aggressive treatment schedules and/or RT on involved fields in front-line treatment may, however, be needed for the more advanced stages IIIB or IV. Large studies with adequate follow-up are needed to evaluate whether, if RT is omitted, higher cumulative doses of more toxic drugs are required and thus compare the long-term toxic effects of different treatment modalities.

Adolescent↗
PubMed1997

Generation of Rhizobium strains with improved symbiotic properties by random DNA amplification (RDA)

To select for bacterial strains with enhanced phenotypes, random fragments of a whole genome, or a defined region of the genome, are cloned in a nonreplicating vector. The resulting plasmids are integrated by recombination into the homologous DNA region of the original strain. Integration gives rise to a nontandem direct duplication of the corresponding DNA region separated by the vector moiety of the plasmid. Recombination between the direct repeats leads to tandem duplication and further amplification of the entire integrated DNA, including the vector. Bacteria harboring the amplified DNA are selected by increasing the dosage of an antibiotic corresponding to a resistance marker of the integrated vector. Pooled strains carrying amplifications are then challenged with a selective pressure for the desired phenotype. After repeated selection cycles, the most fit strains are isolated. We used this process, which we called random DNA amplification, to select Rhizobium strains with increased competitiveness for nodule formation. Derivatives containing randomly amplified DNA regions of the symbiotic plasmid of Rhizobium tropici CFN299 strain were generated. Pools of amplified strains were inoculated onto various tropical legumes. After several cycles of selection through plants, amplified derivatives showing an increased competitiveness for nodule formation with the leguminous plant Macroptilium atropurpureum were obtained.

Cloning, Molecular↗
PubMed1997

Kinetics of allergen expression in cultures of house dust mites, Dermatophagoides pteronyssinus and D. farinae (Acari: Pyroglyphidae).

Laboratory cultures of house dust mites Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 were used to study the population dynamics of the mites and the kinetics of antigen appearance. The analysis of extracts obtained after different incubation periods, carried out by SDS-PAGE, immunoblotting, and enzyme-linked immunosorbent assay, allows for the definition of 3 different growth phases: the latency phase (F1); the exponential growth phase (F2) during which the allergenic proteins, including the Der 1 and Der 2 major allergens, were expressed more intensely and in larger quantities; and a final phase (F3), death, in which the lowest rates of allergenic components with a clearly different pattern were seen. The data obtained from this work demonstrates that mite cultures during the maximum growth phase (F2) contain the largest amount of allergenic components as well as the highest major allergen concentrations.

Allergens↗
PubMed1997

Improvement of fruit allergenic extracts for immunoblotting experiments.

A method based on ion-exchange column chromatography to enhance the protein content of fruit allergenic extracts was found to help make the subsequent SDS-PAGE immunoblotting assays possible; otherwise, they were difficult to achieve due to the high carbohydrate content. Fractionated extracts of apple, pear, and peach (peel and pulp) were obtained by anion-exchange chromatography (Q-Sepharose column), providing clear electrophoretic patterns which allowed IgE detection by enzymatic assays of the transferred membranes. This chromatographic method produced in one single step an enriched extract directly from the standard crude aqueous one, with an increment in the protein content of more than sixfold, on average; thus, it proved to be more suitable than the usual chemical fractionation procedures.

Allergens↗
PubMed1997

Occupational allergy in saffron workers.

Sensitization to the flower of saffron, a plant commonly grown in Spain for commercial purposes, and its clinical significance as an occupational allergen were studied. The prick test and RAST, with saffron pollen, stamen, and pistil extracts, were used to evaluate the cutaneous and specific antibody responses in the studied population. Provocation tests in patients with clinical findings were used to verify the implication of saffron components in these symptoms. Fifty saffron workers were evaluated. Three of them were sensitized to saffron pollen and stamen proteins, giving prick and RAST positive values. One patient presented asthma, showing a positive bronchial provocation test, and two patients rhinoconjunctivitis, showing positive conjunctival provocation tests. Of a general allergic population of 237, 10 patients also presented cutaneous test and IgE positive to saffron. Saffron allergens (from pollen and stamens) were characterized by SDS-PAGE immunoblotting. A relevant allergen of 15.5 kDa with profilinic nature was detected and further purified by high-resolution gel filtration chromatography. No allergenic components were demonstrated in pistils. Cross-reactivity of saffron extracts was evaluated by RAST inhibition with respect to other pollen species commonly causing sensitization in the same area of study. A significant degree of cross-reactivity was demonstrated between saffron and Lolium, Salsola, or Olea. The identification of the protein components involved in the cross-reactions was investigated by blot inhibition.

Adolescent↗
PubMed1997

Gene amplification and genomic plasticity in prokaryotes.

Gene amplification is a common feature of the genome of prokaryotic organisms. In this review, we analyze different instances of gene amplification in a variety of prokaryotes, including their mechanisms of generation and biological role. Growing evidence supports the concept that gene amplification be considered not as a mutation but rather as a dynamic genomic state related to the adaptation of bacterial populations to changing environmental conditions or biological interactions. In this context, the potentially amplifiable DNA regions impose a defined dynamic structure on the genome. If such structure has indeed been selected during evolution, it is a particularly challenging hypothesis.

Gene Amplification↗