A new series of M3 muscarinic antagonists based on the 4-amino-piperidine scaffold.

A series of 4-amino-piperidine containing molecules have been synthesized and structure-affinity relationship toward the M3-muscarinic receptor has been investigated. Chemical modulations provided molecules with K(i) for the human M3-R up to 1 nM with variable selectivity (3- to 40-fold) over the human M2-R. Compounds 2 (pA(2)=8.3, 8.6) demonstrates in vitro on guinea pig bladder and ileal strips potent anticholinergic properties and tissue selectivity.

Mivazerol prevents the tachycardia caused by emergence from halothane anesthesia partly through activation of spinal alpha 2-adrenoceptors.

BACKGROUND: Mivazerol (MIV) is an alpha 2-adrenoceptor agonist designed to prevent adverse cardiac outcome in perioperative patients. The present study was undertaken to determine whether the hyperdynamic state observed at emergence from halothane (HAL) anesthesia in rats could be modulated by MIV and to explore the mode of action of MIV under such conditions. METHODS: Male Sprague Dawley rats were anesthetized with 1% HAL and assisted for respiration (N2O-O2: 70-30%). MIV 2.2-15.3 micrograms.kg-1.h-1 i.v. was infused 30 min before withdrawal of anesthesia and compared for heart rate (HR) and systolic arterial blood pressure (SAP) to control animals treated with saline. In some experiments, animals were pretreated with intrathecal pertussis toxin (T2 level, 0.5 microgram, 7 d), or i.v. rauwolscine (0.34 mg/kg, 5 min) or were bilaterally stellectomized (30 min) prior to withdrawal of HAL. RESULTS: Increases in HR (65 bpm, +20%) and in SAP (25 mmHg, +26%) were observed immediately upon discontinuation of HAL and remained constant for at least 30 min. The increase in HR was abolished by removal of the stellate ganglia. MIV dose-dependently inhibited the increase in HR from 4.8 micrograms.kg-1.h-1 (68% reduction, P < 0.05) without affecting HR or SAP during anesthesia. Inhibition of HR increase was of 98% at 15.3 micrograms.kg-1.h-1. This effect was abolished by rauwolscine, and partially (50%) inhibited by pertussis toxin pre-treatment. CONCLUSION: These results demonstrate that withdrawal of HAL anesthesia in the rat produces a sustained increase in HR due to activation of the sympathetic system and that MIV inhibits this tachycardia via activation of alpha 2-adrenoceptors located at least in part in the spinal cord.

Putative genes of a variant-specific antigen gene transcription unit in Trypanosoma brucei.

In a 7-kilobase (kb) sequence upstream from the 5' barren region, the Trypanosoma brucei AnTat 1.3A expression site carries two putative genes, named ESAG 2 and ESAG 3 for expression site-associated genes, as well as a copy of ESAG 1 (D.F. Cully, H.S. Ip, and G.A.M. Cross, Cell 42:173-182, 1985). At least 3 kb of this expression site exhibits a high degree of homology with the silent telomere carrying the AnTat 1.3A basic copy, whose ESAG 1 is interrupted by stop codons. Like the antigen gene, the region containing the ESAGs is transcribed only in the bloodstream forms, although transcription of 5' barren- and ESAG 2-related sequences also occurs in cultured procyclics. Analysis of steady-state and nascent transcripts suggests a continuous transcription of the whole expression site by an RNA polymerase resistant to alpha-amanitin, possibly initiating at a polymerase I-like promoter located about 17 kb upstream from the antigen gene. This polymerase seems prone to becoming inactivated upon incubation of the trypanosomes at low temperature. The putative protein encoded by ESAG 3 may carry a hydrophobic signal peptide, suggesting interaction with a membrane.

Trypanosoma brucei repeated element with unusual structural and transcriptional properties.

The genome of Trypanosoma brucei contains up to 400 copies of a conserved sequence (TRS, trypanosome repeated sequence). The majority of TRS copies (TRS1) are 5.2 X 10(3) base-pairs (kb) and are flanked by different separate halves of the previously described transposable element RIME (ribosomal mobile element), although a variant copy (TRS2) contains only the central 1.45 kb portion and lacks RIME. TRS1 elements can probably undergo transposition, since they are dispersed in all chromosome size classes and are bordered by direct repeats of about four base-pairs. Some TRS1 elements may contain an open reading frame over almost their entire length (1651 codons), encoding a protein showing homology with reverse transcriptase. TRS probes detect poly(A)+ transcripts of 5 to 9 kb, generated by a polymerase moderately sensitive to alpha-amanitin. Transcription is developmentally regulated. Both TRS and RIME sense transcripts are preferentially synthesized compared to anti-sense transcripts, and are much more abundant in bloodstream forms than in cultured procyclics.

Trypanosome hybrids generated in tsetse flies by nuclear fusion.

Genetic exchange may occur between two particular Trypanosoma brucei clones simultaneously transmitted by the same tsetse fly. We report here that this exchange takes place in the fly, through nuclear fusion. The resulting hybrids appear to be sub-tetraploid, some particular DNA sequences from one of the parental stocks being lost before enough cloned hybrid trypanosomes could be harvested for DNA analysis. A further reduction of the DNA content of these hybrids occurs gradually upon growth and yields near diploid value in a major part of the population. This mode of hybrid generation is different from the fusion of haploid gametes, which is thought to occur normally upon inoculation of metacyclic trypanosomes in their mammalian host. In this respect, the sub-tetraploid hybrids appear to undergo meiosis in the fly, generating sub-diploid metacyclic forms, then fusion in the mammalian blood.

Telomeric reciprocal recombination as a possible mechanism for antigenic variation in trypanosomes.

In African trypanosomes, antigenic variation is achieved through differential gene activation, with one antigen gene being expressed at a time among a large collection of antigen-specific sequences. Transcription of the antigen gene always takes place in a telomere, but different telomeres can alternatively act as the expression site. Telomeric antigen genes can be expressed without apparent DNA rearrangement, but they can also, like non-telomeric genes, have access to the telomeric expression site through a duplicative transposition mechanism resembling gene conversion. We report here that, as previously suggested, telomeric genes may use another route to be activated. This mechanism of gene activation is by reciprocal crossing-over upstream from the gene, in the so-called 'barren' region. This allows the antigen gene to be placed in the previously activated telomere, while inactivating the formerly expressed gene by recombination into a silent environment. At least for the telomeric antigen gene described here, three possible activation mechanisms coexist.

Trypanosoma brucei: a surface antigen mRNA is discontinuously transcribed from two distinct chromosomes.

The mRNAs for variant surface glycoproteins (VSGs) and many other proteins in Trypanosoma brucei start with the same sequence of 35 nucleotides, encoded by a separate mini-exon. There are approximately 200 mini-exon genes per trypanosome and these are highly clustered on large chromosomes. We have found two trypanosome variants that express a VSG gene located on a small, 225-kb chromosome. Each gene yields a mRNA containing the 35-nucleotide sequence even though the 225-kb chromosome does not contain a complete mini-exon gene. These results provide a strong support for the hypothesis that transcription of protein-coding genes in trypanosomes is discontinuous.

Cellular oncogenes (c-erb-A and c-erb-B) located on different chicken chromosomes can be transduced into the same retroviral genome.

Avian erythroblastosis virus has transduced two cellular genes, c-erb-A and c-erb-B. Using fractionated chicken chromosomes, we found that the two genes are located on different chromosomes in the chicken genome: c-erb-A is on a microchromosome, and c-erb-B is on a large chromosome. The locations of two other cellular oncogenes (c-fps and c-myb) were also determined: c-fps is on a microchromosome, and c-myb is on chromosome of an intermediate size. Our results suggest that avian erythroblastosis virus had transduced the two cellular genes independently, conforming to previous indications that cellular oncogenes are dispersed among multiple chromosomes in every species that has been examined.

Induction and properties of beta-adrenergic receptors during erythroid differentiation of Friend leukemic cells.

beta-Adrenergic receptors on Friend erythroleukemic cells were identified by the use of 125I-labeled hydroxybenzylpindolol, a potent beta-adrenergic antagonist. Binding of this ligand was saturable and stereospecific. The relative orders of potency of isoproterenol, epinephrine, and norepinephrine to displace bound hydroxybenzylpindolol indicate that the Friend cells have beta 2-adrenergic receptors. After culture for 6 days in the presence of dimethyl sulfoxide or hexamethylene bisacetamide, both undifferentiated and differentiated cells have a similar number of receptors (1500 per cell), but the density of beta receptors on the cell surface increases during the process of erythroid differentiation. Incubation of the Friend cells for 24 hr with high concentrations of butyric acid, dimethyl sulfoxide, or hexamethylenebisacetamide resulted in a striking increase of th number of beta-catecholamine receptors. The induction of beta-adrenergic receptors also occurred in the presence of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate and dexamethasone.