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Biomedical subjects

R Diaz

Publications and source records attributed to R Diaz.

At least 109 records · Page 6Linked to original sources
PubMed1988

In vitro fusion of endosomes following receptor-mediated endocytosis.

Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.

Adenosine Triphosphate↗
PubMed1988

Olive (Olea europea) pollen allergens--I. Immunochemical characterization by immunoblotting, CRIE and immunodetection by a monoclonal antibody.

The pattern of reactivity of the Olea europea crude extract antigens was analysed after electroblotting to nitrocellulose from SDS-PAGE. The antigens contained in the 17, 19 and 42 K bands were most reactive with specific IgE from individual sera. Following immunization with a crude extract, one monoclonal antibody (OL-1) was raised against components which exhibited IgE binding capacity in electroblotting and crossed radioimmunoelectrophoresis (CRIE). Monoclonal antibody OL-1 reacted with the 17 and 19 K antigens and with three arcs of crossed immunoelectrophoresis (CIE), one of which is considered to contain a major allergen by CRIE.

Allergens↗
PubMed1988

Olive (Olea europea) pollen allergens--II. Isolation and characterization of two major antigens.

A dialyzed extract of olive (Olea europea) pollen was fractionated by anion exchange chromatography on DEAE-Sepharose CL-6B using a discontinuous gradient of ammonium bicarbonate. The most important protein allergen was obtained from the 0.3 M fraction after gel filtration on Sephadex G-100 and separation by lentil-lectin Sepharose-4B. The major allergen of olive pollen was contained in the effluent and was designated Olea Antigen I. This material inhibited the RAST activity of 15 patients' sera that were tested. Analytical IEF demonstrated a major band at pH 5.3 and two minor ones at pH 5.6 and 5.0. When these were run into SDS-polyacrylamide gel electrophoresis in a second dimension, all were separated into two bands of mol. wt 17 and 19 K. A second protein, which is the next most important allergen, Olea Antigen II, was obtained from the 0.5 M fraction by chromatofocusing in a 4-7 pH range followed by filtration on Bio-gel P-30. Olea Antigen II had a mol. wt of 8 K as assessed by SDS-PAGE. IEF analysis displayed one main band at pH 3.6 and two minor bands at pH 3.8 and 4.0, respectively. OL-1, an anti-Olea europea monoclonal antibody (MAb) previously reported by us Lauzurica et al. (1988) reacted with the 17 and 19 K antigens from the crude extract and with Olea Antigen I but not with Olea Antigen II.

Allergens↗
PubMed1987

Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coli.

pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably maintained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30 degrees C. The optimal concentration of template DNA was 40 micrograms/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its derivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.

Cell-Free System↗
PubMed1986

The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA.

The in vitro replication of R1 miniplasmid promoted by purified preparations of the plasmid encoded RepA protein in cell extracts of E. coli is resistant to rifampicin and can be completely inhibited by antibodies against DnaG, the primase of the cell, as well as by antibodies against proteins DnaB and SSB. R1 replication is abolished in extracts deficient in the DnaA protein. This deficiency is efficiently complemented by purified preparations of the DnaA protein. The in vitro replication of plasmid R1 is also abolished in DnaC deficient extracts and by a 10 bp deletion (nucleotides 1463-1472) within the minimal origin region. These data indicate the requirement of the DnaA, DnaB, DnaC, DnaG and SSB replication proteins of the host, as well as of specific oriR1 sequences for the RepA dependent replication of plasmid R1. The implications of these results for the initiation of R1 replication are discussed.

Bacterial Proteins↗
PubMed1986

Cytosine-specific DNA modification interferes with plasmid establishment in Escherichia coli K12: involvement of rglB.

Several chimeric pBR322/328 derivatives containing genes for cytosine-specific DNA methyltransferases (Mtases) can be transformed into the Escherichia coli K12/E. coli B hybrid strains HB101 and RR1 but not into other commonly used E. coli K12 strains. In vitro methylation of cytosine residues in pBR328 and other unrelated plasmids also reduces their potential to transform such methylation sensitive strains, albeit to a lesser degree than observed with plasmids containing Mtase genes. The extent of reduced transformability depends on the target specificity of the enzyme used for in vitro modification. The role of a host function in the discrimination against methylated plasmids was verified by the isolation of K12 mutants which tolerate cytosine methylated DNA. The mutations map in the vicinity of the serB locus. This and other data indicate that the host rglB function is involved in the discrimination against modified DNA.

Cytosine↗
PubMed1986

Maternal social support as a predictor of mother-child stress and stimulation.

There is increasing interest in the role of social support in determining risk for child abuse and neglect. The present study assessed the relationship between maternal social support and two areas: stress in the mother-child relationship: and level of stimulation provided in the home. Maternal social support was assessed prenatally and at a two-year follow-up, while the latter variables were compiled at the two-year follow-up. The data were obtained from 38 urban, low SES (80% on public assistance) mothers and their 2-year-old children. Maternal social support correlated positively with level of stimulation and negatively with level of mother-child stress, and was the best predictor of both, relative to any SES, mother or child variables. In addition, high stress, low support mothers provided significantly less stimulation to their children. The theoretical implications for social support as a mediator variable as well as its implications for early identification and prevention efforts in abuse and neglect are discussed.

Adolescent↗
PubMed1986

Comparison of lipopolysaccharide and outer membrane protein-lipopolysaccharide extracts in an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection.

Brucella ovis hot saline extracts and petroleum ether-chloroform-phenol lipopolysaccharide were compared in an enzyme-linked immunosorbent assay for the diagnosis of B. ovis ram epididymitis. Hot saline extracts detected greater numbers of infected rams. Chemical characterization of the antigens showed that, although both contained lipopolysaccharide, hot saline extracts also contained outer membrane proteins. These proteins were active as antigens in Western blot tests with sera of infected rams, and therefore they explained the better diagnostic results obtained with hot saline extracts. However, compared with lipopolysaccharide, hot saline extracts showed a higher degree of cross-reactivity with sera from smooth B. melitensis-infected animals. This observation might be explained by the presence of B. ovis outer membrane proteins in hot saline extracts which lack the specificity necessary for serological identification of the Brucella species present.

Animals↗
PubMed1986

Demonstration of antibodies against Brucella melitensis 16M lipopolysaccharide and native hapten in human sera by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of human immunoglobulin G (IgG), IgA, and IgM antibodies against Brucella melitensis 16M by using lipopolysaccharide (LPS) and native hapten (NH) as antigens is described. The results obtained with the LPS ELISA were compared with the results of the NH ELISA. A good statistically significant correlation was established between the antibody titers of the IgG class against both antigens. A total of 104 (99%) of the 105 serum samples of patients with brucellosis exhibited specific anti-NH antibodies by the ELISA technique. In 52 (50%) of these positive samples, antibodies against NH were detected by radial immunodiffusion (RID). In 100% of these RID-positive sera, the antibody titers of the IgG class with ELISA-determined anti-NH specificity were equal to or greater than 160. These results point to a higher sensitivity of the ELISA technique as compared with RID. Inhibition experiments revealed that the assay was specific for LPS and NH from B. melitensis 16M.

Antibodies, Bacterial↗
PubMed1985

Definite vs adjuvant radiotherapy. Comparative effects on lymphocyte subpopulations in patients with head and neck squamous carcinoma.

The recent association of alterations in T-lymphocyte subpopulations and impaired cellular immunity prompted an investigation of the effects of radiotherapy (RT) on serial levels of lymphocyte subsets in 30 patients with head and neck squamous carcinoma. Percentage and absolute levels of T3, T4, T6, T8, T10, T11, and Leu 7 cells were measured before, during, and after RT at monthly intervals for six months and compared with levels in 40 normal subjects. Sixteen patients received curative and 14 postoperative adjuvant RT. Before treatment, mean subset levels were similar among the patient and normal groups except for elevated Leu 7 (natural killer) cells in patients with stage I and II disease. There were profound decreases in absolute levels of each subpopulation during and after RT. The percentage of T4 (helper/inducer) cells decreased, whereas that of T8 (cytotoxic/suppressor) and Leu 7 cells tended to increase. Compared with normal values, the mean T4/T8 ratio decreased significantly by six months after RT, when absolute levels of the subsets had rebounded to pretreatment levels in the definitive RT group but remained profoundly decreased in the adjuvant group. The differing recovery patterns suggest that factors other than RT may contribute to persistent immunosuppression following RT.

Carcinoma, Squamous Cell↗
PubMed1985

Entero-pancreatic reflexes revealed by duodenal anesthesia in the dog.

This study was designed to improve our understanding of duodeno-pancreatic reflexes, the existence of which was suggested by the previous observation of a reduction in secretin-stimulated pancreatic secretion during local anesthesia of the duodenal mucosa. The effects on this reduction in secretin-stimulated secretion of cholinergic or adrenergic blocking agents (alone or in combination) and of truncal vagotomy, were studied in conscious dog with gastric and pancreatic fistulae. For each agent and for secretin alone in normal and vagotomized dogs, a comparison was made of pancreatic secretion with and without lignocaine anesthesia of the duodenal mucosa. Lignocaine reduced pancreatic secretion with secretin alone, and stimulated it during infusion of atropine. The changes in both protein bicarbonate secretion were blocked by pentolinium and by phenoxybenzamine whereas propranolol mainly blocked the effects on bicarbonate output. The effect of truncal vagotomy resembled that of atropine. These results suggest the existence of two enteropancreatic reflex mechanisms; an excitory cholinergic vagal reflex and an inhibitory, atropine-resistant non-vagal reflex. Both are blocked by pentolinium (a ganglion blocker) and by phenoxy-benzamine, suggesting the involvement of alpha-adrenergic receptors probably also at the level of the ganglion cell. Beta-adrenergic receptors are also involved in the regulation of bicarbonate and fluid secretion.

Anesthesia↗
PubMed1984

Initiation of plasmid R1 replication in vitro is independent of transcription by host RNA polymerase.

A Sau3A fragment containing most of the repA gene of plasmid R1 has been cloned in the BamH1 site of the expression vector pAS1. One of the recombinants, pSO1, codes for a fused RepA' protein which is efficiently synthesized both in vivo and in vitro from transcriptional and translational signals of the vector. It is shown that following pSO1 promoted accumulation of RepA' in cell-free extracts of E. coli, in vitro replication of the R1 miniplasmid pKN182 can initiate and proceed uncoupled from further protein synthesis. Using this uncoupled system and also a M13mp9 based ori-R1 recombinant, pRD95, it is also shown that RepA' acts at the origin region of R1 to promote initiation of replication that is independent on transcription by host RNA polymerase. This is indicated by the insensitivity of pRD95 and pKN182 replication to rifampicin as well as to RNA polymerase antibodies. The properties of the uncoupled in vitro replication system are further described.

Base Sequence↗
PubMed1984

Studies on the polysaccharide B and native haptene of Brucella and Yersinia enterocolitica serotype 9.

Trichloroacetic (TCA) extraction of B. melitensis 115, or hot saline extraction of B. melitensis 16M yields a polysaccharide component that can be purified free of protein and lipopolysaccharide by gel filtration and TCA precipitation. Antibodies to this polysaccharide can be detected in sera of rabbits infected with virulent Brucella but not with B. abortus 19. Similarly, cattle with active brucellosis and humans with acute brucellosis develop antibodies against this polysaccharide. After extraction and purification, this polysaccharide does not elicit antibody response in rabbits or mice. It does not fix the complement, does not bind to red cells or to polystyrene plates.

Animals↗
PubMed1984

Evaluation of a radial immunodiffusion test for diagnosing brucellosis in sheep and its possible value for differentiating infected from Brucella melitensis REV 1 vaccinated sheep.

The efficiency of 4 serological test: Rose Bengal (RB), complement fixation (CF), gel diffusion (GD) and radial immunodiffusion (RID) for diagnosing Brucellosis in sheep was compared. RID and CF were also used to evaluate sheep serological response following REV 1 vaccination. Crude smooth lipopolysaccharide (LPS-S) and Polysaccharide B antigens obtained from B. melitensis 16 M were used in GD and RID tests respectively. In experiment 1, two hundred and sixty five sera from adult unvaccinated sheep were studied. The animals used belonged to 4 different flocks, in which was bacteriologically proved the existence of B. melitensis infection. RB positive reaction was obtained in 122 sera being 100, 87 and 91 of these, positive to CF, GD and RID respectively. In experiment 2, one hundred and one ewes from 5 to 10 months of age (and CF negative) were subcutaneously inoculated with 2.10(9) viable Rev 1 organisms. All animals were bled at 1, 2, 5, 10, 20, 24, 28 and 52 weeks after being vaccinated, and their serological response to CF and RID tests was studied. The percentage of CF reactors between the 24th and the 52nd weeks decreased from about 30% to less than 8%. The RID reactors in weeks 24 and 52 were 1% and 0% respectively when a concentration of 15 micrograms of Polysaccharide B per ml of gel was used.

Animals↗