Identification and purification of overlapping cosmid clones of the region Xq24-qter of human X chromosome using HPLC.

The assembly of a large physical map of genomes requires simultaneous analysis of many cosmid clones for overlapping regions. The search for overlapping regions may be achieved by various means. High-performance liquid chromatography (HPLC) provides an alternative to gel electrophoresis since microgram amounts of each DNA fragment may be collected into individual test tubes for further analysis. HPLC has been used to identify overlapping cosmid clones from a pool of cosmid DNA containing the terminal portion of the long arm of the human X chromosome (Xq24-qter). Among 400 cosmids analyzed, 3 were shown to overlap.     

Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

Methionine sulfoxide formation in proteins: NMR study

Summary ¹H-NMR spectra of bovine pancreatic trypsin inhibitor (BPTI) both native and oxidized by chloramine T, are reported. The spectrum of the oxidized form is characterized by the appearance of two singlets for methyl group shifted 0.60 and 0.46 ppm downfield with respect to the native form.     

Muscarinic Receptors Modulate Dopamine-Activated Adenylate Cyclase of Rat Striatum

We investigated the effect of acetylcholine (ACh) on the activation of adenylate cyclase by dopamine (DA) in a lysed synaptosomal preparation from rat striatum. ACh reduced both basal and the DA-activated adenylate cyclase with an apparent IC50 of approximately 1 microM. From a kinetic analysis it appeared that ACh reduced the Vmax for activation by DA but not the activation constant for DA. For most preparations the Vmax was reduced by 30-40%. The presence of atropine did not affect the activation of the enzyme by DA but it blocked the inhibition by ACh. Following 6-hydroxydopamine lesion of the nigrostriatal pathway, the enzyme became supersensitive to activation by DA and also more sensitive to inhibition by ACh. Inhibition of adenylate cyclase by ACh appeared to be rather specific for activation by DA, as ACh had no effect on activation of adenylate cyclase by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine. These results indicate that some striatal muscarinic and dopaminergic receptors are probably coupled to the same adenylate cyclase domain. Moreover, they suggest a biochemical model for the dynamic balance of cholinergic and dopaminergic neurons that innervate the striatum.     

An antibody cross-reacting with LA and FOUR antigens of theHL-A system

A volunteer was immunized by planned blood transfusions from an LA and FOUR incompatible donor. Among the several antibodies produced, an antibody population was found that cross-reacts with both LA and FOUR antigens; both these crossreacting determinants were present in the immunizing donor. Monospecificity of the antibody has been proven by adsorptions and elutions. Resistance to lysis experiments excluded the possibility that this antibody does not react against LA and FOUR, but reacts instead against an antigen belonging to another independentHL-A locus with alleles in strong linkage disequilibrium withLA andFOUR alleles. The following hypotheses are therefore formulated: a)LA andFOUR factors have structural analogies; b) LA and FOUR molecules lie in close proximity on the cell surface so that the appearance of a “hybrid” antigenic determinant becomes possible.     

Activation of adenosine A1 receptor by N6-(R)-phenylisopropyladenosine (R-PIA) inhibits forskolin-stimulated tyrosine hydroxylase activity in rat striatal synaptosomes

We investigated the effect of the relatively selective A1 adenosine receptor agonist N6-(R)-phenylisopropyladenosine (R-PIA) on tyrosine hydroxylase activity (TH) of synaptosomes obtained from rat striatum. TH activity was assayed in supernatant obtained following sonication and centrifugation of the tissue preincubated with the test compounds. R-PIA produced a modest decrease of basal enzyme activity, but significantly reduced the activation of the enzyme by submaximal (0.1-0.5 microM) concentrations of forskolin (FSK) a stimulator of adenylate cyclase. The IC 50 value of R-PIA was 17 nM and the maximal inhibition corresponded to 30-40% decrease of the enzyme activity stimulated by FSK. The S-isomer of PIA failed to affect TH activity under control and stimulated conditions. Moreover, the inhibitory effect of R-PIA was completely antagonized by 8-cyclopentyl- 1,3 -dimethylxanthine, an adenosine receptor blocker. R-PIA inhibited both basal and FSK-stimulated adenylate cyclase activity. These results indicate that in striatal dopaminergic terminals TH activity can be modulated in an inhibitory manner by activation of presynaptic A1 adenosine receptors.     

Vasoactive Intestinal Peptide and Forskolin Stimulate Interleukin 6 Production by Rat Cortical Astrocytes in Culture via a Cyclic AMP-Dependent, Prostaglandin-Independent Mechanism

Abstract: In this study we analyzed the involvement of the cyclic AMP (cAMP)-protein kinase A system in the regulation of interleukin 6 production by cultured cortical astrocytes. Vasoactive intestinal peptide strongly increased, in a dose-dependent manner, interleukin 6 production. This effect was reduced when protein kinase A was blocked by KT-5720; it was not affected by calphostin C, a protein kinase C inhibitor. Forskolin caused a concentration-dependent increase in interleukin 6 release that was also inhibited by KT-5720. Because prostaglandins are believed to play a role in interleukin 6 production, we tried to determine whether the stimulatory effects of vasoactive intestinal peptide and forskolin on cytokine release might be mediated by stimulation of prostaglandin production in cortical astrocytes. Vasoactive intestinal peptide did not increase the production of either prostaglandin E₂ or F_2α. Conversely, forskolin concentration-dependently stimulated the production of both prostaglandins, an effect that was blocked by indomethacin. Indomethacin did not affect either vasoactive intestinal peptide- or forskolin-stimulated interleukin 6 production. To exclude the possibility that prostaglandins participate in interleukin 6 production induced by forskolin, we tested prostaglandins E₂ and F_2α. The former was completely ineffective in eliciting the cytokine production, whereas prostaglandin F_2α slightly increased interleukin 6 production only at the highest concentrations. 8-Bromo-cAMP and dibutyryl-cAMP stimulated interleukin 6 production to a lesser extent than vasoactive intestinal peptide and forskolin. In conclusion, we provide evidence that vasoactive intestinal peptide increases interleukin 6 production by astrocytes through the stimulation of the cAMP-protein kinase A pathway, an effect that is reproduced by cAMP analogues. In addition, we point out that prostaglandins are not involved in vasoactive intestinal peptide- and forskolin-mediated induction of interleukin 6 production in cultured astrocytes.     

Bimodal regulation of cyclic AMP by muscarinic receptors. Involvement of multiple G proteins and different forms of adenylyl cyclase

In membranes of rat olfactory bulb, muscarinic receptor agonists stimulate basal adenylyl cyclase activity . This response is inhibited by a number of muscarinic receptor antagonists with a rank order of potency suggesting the involvement of the M4 muscarinic receptor subtype. The stimulatory effect does not require Ca2+ and occurs independently of activation of phosphoinositide hydrolysis. Pretreatment with pertussis toxin completely prevents the muscarinic stimulation of adenylyl cyclase, indicating the participation of G proteins of the Gi/Go family. Immunological impairment of the G protein, Gs, also reduces the muscarinic response, whereas concomitant activation of Gs-coupled receptors by CRH or VIP results in a synergistic stimulation of adenylyl cyclase activity. Although these data suggest a role for Gs, a body of evidence indicates that the muscarinic receptors do not interact directly with this G protein. Moreover, the Ca2+/calmodulin (Ca2+/CaM)- and forskolin-stimulated enzyme activities are inhibited by muscarinic receptor activation in a pertussis toxin-sensitive manner and with a pharmacological profile similar to that observed for the stimulatory response. These data indicate that in rat olfactory bulb M4 muscarinic receptors exert a bimodal control on cyclic AMP formation through a sequence of events that may involve activation of Gi/Go proteins, synergistic interaction with Gs and differential modulation of Ca2+/CaM-independent and -dependent forms of adenylyl cyclase.