Mutations in ASIP and MC1R: dominant black and recessive black alleles segregate in native Swedish sheep populations.

By studying genes associated with coat colour, we can understand the role of these genes in pigmentation but also gain insight into selection history. North European short-tailed sheep, including Swedish breeds, have variation in their coat colour, making them good models to expand current knowledge of mutations associated with coat colour in sheep. We studied ASIP and MC1R, two genes with known roles in pigmentation, and their association with black coat colour. We did this by sequencing the coding regions of ASIP in 149 animals and MC1R in 129 animals from seven native Swedish sheep breeds in individuals with black, white or grey fleece. Previously known mutations in ASIP [recessive black allele: g.100_105del (D5 ) and/or g.5172T>A] were associated with black coat colour in Klövsjö and Roslag sheep breeds and mutations in both ASIP and MC1R (dominant black allele: c.218T>A and/or c.361G>A) were associated with black coat colour in Swedish Finewool. In Gotland, Gute, Värmland and Helsinge sheep breeds, coat colour inheritance was more complex: only 11 of 16 individuals with black fleece had genotypes that could explain their black colour. These breeds have grey individuals in their populations, and grey is believed to be a result of mutations and allelic copy number variation within the ASIP duplication, which could be a possible explanation for the lack of a clear inheritance pattern in these breeds. Finally, we found a novel missense mutation in MC1R (c.452G>A) in Gotland, Gute and Värmland sheep and evidence of a duplication of MC1R in Gotland sheep.

The use of endogenous retroviruses as markers to describe the genetic relationships among local Swedish sheep breeds.

The aim of this study was to describe the genetic relationships among five Swedish sheep breeds using insertional polymorphisms of six endogenous Jaagsiekte retroviruses of sheep. Although the Swedish breeds were found to have genomes of 'primitive' origin, there also are indications of the presence of more recently derived sheep breeds within the ancestries of three of the breeds.

In vivo occupancy of dopamine D3 receptors by antagonists produces neurochemical and behavioral effects of potential relevance to attention-deficit-hyperactivity disorder.

Dopamine D(3) receptors have eluded definitive linkage to neurologic and psychiatric disorders since their cloning over 20 years ago. We report a new method that does not employ a radiolabel for simultaneously defining in vivo receptor occupancy of D(3) and D(2) receptors in rat brain after systemic dosing using the tracer epidepride (N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-iodo-2,3-dimethoxybenzamide). Decreases in epidepride binding in lobule 9 of cerebellum (rich in D(3) receptors) were compared with nonspecific binding in the lateral cerebellum. The in vivo occupancy of the dopamine D(3) receptors was dose dependently increased by SB-277011A (trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide) and U99194 (2,3-dihydro-5,6-dimethoxy- N,N-dipropyl-1H-inden-2-amine). Both antagonists increased extracellular levels of acetylcholine (ACh) in the medial prefrontal cortex of rats and modified brain-tissue levels of ACh and choline. Consistent with these findings, the D(3) receptor antagonists enhanced the acquisition of learning of rats either alone or in the presence of the norepinephrine uptake blocker reboxetine as with the attention-deficit-hyperactivity disorder (ADHD) drug methylphenidate. Like reboxetine, the D(3) receptor antagonists also prevented deficits induced by scopolamine in object recognition memory of rats. Mice in which the dopamine transporter (DAT) has been deleted exhibit hyperactivity that is normalized by compounds that are effective in the treatment of ADHD. Both D(3) receptor antagonists decreased the hyperactivity of DAT(-/-) mice without affecting the activity of wild type controls. The present findings indicate that dopamine D(3) receptor antagonists engender cognition-enhancing and hyperactivity-dampening effects. Thus, D(3) receptor blockade could be considered as a novel treatment approach for cognitive deficits and hyperactivity syndromes, including those observed in ADHD.

Genetic analysis of 31 Swedish type 1 von Willebrand disease families reveals incomplete linkage to the von Willebrand factor gene and a high frequency of a certain disease haplotype.

BACKGROUND: The most common type of von Willebrand disease (VWD), type 1, has in only a few cases been explained by an identified causative mutation in the von Willebrand factor (VWF) gene. The ABO blood group and other modifier loci outside the VWF gene may contribute to the development of type 1 VWD. OBJECTIVES AND METHODS: Our aim was to determine whether there was genetic linkage to the VWF gene in 31 Swedish type 1 VWD families. Stringent diagnostic criteria in accordance with ISTH guidelines were used. Genetic linkage was investigated by using two highly informative dinucleotide microsatellite markers, which we have recently identified, located in introns six and 15 of the VWF gene. We also investigated the existence of common disease haplotypes and the relation between type 1 VWD and ABO blood group. RESULTS: We found genetic linkage to the VWF gene in 27 (87%) of the families. However, in four (13%) of the families, there was clearly no genetic linkage. We found the 4751A>G (Tyr1584Cys) sequence variation in exon 28, which is a common mutation in the Canadian VWD population (14.3%), in only one of the 31 families (3.2%). A possible common mutation was identified in six of the 27 (22%) families with genetic linkage. Blood group O was over-represented among type 1 VWD patients. CONCLUSION: We conclude that there is linkage between the VWF gene and hereditary type 1 VWD in a majority of families.

Co-segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1.

Inherited deficiency of protein S constitutes an important risk factor of venous thrombosis. Many reports have demonstrated that causative mutations in the protein S gene are found only in approximately 50% of the cases with protein S deficiency. It is uncertain whether the protein S gene is causative in all cases of protein S deficiency or if other genes are involved in cases where no mutation is identified. The aim of the current study was to determine whether haplotypes of the protein S gene cosegregate with the disease phenotype in cases where no mutations have been found. Eight protein S-deficient families comprising 115 individuals where previous DNA sequencing had failed to detect any causative mutations were analyzed using four microsatellite markers in the protein S gene region. Co-segregation between microsatellite haplotypes and protein S deficiency was found in seven of the investigated families, one family being uninformative. This suggests that the causative genetic defects are located in or close to the protein S gene in a majority of such cases where no mutations have been found.

Novel (R)-2-amino-5-fluorotetralins: dopaminergic antagonists and inverse agonists.

A series of secondary and tertiary N-alkyl derivatives of (R)-2-amino-5-fluorotetralin have been prepared. The affinities of the compounds for [3H]raclopride-labeled cloned human dopamine (DA) D2 and D3 receptors as well as [3H]-8-OH-DPAT-labeled rat hippocampal 5-HT1A receptors were determined. In order to selectively determine affinities for the high-affinity agonist binding site at DA D2 receptors, the agonist [3H]quinpirole was used. The intrinsic activities of the compounds at DA D2 and D3 receptors were evaluated in a [35S]GTP gamma S binding assay. The novel compounds were characterized as dopaminergic antagonists or inverse agonists. The antagonist (R)-2-(butylpropylamino)-5-fluorotetralin (16) bound with high affinity (Ki = 4.4 nM) to the DA D3 receptor and was the most D3-selective compound (10-fold). (R)-2-[[4-(8-Aza-7, 9-dioxospiro[4.5]decan-8-yl)butyl]propylamino]-5-fluorote tralin (18) bound with very high affinity to both DA D3 and 5-HT1A receptors (Ki = 0.2 nM) and was also characterized as a dopaminergic antagonist. (R)-2-(Benzylpropylamino)-5-fluorotetralin (10) behaved as an inverse agonist at both DA D2 and D3 receptors. It decreased the basal [35S]GTP gamma S binding and potently inhibited the DA-stimulated [35S]GTP gamma S binding. It is apparent that the intrinsic activity of a 2-aminotetralin derivative may be modified by varying the N-alkyl substituents.

Resolved cis- and trans-2-amino-5-methoxy-1-methyltetralins: central dopamine receptor agonists and antagonists.

A series of 35 stereochemically well-defined C1-methyl-substituted derivatives of the potent dopamine (DA) receptor agonist 5-hydroxy-2-(di-n-propylamino)tetralin (5-OH-DPAT) have been synthesized. The compounds were tested for central DA receptor agonistic and antagonistic activity, by use of biochemical and behavioral tests in rats. In addition, the compounds were tested for in vivo interactions with 5,6-dihydroxy-2-(di-n-propylamino)tetralin (DiPr-5,6-ADTN). On the basis of pharmacological activity profiles, the active compounds have been classified into four groups: classical pre- and postsynaptic DA receptor agonists, DA receptor agonists with preferential action at presynaptic receptors, pre- and postsynaptic DA receptor antagonists, and DA receptor antagonists with preferential action at presynaptic receptors. Results obtained indicate that both 2R and 2S enantiomers of C5-oxygenated 2-aminotetralins may be able to bind to DA receptors but that only 2S antipodes are able to activate the receptors. O-Methylation of the C5-oxygenated (1S,2R)-2-amino-1-methyltetralin derivatives tends to increase their DA receptor antagonistic activity, whereas decrease of the size of the N-substituent(s) from n-propyl to ethyl or methyl appears to increase their activity at postsynaptic DA receptors.

Conformational analysis of the dopamine-receptor agonist 5-hydroxy-2-(dipropylamino)tetralin and its C(2)-methyl-substituted derivative.

The conformational preferences of the dopamine (DA) receptor agonist 5-hydroxy-2-(di-n-propylamino)tetralin (1) and the DA-inactive 5-hydroxy-2-methyl-2-(di-n-propylamino)tetralin (2) have been studied by use of molecular mechanics (MMP2) calculations and NMR spectroscopy. A good agreement is demonstrated between the experimentally determined (by NMR) and the calculated (by MMP2) conformational distribution of 1 and 2. In addition, there is a good agreement between bond distances and bond angles in the X-ray structure of the hydrobromide of 1 and those in the corresponding MMP2 conformation. Results obtained demonstrate that the energetically preferred conformations of 1 and 2 are different: Compound 1 preferentially adopts half-chair conformations with a pseudoequatorial nitrogen substituent whereas the low-energy conformations of compound 2 have a pseudoaxial nitrogen substituent. However, the results also indicate that the difference in conformational preferences is too small to account for the dopaminergic inactivity of 2. Therefore it is suggested that the steric bulk of the C(2)-methyl group per se prevents a proper alignment of (2S)-2 with DA receptors.

Derivatives of (R)-1,11-methyleneaporphine: synthesis, structure, and interactions with G-protein coupled receptors.

The design and synthesis of a well-characterized novel ring system, (R)-lambda1,11-methyleneaporphine [(R)-4], and 15 derivatives thereof are presented. The addition of various nucleophiles to (R)-lambda1,11-carbonylaporphine [(R)-11] or to the 1,11-hydroxymethyleneaporphine epimers gave separable mixtures of epimers. The epimeric ratios obtained in most reactions seem to be a result of steric factors directing the nucleophilic attack. The structure of the epimers was determined by a combination of X-ray crystallography (5 derivatives), NMR spectroscopy, and chemical correlation. Interesting and diverse pharmacological profiles of the derivatives were revealed through binding studies at serotonin 5-HT(7) and 5-HT(1A) receptors as well as at dopamine D(2A) receptors. Two derivatives appeared to be selective 5-HT(7) receptor antagonists. It is evident from our results that the novel ring system [(R)-4] provides a useful complement to other scaffolds available to medicinal chemists involved in studies of GPC receptors.

Derivatives of (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin: synthesis and interactions with 5-HT1A receptors.

Analogs of the 5-HT1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin [(S)-1,(S)-UH301] have been prepared. The C8-substituent has been varied, and in some derivatives one of the N-propyl groups has been exchanged for a 4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)-butyl group. The novel compounds have been evaluated for affinity to rat brain 5-HT1A receptors in competition experiments with [3H]-8-OH-DPAT. In addition, the efficacy of the compounds was assessed by their ability to inhibit the VIP-stimulated cAMP formation in GH4ZD10 cells expressing rat 5-HT1A receptors. Varying degrees of intrinsic activity was revealed among the compounds tested, i.e., the profiles ranged from full agonists to antagonists. All R-enantiomers are characterized as full agonists at 5-HT1A receptors, whereas partial agonists or antagonists were found among the corresponding S-enantiomers. Substitution of one of the N-propyl groups for a 4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)butyl group seems to increase efficacy as well as affinity for 5-HT1A receptors. A favorable interaction with an accessory binding site by the N-4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)butyl group may contribute to the increased affinity.

10-substituted 11-oxygenated (R)-aporphines: synthesis, pharmacology, and modeling of 5-HT1A receptor interactions.

Derivatives of the selective serotonin 5-HT1A receptor agonist (R)-11-hydroxy-10-methylaporphine (2) having various substituents in the C10-position or at the nitrogen have been synthesized from natural morphine or 6-O-acetylcodeine, respectively. The C10-substituents were introduced using efficient Stille or Suzuki cross-coupling reactions. The compounds were evaluated for their affinities to 5-HT1A and dopamine (DA) D1 and D2A receptors in vitro. All compounds tested displayed low (micromolar) affinities to D1 and D2A receptors. In addition, changes in steric bulk and/or electronic properties of the C10-substituent as compared to a C10-methyl group, as well as substitution of the N-methyl group for a hydrogen or a larger N-alkyl group, produced a marked decrease in the affinities to 5-HT1A receptors. Selected compounds that displayed moderate to high affinities to 5-HT1A receptors were evaluated for their ability to stimulate 5-HT1A receptors in vivo. The evaluated compounds behaved as agonists at 5-HT1A receptors, except for the N-propyl analogue of 2, (R)-11-hydroxy-10-methyl-N-propylnoraporphine (23), which displayed weak DA receptor agonism at the doses tested. Hence, the substitution pattern of 2 (a C10-methyl, a C11-hydroxy, and an N-methyl group) appears to be optimal for potent interaction of 10,11-disubstituted (R)-aporphines with 5-HT1A receptors. Modeling of ligand-5-HT1A receptor interactions was performed in an attempt to rationalize the observed affinity data. The binding site model suggests the presence of a "methyl pocket" in the 5-HT1A receptor binding ste. The C11-methoxy-substituted aporphines appear to have a different binding mode compared to 2, implying a different accessibility of these compounds to the "methyl pocket".

Synthesis and pharmacology of the enantiomers of cis-7-hydroxy-3-methyl-2-(dipropylamino)tetralin.

The enantiomers of cis-7-hydroxy-3-methyl-2-(dipropylamino)tetralin (3) have been synthesized and evaluated for activity at central dopamine (DA), 5-hydroxytryptamine, and norepinephrine (NE) receptors, by use of biochemical and behavioral tests in rats. In addition, the affinities of the compounds for striatal [3H]spiroperidol and [3H]N-propylnorapomorphine binding sites were determined. The absolute configuration of the enantiomers was determined by X-ray diffraction of (+)-3. The pharmacological effects of both enantiomers are complicated, but (2R,3S)-7-hydroxy-3-methyl-2-(dipropylamino)tetralin [(-)-3] produced biochemical effects in vivo similar to those elicited by classical DA D2-receptor antagonists.

Central dopaminergic and 5-hydroxytryptaminergic effects of C3-methylated derivatives of 8-hydroxy-2-(di-n-propylamino)tetralin.

A number of stereochemically well defined C3-methylated derivatives of the potent 5-hydroxytryptamine (5-HT) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) have been synthesized, and their stereochemical characteristics have been studies by use of NMR spectroscopy, X-ray crystallography, and molecular mechanics calculations. The compounds were tested for activity at central 5-HT and dopamine (DA) receptors, by use of biochemical and behavioral tests in rats. In addition, the ability of the cis- and trans-8-hydroxy-3-methyl-2-(di-n-propylamino)tetralins (15 and 11) to displace [3H]-8-OH-DPAT from 5-HT1A binding sites was evaluated. The stereoselectivity of the interaction of 11 and 15 with 5-HT receptors was much greater than that of 8-OH-DPAT. Observed rank order of potencies in the 5-HT1A binding assay corresponds to that in the in vivo biochemical assay.

C3-methylated 5-hydroxy-2-(dipropylamino)tetralins: conformational and steric parameters of importance for central dopamine receptor activation.

C3-Methyl-substituted derivatives of the potent dopamine (DA) receptor agonist 5-hydroxy-2-(di-n-propylamino)tetralin (5-OH-DPAT) have been synthesized and their conformational preferences have been studied by use of NMR spectroscopy, X-ray crystallography, and molecular mechanics calculations (MMP2). The compounds were tested for activity at central DA receptors, by use of biochemical and behavioral tests in rats. (2R,3S)-5-Hydroxy-3-methyl-2-(di-n-propylamino)tetralin [(-)-8] was demonstrated to be a highly potent DA receptor agonist, while the other new compounds were of low potency or inactive. Results obtained confirmed the hypothesis that the tetralin inversion angle phi and the direction of the N-electron pair (N-H) tau N are conformational parameters of critical importance for DA D2 receptor activation in the 2-aminotetralin series. The high potency of (-)-8 allowed an extension of a previously defined "partial DA D2 receptor excluded volume".

N,N-Dialkylated monophenolic trans-2-phenylcyclopropylamines: novel central 5-hydroxytryptamine receptor agonists.

N,N-Dialkylated monophenolic derivatives of trans-2-phenylcyclopropylamine were synthesized and tested for central 5-hydroxytryptamine (5-HT) and dopamine (DA) receptor stimulating activity by use of a biochemical test method in rats. A hydroxy substituent in the 2- or 3-position of the phenyl ring was required for 5-HT-receptor stimulation. N,N-Diethyl or N,N-di-n-propyl substitution gave the most potent 5-HT-receptor agonists. The 4-hydroxy and 3,4-dihydroxy derivatives of trans-2-phenyl-N,N-di-n-propylcyclopropylamine were inactive at central DA and 5-HT receptors. In contrast, the corresponding 3-hydroxy derivative 18 and some of its derivatives weakly affected both DA and NE synthesis. Two of the most potent 5-HT-receptor agonists, trans-2-(2-hydroxyphenyl)-N,N-di-n-propylcyclopropylamine (8) and the 3-hydroxy isomer 18 were resolved into the enantiomers. The 1R,2S enantiomers of 8 and 18 displayed 5-HT activity, while the 1S,2R enantiomers were inactive. Compound (1R,2S)-18, but not (1R,2S)-8, weakly affected rat brain DA and NE synthesis.

C1-Methylated 5-hydroxy-2-(dipropylamino)tetralins: central dopamine-receptor stimulating activity.

C1-Methylated derivatives of the potent dopaminergic agonist 5-hydroxy-2-(di-n-propylamino)tetralin (6) have been synthesized and tested for central dopamine (DA) receptor stimulating activity, by using biochemical and behavioral tests in rats. Both cis- and trans-5-hydroxy-1-methyl-2-(di-n-propylamino) tetralin (4 and 3) may be classified as central DA-receptor agonists, albeit of lower potency than 6. The results obtained indicate that both 4 and 3 display DA-autoreceptor stimulation capacity. However, only one of the isomers, trans-3, is able to elicit clear-cut postsynaptic DA receptor agonist actions at larger doses. 5-Hydroxy-1,1-dimethyl-2-(n-propylamino) tetralin (5) was found to be inactive.

8-Hydroxy-2-(alkylamino)tetralins and related compounds as central 5-hydroxytryptamine receptor agonists.

A series of 2-(alkylamino)tetralins related to 8-hydroxy-2-(di-n-propylamino)tetralin (21) were prepared and tested as dopamine (DA) and 5-hydroxytryptamine (5-HT) receptor agonists. Several of the compounds were potent 5-HT agonists devoid of DA-mimetic effects. N-Ethyl or N-propyl substitution of 8-hydroxy-2-aminotetralin gave the most potent agonists. It was shown that the most potent compound, (+)-21, has the 2R configuration. 5,8-Di-methoxy-2-(di-n-propylamino)tetralin (31) was found to be a weak DA agonist devoid of 5-HT activity. The corresponding indan derivative, 4,7-dimethoxy-2-(di-n-propylamino)indan (39), has been reported to be active on both DA and 5-HT receptors. The 5-HT-stimulating properties of compounds 21 and 39 as compared to the incapability of compound 31 to activate the 5-HT receptor is tentatively explained by the assumed mode of binding of the compounds to the 5-HT receptor.

Resolved monophenolic 2-aminotetralins and 1,2,3,4,4a,5,6,10b-octahydrobenzo[f]quinolines: structural and stereochemical considerations for centrally acting pre- and postsynaptic dopamine-receptor agonists.

A detailed structure-activity relationship is revealed for resolved, centrally acting dopamine (DA) agonists acting on both pre- and postsynaptic DA receptors. The compounds resolved are 5- and 7-hydroxy-2-(di-n-propylamino)tetralin and cis- and trans-7-hydroxy-4-n-propyl-1,2,3,4,4a,5,6,10b-octahydrobenzo [f]quinoline. By the superimposition of the structures of the more active enantiomers of these compounds with those of known dopaminergic agonists, apomorphine and ergolines, a new DA-receptor model is proposed as an outgrowth of current DA-receptor theories. One of the most important concepts of this receptor model is its emphasis on the possible positions taken by the N-substituents of dopaminergic compounds. One of these positions i sterically well defined while the other direction is sterically less critical. The model has been used to explain the lack of dopaminergic activity of some previously reported structures and also to predict properties of novel structures, including inherent chirality, which should be active at DA receptors. Hopefully, this heuristic DA-receptor model will lead to the discovery of more selective and potent pharmacological tools, which ultimately might lead to the development of therapeutic agents for treating diseases of dopaminergic function in the central nervous system.

C1- and C3-methyl-substituted derivatives of 7-hydroxy-2-(di-n-propylamino)tetralin: activities at central dopamine receptors.

C1- and C3-methyl-substituted derivatives of the potent dopamine (DA) receptor agonist 7-hydroxy-2-(di-n-propylamino)tetralin (7-OH-DPAT) have been synthesized, and their conformational preferences have been studied by use of NMR spectroscopy, X-ray crystallography, and molecular mechanics (MMP2) calculations. The compounds were tested for activity at central DA receptors, by use of biochemical and behavioral tests in rats. (1S,2R)-7-Hydroxy-1-methyl-2-(di-n-propylamino)tetralin [(+)-10] was demonstrated to be sevenfold less potent than (2R)-7-OH-DPAT as a DA receptor agonist. The other new compounds were of lower potency or inactive.

Novel dopamine receptor agonists and antagonists with preferential action on autoreceptors.

The enantiomers of cis-5-hydroxy-1-methyl-2-(di-n-propylamino)tetralin and its methyl ether have been synthesized. The compounds were tested for central dopamine (DA) receptor activity, by using biochemical and behavioral tests in rats. The (1R,2S)-(-) enantiomers of 1 and 2 are characterized as centrally acting DA-receptor agonists while the corresponding (1S,2R)-(+) enantiomers are characterized as centrally acting DA-receptor antagonists. Compounds (+)-1 and (+)-2 differ from classical neuroleptics in being able to increase DA synthesis rate in a wide dose range without reducing locomotor activity, suggesting a pronounced selectivity for DA autoreceptors. Also the (-) enantiomers seem to act preferentially on DA autoreceptors.