Biomedical subjects

G F Santos

Publications and source records attributed to G F Santos.

10 recordsLinked to original sources

PubMed2001

Importance of complement source in measuring meningococcal bactericidal titers.

Complement-mediated bactericidal antibodies in serum confer protection against meningococcal disease. The minimum protective titer is estimated to be between 1:4 and 1:8 when measured by the Goldschneider assay performed with human complement, the assay used in the 1960s to establish the correlation between bactericidal antibodies and protection. A more recently described bactericidal assay standardized by an international consortium uses rabbit complement, which is known to augment bactericidal titers. To define a protective titer measured by the standardized assay, we compared bactericidal titers against serogroup C strains measured by this assay to titers measured by the assay described by Goldschneider et al. A titer of > or =1:128 measured by the standardized assay was needed to predict with > or =80% certainty a positive titer of > or =1:4 as measured by the Goldschneider assay. However, the majority of samples with titers of 1:4 measured by the Goldschneider assay had titers of <1:128 when measured by the standardized assay. Therefore, by the results of the standardized assay such persons would be falsely categorized as being susceptible to disease. In conclusion, high bactericidal titers measured with the standardized assay performed with rabbit complement are predictive of protection, but no threshold titer is both sensitive and specific for predicting a positive titer measured by the Goldschneider assay using human complement. Up to 10% of the U.S. adult population lacks intrinsic bactericidal activity against serogroup C strains in serum and can serve as complement donors. Therefore, use of the Goldschneider assay or an equivalent assay performed with human complement is preferred over assays that use rabbit complement.

Adult↗

PubMed1998

Interaction of bradykinin and angiotensin-(1-7) in the central modulation of the baroreflex control of the heart rate.

OBJECTIVE: Previous studies have shown that angiotensin-(1-7) potentiates the vascular actions of bradykinin. In the present study, we evaluated the interaction of bradykinin and angiotensin-(1-7) in the central modulation of baroreflex control of the heart rate. MATERIALS AND METHODS: Blood pressure and reflex bradycardia, elicited by intravenous injection of phenylephrine, were evaluated in conscious male Wistar rats before and at the end of 1 h of an intracerebroventricular infusion of angiotensin-(1-7) at 0.5 or 1.0 microg/h combined with bradykinin at 2.5 microg/h; or angiotensin-(1-7) at 2.0 microg/h combined with bradykinin at 4.0 microg/h; or angiotensin-(1-7) alone at 2.0 or 4.0 microg/h; or bradykinin alone at 4.0 or 8.0 microg/h; or saline at 8 microl/h. In addition, baroreflex bradycardia was evaluated before and at the end of 1 and 2 h of intracerebroventricular infusion of angiotensin-(1-7) at 4 microg/h for 2 h; or saline at 8 microl/h in the first hour followed by HOE 140 at 90 ng/h in the second hour; or angiotensin-(1-7) at 4 microg/h in the first hour followed by angiotensin-(1-7) at 4 microg combined with HOE 140 at 90 ng/h in the second hour; or HOE 140 at 90 ng/h in the first hour followed by HOE 140 at 90th ng/h combined with angiotensin-(1-7) at 4 microg/h in the second hour; or saline at 8 microl/h for 2 h. RESULTS: The intracerebroventricular infusion of angiotensin-(1-7) or bradykinin alone required a dose of 4.0 and 8.0 microg/h, respectively, to facilitate baroreflex control of the heart. However, a simultaneous infusion of these peptides at subeffective rates was able to produce a significant increase in baroreflex sensitivity. In addition, the facilitation of the baroreflex control of the heart rate induced by angiotensin-(1-7) at 4.0 microg/h was inhibited by HOE 140. CONCLUSIONS: These results suggest that centrally, bradykinin and angiotensin-(1-7) can interact in order to modulate baroreflex control of the heart rate. In addition, our data indicate that the central modulatory effect of angiotensin-(1-7) on the baroreflex is mediated, at least in part, by the release of kinins.

Adrenergic beta-Antagonists↗

PubMed1998

A modified enzyme-linked immunosorbent assay for measurement of antibody responses to meningococcal C polysaccharide that correlate with bactericidal responses.

The standardized enzyme-linked immunosorbent assay (ELISA) for measurement of serum immunoglobulin G (IgG) antibody responses to meningococcal C polysaccharide has been modified to employ assay conditions that ensure specificity and favor detection primarily of high-avidity antibodies. The modified and standard assays were used to measure IgG antibody concentrations in sera of toddlers vaccinated with meningococcal polysaccharide vaccine or a meningococcal C conjugate vaccine. The results were compared to the respective complement-mediated bactericidal antibody titers. In sera obtained after one or two doses of vaccine, the correlation coefficients, r, for the results of the standard assay and bactericidal antibody titers were 0.45 and 0.29, compared to 0.85 and 0.87, respectively, for the modified assay. With the standard assay, there were no significant differences between the geometric mean antibody responses of the two vaccine groups. In contrast, with the modified assay, 5- to 20-fold higher postvaccination antibody concentrations were measured in the conjugate than in the polysaccharide group. Importantly, the results of the modified assay, but not the standard ELISA, paralleled the respective geometric mean bactericidal antibody titers. Thus, by employing conditions that favor detection of higher-avidity IgG antibody, the modified ELISA provides results that correlate closely with measurements of antibody functional activity that are thought to be important in protection against meningococcal disease.

Antibodies, Bacterial↗

PubMed1996

Changes in the baroreflex control of heart rate produced by central infusion of selective angiotensin antagonists in hypertensive rats.

We have recently shown that an angiotensin-(1-7) [Ang-(I-7)] analogue, D-Ala7-Ang-(1-7) (A-779), is a selective Ang-(1-7) antagonist with no significant action on angiotensin type 1 or type 2 receptors. The availability of selective angiotensin antagonists prompted us to evaluate the role of Ang-(1-7) and Ang II on central modulation of the baroreflex control of heart rate in normotensive Wistar rats and spontaneously hypertensive rats (SHR). Blood pressure recording and reflex changes in heart rate elicited by intravenous bolus injections of phenylephrine were made before and within 1 and 3 hours of intracerebroventricular (ICV, lateral ventricle) infusion of saline (8 microL/h), A-779 (4 microg/h), DuP 753 (100 microg/h), or CGP 42112A (50 mu g/h) in conscious rats. The slope of the relationship between changes in pulse interval versus changes in mean arterial pressure was used as an index of the baroreflex control of heart rate. ICV infusion of saline or any of the antagonists did not significantly change basal levels of mean arterial pressure and heart rate in SHR (170 +/- 6 mm Hg nd 360 +/- 9 beats per minute, respectively; n = 29) or Wistar rats (108 +/- 2 mm Hg and 377 +/- 6 beats per minute, respectively; n=29). Three hours of ICV infusion of A-779 markedly decreased baroreflex sensitivity in Wistar rats (from a basal slope of 1.09 +/- O.3). In contrast, A-779 did not significantly alter the depressed baroreflex sensitivity of SHR (0.61 +/- O.l). ICV infusion of DuP 753 produced a significant increase (60 percent) in baroreflex control of heart rate in both Wistar rats and SHR. Saline or CGP 42112A infusions did not significantly alter baroreflex control of heart rate. These results suggest that endogenous Ang II and Ang-(1-7) are differentially affecting central baroreflex modulation, acting probably through distinct receptor subtypes. Although the central Ang II inhibitory effect is mediated by the type 1 receptor subtype, the facilitatory effect of Ang-(1-7) might be mediated by a different, unidentified receptor.

Angiotensin II↗

PubMed1995

Dystrophin-related protein in the platelet membrane skeleton. Integrin-induced change in detergent-insolubility and cleavage by calpain in aggregating platelets.

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 x g) but the membrane skeleton requires 100,000 x g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin alpha IIb beta 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following alpha IIb beta 3-ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate an approximately 140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.

Amino Acid Sequence↗

PubMed1995

cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein.

Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Activation of the cystic fibrosis transmembrane regulator by cyclic AMP is not correlated with inhibition of endocytosis.

Based on the observation (Bradbury et al. (1992) Am. J. Physiol. 262, C752-C759) that conditions known to activate the cystic fibrosis transmembrane regulator protein (CFTR) increase the rate of exocytosis and decrease the rate of endocytosis, it was proposed that activation of the CFTR may involved cAMP-dependent fusion of CFTR containing endosomes with the apical membrane. We have tested this hypothesis in two cell lines derived from epithelia that express defective chloride transport in cystic fibrosis (CF): the human colonic cell line, T84, and the tracheal cell line 9HTEo-. The dose-dependence of forskolin- and CPT-cAMP-induced inhibition of endocytosis were compared with the dose-dependence of chloride channel activation. Endocytosis was determined from the uptake of FITC-dextran, and assayed in purified endosomes. Chloride channel activity was measured from the rate of I-efflux. If the fusion hypothesis is correct: (1) concentrations of agonist that inhibit endocytosis should activate chloride channel activity, and (2) the relationship between endocytosis and channel activation should be similar for forskolin and CPT-cAMP. Results in both cell lines were inconsistent with these postulates, suggesting that either chloride channel activation and the inhibition of endocytosis are separate effects of cAMP, or that the increase in apical CFTR resulting from agonist-dependent inhibition of endosomal fusion is minimal.

Cell Line↗

PubMed1989

Expression of c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes in normal and malignant human breast epithelial cells.

Short-term cultures of normal human mammary epithelial cells were used to determine the extent to which c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon short-term tissue culture. In every sample, proto-oncogene transcript levels increased upon short-term culture of the epithelial cells. These levels often exceeded by 10-fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the proto-oncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for proto-oncogene expression. The effusion metastasis sample expressed high levels of c-erbB-2 messenger RNA, in accord with its amplified gene copy number; otherwise, the levels of proto-oncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these proto-oncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated proto-oncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer.

Blotting, Northern↗

PubMed1988

Estrogen-induced post-transcriptional modulation of c-myc proto-oncogene expression in human breast cancer cells.

The effects of estradiol (E2) on c-myc proto-oncogene expression were studied in the estrogen receptor (ER)-positive human breast cancer cell line, MCF-7. A biphasic modulation in c-myc mRNA levels occurs during the first 24 h of E2 (1 nM) exposure and in the absence of changes in MCF-7 culture growth, with transcript levels rising 4-6-fold within 1 h, returning to near base-line level after 3-6 h, and increasing again after 24 h of exposure. In contrast, the growth-inhibiting antiestrogen, tamoxifen (1 microM), reduces c-myc to 20% of the pretreatment level within 3-6 h of exposure, and this early decline is followed by a gradual return toward base-line level after continuous 72-h treatment. In ER-negative cells there is no change in c-myc expression following E2 exposure. MCF-7 nuclear runon assays show that c-myc transcription rates remain unchanged from base line for 24 h after E2 administration; as well, cycloheximide inhibition of protein synthesis superinduces c-myc expression and prevents E2 modulation of transcript levels. These results indicate that post-transcriptional modulation of c-myc by E2 is mediated by a labile degradative protein or otherwise dependent on active protein synthesis. We also developed MCF/nm and MCF/dm sublines by transfecting normal cells with human c-myc exons 2-3, transcriptionally driven by a retroviral long terminal repeat. Expression of the transfected c-myc genes in these sublines remains constant and elevated 10-fold, while transcript levels from the endogenous proto-oncogene continue to be modulated by E2. These findings suggest that in ER-positive breast cancer cells, E2 can modulate c-myc mRNA levels by a post-transcriptional mechanism that depends on gene sequences upstream from c-myc exon 2.

Breast Neoplasms↗

PubMed1987

Effects of cis- and trans-tamoxifen isomers on RNA incorporation of human breast cancer cells.

Cultured T47-D human breast cancer cells were used to investigate growth-inhibiting effects of the antiestrogen, trans-tamoxifen, on [3H]Cyd incorporation into specific classes of nuclear and cytoplasmic RNA. The steroid agonist, 17 beta-estradiol, and the inactive cis isomer of tamoxifen were used as treatment controls to compare antiestrogen-induced changes in RNA metabolism, independent of estrogen receptor-binding properties. Using a 24-hr labeling interval, trans-tamoxifen produced a 1.4- to 4-fold enhanced incorporation into pre-rRNA species (20S, 32-45S), with slight reduction in mature 18S rRNA incorporation, and a 2- to 3-fold increased incorporation into 5S and 5.8S rRNA and 4-4.5S tRNA. Most notable, trans-tamoxifen enhanced incorporation into the less abundant low molecular weight U1, U3, and 7S RNA species by 3- to 6-fold. These findings were associated with an apparent reduction in pre-rRNA content and little change in U1, U3, or 7S RNA levels in antiestrogen-treated cells, suggesting that trans-tamoxifen independently regulates RNA transcription and turnover. The present study provides new rationale for the choice of molecular probes to study trans-tamoxifen effects on synthesis and turnover of specific nuclear and cytoplasmic RNA species.

Breast Neoplasms↗