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Measurement of responses from Gi-, Gs-, or Gq-coupled receptors by a multiple response element/cAMP response element-directed reporter assay.

We have established a rapid, sensitive, high-throughput assay that requires one assay condition to detect agonist effects from Gi-, Gs-, and Gq-coupled receptors. We utilized a vector containing a promoter with three multiple response elements, the vasoactive intestinal peptide promoter and a cAMP response element controlling the transcription of the luciferase gene. An adrenergic agonist, para-aminoclonidine, inhibited forskolin-stimulated luciferase expression when cells were cotransfected with the Gi-coupled alpha(2)-C adrenergic receptor and the MRE/CRE reporter vector. Further, we demonstrate that gastrin-releasing peptide, which activates a Gq-coupled GRP receptor, isoproterenol, which activates a Gs-coupled beta-adrenergic receptor, calcium ionophores, and phorbol 12-myristate 13-acetate, a stimulator of protein kinase C, can mediate increases in luciferase expression in the presence of forskolin but not in its absence. The effect at Gi-coupled receptor activation correlates with the phosphorylation of the CRE binding protein (CREB); however, the mechanisms mediating the responses to Gq- and Gs-coupled receptors are more complex. We demonstrate that this assay is useful for pharmacological analysis of both agonists and antagonists and has the potential to associate orphan G-protein-coupled receptors with their corresponding ligands.

Base Sequence↗

Induction of a pleiotropic response by phenobarbital and related compounds. Response in various inbred strains of rats, response in various species and the induction of aldehyde dehydrogenase in Copenhagen rats.

The ability of phenobarbital (PB) to induce a "pleiotropic response" which includes both cytochromes P450 (CYP) as well as other drug-metabolizing enzymes was investigated in mice, rabbits, hamsters, and various inbred strains of rats. PB induced similar drug-metabolizing enzymes (CYP2B, CYP3A, and epoxide hydrolase) in rats, mice, rabbits and hamsters. PB and two structural analogues (ethylphenylhydantoin and barbital) induced a variety of drug-metabolizing enzymes (CYP2B, CYP3A, CYP2A, epoxide hydrolase) in a series of inbred strains of rats. In contrast, levels of aldehyde dehydrogenase (ALDH) (propionaldehyde, NAD+) which were expressed constitutively in all strains of rats were induced by PB in only two of the eight strains (ACI, Copenhagen). Further investigations of ALDH induction by structurally diverse compounds in Copenhagen rats demonstrated a strong correlation between the induction of ALDH and other elements of the pleiotropic response (CYP2B, CYP3A, epoxide hydrolase). These results imply that induction of ALDH (propionaldehyde, NAD+) is associated with the PB pleiotropic response in Copenhagen rats.

Aldehyde Dehydrogenase↗

Correlation between increased bronchial responsiveness to histamine and diminished plasma cyclic adenosine monophosphate response after epinephrine in asthmatic children. Diminished plasma cyclic adenosine monophosphate response after epinephrine in moderate childhood asthma.

The respiratory threshold to histamine and the plasma cyclic adenosine monophosphate (AMP) every 5 min for 40 min after subcutaneous epinephrine were determined in 21 children with moderate bronchial asthma who were without symptoms at the time of study. There was a statistically significant correlation between a high respiratory sensitivity to histamine and a low plasma cyclic AMP response to epinephrine. The plasma cyclic AMP response was compared with that in 16 control subjects. The asthmatic patients had significantly diminished responses; the difference was greatest for the values 25 min after stimulation. This study supports the hypotheses that the bronchial hyperresponsiveness in asthma is due partly to a defective beta adrenergic system and that the defect is permanent, existing also during periods without symptoms or medication and in patients with moderate asthma.

Adolescent↗

Beclomethasone given after the early asthmatic response inhibits the late response and the increased methacholine responsiveness and cromolyn does not.

BACKGROUND: Single doses of inhaled beclomethasone or inhaled cromolyn, given before allergen inhalation, inhibit allergen-induced late asthmatic responses (LARs) and increased airway responsiveness (delta log methacholine PC20). We hypothesized that when given 2 hours after allergen, beclomethasone might work better than cromolyn. METHODS: In 10 patients with mild, stable, atopic asthma with LARs or delta log PC20 or both, we performed a double-blind, double-dummy, random-order trial comparing a single dose of inhaled beclomethasone (500 micrograms), cromolyn (20 mg), and placebo, administered 2 hours after allergen challenge on LAR and delta log PC20. RESULTS: The treatment effect on LAR was significant (p < 0.001). The LAR after beclomethasone (7.3% +/- 6.1%) was significantly less than after cromolyn (20.4% +/- 15.2%) or placebo (26.4% +/- 8.2%); cromolyn was not different from placebo. There was a borderline treatment effect on delta log PC20 (p = 0.056) with beclomethasone (0.12 +/- 0.31) less than placebo (0.37 +/- 0.39) but not less than cromolyn (0.34 +/- 0.18). CONCLUSION: Beclomethasone (500 micrograms) administered 2 hours after allergen challenge markedly inhibited the LAR and had a small effect on allergen-induced airway responsiveness. Cromolyn (20 mg) was not effective on maximal LAR; a small effect on the early part of the LAR was suggested.

Adolescent↗

The CYP2B2 phenobarbital response unit contains an accessory factor element and a putative glucocorticoid response element essential for conferring maximal phenobarbital responsiveness.

Hepatic cytochrome P450s play a critical role in the metabolism of hydrophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces hepatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver. By using primary rat hepatocytes for transfection analyses, we previously identified in the CYP2B2 5'-flank a 163-base pair Sau3AI fragment that confers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer. Transfection experiments with sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory element, AF1, was identified and localized. DNase I footprinting analysis revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which are putative recognition sites for known transcription factors. Site-directed mutagenesis indicated that a putative glucocorticoid response element as well as a nuclear factor 1 site and an associated nuclear receptor hexamer half-site are essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that phenobarbital induction of CYP2B2 requires interactions among multiple regulatory proteins and cis-acting elements constituting a phenobarbital response unit.

Animals↗

The immune response to bee venom. Comparison of the antibody response to phospholipase A2 with the response to inhalant antigens.

The antibody response to phospholipase A2 (PLA2) was studied by using radiolabelled antigen-binding techniques for both IgG and IgE antibodies. The results are compared with results for antigen P1 from Dermatophagoides pteronyssinus and for rye I from rye grass pollen. The response to PLA2 differs from the response to the inhalant allergens in several ways: almost all exposed bee keepers produce IgG antibodies to PLA2; among the allergic bee keepers there was no quantitative correlation between IgG and IgE antibodies; and the antibodies to PLA2 are not produced locally in the nose. Bee keepers with hay fever did not appear to have an increased incidence of allergic reactions to bee venom. In addition, the bee keepers with hay fever and the same levels of IgG and IgE antibodies to rye I as other individuals with hay fever. However, the bee keepers who had no history of hay fever were found to have high levels of IgG antibodies to rye I. These antibodies did not cross-react with PLA2 or bee venom, and the results suggest that bee stings may act as an adjuvant for IgG antibodies to pollen antigens.

Allergens↗

Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress.

Cancer chemopreventive agents can act by inhibiting either the acquisition of mutations or the neoplastic processes that occur subsequent to mutagenesis. Compounds that reduce the rate at which mutations arise, referred to as blocking agents, exert their effects largely through their ability to induce the expression of antioxidant and detoxification proteins. This is achieved by the transcriptional activation of a small number of genes that are co-regulated through the presence of an antioxidant responsive element (ARE) in their promoters. Blocking agents can cause gene induction by producing oxidative and/or chemical stress within the cell and, as the inducible proteins act to ameliorate the metabolic insult, the process represents a form of adaptive response. The transcription factors which mediate this response through the ARE are members of the basic leucine zipper superfamily. The mechanism whereby cells sense and respond to the chemical signal(s) generated by chemopreventive blocking agents is discussed.

Adaptation, Physiological↗

[Pressor response to glutamate-injection into hypothalamic paraventricular nucleus and its relation to pressor response of locus coeruleus and depressor response of A1].

In urethane-anesthetized, tubocurarine-immobilized and artificially ventilated rats, microinjection of L-glutamate (Glu) into hypothalamic paraventricular nucleus (NPV) or locus coeruleus (LC) induced a pressor response. The LC-pressor response could be attenuated by preinjection of phentolamine or propranolol into bilateral NPV; Preinjection of phentolamine or bicuculline into bilateral NPV could also attenuate the depressor effect of A1-excitation by Glu, but preinjection of propranolol had no such effect; suggesting that the LC-pressor or A1-depressor effect is mediated partly by NPV, and GABAergic inhibitory interneurons in NPV may be involved in A1-depressor response.

Animals↗

Interferon response heterogeneity: activation of a pro-inflammatory response by interferon alpha and beta. A possible basis for diverse responses to interferon beta in MS.

Interferon gamma (IFN-gamma) stimulates the (pro-inflammatory) type II interferon receptor and is known to exacerbate multiple sclerosis (MS). In contrast, IFN-alpha and IFN-beta are ligands for the (anti-inflammatory) type I interferon receptor and are beneficial in some (but not all) patients with MS. Should IFN-beta elicit a type-II-like pro-inflammatory response, the beneficial effects might be attenuated. These studies were undertaken to test this possibility with the use of quinolinic acid (QUIN) formation as a measure of type II receptor activation. In normal human macrophage cultures, IFN-gamma was the most potent stimulus for QUIN formation. Generally, IFN-beta and IFN-alpha were less potent. However, an unexpected inter-patient variability was observed. In some subjects, IFN-alpha was more potent than IFN-beta. In other subjects, IFN-beta was more potent than IFN-alpha. The present data demonstrate an inter-subject variability for QUIN production following exposure to the interferons. MS patients who demonstrate a pro-inflammatory response to IFN-beta (e.g., increased QUIN) may be less likely to benefit from this therapy.

Adult↗

Characterization of cDNAs representing five abscisic acid-responsive genes associated with somatic embryogenesis in Picea glauca, and their responses their responses to abscisic acid stereostructure.

Five abscisic acid (ABA)-responsive cDNAs that were identified by differential screening of a white spruce [Picea glauca (Moench) Voss] cotyledonary somatic embryo cDNA library are characterized. Three of the cDNAs (PgEMB12, 14, 15) are predicted to encode homologues of different late-embryogenesis-abundant (LEA) proteins, a further two (PgEMB 5 and 23) share little similarity to any known DNA or protein sequences. When suspension cultures were fed 1.5 x 10(5) M S-(+)-ABA and assessed for transcript abundance over 48 h, gene expression corresponding to each lea-like gene was inducible and was evident during the 48-h period. Conversely, transcripts were at a very low abundance when suspensions were fed R-(-)-ABA. When suspensions were treated with a range of S-(+)-ABA and R-(-)-ABA concentrations (10(-7)-10(-4) M), expression was induced at S-(+)-ABA concentrations of 10(-5) M or above. Expression corresponding to PgEMB5 and PgEMB23 indicates less specificity for ABA stereostructure, with similar expression patterns being observed for either enantiomer during the 48 h after feeding. The two corresponding genes also appear more responsive to ABA concentration than the predicted lea genes, within the range examined (10(-7)-10(-4) M).

Abscisic Acid↗

Phytochrome-like responses in Euglena: A low fluence response that reorganizes the spectral dependence of the high irradiance response in long-day photoperiodic induction of cell division.

Irradiance spectra change spatiotemporally, and angiosperms adapt accordingly, mainly through phytochromes. This study challenges the long-held belief that the flagellated alga Euglena gracilis lacks phytochromes and is therefore unaffected by spectral changes. We photoautotrophically cultured the alga under continuous light (LL), then transferred it to darkness. After about 26h in darkness, different irradiations for 3h enabled cell division in dark-arrested G2 cells evoking a high-irradiance response (HIR). The spectral characteristics of the irradiation during the LL period (pre-irradiation) defined the spectral sensitivity in the subsequent dark period. LL with light rich in the red spectrum led to a HIR to the red spectrum (R-HIR), whereas light rich in the far-red spectrum (FR) led to a FR-HIR. Finishing the period of pre-irradiation consisting of continuous cool-white fluorescent light (rich in R) by a FR pulse enhanced the characteristics of the FR-HIR 26h later. By contrast, a R pulse given at the end of the pre-irradiation rich in FR potentiated the R-HIR. The effects were completely photoreversible between R and FR with critical fluences of about 2mmolm(-2), satisfying the classic diagnostic feature of phytochromes. The action spectrum of the FR effect at the end of pre-irradiation consisting of continuous cool-white fluorescent light (rich in R) had a main peak at 740nm and a minor peak at 380nm, whereas antagonization of the FR effect had a main peak at 640nm and a minor peak at 480nm. Wavelengths of 610 and 670nm appeared in both spectra. We also demonstrated the photoreversibility of 380/640, 480/740, and (610 and 670)/(640 and 740) nm. We conclude that Euglena displays phytochrome-like responses similar to the 'shade avoidance' and 'end-of-day FR' effects reported in angiosperms.

Animals↗

Identification and characterization of a versatile retinoid response element (retinoic acid receptor response element-retinoid X receptor response element) in the mouse tissue transglutaminase gene promoter.

Tissue transglutaminase (transglutaminase type II) is an intracellular protein cross-linking enzyme that accumulates in connective tissue and in cells undergoing apoptosis. Retinoids regulate the transcription of the mouse tissue transglutaminase gene via activation of regulatory elements contained within 4 kilobases of the 5'-end of the gene. Co-transfection studies with retinoid receptor expression vectors in CV-1 cells demonstrated that the mouse tissue transglutaminase promoter is activated by ligand activation of either retinoic acid receptor-retinoid X receptor (RAR.RXR) heterodimers or RXR homodimers. Optimal induction is achieved with retinoid receptor panagonists; partial activation can also be achieved with either RAR-specific or RXR-specific retinoids. Retinoid-dependent activation of the tissue transglutaminase promoter depends on both a proximal regulatory region containing sequences highly conserved between the human and the mouse tissue transglutaminase promoters and a distal region that includes a 30-base pair retinoid response element (mTGRRE1). mTGRRE1 contains three hexanucleotide half-sites (two canonical and one non-canonical) in a DR7/DR5 motif that bind both RAR*RXR heterodimers and RXR homodimers. These studies suggest that retinoid-dependent expression of the mouse tissue transglutaminase gene is mediated by a versatile tripartite retinoid response element located 1.7 kilobases upstream of the transcription start site.

3T3 Cells↗

Pathophysiologic mechanisms responsible for the reversible (thiamine-responsive) and irreversible (thiamine non-responsive) neurological symptoms of Wernicke's encephalopathy.

Chronic alcoholism results in thiamine deficiency as a result of poor nutrition and impaired gastrointestinal absorption of the vitamin. Pyrithiamine-induced thiamine deficiency in the rat reproduces a neurological syndrome and ultimately neuropathological damage of a nature and distribution that is similar to that encountered in Wernicke's encephalopathy in humans. Pyrithiamine-induced thiamine deficiency results in selective reversible decreases in activity of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase and concomitant reversible changes in brain amino acids. It is proposed that these changes constitute "the biochemical lesion" in thiamine deficiency encephalopathy. If sufficiently severe and prolonged, decreased activities of alpha-ketoglutarate dehydrogenase may result in compromised brain energy metabolism and in lactate accumulation in brain, both of which could be responsible for neuronal cell death in this condition. In addition, it has been suggested that cell death results from NMDA-receptor mediated excitotoxic damage. Similar pathophysiologic mechanisms could be responsible for brain cell death in Wernicke's encephalopathy in humans.

Journal Article↗

Xenogeneic human anti-mouse T cell responses are due to the activity of the same functional T cell subsets responsible for allospecific and major histocompatibility complex-restricted responses.

Human T cells respond strongly to mouse major histocompatibility complex (MHC) antigens. The response is directed predominantly to the polymorphic determinants of the MHC antigens and there is little or no response to the nonpolymorphic determinants or to non-MHC antigens. Human cytotoxic T lymphocytes (CTL) are generated specific for the mouse class I MHC antigens and the CTL effectors are blocked by anti-Leu-2a antisera. Human interleukin 2-producing T cells are generated specific for mouse class II antigens and their induction is blocked by anti-Leu-3a antisera. These and other considerations lead us to propose a model for the T cell receptor that provides an explanation for several of the features of T cell recognition. In this model, the recognition of the "class" (I or II) of MHC antigen is separate from the recognition of the polymorphic determinants. We suggest that the initial recognition of the conserved "class" determinants positions another domain of the receptor so that it can only engage with the part of the MHC molecule carrying the polymorphic determinants.

Animals↗

The relationship between the acute phase response and antibody production in the rabbit. II. The stimulation of Cx-reactive protein response by certain adjuvants and the relation of this response to the enhancement of antibody formation.

The ability of an adjuvant and its individual constituents to induce the production of Cx-reactive protein in rabbits has been studied. It was found that the adjuvant stimulated rabbits to produce large amounts of the acute phase protein for 3 to 6 days. Melted aquaphor blended with saline stimulated the production of Cx-reactive protein for 3 or 4 days. Mineral oil was less effective in stimulating the production of the protein than either adjuvant or aquaphor. Heat-killed Jamaica strain tubercle bacilli suspended in mineral oil did not induce the Cx-protein response. The ability of subcutaneously administered adjuvant without antigen incorporated in the saline phase to potentiate the antibody response of rabbits to the intravenously administered antigens, C-reactive protein and human gamma globulin, was investigated. It was found that the adjuvant-treated animals produced more precipitating antibody to the two intravenously administered antigens than did the control animals given intravenous antigen alone.

Acute-Phase Reaction↗

Appropriate public health responses to clusters: the art of being responsibly responsive.

Between 1981 and 1988, the Minnesota Department of Health actively responded to over 400 reports from persons concerned about disease occurrence in their community, school, or workplace. Almost all of these reports involved perceived excesses of cases of cancer. Although there is little potential for identifying unsuspected public health problems or developing new etiologic insights, the Minnesota Department of Health has found that responding to reported clusters is a legitimate and necessary public health activity. To be responsibly responsive to these concerns, the Department has developed four steps to prioritize investigation of reported disease clusters, as well as six criteria for determination of the feasibility of environmental epidemiologic investigations. Approximately 95% of all concerns have been handled within the first two steps of this approach, generally requiring only education, or sometimes examination of readily-available data. Less than 5% of the concerns have required additional data collection and evaluation, and only about 1% have resulted in full-scale epidemiologic studies. Successful conclusions at all levels of this process require that public health officials develop effective communication, maintain objectivity, and provide leadership for controversial and difficult issues.

Clinical Protocols↗

The role of the CD8-positive subset of T cells in proliferative responses to soluble antigens. II. CD8-positive cells are not responsible for DR-associated differences in responsiveness to mumps and Coxsackie B4.

The role of CD8 (T8, Leu 2)-positive T lymphocytes in the proliferative T-lymphocyte response to mumps and Coxsackie B4 viral antigens in vitro was investigated. The frequency among enriched T-lymphocyte blasts of antigen-reactive T lymphocytes (ARTL) restricted by different DR-associated elements was investigated, using antigenic restimulation with allogeneic antigen-presenting cells in a limiting dilution assay. A decreased frequency of DR3-restricted and an increased frequency of DR4-restricted mumps and Coxsackie B4 ARTL were seen in the limiting dilution assay, whether or not HLA class I determinants were shared in the antigenic restimulation. Removal of CD8-positive cells did not increase the primary in vitro responsiveness to mumps and Coxsackie B4 viral antigens, and did not change the DR-associated differences in the frequency of ARTL seen in the limiting dilution assay.

Antigen-Presenting Cells↗

Response compatibility and the relationship between event-related potentials and the timing of a motor response.

1. Earlier studies have shown that changes in the difficulty of sensory discrimination in a choice reaction time task result in a prolongation of the peak latency for several components of the long-latency event-related potential (ERP). With the use of the technique of response-locked averaging, we have previously shown that manipulation of the difficulty of sensory discrimination also affects response execution as assessed by the interval between the ERP and onset of the response. In the present paper we examine the hypothesis that changing the compatibility of the responses may also affect the difficulty of the discrimination, as well as the execution of the response, as assessed by the interval between stimulus onset and the ERP. Such an effect of response compatibility would provide further evidence for the close integration of motor and sensory processes in the performance of choice reaction time tasks. 2. We continuously recorded the electroencephalogram (EEG) from the scalp and the electromyogram (EMG) from the responding muscles in both compatible and noncompatible visual choice reaction time tasks. In the compatible task subjects responded to a lateralized visual stimulus with the hand ipsilateral to the stimulus, whereas in the noncompatible task they responded with the contralateral hand. EEG and EMG responses were analyzed and averaged off-line, aligning the waveforms by either stimulus onset (stimulus-synchronized averages) or response onset (response-synchronized averages), and averaged separately for both correct and incorrect response outcomes. 3. Response times were significantly faster for frequent stimuli than rare stimuli and were significantly faster to rare stimuli in the compatible than the noncompatible condition. In responses to the frequent stimuli (where both hands were required to respond), the right hand was slightly but consistently faster than the left hand. The right hand also accounted for 83% of the errors made. 4. Stimulus-synchronized and response-synchronized ERPs to either frequent or rare stimuli had a similar appearance for correct responses in both the compatible and noncompatible conditions. The coupling of the response to the ERP for the rare stimuli, however, was different for the two conditions: the response occurred later relative to the ERP components in the response-synchronized average in the noncompatible condition compared with the compatible condition. By contrast, the coupling of the ERPs to the onset of the stimulus was the same in the two conditions. 5. Stimulus-synchronized averages for error responses in which the rare tone was mistaken for a frequent tone showed early sensory processing (as judged by the ERPs) that was similar to that of correct responses to the rare stimuli. After the apparent positive (P2) component of the cerebral response, however, the processing differed, with a superimposed broad negativity possibly reflecting awareness by the subject that a mistake had been made. By contrast, the response-synchronized averages for these error trials appeared like those to frequent stimuli, with the response being coupled to the P2 component of the cerebral response. 6. These results suggest that response compatibility affects response selection processes but does not alter sensory discrimination. However, despite the similarly tight coupling of the response to the ERP in both the compatible and noncompatible conditions, the response occurred later relative to the ERPs in the noncompatible condition. This suggests that different components of the ERP are responsible for triggering the response in different circumstances. Our observations on the error trials suggests that the decision to respond (on these trials) is based on the occurrence of cerebral events that are evoked by either rare or frequent stimuli, whereas this decision (on correct response trials) is based on cerebral events elicited only by the rare stimuli.

Adult↗