PubMed Health⌕ Search

Biomedical subjects

M de Frutos

Publications and source records attributed to M de Frutos.

15 recordsLinked to original sources
PubMed2006

Solubility and charge inversion of complexes of DNA and basic proteins.

The basic proteins, protamines and histones H1, are known to condense DNA in vivo. We examine here their ability to condense and solubilize in vitro linear DNA [and a synthetic polyanion, Poly(Styrene-Sulfonate) or PSS] at low ionic concentrations by varying the charge concentration ratio. Phase separation is observed in a very narrow range of ratios for short DNA and PSS; on both sides of this range, polydisperse and charged complexes are formed. A charge inversion is detected. For long DNA chains however, a different behavior is observed: the complexes are not soluble in excess of proteins.

Binding Sites↗
PubMed2005

Effect of spermine and DNase on DNA release from bacteriophage T5.

Bacterial viruses (bacteriophages) consist of nucleic acid protected by a protein envelope called capsid. At the start of infection, the phage genome is translocated into the bacterial cytoplasm. In vitro (and also in vivo), this DNA release can be triggered by binding a specific receptor protein to the phage tail. The force responsible for the release arises from energy stored in the capsid due to strong confinement of the DNA. We show that this force can be modified by adding molecules like spermine that affect DNA conformation. The tetravalent cation spermine can reduce the pressure inside the capsid and induce condensation of the released DNA. We examine the effect of spermine on DNA ejection from phage T5 by using light scattering and gel electrophoresis to measure the amount of DNA remaining in the capsid at the end of ejection. We discuss the results in terms of free energy minimization and we demonstrate that the presence of a DNA condensate outside the phage generates an additional force pulling passively on the DNA remaining inside the capsid.

DNA Packaging↗
PubMed2001

Feasibility of high-performance immunochromatography as an isolation method for PCBs and other dioxin-like compounds.

A high performance immunochromatographic procedure to isolate polychlorinated biphenyls (PCBs) and other dioxin-like compounds from a sample is shown. Development of the procedure includes (i) synthesis of the hapten, binding it to the spacer arm and to the carrier protein to make the immunizing molecule; (ii) raising and purification of anti-PCB antibodies; (iii) building of the immunocolumn; (iv) selection of the binding, rinsing, and elution conditions adequate for these highly lipophilic compounds; (v) study of the influence of the concentration and volume of sample on recovery; and (vi) study of the selectivity of the immunosystem for dioxins, furans, PCBs, and several insecticides of different toxicity. Evaluation of the method is carried out by analyzing the fractions retained and nonretained in the immunocolumn by GC/MS. The immunochromatographic system that is developed shows itself to be feasible as cleanup and isolation steps carried out prior to GC/MS analyses. When compared to classical cleanup and isolation methods traditionally used for analysis of PCBs in water, the immunochromatographic method is > 20x faster and uses 100x less organic solvents, and its selectivity is enormously enhanced. Good recoveries are obtained with both kinds of methods. The immunochromatographic procedure fulfils the acceptance criteria indicated by the EPA, even for sub-parts-per-billion concentrations.

Journal Article↗
PubMed2001

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.

Animals↗
PubMed2000

[Laboratory diagnosis and serologic course in patients with tularemia].

BACKGROUND: Tularemia was practically unknown in Spain until the end of 1997, when an epidemic outbreak was declared. This paper presents the data on microbiological diagnosis of 55 patients who suffered from tularemia. PATIENTS AND METHODS: Thirty-two samples from 19 patients and 151 serum samples from 55 patients were obtained for culture. Serologic diagnosis was performed by tube sero-agglutination and microagglutination. Three types of tests were performed on all sera: Wright sero-agglutination (WSA), Coombs test against Brucella spp. and sero-agglutination against Yersinia enterocolitica O:3, Yersinia enterocolitica O:3, and Proteus OX 19. RESULTS: F. tularensis was found in two samples (6.25%) of the 32 received. Titers > or = 1/160 were obtained in 78.2% and 74.5% of the initial sera by tube sero-agglutination and microagglutination, respectively. Correlation between the two tests was 0.80 (p < 0.001). Prozone phenomenon was observed in 59.9% of the sera, while crossed reactivity to Brucella spp. and Proteus spp. was found in 9.3% and 22.8%, respectively. No crossed reactivity was observed with Yersinia spp. CONCLUSIONS: Culture of F. tularensis has low sensitivity. The correlation obtained between tube sero-agglutination and microagglutination is good. Both techniques are useful in routine diagnosis of tularemia, although microagglutination has some advantages over tube agglutination.

Adult↗
PubMed1999

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis.

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78-4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.

Animals↗
PubMed1999

First molecular characterization of a uridine diphosphate galacturonate 4-epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1.

Uridine diphosphate galacturonate 4-epimerases (UDPGLEs) are enzymes that convert UDP-glucuronate into UDP-galacturonate. Although the presence of UDPGLEs has been reported in prokaryoic and eukaryotic organisms, the genes coding for these enzymes are completely unknown. The galacturonic acid-containing capsular polysaccharide of Streptococcus pneumoniae type 1 is synthesized through the action of a specific UDPGLE. We have constructed a defined deletion mutant in the cap1J gene (one of the 15 cap1 genes responsible for the synthesis of the type 1 capsule) that exhibited an unencapsulated phenotype. This mutant was unable to synthesize UDPGLE, suggesting that Cap1J was the type 1-specific UDPGLE of S. pneumoniae. Escherichia coli cells harbouring the recombinant plasmid pRMM38 (cap1J) overproduced a 40 kDa protein, characterized as Cap1J on the basis of the N-terminal amino acid sequence analysis, and expressed high levels of enzymatically active Cap1J epimerase. Cap1J was partially purified, although purification to electrophoretic homogeneity inactivated the enzyme irreversibly. The enzyme has the following characteristics: K(m) for UDP-glucuronate, 0.24 mM; pH optimum, 7.5; equilibrium constant (in the direction of UDP-galacturonate formation), 1.3; and an approximate M(r) of 80,000 for the active form. The Cap1J protein exhibited a fluorescence emission spectrum similar to that of NADH. Upon inactivation with p-hydroxymercuribenzoate, the addition of NAD+ and 2-mercaptoethanol were sufficient to reactivate the enzyme. Among several compounds tested, UDP-galactose and UDP-xylose exhibited the highest inhibition of the UDPGLE activity. Inactivation of UDPGLE activity was also observed in the presence of UMP and several reducing sugars. To our knowledge, this is the first example of a thoroughly molecular characterization of a UDPGLE.

Amino Acid Sequence↗
PubMed1997

Multiple peaks in high-performance liquid chromatography of proteins. beta-Lactoglobulins eluted in a hydrophobic interaction chromatography system.

The chromatographic behavior of beta-lactoglobulins when eluted in hydrophobic interaction chromatography systems is studied. By modifying some factors, such as pH and temperature, the relationship between shape of the chromatographic peak and protein structure is shown. At pH 4.5 and low temperature multiple peaks for beta-LG A and beta-LG B are observed and assigned to aggregates. The effects of other parameters, besides pH and temperature, such as volume and concentration of injected sample, contact time between protein and stationary and/or mobile phases, and nature and concentration of mobile phase upon aggregation are studied. Comparison of the chromatographic behavior of both variants of beta-lactoglobulin is made.

Animals↗
PubMed1995

Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries.

A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), alpha-LA (alpha-lactalbumin), beta-LG A (beta-lactoglobulin A) and beta-LG B (beta-lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.

Animals↗
PubMed1993

Liquid chromatography based enzyme-amplified immunological assays in fused-silica capillaries at the zeptomole level.

Through the use of fused-silica capillaries it was shown that reducing the liquid volume of an enzyme-amplified immunological assay increases the rate of amplification and sensitivity of the assay by several orders of magnitude. Human immunoglobulin G (hIgG) captured on protein G-coated 100-microns-i.d. columns was saturated with F(ab) anti-hIgG conjugated to alkaline phosphatase (ALP). Conjugated enzyme captured by the antigen was subsequently assayed in a stop-flow incubation with p-nitrophenyl phosphate. p-Nitrophenol produced in the stop-flow incubation was then swept to the detector and quantitated at 405 nm. The detection limit in the stop-flow mode was approximately 3 fmol. Three problems were identified in this flow-through, capillary assay format. The first was that the rate of immunological complex formation within the capillary was too slow. Preforming the immunological complex before application to the column increased the sensitivity by 2 orders of magnitude. Another problem was that the rate of mass transfer within the capillary limited capture of the preformed immunological complex. This problem was solved by stop-flow incubation of the complex in the column. The combination of preformation of the immunological complex and stop-flow binding within the column reduced the detection limit to approximately 3 amol. Finally, reducing the amount of F(ab)-ALP used in the assay minimized nonspecific binding of the conjugated enzyme and reduced the detection limit further to 333 zmol.

Alkaline Phosphatase↗
PubMed1993

Tandem chromatographic-immunological analyses.

The function of conventional immuno-logical assays is to determine the presence and amount of known substances. Cross-reactivity of antibodies with unknown species of similar structure is a problem that can be circumvented by using chromatography in a second level of discrimination. Tandem LC-IA systems increase the selectivity and the sensitivity of assays by removing interfering species. For cases in which the goal is to prove that similar species are absent from a sample or to identify antigen variants, conventional assays fail. LC-IA systems are adept at dealing with these tasks. The necessary steps of metering reagents, separating Ag:Ab complex from other species, and enhancing detection in an immunological assay are easily accommodated by the LC system. In fact, the LC system provides a wide variety of new, high-sensitivity, high-speed methods for carrying out immunological assays. On the basis of the enormous versatility, selectivity, and sensitivity that LC-IA brings to analytical chemistry, we believe this technique will become increasingly important for monitoring analytes in complex biological matrices.

Amino Acids↗
PubMed1992

Rapid analysis of whey proteins from different animal species by reversed-phase high-performance liquid chromatography.

This paper explores the possibilities of reversed-phase high performance liquid chromatography (RP-HPLC) for analysing whey proteins from the milk of cows and other animal species. An RP-HPLC method is proposed to separate and quantify bovine whey proteins. Using this method, bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin A and beta-lactoglobulin B were separated in less than 7 min. It is demonstrated that irreversible adsorption of bovine whey proteins occurs on unused columns (i.e. those not previously used to separate proteins). Therefore, prior conditioning of the column with whey proteins is required for valid protein quantification. Conditioning can be achieved by eluting a large amount of at least one of the bovine whey proteins through the unused column. The calibration curve (peak area vs. protein concentration) for the main bovine whey proteins was linear. This method also allowed good separation of caprine and ovine whey proteins and separation of some homologous whey proteins of different animal species. Detection of milk mixtures from different animal species was carried out using this method.

Animals↗