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Biomedical subjects

A Pascual

Publications and source records attributed to A Pascual.

At least 163 records · Page 9Linked to original sources
PubMed1990

Surface proteins of coagulase-negative staphylococci: their role in adherence to biomaterials and in opsonization.

During the last decade coagulase-negative staphylococci (CN-Staph) have clearly emerged as pathogens in patients equipped with foreign devices. This has fueled the interest in these bacteria considerably and as a result, knowledge of the biology of CN-Staph as well as insight into the pathogenesis of biomaterial-associated infections due to these bacteria are rapidly expanding. Adherence of bacteria to biomaterials is a necessary step in the process of these infections. Evidence is accumulating that surface proteins of CN-Staph are essential in the early phases of adherence to biomaterials. By using monoclonal antibodies in immunoblotting and immune electron micrography we have identified a cell wall protein complex apparently located on the surface of CN-Staph and involved in adherence to biomaterials. Further studies will be oriented at characterization of the protein adhesin(s) and at identification of the surface structures with which the adhesion is associated.

Bacterial Adhesion↗
PubMed1990

[Spleen abscess].

3 cases of splenic abscess (SA), all of them with similar clinical findings; abdominal pain, liver and splenic enlargement and leukocytosis, are presented. The diagnosis was suspected by abdominal ultrasound and confirmed by CTR. We focused the interest on the etiopathogenesis, diagnosis and the rareness of the bacteria isolated: Salmonella group D (two cases) and Bacteroides Fragilis.

Abscess↗
PubMed1989

Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: evidence that changes in receptor levels are not associated with changes in receptor distribution.

The association of [125I]T3-receptor complexes with C6 cell chromatin was analyzed after a limited digestion with micrococcal nuclease (MN) or DNase I. Both nucleases solubilized up to 60-70% of receptor and 0.4 M KCl extracted 70% of the non-digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix. With DNase I the receptor was released 2-3-fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN. The digestion of receptor with DNase I and MN occurred 14- and 6-fold faster, respectively, than the appearance of PCA-soluble chromatin. Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases. These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin.

Cell Line↗
PubMed1989

Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate.

The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells.

Animals↗
PubMed1989

Sodium butyrate induces major morphological changes in C6 glioma cells that are correlated with increased synthesis of a spectrin-like protein.

Butyrate induced flattening and development of cell processes in rat glioma (C6) cells and this change was correlated with an increase in the synthesis of a polypeptide doublet with an apparent molecular weight of about 200 kDa. Blot analysis revealed that at least one of these polypeptides was a spectrin-like protein. Indirect immunofluorescence studies with the spectrin antiserum indicated that the antigen was present in the cell bodies, and also in the cell processes. Thus fodrin may be one the major targets for the action of butyrate on C6 cells.

Autoradiography↗
PubMed1989

Effect of amoxycillin and clavulanic acid, alone and in combination, on human polymorphonuclear leukocyte function against Staphylococcus aureus.

The effect of amoxycillin and clavulanic acid on the interaction in vitro of human polymorphonuclear leukocytes with Staphylococcus aureus was examined. The exposure of a non-penicillinase producing Staphylococcus aureus strain to one-fourth the MIC of amoxycillin or clavulanic acid alone significantly increased the uptake of both unopsonized and opsonized bacteria by human polymorphonuclear leukocytes. This effect was also observed when bacteria were exposed to one-fourth the MIC of different proportions of the combination of amoxycillin and clavulanic acid (4/1, 1/1, 1/8 and 1/32). When a penicillinase-producing Staphylococcus aureus strain was used, only clavulanic acid significantly increased the uptake of unopsonized bacteria. The production of superoxide radicals by human polymorphonuclear leukocytes was impaired only by the presence of high concentrations (100 mg/l) of both clavulanic acid or the combination amoxycillin/clavulanic acid (4/1). At this high concentration, however, amoxycillin/clavulanic acid (4/1) significantly increased the intracellular killing of Staphylococcus aureus.

Amoxicillin↗
PubMed1989

Effects of cyclic AMP elevation on the levels of insulin receptors in glial C6 cells.

The regulation of the insulin receptor by cAMP has been examined in glial C6 cells. Incubation for 48 h with dibutyryl cyclic AMP produced a dose-dependent inhibition of 125I-insulin binding to the cells. Other agents that elevate intracellular cAMP levels such as forskolin and cholera toxin mimicked the effect of this cyclic AMP analog. With all compounds the maximal decrease of binding was found between 24 and 48 h and normally varied between 40 and 60%. Forskolin, cholera toxin and dibutyryl cyclic AMP also affected C6 cell proliferation, and the dose-response for decreasing the receptor was very similar to that observed for the inhibition of cell growth, suggesting a relationship between both phenomena. Scatchard analysis showed that cAMP did not produce major changes in the affinity of the receptor for insulin, but rather decreased receptor number. An accumulation of receptors at the cell surface was observed in the absence of de novo protein synthesis, since cycloheximide caused a significant increase in insulin binding to the cells. This inhibitor almost totally blocked the cAMP effect both when added simultaneously to the cells with the agents which increase cAMP and when added to cells pre-treated for 48 h with the same compounds.

Animals↗
PubMed1989

Fluorometric measurement of ofloxacin uptake by human polymorphonuclear leukocytes.

A fluorometric assay, based on the natural fluorescence of the quinolone nucleus, was used to determine the uptake of ofloxacin by human polymorphonuclear leukocytes. The ratio of cellular concentration to extracellular concentration (C/E) at 20 min and 37 degrees C was 7.2, using an extracellular concentration of 5 micrograms/ml. Uptake was rapid and was not affected by pH (5 to 9), but required elevated environmental temperature and cell viability. The metabolic inhibitors sodium fluoride and sodium cyanide significantly decreased the uptake of ofloxacin. The penetration of ofloxacin was not affected by the presence of glucose or adenosine, but was decreased by L-amino acids (lysine, leucine, and glycine). These results suggest that ofloxacin could be transported via an amino acid transport system and that the fluorometric assay is a useful method for determining the intracellular penetration of fluoroquinolones, avoiding the use of radiolabeled antimicrobial agents.

Adenosine↗
PubMed1989

Effect of ciprofloxacin and ofloxacin on human polymorphonuclear leukocyte activity against staphylococci.

The interaction of ciprofloxacin and ofloxacin with the bactericidal capability of human polymorphonuclear leukocytes against staphylococci was investigated. Exposure of Staphylococcus aureus and Staphylococcus epidermidis to subinhibitory concentrations of ciprofloxacin and ofloxacin did not significantly affect the uptake and killing by human polymorphonuclear leukocytes. In our assay, ciprofloxacin and ofloxacin in concentrations of one time the minimal inhibitory concentration induced a significant reduction in viable intraphagocytic S. aureus (percentages of survival: 43 and 51%, respectively) compared to the controls without antibiotic (percentage of survival: 65%). In contrast, both antimicrobials failed to produce a reduction in viable intraphagocytic S. epidermidis.

Ciprofloxacin↗
PubMed1989

[Adhesion capacity and surface properties of Staphylococcus epidermidis and Staphylococcus saprophyticus].

We have comparatively studied the adherence of Staphylococcus epidermidis and S. saprophyticus to epithelial cells of a continuous cellular line of urinary origin (MDCK). The adherence of S. saprophyticus was significantly higher than that of S. epidermidis and this phenomenon was not related to the bacterial surface hydrophobicity or the ability of producing slime in vitro. The pre-incubation of the bacteria with sub-inhibitory concentrations of vancomycin, teicoplanin, amikacin, clindamycin, and roxitromycin did not modify the adherence ability of the microorganisms. Of the antimicrobial agents evaluated, only clindamycin significantly agents evaluated, only clindamycin, significantly inhibited the ability of producing slime of S. saprophyticus strains.

Animals↗
PubMed1989

[Evaluation of 2 methods for studying slime production by coagulase-negative Staphylococcus strains].

The surface hydrophobicity and the slime production of 46 coagulase negative staphylococci isolated from the urine, blood and skin were evaluated. The hydrophobicity values were very heterogeneous and did not correlate with the species, origin or slime production. Slime production was evaluated by the classical tube method and by a micromethod with spectrophotometric readings. There was a high correlation between both methods, but the results were more reproducible and quantifiable with the latter. Twenty-six of the evaluated species produced some degree of slime. This feature did not correlate with their origin or species. The preincubation of 5 strains of Staphylococcus epidermidis with subinhibitory concentrations of vancomycin, clindamycin and amikacin did not influence slime production, whereas ciprofloxacin slightly inhibited it in two strains and oxacillin completely inhibited slime production in the five evaluated strains.

Anti-Bacterial Agents↗
PubMed1989

[Evaluation of 2 commercial monoclonal antibody kits for the identification of herpes simplex virus].

Two commercial monoclonal antibody kits (Virgo and Mikrotrak for the identification of Herpes simplex virus (HSV) have been assessed. In 65 positive HSV cultures from clinical samples, 25 strains (38%) were identified as HSV1 with both kits. The Virgo kit identified 37 strains (57%) as HSV2, and did not identify the remaining 3 (5%). With the Microtrak kit, 34 strains (52%) corresponded to type 2, 2 (3%) strains remained nonidentified, and in the remaining 4 isolates (6%) both viral types were identified. The concordance between the two evaluated kits was complete in 86% of strains. In the five strains not identified by immunofluorescence, genome analysis was carried out with endonucleases: Hind III and Xho I. Two of these isolates were identified as HVS1, another as HSV2, and in the remaining two both viral types were identified. Overall 6 mixed infections (9%) were detected, 4 with the Microtrak kit and endonucleases and 2 with only the latter method. The Microtrak system shows some advantages such as quickness, easy readings and the possibility to diagnose infections where both types of virus participate.

Antibodies, Monoclonal↗
PubMed1989

[Genital herpes of mixed etiology].

The relevance of mixed genital infections in our area was evaluated, and we report their clinical and epidemiological features for both viral types. We evaluated overall 54 Herpes simplex virus (HSV) isolates from 44 patients seen at the clinic of sexually transmitted diseases (STD) of the Sevilla School of Medicine during 1986 and 1987. The identification of the isolated strains was made by direct immunofluorescence with type-specific monoclonal antibodies. In those isolates where we detected both viral types confirmation was obtained with the endonuclease analysis: Hind III and Xho I. Four mixed infections were diagnosed, representing 7% of all HVS isolates of genital origin. Two were classified as initial nonprimary infections, and the remaining two were asymptomatic infections. Two patients showed simultaneous infections by other organisms.

Adult↗
PubMed1988

Proliferation and differentiation are not directly related to H1(0) accumulation in cultured glial cells.

A basic nuclear chromatin protein with electrophoretic mobility of H1(0) histone is present in C6 rat glial cells and in primary cultures of rat brain astroglial cells. That this protein is identical to H1(0) is further demonstrated by the finding that it accumulates in C6 cells in a time- and dose-dependent manner in response to butyrate, an agent which is known to induce this protein in other cell types. Other short-chain fatty acids were found to influence H1(0) levels similarly although to a lesser extent than butyrate. There was a very close correlation between the induction of H1(0) and the inhibition of growth induced by different concentrations of short-chain fatty acids which supports the idea that the concentration of this protein is higher in non-proliferating cells. However, when cell growth was inhibited by dexamethasone or agents that increase intracellular cyclic adenosine monophosphate levels, H1(0) levels were not affected, even though these compounds also blocked DNA synthesis and induced morphologic changes in C6 cells. These observations suggest that, at least in glial cells, the accumulation of H1(0) is specifically caused by short-chain fatty acids and that suppression of cell division or commitment to differentiation are not sufficient 'per se' for the induction of this protein.

Animals↗
PubMed1988

Rat growth hormone gene expression. Both cell-specific and thyroid hormone response elements are required for thyroid hormone regulation.

The elements involved in mediating cell-specific and thyroid hormone stimulation of rat growth hormone gene expression have been defined by transfection studies and by nuclease footprinting. 5'-Flanking DNA extending to -104 can mediate cell-specific expression, and this is enhanced 3- to 4-fold with DNA extending to -145. Cell-specific factors, found only in rat growth hormone producing cells, bind within the -137/-107 and -95/-65 regions, and competition studies suggest that the same factor binds to both sites. The sequence A (A or T) TAAAT is found at the center of both footprints at -80 and -122, suggesting that it is a core component of the recognition sequence of the cell-specific factor. Disruption of the spatial and/or distance relationships between the two regions eliminates the enhanced level of cell-specific expression, suggesting a cooperative interaction of the proteins which bind to these elements. Sequences located between -208 and -178 can confer thyroid hormone-regulated expression when linked in either orientation in close proximity to one or both cell-specific elements. The thyroid hormone and cell-specific elements function as an enhancer-like unit and are both required to confer regulated expression to heterologous promoters. We propose that thyroid hormone acts via its receptor to enhance the function of the cell-specific element by forming a more "active" transcription complex which stimulates the level of gene expression.

Animals↗
PubMed1988

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations.

The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.

Affinity Labels↗