Biomedical subjects

V G Wilson

Publications and source records attributed to V G Wilson.

At least 55 records · Page 3Linked to original sources

PubMed1995

Agmatine recognizes alpha 2-adrenoceptor binding sites but neither activates nor inhibits alpha 2-adrenoceptors.

It has been suggested that agmatine (decarboxylated arginine) is an endogenous clonidine-displacing substance (CDS) which recognizes alpha 2-adrenoceptor and non-adrenoceptor, imidazoline binding sites. We have examined the effect of agmatine at alpha 2-adrenoceptor binding sites and pre- and postjunctional alpha 2-adrenoceptors. Agmatine produced a concentration-dependent inhibition of 1 nmol/l 3H-clonidine binding to both rat (pKi-5.10 +/- 0.05) and bovine (pKi-4.77 +/- 0.38) cerebral cortex membranes. However, agmatine (0.1-100 microM) failed to activate pre-junctional alpha 2-adrenoceptors regulating transmitter release in the guinea-pig isolated ileum and rat isolated vas deferens, nor did it activate postjunctional alpha 2-adrenoceptors of the porcine isolated palmar lateral vein which mediate contraction or inhibition of forskolin-stimulated cyclic AMP formation. High concentrations of agmatine (10-30-fold the pKi at alpha 2-adrenoceptor binding sites) failed to influence alpha 2-adrenoceptor activation by either clonidine or UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate) in any of the peripheral preparations examined. Moreover, even in a preparation where an interaction with alpha 2-adrenoceptor binding sites on cell membranes can be demonstrated, the rat cerebral cortex, agmatine failed to inhibit forskolin-stimulated cyclic AMP in the intact tissue or affect the inhibition produced by the selective alpha 2-adrenoceptor agonist UK-14304. Agmatine was also devoid of agonist activity in two preparations, the rat isolated thoracic aorta and the rat isolated gastric fundus, in which CDS has been reported to produce non-adrenoceptor effects. Thus, we have confirmed that agmatine recognizes alpha 2-adrenoceptor binding sites and, therefore, is a CDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗

PubMed1995

Evidence for the presence of a non-catecholamine, clonidine-displacing substance in crude, methanolic extracts of bovine brain and lung.

In the present study we have prepared crude, methanolic extracts of bovine lung and bovine brain and, using radioligand binding assays in conjunction with a number of simple chromatographic techniques, provided evidence for the presence of a non-catecholamine 'clonidine-displacing substance' (CDS). The level of CDS in lung extracts (9 units/g wet weight n = 11) is approximately 3 times that in the brain extracts. Furthermore, the effect of the crude, methanolic extracts are selective for non-adrenoceptor, imidazoline (labelled by [3H]-idazoxan) and alpha 2-adrenoceptor binding sites (labelled by [3H]-clonidine); both extracts are 5-10-fold more potent displacers of ligand binding to alpha 2-adrenoceptors compared with binding to opiate receptors (labelled by [3H]-etorphine) and practically inactive against alpha 1-adrenoceptor and muscarinic binding sites (labelled by [3H]-prazosin and [3H]-quinuclidinyl benzilate, respectively). With the exception of the non-adrenoceptor, imidazoline binding assay, which used rat kidney membranes labelled by [3H]-idazoxan in the presence of the alpha 2-adrenoceptor antagonist RS-15385-197, all radioreceptor assays involved bovine cerebral cortex membranes. Although the extracts contain catecholamines (brain only), histamine (lung only) and monovalent cations (both), which have the potential to interfere with the radioligand binding assays, their concentrations were too low to account for the effects observed. Preliminary attempts at purification of the extracts revealed that CDS activities from the two tissues are similar, i.e., practically insoluble in organic solvents at room temperature, not affected by either Sep-Pak C18 column or anion exchange resins but retained (along with the monovalent cations) by cation exchange resin. However, following chromatographic separation on a Biogel P2 column, the CDS-containing eluates are cation-free and exhibit qualitatively similar elution profiles. Future experiments will involve further purification of 'clonidine-displacing substance' to characterize its interaction with alpha 2-adrenoceptor binding sites in greater detail and establish whether it has biological activity consistent with the properties implied by its effects in radioligand binding assays.

Animals↗

PubMed1995

Alpha 2-adrenoceptor mediated inhibition of forskolin-stimulated cyclic AMP accumulation in isolated porcine palmar lateral veins.

The aim of this study was to use a 3H-adenine pre-labelling technique to characterise the effect of alpha 2-adrenoceptor activation on forskolin-stimulated cyclic AMP accumulation in the isolated porcine palmar lateral vein. Forskolin (10(-7)-10(-4) M) stimulated 3H-cyclic AMP accumulation in the isolated porcine palmar lateral vein in a biphasic and concentration-dependent manner. In the absence of the cyclic AMP-selective phosphodiesterase inhibitor rolipram, forskolin stimulated 3H-cyclic AMP accumulation approximately 7-8 fold. The response reached a peak after 5 min. In the presence of rolipram (10(-5) M), basal 3H-cyclic AMP levels were approximately 70% higher than in its absence (basal: 1823 +/- 57 dpm; rolipram: 3088 +/- 229, n = 3) and forskolin (3 x 10(-5) M) stimulated 3H-cyclic AMP accumulation approximately 8 fold. The latter response reached a plateau 10 min after the addition of forskolin. In all subsequent studies, the tissues were incubated with forskolin (3 x 10(-5) M) for 5 min in the absence of rolipram. Noradrenaline (NA; 10(-9)-10(-4) M) and UK14304 (10(-9)-10(-4) M) inhibited forskolin-stimulated 3H-cyclic AMP accumulation in a concentration-dependent manner with mean pIC50 values of 7.61 +/- 0.37 (n = 4) and 7.76 +/- 0.23 (n = 5), respectively. With either NA or UK14304, the maximal inhibition of the forskolin response obtained was approximately 75%. Neither NA (10(-4) M) nor UK14304 (10(-4) M) altered basal 3H-cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Examination of the role of inhibition of cyclic AMP in alpha 2-adrenoceptor mediated contractions of the porcine isolated palmar lateral vein.

1. We have examined the effect of elevation of cellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on alpha 1- and alpha 2-adrenoceptor-mediated contraction of the isolated palmar lateral vein of the pig. Cellular cyclic AMP was increased by either inhibition of phosphodiesterase by rolipram, or direct activation of adenylyl cyclase by forskolin. 2. Noradrenaline (1 nM-10 microM) caused concentration-dependent contractions of the porcine isolated palmar lateral vein (pD2 7.32 +/- 0.07, n = 10). The selective alpha 1-adrenoceptor antagonist, prazosin (0.1 microM) and the selective alpha 2-adrenoceptor antagonist, rauwolscine (1 microM) caused a 10 fold rightward displacement of the concentration-response curve and a combination of the two antagonists caused a 200 fold rightward displacement of the concentration-response curve. The selective alpha 2-adrenoceptor agonist, UK-14304, also produced concentration-dependent contractions of the palmar lateral vein (pD2 7.70 +/- 0.15, n = 5), but the maximum response was 55.5 +/- 7.6% (n = 5) of that produced by noradrenaline. Prazosin (0.1 microM) failed to affect responses to UK-14304 but rauwolscine, 1 microM, caused a 200 fold rightward displacement. The estimated pKB value for rauwolscine (8.28 +/- 0.19, n = 10) is consistent with inhibition of alpha 2-adrenoceptors. Thus, the porcine isolated palmar lateral vein has a population of alpha 1- and alpha 2-adrenoceptors capable of producing a contraction. 3. Rolipram, 10 micro M, and forskolin, 1 micro M, caused a 2-3 fold rightward displacement of the noradrenaline concentration-response curve (CRC), but 1,9-dideoxyforskolin, 1 micro M, a forskolin analogue which does not activate adenylyl cyclase, failed to produce a significant inhibition of noradrenaline induced contractions. The combination of forskolin (1 micro M) and rolipram (10 micro M) were additive, producing a 20 fold rightward displacement of the noradrenaline CRC.4. Responses to noradrenaline were similarly affected by a combination of rolipram (10 micro M) and prazosin (0.1 micro M) (isolation of alpha 2-adrenoceptors) and the combination of rolipram (10 micro M) and rauwolscine(1 micro M) (isolation of alpha l-adrenoceptors), resulting in a 100 fold rightward displacement of the noradrenaline CRC. Although forskolin inhibited both alpha l- and alpha 2-adrenoceptor-mediated contractions,the effects produced were not similar. In particular, noradrenaline, 0.3-3 micro M, produced a significant contraction in the presence of forskolin (1 micro M) and prazosin (0.1 micro M) (an alpha 2-adrenoceptor-mediated response) but not in the presence of forskolin (1 micro M) and rauwolscine (1 micro M) (an alpha l-adrenoceptor mediated response).5. Five minute exposure to either rolipram (10 micro M) or forskolin (1 micro M) elevated [3H]-cyclic AMP of the porcine isolated palmar lateral vein by approximately 70% and 150-200%, respectively. Neither noradrenaline (1 nM- 100 micro M) nor UK-14304 (1 nM- 100 micro M) affected basal levels of [3H]-cyclic AMP,but both produced a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP accumulation with a pKi of 7.43 +/- 0.1 (n = 3) and 7.97 +/- 0.18 (n = 3), respectively. The effect of noradrenaline against forskolin-stimulated [3H]-cyclic AMP accumulation was reversed by rauwolscine(1 micro M) but not by prazosin (0.1 micro M). In contrast, alpha 2-adrenoceptor activation did not affect rolipram induced elevation of [3H]-cyclic AMP.6. These findings indicate that M2-adrenoceptor contractions of the porcine isolated palmar lateral vein are not produced by reduction in cellular cyclic AMP per se. It is proposed that this response involves a novel signal transduction mechanism. However, when cellular cyclic AMP has been elevated by agents that stimulate adenylyl cyclase, rather than through inhibition of phosphodiesterase, the ability of alpha 2-adrenoceptors to inhibit cyclic AMP formation may be of functional importance in vascular smooth muscle.

Adrenergic alpha-2 Receptor Agonists↗

PubMed1995

Comparison of the interaction of agmatine and crude methanolic extracts of bovine lung and brain with alpha 2-adrenoceptor binding sites.

1. In the present study we have evaluated whether alpha 2-adrenoceptor binding sites on bovine cerebral cortex membranes labelled by [3H]-clonidine, [3H]-idazoxan and [3H]-RX-821002 can distinguish between known agonists and antagonists. This model has then been used to compare the binding profiles of the putative non-catecholamine, clonidine-displacing substance (CDS), agmatine and crude methanolic extracts of bovine lung and brain. 2. Saturation studies carried out in the presence and absence of noradrenaline, 10 mumol 1(-1), revealed that the maximum number of binding sites on bovine cerebral cortex membranes for [3H]-idazoxan and [3H]-RX-821002 were approximately 60-80% greater than those for [3H]-clonidine (62.6 fmol mg-1 protein). Rauwolscine, the selective alpha 2-adrenoceptor antagonist, was approximately 100 fold more potent against each of the ligands than the selective alpha 1-adrenoceptor diastereoisomer, corynanthine. Also, the pKi value for the selective alpha 1-adrenoceptor prazosin against each ligand was less than 6. 3. Adrenaline, UK-14034, rauwolscine, corynanthine, RX-811059 and prazosin produced concentration-dependent inhibition of binding of all three 3H-ligands. The agonists, adrenaline and UK-14304, were approximately 5 and 10 fold less potent against [3H]-idazoxan and [3H]-RX-821002, respectively, than against [3H]-clonidine. In marked contrast, the antagonists, rauwolscine, corynanthine, RX-811059 and prazosin exhibited a different profile, being approximately 2-3 fold more potent against sites labelled by [3H]-RX-821002 and [3H]-idazoxan compared to sites labelled by [3H]-clonidine. 4. Agmatine and histamine produced a concentration-dependent displacement of [3H]-clonidine, [3H]-idazoxan and [3H]-RX-821002 binding to bovine cerebral cortex membranes. The pKi values for agmatine and histamine were independent of the 3H-ligand employed, approximately 4.8 and 4.5,respectively.5. Crude methanolic extracts of bovine brain and lung produced a concentration-dependent inhibition of [3H]-clonidine binding to bovine cerebral cortex membranes (>90%). Based on the volume of the extract that caused 50% inhibition of [3H]-clonidine binding, bovine lung contains 3 fold more CDS than bovine brain. Both extracts were at least 5 fold more potent against a2-adrenoceptor sites labelled by[3H]-clonidine than those labelled by [3H]-idazoxan and [3H]-RX-821002.6. All three 3H-ligands label the same population of alpha2-adrenoceptor binding sites on bovine cerebral cortex membranes, but [3H]-clonidine appears to label selectively the 'agonist' state of the sites: for which known agonists, adrenaline and UK-14304, exhibit a higher affinity. Our results indicate that neither agmatine nor histamine can account for the CDS activity present in crude extracts of bovine brain and lung. Moreover, these extracts appear to possess a binding profile similar to that of adrenaline and UK-14304, suggesting that they may possess agonist activity.

Adrenergic alpha-Agonists↗

PubMed1995

The relationship between density of alpha-adrenoceptor binding sites and contractile responses in several porcine isolated blood vessels.

1. The aim of this study was to investigate constrictor alpha-adrenoceptors in three isolated blood vessels of the pig, the thoracic aorta (TA), the splenic artery (SA) and marginal ear vein (MEV) and then compare the functional response with the densities of alpha 1- and alpha 2-adrenoceptor binding sites in these and several other porcine vascular tissues, palmar common digital artery (PCDA), palmar lateral vein (PLV) and ear artery (EA). 2. Noradrenaline (NA), phenylephrine (PE) and UK14304 (all at 0.03-10 microM) elicited concentration-dependent contractions in the TA and MEV, with a rank order of potency of UK14304 > NA > PE. UK14304 produced maximal responses which were 58% (TA) and 65% (MEV) of that of NA. In the SA, UK14304 and PE produced maximal responses which were less than 10% and 50% of the NA-induced maximal response respectively, with an order of potency of NA > PE. In the SA, NA-induced contractions were competitively antagonized by prazosin (pA2 = 8.60 +/- 0.15). Further, rauwolscine (1-10 microM) antagonized NA-induced contractions with an apparent pKB of 6.09 +/- 0.11 (n = 6), indicating an action at alpha 1-adrenoceptors. The combination of the two antagonists at concentrations selective for alpha 1- (0.1 microM) and alpha 2-adrenoceptors (1 microM) had no greater effect than either antagonist alone. This suggests that the SA expresses only post-junctional alpha 1-adrenoceptors. 3. In the TA, prazosin produced non-parallel shifts in the NA-induced CRC and this was also observed with rauwolscine, where reductions in the maximal responses were also observed. In the MEV, prazosin was largely inactive in antagonizing NA-induced contractions. In both these vessels a combination of these two antagonists had a greater effect than either alone, indicating the presence of functional alpha 1- and alpha 2-adrenoceptors. The post-junctional alpha 2-adrenoceptors in all of these vessels were resistant to prazosin, suggesting the alpha 2-adrenoceptor to be of the alpha 2A/2D subtype. The expression of functional alpha 2-adrenoceptors was MEV > TA > PLV > PCDA > SA. 4. In radioligand binding studies using TA P2 pellet membranes, [3H]-prazosin and [3H]-RX821002 ([1,4-[6,7(n)-3H] benzodioxan-2-methoxy-2-yl)-2-imidazole) labelled different high affinity sites, and in competition studies using identical membranes corynanthine displaced [3H]-prazosin with 10 fold higher affinity than rauwolscine, indicating that [3H]-prazosin was selectively binding to alpha 1-adrenoceptor sites. Further, rauwolscine displaced [3H]-RX821002 with approximately 100 fold greater affinity compared to corynanthine, which is indicative of selective alpha2-adrenoceptor binding.5. Separation of the P2 pellet into plasma membrane and mitochondrial fractions was carried out using a differential sucrose density gradient. [3H]-prazosin and [3H]-RX821002 binding sites were found in both the plasma membrane and mitochondrial fractions.6. In saturation studies all tissues produced single site saturation curves with no difference in the Kd(range 0.13-0.20nM) of the alpha1-adrenoceptor sites for [3H]-prazosin. However, there was considerable variation in Bmax of alpha 1-adrenoceptor sites; the highest density was found in the TA (397.9 =/- 52.7 fmol mg-1, n = 4), followed by the PCDA (256.7 +/- 22.7 fmol mg-1, n = 4), the PLV and SA having approximately equal density (143.6 +/- 3.9 and 159.1 +/- 7.0 fmol mg-1 respectively, n = 4 for both), followed bythe EA (91.3 +/- 10.5 fmol mg-1, n = 3) and the MEV had the lowest density (48.9 +/- 11.4 fmol mg-1,n = 3).7. In saturation studies using [3H]-RX821002, all tissues produced single site saturation curves with no differences in the Kd values (range 1.31 +/- 2.16 nM) but the highest densities were found in the TA and MEV (545.3 +/- 36.2 and 531.0 +/- 40.9 fmol mg-1 respectively), followed by the PLV (418.4 +/- 39.4 fmol mg-1), then the EA (266.3 +/- 40.0 fmol mg-1), and low densities of [3H]-RX821002 binding being found in the PCDA and SA (155.9 +/- 18.1 and 117.5 +/- 19.3 fmol mg-1 respectively).8. The pattern of binding site distribution for alpha l- and alpha 2-adrenoceptors is in reasonable agreement with functional studies carried out in these porcine vascular tissues; the TA has the highest densities of alpha 1-and alpha2-adrenoceptors; in the SA and PCDA there is a predominance (although small) of alpha l-adrenoceptor binding sites, the reverse of which is observed both in the PLV and MEV (i.e. greater density of alpha2-adrenoceptor sites). Thus, it would appear that alpha 1- and alpha2-adrenoceptor densities play a role in the expression of functional responses via these receptor subtypes; although it is interesting to note that the SA did have a small density of alpha 2-adrenoceptor binding sites, no functional response was observed after alpha2-adrenoceptor activation.

5'-Nucleotidase↗

PubMed1995

Pharmacological characterization of noradrenaline-induced contractions of the porcine isolated palmar lateral vein and palmar common digital artery.

1. The aim of this study was to examine the pharmacological characteristics of alpha-adrenoceptor-mediated contractions in two porcine isolated blood vessels, the palmar lateral vein (PLV) and the palmar common digital artery (PCDA). This was carried out with noradrenaline used as the agonist throughout, and either phentolamine (non-selective alpha-adrenoceptor antagonist), prazosin and YM-12617 (selective alpha 1-adrenoceptor antagonists) or rauwolscine and CH-38083 (selective alpha 2-adrenoceptor antagonists). 2. Noradrenaline (0.003-10 microM) produced concentration-dependent contractions in both vessels, with the PCDA (pD2 = 6.33 +/- 0.07, n = 10) being approximately 10 fold less sensitive to noradrenaline compared to the PLV (pD2 = 7.39 +/- 0.09, n = 8). Also, the maximal response to noradrenaline was greater in the PCDA compared to the PLV. Phentolamine (0.03-30 microM) produced parallel rightward shifts in the CRC to noradrenaline in both tissue preparations. The pA2 values were similar and slopes of the Schild plots were not significantly different from unity, indicating an interaction between phentolamine and a single receptor in each preparation. 3. In the PCDA the alpha 1-adrenoceptor antagonists, prazosin (0.01-1 microM) and YM-12617 (0.01-1 microM) produced non-parallel rightwards shifts in the CRC to noradrenaline, with the lower 10-15% of the CRC exhibiting greater resistance to the effects of these antagonists compared to the upper part. In contrast, rauwolscine (1-10 microM) and CH-38083 (10 microM) produced parallel displacement of the CRC to noradrenaline. In the PLV, low concentrations of either alpha l- (0.01 microM) or alpha2-adrenoceptor antagonists(0.1-1 microM) produced a large shift in the CRC, but subsequent higher concentrations had only small additional effects. Based upon pKB values estimated from the effects of the lower concentrations of antagonists, the results are consistent with a large population of alpha1-adrenoceptors in the PCDA and a mixture of alpha l- and alpha2-adrenoceptors in the PLV.4. In both tissues, when an ac,- and an a2-adrenoceptor antagonist were used in combination the effect produced was greater than that with either agent alone. In contrast, the combination of the alpha1-adrenoceptor antagonists (prazosin and YM-12617 together) or the alpha2-adrenoceptor antagonists (CH-38083 and rauwolscine together) were no more effective than that produced by the individual antagonists. These findings suggest the presence of functional alpha l- and alpha2-adrenoceptors in the PLV andPCDA.5. Phenoxybenzamine (0.3-3 microM, 60min exposure) produced a concentration-dependent reduction in the maximal response to noradrenaline which was more pronounced in the PCDA than the PLV. After a 60 min exposure to a combination of phenoxybenzamine (1 microM) and rauwolscine (1 microM), the remaining NA-induced contraction after washout was resistant to prazosin (0.1 microM) and sensitive to rauwolscine(1 microM) in both tissue preparations, indicating the existence of functional alpha2-adrenoceptors in both vessels.6. Evidence suggests that post-junctional alpha l- and alpha2-adrenoceptors contribute to noradrenaline-induced contractions in the PCDA and PLV, with the latter possessing a larger population of functional alpha2-adrenoceptors.

Adrenergic alpha-Antagonists↗

PubMed1995

Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.

Adenovirus E1A Proteins↗

PubMed1994

Overnight storage of the porcine isolated splenic artery enhances endothelium-dependent contractions to NG-nitro-L-arginine methyl ester without impairing endothelium-dependent dilator function.

This study examined the influence of overnight storage on endothelium-independent contractions to 5-hydroxytryptamine (5-HT), endothelium-dependent contractions to NG-nitro-L-arginine methyl ester (L-NAME), and endothelium-dependent relaxations to substance P (SP) and L-arginine, using the porcine isolated splenic artery. In endothelium-intact (E+) segments from fresh porcine isolated splenic arteries or segments from the same vessels stored overnight at 4 degrees C, either in Krebs-Henseleit saline or in Krebs-Henseleit saline containing 1 mM L-arginine, 5-HT caused concentration-related contractions that were similar under all three conditions. Overnight storage enhanced contractions of the splenic artery to L-NAME, an effect not observed if the vessels were co-stored with 1 mM L-arginine. L-NAME failed to contract endothelium-denuded (E-) segments from fresh tissues or tissues stored overnight, indicating that its constrictor effects were endothelium-dependent. SP caused concentration-related, endothelium-dependent relaxations of the splenic artery that were inhibited by 100 microM L-NAME, indicating that the relaxations could be attributed to the stimulated release of NO from endothelial cells. Established contractions to 100 microM L-NAME in E+ segments from fresh tissues, or segments from the same tissues stored overnight at 4 degrees C, either in Krebs-Henseleit saline or in Krebs-Henseleit saline containing 1 mM L-arginine, were all reversed by 1 mM L-arginine to similar extents, indicating that overnight storage did not affect endothelium-dependent dilator responses to L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

The bovine papillomavirus type 1 genome contains multiple loci of static DNA bending, but bends are absent from the functional origin of replication.

Twenty-four overlapping restriction fragments spanning the entire bovine papillomavirus type 1 (BPV-1) genome were analyzed by electrophoresis to determine the extent of static DNA bending in the BPV-1 genome. Thirteen of 24 fragments contained static bends. Based on known locations of previously mapped bend loci and the overlapping pattern of these 13 fragments, we estimate that there are 8-11 distinct static bend loci in the BPV-1 genome. The bend loci were not uniformly distributed on the genome and with one exception, were clustered from nucleotides 5816 to 2621 on the BPV-1 map. This portion of the BPV-1 genome contains most of the transcriptional regulatory sequences as well as the origin of replication. The concordance between the genomic distribution of DNA bends and cis-active elements is consistent with the possibility that bent sequences may contribute to the function of at least some of these elements. However, unlike SV40, there was no static bend at that functional origin of replication for BPV-1. The nearest bends to the origin were approximately 120 bp to the 5' side and 300 bp to the 3' side. As both of these bends were outside of the sequences required for origin function, it is unlikely that static bending plays a critical role in BPV-1 replication.

Base Sequence↗

PubMed1994

Genetically defined nuclear localization signal sequence of bovine papillomavirus E1 protein is necessary and sufficient for the nuclear localization of E1-beta-galactosidase fusion proteins.

The 605 amino acid E1 protein of bovine papillomavirus type 1 (BPV-1) is a multifunctional nuclear protein required for viral DNA replication. A nuclear localization signal (NLS) sequence was previously defined by point mutations in three short adjacent clusters of basic amino acids located in the amino-terminal region of the E1 protein. In this study, we used a fusion protein approach to evaluate the contribution of other regions of the E1 protein to nuclear transport. The nearly full-length E1 gene and six non-overlapping subfragments were each fused in-frame with the lacZ gene in a eukaryotic expression vector. Each clone was electroporated into COS-1 cells, and the intracellular location of the E1-beta-galactosidase fusion proteins was determined by immunofluorescence. Only the constructs containing the full-length E1 or a single subregion (E1-259; amino acids 84 to 166) produced fusion proteins that entered the nucleus. Point mutations in the NLS sequences of the E1-259-lacZ construct prevented nuclear translocation of the corresponding fusion protein. This confirms the previous result that the cluster of basic amino acids is critical for nuclear transport. Furthermore, the data obtained in this investigation indicated that the region of E1 containing the NLS sequence was not only necessary, but was also sufficient for nuclear localization. No other region of E1 contained independent nuclear localization activity.

Amino Acid Sequence↗

PubMed1994

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.

Base Sequence↗

PubMed1993

Endothelium-dependent contractions to NG-nitro-L-arginine methyl ester in the porcine isolated splenic artery are sensitive to cyclooxygenase and lipoxygenase inhibitors.

The nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), produced large endothelium-dependent contractions in isolated segments of the porcine splenic artery, equivalent to approximately 30% of the maximum responses to 5-hydroxytryptamine (5-HT). These responses were inhibited by 1mM L-arginine, but not by either 1mM D-arginine or the superoxide anion scavenger, superoxide dismutase. However, L-NAME-induced contractions were markedly inhibited by the cyclooxygenase inhibitor, flurbiprofen, and the lipoxygenase inhibitor, 2,3,5-tri-methyl-6-(12-hydroxy-5,10-dodecadiynyl)1,4-benzoquinone (AA-861). The combined cyclooxygenase and lipoxygenase inhibitor 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW-755C) abolished L-NAME-induced contractions. These findings suggest that suppression of endothelial nitric oxide synthase in the porcine isolated splenic artery results in activation of arachidonic metabolism and production of vasoconstrictor eicosanoids.

Amino Acid Oxidoreductases↗

PubMed1993

Species-selective binding of [3H]-idazoxan to alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in the central nervous system.

1. We have used the imidazoline derivative [3H]-idazoxan to define alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in cerebral cortex membranes of calf, mouse, rat, guinea-pig and man. 2. Competition experiments using the selective alpha-adrenoceptor drugs, rauwolscine and corynanthine, indicated that [3H]-idazoxan bound to a single population of sites in the calf and mouse membranes. However, [3H]-idazoxan also labelled non-adrenoceptor, imidazoline binding sites in the rat (15%), guinea-pig (30%) and human (40%) cerebral cortex membranes. 3. Competition experiments with adrenaline and cirazoline in the guinea-pig cortex, verified [3H]-idazoxan binding to both alpha 2-adrenoceptors and to non-adrenoceptor, imidazoline binding sites. 4. It has been postulated by several groups that [3H]-idazoxan may possess partial agonist activity. To investigate this further, saturation experiments were performed in the cerebral cortex membranes of all five species in the absence and presence of 300 microM guanosine triphosphate (GTP). GTP had no effect on [3H]-idazoxan binding in guinea-pig cerebral cortex; in both rat and mouse membranes 300 microM GTP increased the dissociation constant for [3H]-idazoxan by 2-3 fold without significantly affecting the Bmax. GTP reduced the Bmax by approximately 30% and 60% in calf and human cerebral cortex membranes, respectively, without significantly altering the Kd. 5. Saturation experiments were performed in the calf cerebral cortex membranes in the absence and presence of 300 microM GTP with the selective alpha 2-adrenoceptor agonist [3H]-clonidine and the selective muscarinic antagonist [3H]-quinuclidinyl benzilate (QNB). GTP reduced the Bmax for [3H]-clonidine without altering the Kd, but failed to affect either the Bmax or the Kd for [3H]-QNB.6. Saturation experiments were performed in human cerebral cortex membranes in the presence of alpha2-adrenoceptor blockade with and without GTP. GTP 300 microM reduced the Bmax for [3H]-idazoxan at the non-adrenoceptor, imidazoline binding sites, without affecting the Kd. GTP did not affect [3H]-QNB binding to muscarinic sites.7. Thus, there is a need to investigate further the pharmacological actions of [3H]-idazoxan in view of its ability to recognise both alpha2-adrenoceptors and non-adrenoceptor, imidazoline binding sites and because it might possess agonist activity at some of these sites.

Adrenergic alpha-Antagonists↗

PubMed1993

Evidence for functional dissociation of dependence and tolerance in guinea-pig isolated ileal segments following 20 hour exposure to morphine in vitro.

1. In the present study we have examined the relationship between tolerance and dependence in isolated ileal segments from the guinea-pig under three different conditions: fresh preparations not previously exposed to morphine (fresh/morphine naive); preparations stored overnight at 4 degrees C in modified Krebs-Henseleit saline (overnight-stored/morphine-naive); preparations stored overnight at 4 degrees C in Krebs-Henseleit saline containing 10 microM morphine and extensively washed with modified Krebs-Henseleit saline to remove residual morphine (overnight-stored/morphine-exposed). 2. Morphine produced a concentration-dependent inhibition of the response of ileal segment to 0.1 Hz, 1 ms and 10 V transmural field stimulation in fresh/morphine-naive, overnight-stored/morphine-naive and overnight-stored/morphine-exposed preparations. The maximum effect observed was similar in all three preparations-approximately 80% inhibition. Although, morphine was significantly more potent in the fresh/morphine-naive preparations (pD2 6.72 +/- 0.05, n = 8) than either the overnight-stored/morphine-native (pD2 6.42 +/- 0.11, n = 8) or the overnight-stored/morphine-exposed (pD2 6.44 +/- 0.14, n = 8), there was no significant difference between the overnight exposure to ileal segments to 10 microM morphine at 4 degrees C failed to induce tolerance to morphine. 3. The mu opiate receptor antagonist, naloxone (10 microM), produced contractions in both fresh/morphine-naive and overnight-stored/morphine-naive ileal segments following acute exposure to 10 microM morphine. Naloxone (10 microM) also produced contractions in 2/9 fresh/morphine-naive, 1/9 overnight-stored/morphine-naive and 7/9 overnight-stored/morphine-exposed preparations in the absence of morphine. The greater incidence of naloxone-induced contractions in overnight-stored/morphine-exposed preparations,suggests that dependence in this model is the product of adaptive changes that outlive the presence of morphine.4. The selective alpha2-adrenoceptor agonists, clonidine (0.3 microM) and 5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304, 1 microM), inhibited naloxone-induced contractions in overnight-stored/morphine-exposed preparations of ileal segments (n = 4 preparations for each agonist), suggesting that the response is due to transmitter release from the myenteric plexus.5. The findings in the present study indicate that tolerance and dependence to morphine in ileal segments of the guinea-pig can be functionally dissociated by overnight exposure to morphine at 4 degrees C.The development of tolerance to morphine, unlike dependence, appears to be a temperature-dependent process. This also raises the possibility that naloxone possesses intrinsic negative agonism at morphine sensitive receptors, which is manifested as a functional response only after adaptive changes in the myenteric plexus following exposure to morphine.

Animals↗

PubMed1993

Organ distribution of avian polyomavirus DNA and virus-neutralizing antibody titers in healthy adult budgerigars.

Tissue specimens and serum samples obtained from adult budgerigars in various stages of reproduction housed in an aviary with enzootic avian polyomavirus (APV) disease were examined by means of polymerase chain reaction techniques for APV DNA. Although the birds were apparently healthy, APV DNA could be detected in all 40 birds examined (inapparent infection rate, 100%). Viral DNA was found in most organ systems examined. Analysis of data suggested that organ virus concentrations were lower in breeding than in nonbreeding birds. Serum samples from 144 birds were examined for virus-neutralizing (VN) antibody. All serum samples had detectable VN antibody titers. Determining VN titer had a sensitivity of 100% for detection of APV infection in birds and was more sensitive than analysis of droppings by use of polymerase chain reaction techniques to detect APV infection in 6-month-old birds. Analysis of the data suggested that lower VN antibody titers were associated with longer duration of continuous breeding.

Animals↗

PubMed1992

Partite expression of the bovine papillomavirus E1 open reading frame in Escherichia coli.

Six recombinants were constructed which expressed portions of the bovine papillomavirus E1 open reading frame as OmpF/E1/beta-galactosidase tribrid fusion proteins in Escherichia coli. Rabbit sera containing E1-specific antibodies were generated against five of these six fusion proteins (which together constitute 74% of the full-length E1 open reading frame). The individual fusion proteins and their cognate antisera will be useful reagents for defining the structure and function of the BPV E1 protein(s).

Bacterial Outer Membrane Proteins↗

PubMed1992

Evidence for direct vasoconstrictor activity of melatonin in "pressurized" segments of isolated caudal artery from juvenile rats.

Responses of isolated, 60 mmHg 'pressurized' segments of the distal caudal artery of adult and juvenile Wistar rats to melatonin and the selective alpha 2-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304) were examined using the Halpern pressure myograph. Melatonin showed no direct vasoconstrictor activity in vessels from adult rats, whereas UK-14304 produced moderate vasoconstriction (pD2-7.43 +/- 0.09). In the presence of phenylephrine-induced tone, melatonin produced a variable but small constrictor response (less than 10 microns reduction in diameter) in some vessels; the response to 1 mumol/l UK-14304 was less than in the absence of tone. In vessels isolated from juvenile rats, melatonin caused concentration-dependent vasoconstriction with a maximum response about 70% of the maximum response elicited by UK-14304. Vessels from juvenile rats were more sensitive to melatonin (pD2-9.40 +/- 0.07) than they were to UK-14304 (pD2-8.12 +/- 0.14). In the presence of phenylephrine-induced tone, the vasoconstrictor responses to both melatonin and IK-14304 were markedly less; the sensitivity to melatonin was not different from that seen in the absence of tone. These findings indicate that 'pressurized' segments of the isolated distal caudal artery may provide a simple and convenient, functional model of melatonin receptors. The findings also appear to implicate melatonin in thermoregulatory processes in juvenile rats.

Aging↗