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Biomedical subjects

A Da Silva

Publications and source records attributed to A Da Silva.

At least 19 recordsLinked to original sources
PubMed2000

Exclusion limits on the WIMP-nucleon cross section from the cryogenic dark matter search.

The Cryogenic Dark Matter Search (CDMS) employs Ge and Si detectors to search for weakly interacting massive particles (WIMPs) via their elastic-scattering interactions with nuclei while discriminating against interactions of background particles. CDMS data, accounting for the neutron background, give limits on the spin-independent WIMP-nucleon elastic-scattering cross section that exclude unexplored parameter space above 10 GeV/c2 WIMP mass and, at >75% C.L., the entire 3sigma allowed region for the WIMP signal reported by the DAMA experiment.

Journal Article↗
PubMed1999

Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis.

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing beta(2)-adrenergic receptors (beta(2)ARs), sustained agonist treatment caused a time-dependant decrease in both beta(2)AR binding sites and immuno-detectable receptor. Blocking beta(2)AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasome-associated, were ineffective in preventing beta(2)AR down-regulation. Further evidence for an endocytosis-independent pathway of beta(2)AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous beta(2)AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block beta(2)AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for beta(2)AR down-regulation and that the inactivation of beta(2)ARs, leading to a reduction in binding sites, may occur at the plasma membrane.

Adrenergic beta-2 Receptor Agonists↗
PubMed1999

Strategies for safe injections.

In 1998, faced with growing international concern, WHO set out an approach for achieving injection safety that encompassed all elements from patients' expectations and doctors' prescribing habits to waste disposal. This article follows that lead and describes the implications of the approach for two injection technologies: sterilizable and disposable. It argues that focusing on any single technology diverts attention from the more fundamental need for health services to develop their own comprehensive strategies for safe injections. National health authorities will only be able to ensure that injections are administered safely if they take an approach that encompasses the whole system, and choose injection technologies that fit their circumstances.

Disposable Equipment↗
PubMed1998

Effect of WIN 64338, a B2 bradykinin receptor antagonist on guinea-pig tracheal smooth muscle cells in culture.

This study investigated the effect of the non-peptidic B2 bradykinin (BK) receptor antagonist WIN 64338 on BK binding and BK-induced inositol phosphate formation on guinea-pig tracheal smooth muscle cells in culture. The presence of specific and saturable binding sites for BK was demonstrated using [3H]BK. Scatchard analysis indicates a single population of binding sites for [3H]BK with a maximal density (Bmax) of 245.4 +/- 71 fmol/mg of protein and an equilibrium dissociation constant (Kd) of 87.7 +/- 0.12 pM. The order of potency of] B2 BK receptor ligands was Hoe 140 = NPC 17731 > BK > WIN 64338 > D- Arg0[Hyp3, D-Phe7]-BK > > des-Arg9-BK, while B1 BK receptor antagonist, des-Arg9-[Leu8]-BK, was without effect on [3H]BK binding. These results demonstrate the presence of B2 Bk receptors on cultured tracheal smooth muscle cells. The cells were stimulated by BK, and inositol phosphate formation was determined by anion exchange chromatography. The stimulating effect of BK on inositol phosphate formation was concentration dependent (1 nM to 10 microM). The B1 BK agonist des-Arg9-BK did not induce inositol phosphate formation. The order of potency of B2 antagonists to inhibit BK-induced inositol phosphate formation was Hoe 140 = NPC 17731 > WIN 64338 > D-Arg0[Hyp3, D-Phe7]-BK. This study demonstrates that WIN 64338 is able to displace [3H]BK binding and to inhibit B2-BK-induced inositol phosphate formation on cultured guinea-pig tracheal smooth muscle cells.

Adrenergic beta-Antagonists↗
PubMed1997

Influence of receptor density on the patterns of beta2-adrenocepter desensitization.

Sustained stimulation of the beta2-adrenoceptor leads to a desensitization of the receptor-mediated adenylyl cyclase stimulation. While desensitization promoted by nanomolar concentrations of isoproterenol involves the phosphorylation of the beta2-adrenoceptor by protein kinase A alone, both protein kinase A- and beta-adrenoceptor kinase-mediated phosphorylation leading to the binding of beta-arrestin contribute to the desensitization evoked by micromolar concentrations of agonist. In the present study, we assessed the influence of receptor density on the patterns of desensitization induced by these two different levels of stimulation. Murine L cells were transfected with a cDNA encoding the human beta2-adrenoceptor and clonal cell lines expressing various levels of beta2-adrenoceptor were used for the study. In cell lines expressing the highest number of receptor, approx. 150000 sites/cell (approx. 3000 fmol/mg of membrane proteins), pretreatment with micromolar concentrations of isoproterenol causes a desensitization pattern characterized by a reduction in both the potency and the efficacy of isoproterenol to further stimulate the adenylyl cyclase activity. In contrast, desensitization induced by 10 nM isoproterenol resulted only in a decrease in the potency of isoproterenol. This distinct pattern of desensitization is not seen in cells expressing 12000 receptors/cell (approx. 200 fmol/mg of membrane proteins) and, in that case, pretreatment with 10 nM isoproterenol leads to a reduction in both the sensitivity and the maximal response. Similar effects on the beta-adrenoceptor-stimulated adenylyl cyclase were observed in these cells following treatment with dibutyryl cAMP. Receptor density therefore dramatically influences the pattern of desensitization evoked by low level of stimulation. The results also demonstrate that although different molecular events are involved in the desensitization evoked by different levels of stimulation, its phenotypic expression can be qualitatively identical in cells expressing a relatively small number of receptors. Hence, protein kinase A-mediated desensitization cannot be qualitatively distinguished from the beta-adrenoceptor kinase-mediated process in these cells.

Adenylyl Cyclases↗
PubMed1996

New molecular and structural determinants involved in beta 2-adrenergic receptor desensitization and sequestration. Delineation using chimeric beta 3/beta 2-adrenergic receptors.

As the beta 3-adrenergic receptor (beta3AR) is resistant to short term agonist-promoted desensitization and sequestration, chimeric beta3/beta2 receptors were generated to identify the molecular determinants responsible for these regulatory processes in the beta2AR. By exchanging single or multiple intracellular domains of the beta3AR for the corresponding regions of the beta2AR, we show that specific domains can be identified as additive determinants for desensitization, while sequestration is more dependent on global structural conformation. The carboxyl-terminal tail, the third and the second intracellular loops of the beta2AR provided additive contributions to the desensitization observed upon short term agonist stimulation. The second intracellular loop plays a role which is as important as that of third cytoplasmic loop and carboxyl-terminal tail which had previously been identified as the major determinants of agonist-promoted desensitization. Additive contributions of the cytoplasmic domains of the beta2AR were also observed for agonist-promoted sequestration. The substitution of the first and second intracellular loops and the carboxyl tail were associated with a beta2-like sequestration phenotype. However, in contrast to what is observed for desensitization the co-substitution of the third cytoplasmic loop with any of the other domains completely suppressed sequestration. These results suggest that sequestration depends not only on appropriate interactions of multiple molecular determinants within the cytoplasmic region of the beta2AR but also on conformational determinants that may influence their orientation.

Adenylyl Cyclases↗
PubMed1995

Involvement of B2 receptors in the bradykinin-induced relaxation of guinea-pig isolated trachea.

1. The aim of this study was to determine the receptor type and involvement of arachidonic acid metabolites in bradykinin-induced relaxation of the guinea-pig isolated trachea. 2. In the resting tracheal preparation, bradykinin (0.1 nM-30 microM induced a concentration-related contractile response (pD2 = 8.8 +/- 0.3). The maximal tension (1056 +/- 321 mg) was observed at 0.3 microM bradykinin. In contrast, when tracheal preparations were pre-contracted with histamine (30 microM leading to a half-maximum response), a concentration-related relaxation was observed with bradykinin. At the highest concentration of bradykinin used (3 microM), a reversal of 63 +/- 13% of the contractile response to histamine was observed. Both effects of bradykinin were inhibited by the cyclo-oxygenase inhibitor, indomethacin (1 microM). In concentration-response curves, melittin (10 nM-1 microM), a direct activator of phospholipase A2, mimicked both effects of bradykinin. The highest concentration of melittin used (1 microM), induced a tension of 813 +/- 120 mg and led to the reversal of 41 +/- 8% of the contractile response to histamine. The contractile effect of melittin was inhibited in the presence of both indomethacin (1 microM) and AA861 (1 microM), a 5-lipoxygenase inhibitor. 3. [Des Arg9]-bradykinin (1 nM-3 microM), a B1-receptor agonist, was unable to relax precontracted guinea-pig tracheal preparations. The relaxation induced by bradykinin was antagonized by the B2 receptor antagonists, Hoe 140 (D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin) and NPC 17761 (D-Arg0[Hyp3,D-HypE(trans-thiophenyl)7,Oic8]bradykinin ). Hoe 140 (0.1 microM to 0.6 microM) behaved as a non-competitive antagonist with an apparent pA2 = 7.2 +/- 0.4, whereas NPC 17761 (0.3 to 1 microM) competitively antagonized bradykinin-induced relaxation with a pKB = 7.3 +/- 0.2. The Schild regression slope did not differ from unity, 0.96 +/- 0.20, P<0.05.4. These data demonstrate that bradykinin-induced relaxation of guinea-pig trachea occurs via the activation of bradykinin B2-receptors. The stimulation of B2-bradykinin receptors induces the activation of the cyclo-oxygenase pathway, leading either to contraction or relaxation depending on the tone of the trachea.

Animals↗
PubMed1994

Biphasic increase in cytosolic free calcium induced by bradykinin and histamine in cultured tracheal smooth muscle cells: is the sustained phase artifactual?

The effects of bradykinin (BK) and histamine on intracellular Ca2+ ([Ca2+]i) were studied in fura-2-loaded guinea-pig tracheal smooth muscle cells in culture. BK, at 10 nM, and histamine, at 100 microM, induced a rise in [Ca2+]i which was inhibited by the B2 antagonist Hoe 140 and by the H1 antagonist triprolidine, respectively. This rise in [Ca2+]i is biphasic, consisting of a rapid transient phase followed by a sustained phase. The transient phase, induced by either BK or histamine, was strongly inhibited by thapsigargin, a microsomal Ca(2+)-ATPase inhibitor, usually used to deplete certain intracellular Ca(2+)-stores. Ni2+ (4 mM) did not affect the transient phase but abolished the sustained phase when cells were stimulated by BK, further supporting the fact that the transient phase was only dependent on intracellular Ca2+ pools. The sustained phase was partially (for BK) and completely (for histamine) inhibited by 30 microM Mn2+. This effect could be completely reversed by the addition of DTPA, a cell-impermeant chelator of Mn2+, indicating that the Mn2+ exerted its effect extracellularly. The presence of 1 mM probenecid (an inhibitor of a membrane organic anion transporter that extrudes fura-2) drastically inhibited the sustained phase by more than 77% for BK and 88% for histamine. Our results suggest that the effects of BK and histamine on airway smooth muscle cells are mediated via bradykinin B2 receptors and histamine H1 receptors, respectively whose activation allows the rapid transient rise in [Ca2+]i from thapsigargin-sensitive intracellular Ca2+ pools. The sustained phase is proposed to be drastically influenced by an acceleration of fura-2 extrusion during the increase of [Ca2+]i via a probenecid-sensitive mechanism.

Animals↗
PubMed1994

Epithelium modulates the kinetics of the response to substance P and its intrinsic activity in the guinea-pig trachea.

The contractile response of guinea-pig tracheal preparations with or without epithelium to substance P has been studied in the presence or absence of thiorphan, an endopeptidase 24.11 inhibitor, paying special attention to the kinetics of the response. Without thiorphan, the response to substance P was greater in tracheal preparations without epithelium than in tracheal preparations with epithelium. The concentration-response curve was shifted to the left in the absence of the epithelium. In the presence of 10 microM thiorphan, the maximal contractile response induced by single doses of substance P (0.1 to 10 microM) was lower in tracheal preparations without epithelium. The maximal responses required 10 min in tracheal preparations with epithelium and 2 min in tracheal preparations without epithelium. These epithelium-dependent differences of reactivity remained in the presence of lipoxygenase or cyclooxygenase inhibitors and of selective antagonists of muscarinic, serotoninergic and histaminergic receptors, after the pre-treatment of tissues with capsaicin or compound 48/80 and in the presence of tetrodotoxin. The profile of the cumulative concentration-response curves for substance P was largely dependent on the time between two successive doses. When this time was short (2-4 min), curves established with or without the epithelium were parallel and both reached similar maximal values (2696 +/- 214 mg and 2780 +/- 62 mg, respectively). The curve in tracheal preparations without epithelium was slightly shifted to the left (EC50s: 24 +/- 10 nM and 78 +/- 19 nM). When this time was longer (10 min, ie corresponding to the time required for a full response to a single dose in intact trachea) the potency of substance P was not modified (EC50s: 13 +/- 3 nM and 52 +/- 11 nM), but a lower maximal response was observed with tracheal preparations without epithelium (1440 +/- 182 mg and 2832 +/- 209 mg). Similar results were observed with neurokinin A and neurokinin B. Thus, the removal of the epithelium led to a more rapid contraction and to a decrease of the maximal response to neurokinins, ie a decreased intrinsic activity, a property known to be drug- and tissue-dependent. These data suggest that the intrinsic activity of drugs depends on the cellular environment of the target cells in a tissue and is partly related to the diffusion and metabolism of drugs and to drug-induced hyporeactivity of the target cells.

Animals↗
PubMed1993

An epithelial-dependent contracting factor induced by calcium influx in guinea-pig trachea.

The effects of epithelium removal were studied on the contraction induced by Ca2+ in K(+)-depolarizing solution, and by the calcium ionophore A 23187 in guinea-pig isolated tracheal strips. Epithelium removal reduced the maximal response to Ca2+ in K(+)-depolarizing solution and caused a significant shift to the right of the Ca2+ concentration-response curves. The contraction induced by the calcium ionophore A 23187 (10(-6) M) was also markedly reduced by epithelium removal. These results suggest the occurrence of an epithelium-derived contracting factor. The effects of hexamethonium, atropine, spantide and thiorphan showed that acetylcholine and neurokinins play a minor role in the Ca(2+)-induced contraction. The epithelium-dependent potentiation of the calcium- and of the A 23187-induced contractions was inhibited by an antibody selective for rat calcitonin gene-related peptide (rCGRP alpha). Therefore, CGRP-like immunoreactive material may be part of the epithelium-dependent contracting factor of guinea-pig trachea. Comparison of concentration-response curves for rCGRP alpha in epithelium-free and in intact guinea-pig tracheal strips suggests that an epithelium-dependent contracting factor may be mobilized by CGRP.

Acetylcholine↗
PubMed1993

Kinins and respiratory tract diseases.

Bradykinin and related kinins are peptidic hormones, formed in tissues and fluids during inflammation. Various functional sites have been proposed as mediators of the biological effects of kinins, including the B1, B2 and B3 receptors. The existence of the B1 and the B2 receptor has largely been confirmed, whilst that of the B3 receptor is controversial and needs further confirmation. The role of bradykinin in the pathophysiology of asthma is not well understood, but bradykinin was proposed as a putative mediator of asthma, since asthmatic subjects are hyperresponsive to bradykinin, and since immunoreactive kinins are increased in the bronchoalveolar lavage fluids of asthmatic patients. Kinins could provoke bronchoconstriction by acting directly on smooth muscle and/or indirectly by their inflammatory properties. They may also contribute to the symptomatology of allergic and viral rhinitis, since they are the only mediators detected to date that are generated in nasal secretion during experimental and natural rhinovirus colds. Moreover, they can induce relevant symptoms when applied to airway mucosa. It has also been proposed that coughing during treatment with angiotensin-converting enzyme (ACE) inhibitors is linked to the action of kinins, since ACE is able to degrade kinins, and since the effects of ACE inhibitors are reduced by kinin antagonists. Due to their mitogenic properties, kinins have been proposed to regulate lung carcinoma growth. Their action remains speculative, but some findings are of great interest in order to define their role in these pathologies. Despite many studies in animals and in humans, the mode of action of kinins in airways is still poorly understood.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1992

Natural polyamines stimulate G-proteins.

The natural polyamines spermine and spermidine, the biosynthetic precursor putrescine and their analogues cadaverine and tyramine stimulate the GTPase activity of purified GTP-binding proteins (Go/Gi) from calf brain reconstituted into phospholipid vesicles. The order of potency was spermine greater than spermidine greater than putrescine = cadaverine greater than tyramine. The physiological relevance of this observation was assessed, showing the same order of potency of polyamines in the stimulation of peritoneal and tracheal rat mast cells. The activation of rat mast cells by polyamines was inhibited by benzalkonium chloride or by a 2 h pretreatment of the cells with pertussis toxin. The increase in inositol phosphates evoked by polyamines was also inhibited by pertussis toxin. Therefore we propose that intracellular polyamines might control the basal level of second messengers and modulate extracellular signals transduced through G-protein-coupled receptors.

Animals↗
PubMed1992

Endopeptidase 24.15 modulates bradykinin-induced contraction in guinea-pig trachea.

Guinea-pig tracheal strips were contracted with cumulative concentrations of bradykinin or substance P in the presence of thiorphan, an inhibitor of endopeptidase 24.11 and of angiotensin-converting enzyme, or in the presence of N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Ala-Phe-para-aminobenzoate (cFP-AAF-pAB), a selective inhibitor of endopeptidase 24.15. The concentration-effect curve of bradykinin was shifted to the left in the presence of each inhibitor whereas the curve of substance P was sensitive to thiorphan but not to cFP-AAF-pAB. These results show that endopeptidase 24.15 may modulate the contractile effect of bradykinin but not that of substance P in the guinea-pig trachea.

Amino Acid Sequence↗
PubMed1992

Effect of Hoe 140, a new B2 noncompetitive antagonist, on guinea pig tracheal bradykinin receptors.

We investigated the effect of the new B2 antagonist D-Arg0[Hyp3,Thi5, D-Tic7, Oic8]bradykinin (BK) (Hoe 140) on the binding of [3H]BK to membranes from guinea pig trachea with respect to the presence of the epithelium. Scatchard analysis of equilibrium data with [3H]BK revealed a single class of binding sites in the epithelium denuded trachea membrane preparation (E-) with a dissociation constant (Kd) of 55 pM and a Bmax of 0.71 fmol.mg tissue-1. When intact trachea (E+) was used, two binding sites were detected: a saturable high-affinity one (Kd of 40 pM and Bmax of 0.69 fmol.mg tissue-1) and a low-affinity one, not really saturable, with a Kd over 180 nM and a Bmax over 18 fmol.mg tissue-1. In guinea pig ileum, a tissue thought to contain B2 receptors, one class of binding sites was detected with a Kd of 209.3 pM and a Bmax of 16.2 fmol.mg tissue-1. In competition experiments ([3H]BK from 0.3 to 0.5 nM), similar results were obtained in (E +/-) and in ileum membrane preparations. B1 ligands did not displace [3H]BK binding, demonstrating the lack of B1 receptors. BK and B2 antagonist, except Hoe 140, fully displaced [3H]BK with Hill coefficients close to the unity. In competition studies only the high-affinity site was labeled by [3H]BK, in the (E+) preparation, as suggested by the inhibition constant value of unlabeled BK. Hoe 140 fully displaced [3H]BK in competition experiments, but with a Hill coefficient significantly less than unity, suggesting the presence of two binding sites for this compound in the three preparations used.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

[Systemic thrombolysis of arterial occlusions of the lower extremities. Comparison of various treatment schedules].

From 1971-1982 121 patients with arterial occlusions of the lower limbs underwent systemic thrombolysis treatment at the Kantonsspital Basel. During 4 time-periods, 3 different treatment schedules were evaluated consecutively: a) individually titrated high dose streptokinase (SK), b) individually titrated low dose SK and c) p-plasmin, followed by low dose SK-infusion. Thrombolytic success rates did not differ significantly with the 3 treatment schedules. Nevertheless, the p-plasmin-SK scheme tended to the thrombolytically more effective (68%) than high-dose (58%) or low-dose (50%) SK. The most frequent side effects were bleeding complications. In 6 out of the 121 patients, intracranial bleeding occurred and was lethal in 1 of the patients. The incidence of this most serious complication of 4/47 during the sequential p-plasmin-SK schedule led the authors to abandon this scheme for the treatment of arterial occlusions. The intracranial bleeding complications are much less frequent in patients with deep venous thrombosis undergoing systemic thrombolysis, and hence seem to be due in part to the generalized arteriopathy often present in patients with arterial occlusions. The p-plasmin-SK schedule induced the strongest systemic proteolysis in the light of thromboplastin time and factor V values. Comparison of these data with those of other authors is very difficult because of differences in patient selection, treatment schedules and observance of contraindications. The serious prognosis for patients with acute arterial occlusions, with an overall hospital mortality of 26% (experience at the Kantonsspital Basel, 1978-1982) relativizes the importance of the side effects due to systemic thrombolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗