Neurotransmitter, receptor and signal transduction disturbances in Alzheimer's disease.
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Alzheimer Disease↗
Biomedical subjects
C O'Neill
Publications and source records attributed to C O'Neill.
Biosynthesis of platelet-activating factor by two-cell mouse embryos.
Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.
Alkyl and Aryl Transferases↗
Preservation of 5-hydroxytryptamine1A receptor-G protein interactions in the cerebral cortex of patients with Alzheimer's disease.
The coupling of 5-hydroxytryptamine1A (5-HT1A) receptors to guanine nucleotide binding (G) proteins was investigated in membranes prepared from frontal and parietal cortices of control and Alzheimer's disease brains by characterising the effect of guanosine 5'-[beta gamma-imido]diphosphate (Gpp[NH]p) on [3H]8-hydroxy-2-(di-n-propylamino)-tetralin ([3H]8-OH-DPAT) binding parameters. In the absence of guanine nucleotides, [3H]8-OH-DPAT bound to a single high affinity binding site in all membrane types. The number of [3H]8-OH-DPAT binding sites was significantly decreased in the parietal cortex of Alzheimer's disease samples compared with controls, whereas in the frontal cortex the number of binding sites remained unchanged. Gpp[NH]p reduced the [3H]8-OH-DPAT binding affinity and the number of binding sites to the same degree in both regions in control and Alzheimer's disease cases. [3H]8-OH-DPAT binding was inhibited in a concentration dependent manner with an IC50 value of approximately 1 microM in all cases. These results suggest that the 5-HT1A receptor-G protein complex is functionally intact in these regions in Alzheimer's disease brain.
8-Hydroxy-2-(di-n-propylamino)tetralin↗
Coupling of human brain cerebral cortical alpha 2-adrenoceptors to GTP-binding proteins in Alzheimer's disease.
The coupling of alpha 2-adrenoceptors to guanine nucleotide binding G-proteins was investigated in cerebral cortical membranes from control and Alzheimer's disease brain by characterizing the effects of MnCl2 and Gpp[NH]p on [3H]clonidine binding. The manganese induced elevation of [3H]clonidine binding was apparent in both control and Alzheimer's disease samples and the effect showed no significant difference between the two groups in the frontal cortex. However, the MnCl2 concentration dependence curves for control and Alzheimer's disease samples were significantly different from one another in the temporal cortex, whereas the pattern of stimulation by MnCl2 remained the same. The guanine nucleotide analogue, Gpp[NH]p inhibited [3H]clonidine binding in a concentration-dependent manner, the profiles of inhibition showing no significant differences between control and Alzheimer's disease samples. Analysis of the effect of Gpp[NH]p on [3H]clonidine saturation binding curves showed no significant differences between control and Alzheimer's disease samples in either frontal (Kd = 9.68 +/- 1.38, 9.1 +/- 2.6 nM; Bmax = 40.23 +/- 4.33, 44.3 +/- 9.4 fmol/mg, control and Alzheimer's disease values, respectively), or temporal (Kd = 11.61 +/- 4.04, 5.38 +/- 2.5 nM; Bmax = 52.0 +/- 14.0, 31.07 +/- 8.00 fmol/mg control and Alzheimer's disease values, respectively) cortices.(ABSTRACT TRUNCATED AT 250 WORDS)
Aged↗
Assay of a phosphatidylinositol bisphosphate phospholipase C activity in postmortem human brain.
The activity of a phospholipase C which hydrolyses exogenous phosphatidylinositol-4,5-bisphosphate [( 3H]PtdIns(4,5)P2) in membranes prepared from frozen postmortem human brain and rat brain was investigated. Enzyme characteristics were essentially similar in membranes prepared from frozen postmortem brain and fresh or frozen rat brain. The [3H]PtdIns(4,5)P2 solubilization and assay procedure employed resulted in an efficient availability of the substrate for the enzyme. The non-hydrolysable guanosine triphosphate analogue guanosine 5'-[beta gamma-imido]diphosphate (Gpp[NH]p) stimulated hydrolysis rapidly with a half maximum activity of approximately 25 microM. This stimulation was not specific for guanine nucleotides as ATP, imidodiphosphate and pyrophosphate also caused enzyme activation. However these activation effects could be distinguished by the polyanion spermine. The non-hydrolysable guanine dinucleotide analogue guanosine 5'-[beta-thio]diphosphate acted as a partial agonist thereby inhibiting the stimulatory effect of Gpp[NH]p. Gpp[NH]p-stimulated enzyme activity showed a maximum response in the presence of 1 mM deoxycholate and displayed a pH optima in the range 7.0-7.5. PtdIns(4,5)P2 hydrolysis was observed in the absence of added calcium, but hydrolytic cleavage was inhibited in the presence of divalent ion chelators. Magnesium inhibited PtdIns(4,5)P2 hydrolysis in a concentration-dependent manner. Elucidation of these aspects of the phosphatidylinositol cycle in normal human postmortem brain will permit comparative studies in CNS disease states.
Aged↗
The effect of inhibitors of platelet aggregation on the metabolism of platelet-activating factor (PAF) in washed rabbit platelets.
The rabbit platelet metabolizes platelet-activating factor (PAF) intracellularly. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I2 (PGI2), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI2) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor.
Animals↗
Platelet-activating factor (PAF) receptor antagonists inhibit arachidonic acid induced platelet aggregation in rabbit whole blood.
The potency of several platelet-activating factor (PAF) receptor antagonists was measured by observing their inhibitory effects against PAF induced platelet aggregation. Their selectivity was assessed by monitoring their effect on platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP). The antagonists inhibited platelet aggregation induced at the PAF EC50 (0.023 microM) with the following rank order of potency: WEB 2086 greater than WEB 2170 greater than SRI 64-412 greater than SRI 63-675 greater than BN 52021 greater than kadsurenone greater than SRI 63-441 greater than alprazolam. While the antagonists had no inhibitory effect at the EC50 for ADP (10 microM), they did inhibit platelet aggregation induced at the EC50 for AA (55 microM). However, there was considerable variability in the slope of the inhibitory response and the relative potency of each antagonist against PAF induced platelet aggregation as compared to AA induced platelet aggregation. The antagonist IC50 (microM) against PAF and AA were as follows, with those that showed significantly different (p less than 0.01) slopes indicated by an asterisk: SRI 63-441* (3.8, 15.1); SRI 63-675 (1.4, 36.2); SRI 64-412 (0.5, 10.5); BN 52021* (2.4, 58.9); kadsurenone* (2.8, 28.3); alprazolam* (10, 25); WEB 2086 (0.055, 0.220), and WEB 2170 (0.107, 0.534). Therefore, in rabbit whole blood the antagonists were potent, although not completely selective, inhibitors of PAF induced platelet aggregation. These results suggest that the mode of action of PAF and AA induced platelet aggregation may share some common features. However, since the slope of the inhibitory response against PAF and AA for some antagonists differed, mechanistic differences in their action appear to exist.
Adenosine Diphosphate↗
Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.
IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.
8-Hydroxy-2-(di-n-propylamino)tetralin↗
Rapid and selective measurement of platelet-activating factor using a quantitative bioassay of platelet aggregation.
A bioassay for the measurement of platelet-activating factor (PAF) based on the quantification of platelet aggregation was developed. The method used a platelet analyzer in conjunction with a multiwelled micromixing device and whole blood collected from male New Zealand White rabbits. This bioassay can be nonselective and used to quantitate platelet aggregation induced by any activator. The EC50 for platelet-activating factor, arachidonic acid, and adenosine diphosphate were 0.0232, 55, and 10 microM, and at these concentrations the half maximal aggregation response occurred at 7.5, 10.0, and 12.5 min, respectively. The bioassay was capable of the sensitive quantification of platelet aggregation in small volumes (50 microL) of citrated rabbit whole blood, using a short (i.e., 15-min) incubation period. This enabled multiple bioassays to be performed without the need for a large volume of whole blood to be collected from the rabbit. Selective measurement of platelet-activating factor was achieved by adding inhibitors of arachidonic acid- and adenosine diphosphate-dependent pathways of platelet activation, that is, acetylsalicyclic acid and phosphoenolpyruvate/pyruvate kinase, respectively, to the citrated rabbit whole blood immediately before bioassay. These inhibited arachidonic acid- and adenosine diphosphate-induced platelet aggregation, but had no effect on platelet aggregation induced by platelet-activating factor. Platelet-activating factor was selectively inhibited by its receptor antagonist BN 52021. This method for measuring platelet-activating factor was reproducible; at the EC50, the inter- and intrabioassay coefficients of variation were within acceptable limits at 13.17% and 9.75%, respectively.
Animals↗
A physiological role for PAF in the stimulation of mammalian embryonic development.
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Animals↗
A consideration of the factors which influence and control the viability and developmental potential of the preimplantation embryo.
The advent of the new reproductive technologies (including in vitro fertilization) has led to a revolution in the treatment of infertility. It has not yet led to a marked improvement in our understanding of the control of development and of viability of the early embryo. It is the poor viability of embryos, with consequent implantation failure, which is the major limiting factor to successful outcomes. While much of the research in this area has concentrated on strategies of ovulation induction, experimental models have shown that the major cause of reduced embryo viability is due to the actual process of fertilization in vitro and subsequent culture of the preembryo in synthetic culture medium. It is likely that this is due to the absence of critical nutrients or trophic factors of maternal origin and work with co-culture of embryos with somatic cells suggests improved viability can be achieved. Such co-culture is not an option for routine clinical use, however. It is essential therefore to understand by detailed study of the physiology of embryonic development their requirements for optimal development. The empirical approach of comparing different formulations of culture media is unlikely to be successful because of the vast range of parameters to be tested and the large number of pregnancies required to demonstrate a significant improvement in outcome. The strategy that is most likely to be successful in the future, therefore, is the use of appropriate experimental models, such as the developing rodent embryo, to understand the essential physiological changes in the embryo during its development, the control processes in place, and the effect of the embryo's environment on the processes. This will allow the rational design of culture media which can then be rigorously tested for improved outcome. An example of successful application of this approach is the discovery of embryo-derived platelet activating factor (PAF). The production of embryo-derived PAF was first described and validated in the rodent. In the same species it was shown to have an essential role in pregnancy and to act as an autocrine mediator of embryo viability. This fundamental observation in rodents was then confirmed in humans, and recent work has shown that supplementation of culture human embryo media with PAF results in a dramatic increase in their developmental and pregnancy potential. This example should be the first of many such improvements based on a more fundamental understanding of the embryo's developmental requirements.
Animals↗
The effects of cryopreservation and thawing on the development in vitro and in vivo of biopsied 8-cell mouse embryos.
The possible impact of cryopreservation on biopsied 8-cell mouse embryos was investigated. Biopsied and control 8-cell embryos were cryopreserved using a slow freezing and quick thawing protocol with 1,2-propanediol as a cryoprotectant. The cryopreservation process did not affect either the recovery or the survival of biopsied embryos, when compared with intact controls; however, sham controls survived significantly better than biopsied 8-cell embryos (88.6 versus 74.2%, P less than 0.001). When fully and partially intact surviving embryos were cultured in vitro to the blastocyst stage, there was no difference in the proportions of embryos which formed blastocysts (biopsy 97.2%, intact control 98.4% and sham control 93.7%). The developmental potential and fetal development in vivo following embryo transfer were not impaired when assessed on day 17 of pregnancy. Cryopreservation of biopsied 8-cell mouse embryos is therefore a feasible approach to storing embryos while analysis of the biopsied material is carried out.
Animals↗
Comparison of a radioimmunoassay and bioassay for embryo-derived platelet-activating factor.
The pre-embryos of a number of species produce platelet-activating factor (PAF). This study compared the sensitivity, reproducibility and cross-reactivity of a radioimmunoassay (RIA) for PAF with a quantitative PAF bioassay and assessed the use of both assays for the measurement of murine and human embryo-derived PAF. PAF was extracted and partially purified by thin-layer chromatography (TLC) from medium in which mouse (30 x 2-cell) or individual human embryos [produced by in-vitro fertilization (IVF)] had been cultured for 24 h. Dose-response curves were generated with synthetic PAF standards over a concentration range of 0.3-30 ng/ml. The RIA was slightly more sensitive than the bioassay since the concentration which gave 50% response in the RIA was 3 ng/ml compared to 6 ng/ml in the bioassay. The RIA showed greater reproducibility than the bioassay, since at these concentrations the coefficients of variation were 1.97 and 6.77% intra-assay and 4.96 and 10.93% inter-assay, respectively. Both assays had a detection limit of 0.3 ng/ml. C-PAF, a non-metabolizable bioactive analogue of PAF, showed 5.16 and 0.85% cross-reactivity in the bioassay and RIA, respectively, while lyso-PAF and phosphatidylcholine had no effect. Following extraction and TLC, the average PAF concentration was 14.52 ng/ml (range 0.10-141.09) and 20.06 ng/ml (range 0.07-106.15) for mouse embryos, and 16.15 ng/ml (range 0.40-78.31) and 23.70 ng/ml (range 2.00-119.10) for human embryos, as measured by bioassay and RIA respectively. Therefore, both the bioassay and RIA were capable of the sensitive quantitative measurement of embryo-derived PAF.
Animals↗
Home care of the elderly: a family perspective.
Family home care of the elderly has become a primary focus of nursing theory, practice, and research. Traditional study of home care of the elderly, particularly the cognitively impaired, has focused on the experience of the caregiver. There is a need for consideration of the perception of the care recipient and the entire family unit. A model to explain the characteristics of the elderly care recipient, as well as theoretic and methodologic implications, are offered.
Aged↗
Production of platelet-activating factor by the pre-implantation sheep embryo.
Embryos were collected from superovulated ewes on Day 2 (2-8 cell), Day 4 (8-16 cell) and Day 6 (morula/early blastocyst). Two embryos were cultured in 1 ml of one of four media: (i) Ham's F10 + 4 mg bovine serum albumin (BSA)/ml, (ii) synthetic oviduct fluid medium + 20% human serum, (iii) Quinn's human tubal fluid medium (HTF) + 3 mg BSA/ml or (iv) HTF + 10% acid-treated fetal calf serum for 24 h. They were transferred to fresh media of the same type and their further development was monitored. A quantitative bioassay and radioimmunoassay was used to measure the concentration of platelet-activating factor (PAF, 1-o-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine) produced. Following extraction and partial purification, 21/95 (22.1%) of the embryo-conditioned media samples had PAF concentrations greater than that measured in corresponding control media. This was designated as embryo-derived PAF and the corresponding cultures were termed 'PAF-positive'. PAF was produced by embryos at all three developmental stages examined and in each of the four media used, and the average amount of PAF produced was 60.9 +/- 9.8 pmol/embryo/24 h. However, neither the developmental stage of the embryo, nor the type of media affected the proportion of PAF-positive cultures nor the amount of PAF produced during culture. Thus, it is demonstrated for the first time that early ovine embryos can secrete PAF in vitro, and that there is considerable variability in their capacity for PAF secretion.
Animals↗
The development of the radiography degree course at the University of Ulster.
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Curriculum↗
Effect of monovalent ions upon G proteins coupling muscarinic receptors to phosphoinositide hydrolysis in the rat cerebral cortex.
It has been suggested that K+, Li+ and Fl- affect the function of G proteins coupled to signal transducing enzymes. Lithium, at concentrations which were found to reduce forskolin-stimulated adenylate cyclase activity, was without effect on either membrane [3H]phosphatidylinositol-4,5-bisphosphate ([3H]PIP2) hydrolysis measured in the absence or presence of 5'-guanylyl-imidodiphosphate (Gpp(NH)p), or (at greater than or equal to 2.3 mM Li+) upon the stimulation of rat cerebral cortical inositol phospholipid breakdown by either carbachol, noradrenaline or NaF measured at either 6 or 18 mM K+. The increase in assay [K+] greatly enhanced the inositol phospholipid response to carbachol but not to NaF. The inhibitory effect of carbachol upon forskolin-stimulated adenylate cyclase was not affected by raising the [K+] from 6 to 18 mM. At 6 mM K+ (both in the absence and presence of 15 microM AlCl3), the effects of carbachol and NaF upon inositol phospholipid breakdown were essentially additive, whereas at 18 mM K+, the breakdown response to carbachol (antagonised by pirenzepine with a pA2 value of 7.6) was similar in the absence and presence of NaF. It is concluded that in the rat cerebral cortex: (a) Li+ does not affect the function of either the phosphoinositide-specific phospholipase C enzyme itself or the Gp coupled to this enzyme; (b) the difference between the additivity between NaF and carbachol seen at different assay [K+] may reflect the K(+)-dependent changes in the tetrodotoxin-resistant and tetrodotoxin-sensitive pathways of carbachol stimulation of inositol phospholipid breakdown reported by Gurwitz and Sokolovsky (1987, Biochemistry 26, 633); and (c) the effect of K+ on muscarinic receptor-coupled inositol phospholipid breakdown is not found for muscarinic receptors inhibitorily coupled to adenylate cyclase. Evidence is also presented to suggest that NaF affects the dephosphorylation of the formed [3H]inositol polyphosphates.
Adenylyl Cyclases↗
Exclusion of beta-thalassaemia by biopsy and DNA amplification in mouse pre-embryos.
Exclusion of beta-thalassaemia in mice was undertaken by pre-implantation DNA diagnosis. Biopsies of approximately five cells were obtained from mouse pre-embryos. DNA prepared from this material was amplified by the polymerase chain reaction (PCR), enabling identification of the normal beta-major globin gene in 11 out of 20 cases where it was known to be present. The expected negative result was obtained in 14 out of 16 homozygous thalassaemic embryos, with two false positives. Results are promptly available following PCR, thereby facilitating re-implantation of embryos which have had homozygous beta-thalassaemia excluded. The mouse model illustrates a useful approach to pre-implantation diagnosis of genetic disease in the human.
Animals↗