ABSTRACT
Corticotropin-releasing factor is a neuropeptide associated with the integration of physiological and behavioural responses to stress and also in the modulation of affective state and drug reward. The selective, centrally acting corticotropin-releasing factor type 1 receptor antagonist, antalarmin, is a potent anxiolytic and reduces volitional ethanol consumption in Fawn-Hooded rats. The efficacy of antalarmin to reduce ethanol consumption increased with time, suggestive of adaptation to reinforcement processes and goal-directed behaviour. The aim of the present study was to examine the effects of chronic antalarmin treatment on reward-related regions of Fawn-Hooded rat brain. Bi-daily antalarmin treatment (20 mg/kg, i.p.) for 10 days increased tyrosine hydroxylase messenger RNA expression throughout the ventral mesencephalon. Following chronic antalarmin the density of dopaminergic terminals within the basal ganglia and amygdaloid complex were reduced, as was dopamine transporter binding within the striatum. Receptor autoradiography indicated an up-regulation of dopamine D2, but no change in D1, binding in striatum, and Golgi-Cox analysis of striatal medium spiny neurones indicated that chronic antalarmin treatment increased spine density. Thus, chronic antalarmin treatment modulates dopaminergic pathways and implies that chronic treatment with drugs of this class may ultimately alter postsynaptic signaling mechanisms within the basal ganglia.
Subject(s)
Brain/drug effects , Corticotropin-Releasing Hormone/metabolism , Dopamine/metabolism , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Reward , Amygdala/drug effects , Amygdala/metabolism , Animals , Basal Ganglia/drug effects , Basal Ganglia/metabolism , Brain/metabolism , Brain/physiopathology , Dendritic Spines/drug effects , Dendritic Spines/metabolism , Drug Administration Schedule , Male , Mesencephalon/drug effects , Mesencephalon/metabolism , Neural Pathways/drug effects , Neural Pathways/metabolism , Presynaptic Terminals/drug effects , Presynaptic Terminals/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Receptors, Corticotropin-Releasing Hormone/metabolism , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D1/metabolism , Reinforcement, Psychology , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Tyrosine 3-Monooxygenase/genetics , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
The ability to display functional T-cell receptors (TCR) on the surface of bacteriophage could have numerous applications. For instance, TCR phage-display could be used to develop new strategies for isolating TCRs with unique specificity or it could be used to carry out mutagenesis studies on TCR molecules for analyzing their structure-function. We initially selected a TCR from the murine T-cell hybridoma, DO11.10, as our model system, and genetically engineered a three domain single-chain TCR (scTCR) linked to the gene p8 protein of the Escherichia coli bacteriophage fd. Immunoblotting studies revealed that (1) E. coli produced a soluble scTCR/p8 fusion protein and (2) the fusion protein was packaged by the phage. Cellular competition assays were performed to evaluate the functionality of the TCR and showed the DO11.10 TCR-bearing phage could significantly inhibit stimulation of DO11.10 T hybridoma cells by competing for binding to immobilized MHC/peptide IA(d)/OVA(323-339). Flow cytometric analysis was carried out to evaluate direct binding of DO11.10 TCR-bearing phage onto the surface of cells displaying either IAd containing irrelevant peptide or OVA peptide. The results revealed binding of DO11.10 TCR-bearing phage only on cells expressing IA(d) loaded with OVA peptide showing TCR fine specificity for peptide. To illustrate the generality of TCR phage-display, we also cloned and displayed on phage a second TCR which recognizes a peptide fragment from human tumor suppressor protein p53 restricted by HLA-A2. These findings demonstrate functional TCR can be displayed on bacteriophage potentially leading to the development of novel applications involving TCR phage-display.
Subject(s)
Inovirus/metabolism , Receptors, Antigen, T-Cell, alpha-beta/physiology , Animals , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Escherichia coli/virology , Humans , Hybridomas , Inovirus/genetics , Mice , Rats , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/genetics , Substrate Specificity , Viral Fusion Proteins/biosynthesis , Viral Fusion Proteins/genetics , Viral Fusion Proteins/physiologyABSTRACT
The temperature coefficient of the δ(13)C value over the temperature range 14 to 40° has been measured for plants with different photosynthetic pathways. C3 plants (6 species) had a temperature coefficient of-0.0125/(o), C3 plants (5 species)-0.046/(o), "C4-like" CAM (2 species)-0.13/(o) and "C3-like" CAM (2 species)-0.019/(o). The relative temperature insensitivity of the δ(13)C value is in contrast with the measured temperature dependence of δ(13)C values for ribulose-diphosphate carboxylase in vitro, and to the temperature response of photosynthesis. The temperature sensitivity of the "C4-like" CAM plants was the relatively greatest; this is consistent with the evidence that high night temperature inhibits dark CO2 fixation. The same inhibition may occur in the "C3-like" CAM plants but it would not have as significant an effect on the δ(13)C value as these plants are primarily dependent on primary fixation of exogenous CO2 via the ribulose-diphosphate carboxylase reaction.