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Geranylgeranyl diphosphate (GGPP) synthase was purified to homogeneity from bovine brain in a one-step affinity column procedure. For the construction of the affinity column, a farnesyl diphosphate (FPP) analog, CM6-amino-l-hexyl)-P- -farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affigel 10 via the amino group. The native enzyme appeared to be homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pi for the enzyme was 6.2. The Km values for dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) and FTP were estimated to be 33 uM, 0.80 uM and 0.74 uM, respectively. The Km value for isopentenyl diphosphate (IPP) in the presence of both IPP and FPP mixture was 2|iM. The ratio of the reaction velocity for formation of GGPP from DMAPP, GPP or FPP was 0.004:0.145:1. The intermediate FPP was formed in the reaction with GPP as an allylic primer. FPP synthase catalyzing the formation of FPP from DMAPP and IPP was also purified to homogeneity from the same organ by a similar affinity chromatography procedure using a GPP analog, 0-(6-amino-l-hexyl)- -P-geranylmethyl phosphonophosphate as a ligand. The enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that GGPP, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from DMAPP and IPP by the action of FPP synthase catalyzing the reactions C5->C15 followed by the action of GGPP synthase catalyzing the reaction C15->C20.
The neural bases of appetitive and aversive conditioning are different, and at various stages of learning may engage distinct cortical and subcortical networks. Using [14C]2-deoxyglucose (2-DG) autoradiography we examined brain activation in mice during classical conditioning involving stimulation of whiskers on one side of the muzzle paired with an aversive or appetitive UCS. Both variants result in modifi cation of cortical representations of vibrissae activated during the conditioning. Analysis of autoradiograms revealed that the nucleus basalis of Meynert (NBM) and ventral pallidum showed stronger labeling during appetitive training while the lateral hypothalamus (LH) and basolateral amygdala (BLA) were activated only by aversive learning. Apart from that, classicalconditioning with appetitive or aversive UCS increased 2-DG uptake in a similar set of brain structures – the posterior parietal cortex (PPC), cingulate (CG) and retrosplenial gyrus (RET), caudate nucleus (CPU) and nucleus accumbens (NA). Formation of sensory association, compared to pseudoconditioning, induces more activity in the subcortical sensory processing pathway (ventral postero-medial and posterior nuclei of the thalamus) but not in the barrel cortex. Also, conditioning contrasted with pseudoconditioning increases activity in structures important for cognitive and attentional functions (PPC, NA, CG, RET, CPU), which might provide the enhancing input necessary for memory trace formation.
To understand how sensory experiences are stored in the brain, we examined neuronal fi ring in hippocampus during tactile behavior. Rats learned to associate stimulus texture with reward location; multiple textures were associated with the same reward location and thus formed a behavioral category. Rapid fi ring rate modulation carried texture identity information (10% of neurons), free from spatial and behavioral confounds; slow fi ring rate modulation carried behavioral category information (63% of neurons). Category information appeared during texture contact, simultaneous with an increase in theta power in the local fi eld potential; it persisted or recurred during reward collection, when theta power was suppressed and “reward neurons” (8%) fi red. Reward-triggered recurrence of category information could be a mechanism to link stimulus, action, and outcome when separated in time.
GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic anti­depressants on its activity was studied. The results indicated that amitripyline and doxepin — derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine — derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are non- competitive inhibitors of the enzyme with respect to reduced glutathione and non- competitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.
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