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Biomedical subjects

F Acevedo

Publications and source records attributed to F Acevedo.

At least 19 recordsLinked to original sources
PubMed1999

Simple quantification of complement factors C3 and C3b using separation by isotachophoresis.

The separation of complement factors C3 and C3b by isotachophoresis in 1% agarose gel followed by immunoprecipitation and quantification is presented. Glycine was used as spacer in a nonequilibrium isotachophoresis (Acevedo, F., J. Chromatogr. 1991, 545, 391-396). Tricine, beta-alanine and Tris were the leading ion, terminating ion and counter ion, respectively. After electrophoresis the gel was incubated in rabbit anti-human complement factor C3c. The amounts of C3 and C3b in the sample were measured by optical densitometry of the Coomassie Brilliant blue-stained immunoprecipitates in the agarose gel. The correlation coefficient obtained for the logarithm of the integrated densitometric measurement vs. the logarithm of the amount of applied C3 was higher than 0.98 in calibration experiments. The extent of complement factor C3 activation is calculated as the ratio between the amount of C3b and the amount of C3b plus C3 and expressed as percent. The progress of complement activation from human blood plasma samples induced by Mg2+ and zymosan are presented as examples.

Animals↗
PubMed1998

Visualization and quantification of birch-pollen allergens directly on air-sampling filters.

A new technique is presented to detect and quantify birch (Betula pendula)-pollen allergens directly on air-sampling filters. It is based on the use of specific antibodies, enzymatic reactions, and measurement by chemiluminescence or densitometry. The major pollen antigens are the most important birch allergens. The antibodies used recognize birch-pollen antigens which thus correspond to allergens. Calibration was done with a standardized extract of birch pollen for skin prick testing. The correlation coefficient for the logarithms of luminescence and the amount of birch-pollen allergen applied on filters was > 0.98 in the range 0.04-200 SQ units. A similar correlation was found for the logarithms of the integrated densitometry values and the amount of birch-pollen allergen applied on filters. The number of major pollen-antigen particles or grains on filters could be estimated by counting the major stained spots produced by precipitated enzymatic products. The correlation coefficient was 0.90 for the logarithms of the number of counted major antigen spots and the calculated antigen amount obtained by luminometric measurements. Our results demonstrate that birch-pollen allergens can be determined directly on air-sampling, Teflon-based filters by luminometry, optical density, or particle counting.

Air Pollutants↗
PubMed1997

Different patterns of soluble adhesion molecules in systemic and cutaneous lupus erythematosus.

Circulating isoforms of cellular adhesion molecules (CAMs) have been described recently, and elevated levels of certain sCAMs have been reported in various inflammatory diseases such as systemic lupus erythematosus (SLE). There are previously no reports on sCAMs in cutaneous LE. Sera from 61 patients with LE: systemic (SLE: n=24), chronic cutaneous (discoid LE, DLE: n= 19) or subacute cutaneous (SCLE: n=8), chronic biologically false positive (CBFP) reactors for syphilis (n= 10) and 32 controls were examined for sICAM-1, sVCAM-1 and sE-Selectin with specific ELISA kits. Protocol forms were reviewed. We found significantly elevated levels of sE-Selectin in patients with DLE and widespread cutaneous symptoms, and a correlation between active cutaneous disease as well as polymorphous light eruption (PLE) and elevated levels of sE-Selectin. In contrast, patients with systemic LE did not have elevated levels of sE-Selectin, but in concordance with earlier reports, sICAM-1 and sVCAM-1 levels were elevated compared to controls in SLE, as well as in SCLE patients, which has not been reported previously. Since activated endothelial cells are the only source for E-Selectin, the elevated sE-Selectin level in patients with widespread and active cutaneous disease suggests a more important role for endothelial cells in the pathogenesis of cutaneous LE than previously assumed.

Adult↗
PubMed1996

[Dengue in Nicaragua, 1994: reintroduction of serotype 3 in the Americas].

The principal aim of this work was to report the reintroduction of dengue virus serotype 3 in the Americas after an absence of 17 years. In addition, it describes the most common symptoms associated with classical dengue and hemorrhagic dengue and presents data on the distribution of the epidemic in the various comprehensive local health care systems of Nicaragua. The study group consisted of 39 patients hospitalized in Managua and León for dengue with hemorrhagic manifestations and hemorrhagic dengue. Of these patients, 34 were classified as probable or confirmed cases of dengue. The most frequent symptoms were fever, headache, vomiting, and muscle and joint pains. The tourniquet test was positive and thrombocytopenia was confirmed in 56% and 44% of the patients, respectively. Epistaxis (67%) was the most common hemorrhagic sign. Of the 356 serum samples received through the dengue surveillance systems in October 1994, IgM antibodies were detected in 43%. The virus was isolated from 5 of 24 samples tested (serotype 3 from 3 and serotype 1 from 2). The reintroduction of serotype 3 of dengue into the Region was demonstrated, along with its ability to produce epidemics of hemorrhagic dengue. The countries are warned that if they do not quickly take the measures described in the guidelines for the prevention and control of dengue and dengue hemorrhagic fever, new epidemics may occur in the Americas, given the large number of persons susceptible to this serotype and the high density of the mosquito vector in most of the countries of the Region.

Adolescent↗
PubMed1995

Sensitive quantification of proteins by electrophoresis in gels by use of chemiluminescence.

For protein quantitation in gels or blotting membranes, chemiluminescence (CL) offers the advantages of a substantial improvement of detection limits. Easy-to-use CL-chemicals and specific probes such as antibodies conjugated to enzymes, e.g. alkaline phosphatase (AP) may be used in combination with a newly developed luminometer. CL was found to have low detection limits and a linear relation between relative light units (RLU) and the concentration of the antibody enzyme complex present over a wide concentration range. Measurements of the immunoglobulin IgE in dot blots and in blots after sodium dodecyl sulfate (SDS) electrophoresis under nonreducing conditions in agarose gels are described.

Alkaline Phosphatase↗
PubMed1995

Electrophoretic size separation of proteins treated with sodium dodecyl sulfate in 1% agarose gels.

Separation of proteins treated with sodium dodecyl sulfate (SDS) according to molecular size was achieved by discontinuous electrophoresis in vertical low-concentration agarose gels. A linear relationship was found between the migration distance and the square root of the molecular weight. This holds for proteins in the range of 7-200 kDa separated in 1.25% w/v agarose gel slabs (7 x 7 x 0.15 cm) with 0.1% w/v SDS and sulfate as leading ion. The linear regression coefficient was 0.998. The molecular weight and charge of coions influenced the separation. Small ions with low pKa values were found suitable as coions. The migration distance of proteins treated with SDS varied linearly with the agarose concentration of the gel. The agarose type and quality affected the resolution of the SDS-protein bands. We conclude that agarose gels can substitute polyacrylamide gels for the separation of proteins treated with SDS. A homogeneous agarose gel at a concentration of about 1% is a nonsieving support for electrophoresis. Therefore, the separation described here cannot be explained by the pore size of the gel. The results suggest that the separation is mainly due to the relative migration velocities of the coion and the proteins treated with SDS.

Blood Proteins↗
PubMed1993

Isotachophoresis of proteins in sucrose density gradients.

The separation of proteins from human serum by isotachophoresis in sucrose density gradients, with mixtures of discrete amphoteric substances as spacers, is described. Open columns and columns with a dialysis membrane to hold the sucrose gradients were used. A simple algorithm based on the Kohlrausch function was used to calculate the amount of each spacer. The pH gradients generated in open columns were found to be in agreement with the calculations. The load was up to two gram proteins. The analysis of the fractions obtained after the separation showed a distribution of components similar to as analytical isotachophoresis. It is concluded that sucrose density gradients are suitable as supporting media for the preparative separation of proteins by isotachophoresis. The high resolution attained and the possibility of scaling-up the separation systems are major advantages of this system. In addition, the sample is easily and completely recoverable.

Algorithms↗
PubMed1990

Transferrin and epidermal growth.

Growth of keratinocytes in explant culture of mouse ear epidermis was studied. The addition of transferrin to the culture media improved growth. Transferrin fractionated from human and fetal calf serum increased outgrowths of the cultures when compared with commercially available transferrin. An acidic transferrin fraction was present in greater amount in human serum and in fetal calf serum than that found in commercial transferrin. This fraction was more abundant in serum from psoriatic patients than in serum of healthy subjects as shown by isotachophoresis. For the culture studies, preparation of this material was done by chromatography on DEAE-Sepharose 6B-CL columns. Further on, diferric transferrin was preferentially used in order to abolish variation due to iron saturation. Iron concentration higher than 5 microM was deleterious to cell growth. The basal culture medium contained transferrin depleted fetal calf serum in RPMI 1640 with 2 microM glutamine and antibiotics. Serum-free medium was used in some experiments. The additions were 1.7 microM insulin, 1.4 microM hydrocortisone, 10 microM ethanolamine and 10 microM phosphoethanolamine. A partially purified fraction of the acidic forms of transferrin (10-20 micrograms/ml medium) improved outgrowth when compared with a neutral fraction under these circumstances.

Animals↗
PubMed1989

Isotachophoresis of proteins.

The analytical separation of proteins by isotachophoresis (ITP) was achieved in a short electrophoretic path and with a resolution comparable to that of isoelectric focusing by the appropriate selection of (1) a mixture of ampholytes as spacers to generate linear gradients of electrophoretic mobility and (2) the counter ions chosen to buffer the complete pH gradient generated. This ITP technique is exemplified by the analysis of plasma proteins in agarose gels. Upto 46 samples in the same gel plate were analysed. The resolution was such that at least 30 clear and discrete bands per sample could be observed after staining with Coomassie Brilliant Blue. The resolving power of ITP could be further increased for the study of a particular protein or zone by the selection of suitable spacers and counter ions.

Blood Proteins↗
PubMed1989

Complement C3 proteins in psoriasis.

A new polymorphism of the complement factor C3 in human plasma was demonstrated by isotachophoresis in agarose gels followed by immunodetection with rabbit anti-human C3c and C3d immunoglobulins. Four bands were detected in the immunoprint of freshly drawn EDTA-plasma, which were C3s1, C3s2, C3f1 and C3f2. At least four additional C3 components in Mg2+ -zymosan activated plasma were present, which were C3b1 to C3b4. The different forms of C3 in frozen and thawed heparin-plasma from 20 patients with psoriasis and 20 healthy individuals were studied from the immunoprint. The total content of C3 components was 29% greater in the patients with psoriasis than controls. The major difference was in the C3b components which were increased by 46%. In psoriatic patients, the two slow C3 components C3s1 and C3s2 were increased by 24 and 56% respectively, when compared with controls. The two fast C3 components C3f1 and C3f2 were decreased to 29 and 37%. The results suggest a direct involvement of the complement factor C3 in psoriasis.

Adult↗
PubMed1988

Predictive ability of various nutritional variables for mortality in elderly people.

Nutritional indices (percentage ideal body weight [IBW], serum albumin, serum transferrin, total lymphocyte count [TLC] and delayed cutaneous hypersensitivity [DH] response) were assessed in 80 consecutive patients (aged 85-100 y) within 24 h of admission to determine their predictive value for mortality. Nine patients died. Pearson correlation analysis demonstrated that death was significantly (p less than 0.05 to less than 0.01) associated with sepsis, serum albumin less than 30 g/L, TLC less than or equal to 1500 cells/mm3, and percentage IBW less than or equal to 90%. However, when serum albumin was controlled for, logit regression analyses demonstrated that the impact of other nutritional indices on death was insignificant. The effect of serum albumin remained significant (p less than 0.05 to less than 0.01) even when age and physician's diagnosis were held constant. With the logit model, serum albumin greater than or equal to 30 g/L had a sensitivity of 0.33, specificity of 0.99, and overall predictive power of 0.91. Serum albumin is thus the simplest and best single predictor of mortality and can provide early identification of elderly people at increased risk of death.

Aged↗
PubMed1981

The stereospecific D-glucose transport activity of cholate extracts from human erythrocyte membranes.

The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at -70 degrees C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high D-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90000, 23% of the polypeptides of molecular weight around 55000, 30% of the phospholipids and at least 6% of the stereospecific D-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4 degrees C the activity of the extract decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.

Blood Glucose↗
PubMed1980

The glucose transport activity of human erythrocyte membranes. Reconstitution in phospholipid liposomes and fractionation by molecular sieve and ion exchange chromatography.

Human erythrocyte membranes, at a protein concentration of 1-2 g/l, were solubilized with 0.12 M cholate in the presence of 0.06 M phospholipid (egg yolk phospholipids or phosphatidylcholine). More than 40% of the protein was solubilized. Cholate was removed by molecular sieve chromatography, whereby liposomes formed. These liposomes exchanged D-glucose faster than L-glucose. The recovery of glucose transport activity in the reconstituted system was estimated to be higher than 16%. The liposomes were heterogenous in size, as shown by molecular sieve chromatography on Sepharose 4B, and small liposomes predominated. In liposomes formed with phosphatidylcholine, the distribution of glucose transport activity did not parallel the distribution of protein or phospholipid, and the activity was found mainly in the smallest liposomes. The proteins were incorporated mainly in the liposomes that eluted at the lowest ionic strength upon ion exchange chromatography. The glucose transport activity separated into three main peaks upon ion exchange chromatography of egg yolk phospholipid liposomes. The activity eluted at low ionic strength. The liposomes contained proteins mainly from the 3- and 4.5-regions (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1-19). The activity peaks were highest in the first part of the chromatogram. The protein distribution did not coincide with the variation in activity over each peak. Therefore, it cannot be excluded that a minor component not seen in the electrophoretic analyses might be responsible for the glucose transport activity.

Biological Transport, Active↗
PubMed1980

A molecular sieving method for preparing erythrocyte membranes.

The most widely used methods for preparing erythrocyte membranes are time-consuming, since they involve repeated steps of centrifugation and washing. We have found that membranes can be prepared in a rapid and simple way by molecular-sieve chromatography on a Sepharose 4B column. Two sizes of columns have been used, one 9-ml column to prepare about 1 mg of membrane proteins in 10 min and one 2-litre column for larger amounts (up to 200 mg in 15 min). The optimal pH and phosphate concentration for the separation of hemoglobin from the membranes were found to be 7.5 and 5 mM, respectively. Sodium dodecyl sulphate (SDS) electrophoretic analysis of the membrane proteins prepared by centrifugation and by this new method shows similar protein compositions.

Blood Proteins↗
PubMed1977

Theoretical conversion yields for penicillin synthesis.

The efficiency of conversion of the carbon-energy source to product is of primary importance in many fermentation processes. In order to assess the efficiency of a process, one must know how close the actual conversion yield is to the theoretical maximum. Theoretical conversion yields are useful, therefore, as guides in improving a process. This knowledge is particularly important today because the cost of raw materials is rapidly rising. In this study, the biochemical pathway of penicillin synthesis was used to estimate the theoretical yield of penicillin from glucose, ammonia, and sulfate. These values are compared with experimental data from the literature. An analysis of the role of glucose in the synthesis of cell mass and penicillin and in the maintenance of cells makes it possible to assess the efficiency of carbon-source utilization and to direct further advances in penicillin fermentations.

Ammonia↗