Molecular cloning and characterization of a mouse alpha 2C2 adrenoceptor subtype gene.

A clone MA2C2 encoding the murine homologue of adrenoceptor alpha 2C2 was isolated from a mouse genomic library using a subtype specific probe. The nucleotide and the deduced amino acid sequences derived from an ApaI fragment (2 kb) of the clone reveal a single open reading frame encoding a putative receptor protein of 455 amino acids. The 5' untranslated region (0.5 kb) sequenced is characterized by high GC content and CpG island count.     

CGS 7525A, a new, centrally active alpha 2 adrenoceptor antagonist

CGS 7525A, a new tetracyclic compound, was evaluated for alpha 2 adrenoceptor antagonism in receptor binding assays and in behavioral and electrophysiological tests. 3H-Clonidine, but not 3H-prazosin, binding was potently inhibited in vitro by CGS 7525A. In vivo, CGS 7525A attenuated the suppressant action of clonidine on phenylquinone-induced writhing and on locus coeruleus neuronal firing rate. Mianserin was nearly equipotent with CGS 7525A in the 3H-clonidine binding assay, but considerably less potent in the measures of alpha 2 adrenoceptor antagonism in vivo. Both CGS 7525A and mianserin displaced 3H-spiroperidol binding from frontal cortex 5-HT2 binding sites. Although yohimbine resembled CGS 7525A in most respects, its activity at 5-HT2 binding sites was relatively low, CGS 7525A was not associated with any appreciable blockade of norepinephrine or serotonin uptake in vitro. Thus, CGS 7525A appears to be a promising new pharmacological tool for investigating the behavioral function of brain alpha 2 adrenoceptors.     

Characterization of a new radioiodinated probe for the alpha2C adrenoceptor in the mouse brain

[125I]17alpha-hydroxy-20alpha-yohimban-16beta-(N-4-p6 hydroxyphenethyl)carboxamide or [125I]rauwolscine-OHPC, a new radioiodinated probe derived from rauwolscine was synthesized and its binding characteristics investigated on sections of the mouse caudate putamen. [125I]rauwolscine-OHPC binding was saturable and revealed interaction with a single class of binding sites (KD= 0.171 nM, Bmax = 3082 pCi/mg of tissue). The kinetically derived affinity was in close agreement with the affinity evaluated by saturation experiments: k(-1)/k(+1)(0.0403 min(-1)/114 10(6) M(-1) min(-1))=0.35 nM. Competition studies revealed interaction with one single class of binding sites for each of the twelve compounds tested. The rank of potency suggested an interaction with alpha2 adrenoceptors (atipamezole > or = RX 821002 > yohimbine > (-)epinephrine). Moreover, the good affinity of [125I] rauwolscine-OHPC binding sites for spiroxatrine, yohimbine, WB 4101, the relatively good affinity for prazosin (Ki =37.4 nM) and the affinity ratio prazosin/oxymetazoline (37.4/43.4=0.86) were consistent with an alpha2C selective labelling of [125I]rauwolscine-OHPC. The distribution of [125I]rauwolscine-OHPC binding sites in mouse brain was characterized by autoradiography. The density of binding sites was high in the islands of Calleja, accumbens nucleus, caudate putamen and olfactory tubercles, moderate in the hippocampus, amygdala and anterodorsal nucleus of the thalamus. These findings demonstrated that [125I]rauwolscine-OHPC is a useful radioiodinated probe to label alpha2C adrenoceptors in mouse brain.     

Influence of sympathectomy on alpha 2 adrenoceptor binding sites in canine blood vessels

The effects of denervation of alpha 2 adrenoceptor binding sites were examined in canine arteries and veins. Denervation of the lower abdominal aorta, renal and femoral arteries and femoral veins marked reduced vessel norepinephrine concentrations. Denervation had little effect on the concentration of alpha 2 adrenoceptor binding sites or the affinity of (3H)yohimbine for these sites. The apparent lack of any significant reduction in receptor binding sites suggests that the majority of these sites are located on smooth muscle cells of blood vessels. The failure of any appreciable rise in receptor concentration following denervation is consistent with the hypothesis from functional studies that postsynaptic alpha 2 adrenoceptors on blood vessels are located extra-synaptically and hence not influenced by neurally released norepinephrine.     

Theoretical proton affinities of alpha1 adrenoceptor ligands

A systematic study has been performed of the proton affinity of a large family of agonists and antagonists of the alpha1-adrenoceptor at the B3LYP/6-31G* level of theory. After a conformational search, all the N atoms were considered as protonation sites and protonation energy values were determined. The inclusion of solvation by means of the Onsager model yielded stabilization in the proton affinity values obtained. In addition, a good correlation was found between the proton affinity values corresponding to the first protonation in gas phase of some of the compounds and their corresponding experimental affinity constants K(i) for the alpha1A adrenergic receptor.     

Prostaglandin E2 and alpha 2 adrenoceptor agonists inhibit the pentose phosphate shunt in pancreatic islets

Glucose utilization in isolated pancreatic islets of the rat was inhibited by prostaglandin (PG) E2 and the alpha 2 adrenoceptor agonist, clonidine, to a similar extent; other prostaglandins did not affect glucose utilization. Islet oxidation of [1-14C]glucose and [6-14C]glucose demonstrated that the pentose phosphate shunt was inhibited by PGE2 and clonidine. Pertussis toxin antagonizes the effects of clonidine and PGE2 on total glucose utilization and pentose phosphate shunt activity. The results suggest that PGE2 and alpha 2 adrenoceptor agonists may regulate glucose metabolism through similar transduction mechanisms, and that a guanine nucleotide binding regulatory (G) protein modulates certain metabolic effects of prostaglandins and adrenergic agonists.     

Isolation and characterization of horse alpha 2-macroglobulin protease inhibitor

Several publications have described in the past properties of partly purified horse alpha 2-macroglobulin (alpha 2M) which are strikingly different from the human alpha 2M. Horse alpha 2M was therefore isolated to purity by classical procedures, i.e. affinity chromatography, ion exchange chromatography and gel filtration, and its properties are compared with those of its human counterpart. The molecular weight of the native protein and its subunits, the isoelectrofocusing pattern and the change in electrophoretic mobility caused by interaction with protease were similar to those of human alpha 2M. Horse alpha 2M had a broad enzyme specificity and inhibited enzymatic action on macromolecules but not on small molecular weight synthetic substrates. In addition the horse and human alpha 2M were found to be immunochemically related when examined by specific antisera to human as well as to horse alpha 2-macroglobulin.     

Isolation and partial characterization of a alpha 2-beta 1-glycoprotein from horse plasma

A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins.     

Novel 2-imidazoles as potent and selective alpha1A adrenoceptor partial agonists

Novel 2-imidazoles have been identified as potent partial agonists of the α_1A adrenergic receptor, with good selectivity over the α_1B, α_1D and α_2A receptor sub-types. Sulfonamide 23 possessed attractive drug-like properties with respect to physicochemical and ADME properties and wide ligand selectivity.     

Multiple characterizations of Listeria monocytogenes sensitive and insensitive variants to divergicin M35, a new pediocin-like bacteriocin

AIMS: Divergicin M35 is a new class IIa bacteriocin produced by Carnobacterium divergicin M35. The bactericidal activity of this antimicrobial peptide was tested against a set of 11 strains of Listeria monocytogenes isolated from food. METHODS AND RESULTS: The minimal inhibitory concentration (MIC) was determined by the microdilution method. The strains tested displayed a different level of sensitivity to divergicin M35. L. monocytogenes LSD530, referred to as DivS strain, was the most sensitive and appeared to be inhibited by concentration of divergicin M35 below 0.13 microg ml(-1). The mutant resistant to divergicin M35, called DivM, was obtained from L. monocytogenes LSD530 (DivS) by gradually increasing the amounts of divergicin M35 until 1.3 microg ml(-1). Notably, DivM was stable after 50 generations. DivS parental strain was inhibited by a concentration of 4 microg ml(-1). L. monocytogenes LSD530 was shown to be resistant to divergicin M35 at 1.3 microg ml(-1). Remarkably, in the presence of divalent cations such as Ca(2+), Mg(2+) and Mn(2+), the lethality caused by divergicin M35 was reduced by 0.48, 0.54 and 0.63 log CFU per ml (after 18 h at 30 degrees C), respectively. The total DNA profiles of DivS and DivM were similar. DivS and DivM showed variable sensitivity to antibiotics. The two-dimensional (2-D) electrophoresis of cell wall proteins did not show any significant difference between DivS and DivM strains but their fatty acid composition showed a significant difference in C(16:0) content. CONCLUSIONS: Resistance to divergicin M35 is likely ascribed to modification in cell wall fatty acid composition rather than protein modification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides original results contributing to understanding of the resistance of L. monocytogenes to divergicin M35, a new class IIa bacteriocin.     

Interferon sensitive and insensitive MHC variants of a murine thymoma differentially resistant to methotrexate-containing antibody-directed liposomes and immunotoxin

We evaluated the ability of methotrexate-containing liposomes or a ricin alpha-chain immunotoxin, both associated with monoclonal antibodies specific for the major histocompatibility complex-encoded class I molecule H-2Kk, to kill cells of the murine k haplotype thymoma RDM4. Cells were incubated with liposomes or immunotoxin in the presence or absence of interferon-gamma, which is known to augment the expression of the target class I molecules. The great majority of cells were killed by either of these reagents. Two types of mutant cells were obtained: type 1 cells, selected by methotrexate-containing liposomes, failed to express sufficient target H-2k molecules to be killed by liposomes in the absence of interferon-gamma. In the presence of interferon-gamma, these cells increased expression of all H-2 class I molecules and could be killed by targeted liposomes. Type 2 cells were immunoselected from cloned type 1 cells by liposomes in the presence of interferon. These cells failed to respond to interferon with expression of the H-2Kk molecule, but continued to augment H-2Dk expression in response to interferon. A third variant (type 3) selected from the wild type population by an H-2Kk specific immunotoxin in the absence of interferon phenotypically resembled type 1 cells. Type 1 but not type 2 cells respond to interferon by augmented synthesis of H-2Kk specific mRNA. The results suggest that for interferon-sensitive cell surface molecules of tumor cells, use of interferon improves the efficacy of targeted chemotherapy, but does not prevent development of mutants lacking the target molecule.     

Conformational variants of human alpha2-macroglobulin are reflected in a C-terminal 'switch region'.

Human alpha2-macroglobulin displays extensive conformational changes when induced to transform into new quaternary structures, which are eliminated from the systemic circulation by receptor-mediated endocytosis. One major region involved in these conformational changes is located in a segment of 30 amino acids from Glu1314 to Ser1343 (-Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Gln-Thr-Leu-Pro-Gln-Thr-Cys-Asp -Glu-Pro-Lys-Ala-His-Thr-Ser-Phe-Gln-Ile-Ser-Leu-Ser-), which we term the 'switch region' of alpha2-macroglobulin, as deduced by immunochemical techniques. Monoclonal antibodies were generated using either native, methylamine-treated or the 18-kDa C-terminal receptor-binding fragment as the immunogen. From an extensive number of obtained hybridomas, 11 mAbs were selected because of their capacity to bind to the C-terminal fragment. Irrespective of the original configuration of the antigen used for immunization, seven of the antibodies were shown to be reactive with a set of overlapping epitopes, closely positioned within the 'switch region', as confirmed by the use of synthetic peptides covering the entire C-terminal fragment. The specificities of the seven individual antibodies, as determined by ELISA and BIAcore technologies, revealed a pronounced conformational pleomorphism in the 'switch region'. The results indicate that the 'switch region' may be involved in the exposure of the receptor recognition site and can be used as an indicator region for different conformational states of alpha2-macroglobulin.     

Design and synthesis of selective alpha1B adrenoceptor antagonists

A series of novel indolylpiperidine derivatives were synthesized and assessed for their pharmacological profiles at alpha1 adrenoceptor subtypes by in vitro binding studies at rat alpha1A and alpha1B receptors. Compound 11 was a potent (Ki=0.63 nM) and selective (approximately 30-fold more selective for the alpha1B receptor than for the alpha1A receptor) alpha1B adrenoceptor antagonist.     

Effects of chronic alpha 2-adrenoceptor blockade on platelet and lymphocyte adrenoceptor binding in normal volunteers.

Platelet and lymphocyte adrenoceptor binding was measured in 12 healthy male volunteers before and after 22 days treatment with the alpha 2-adrenoceptor antagonist idazoxan 40 mg tds. Platelet alpha 2-adrenoceptor number assessed by the agonist 3H-UK 14304 [correction of UK 14303] was significantly increased following idazoxan, with a smaller increase in antagonist binding (3H-rauwolscine). Lymphocyte beta-adrenoceptor number was unaltered by idazoxan, although the variance within the sample was significantly increased. Plasma MHPG levels were significantly reduced by chronic idazoxan. These data indicate upregulation of the platelet alpha 2-adrenoceptor in response to chronic blockade and suggest that this may reflect a similar change in presynaptic alpha 2-adrenoceptors which regulate norepinephrine release.     

Distribution of alpha 1-antitrypsin variants in a US white population

A white population from the State of Minnesota of primarily German and Scandinavian heritage was subtyped for alpha 1-antitrypsin variants using isoelectric focusing. The frequencies of the genes PI*M1 (0.724), PI*M2 (0.137) and PI*M3 (0.095) were consistent with those for white populations documented in the literature from Northern Europe. Other genes identified in the study were PI*F, PI*I, PI*P, PI*S and PI*Z.     

Electrophoretic variants of alpha 2u-globulin in the livers of adult male rats: a possible polymorphism

Two-dimensional polyacrylamide gel electrophoresis has been used to examine the microsomal fractions from the livers of 32 adult male Alpk/AP (Wistar-derived) rats for the presence of alpha 2u-globulin variants of differing isoelectric point. Three major such isoelectric variants are described. Different combinations of these three forms were found in the population examined, with one-half of the animals expressing all three variants in approximately equal proportions and with one variant being present in all the animals examined. An understanding of the relevance of such different alpha 2u-globulin profiles to the individual animal must now await the assignment of a biological role for alpha 2u-globulin.     

Genetic polymorphism of alpha 2HS-glycoprotein in a French population. Description of two new rare variants

A French population was investigated for genetic polymorphism of alpha 2HS-glycoprotein (A2HS; nomenclature according to Human Gene Mapping 7, Los Angeles, 1983) using isoelectric focusing and immunoblotting. Three variants were observed together with two common alleles A2HS 1 and A2HS 2, whose frequencies were significantly different from the data in Canadians and Egyptians. An anodal variant to A2HS 1 was identical to a variant with two different nomenclatures reported by three different groups, indicating that there is a confusion in the A2HS nomenclature. The others were new variants with cathodal isoelectric points to A2HS 2 in the native state.     

A correction to the sequence of the alpha chains of horse haemoglobin

An atomic model of horse aquomethaemoglobin extensively refined against 2.0 Å X-ray data suggested that an Ala and a Gly residue in the published sequence of the α chain were interchanged. Chemical analysis has confirmed our interpretation of the model.     

Quantitative structure-activity relationship study of novel alpha1a-selective adrenoceptor antagonists

Two series of compounds were recently reported as novel alpha1a-selective adrenoceptor antagonists. In the first series, a dihydropyrimidone moiety is attached to a 4-phenyl piperidine containing side chain, while in the second, it is linked to a 4-substituted phenyl piperazine containing side chain. These compounds having potential for the treatment of benign prostatic hyperplasia, a urological disorder in the older age male population, were subjected to a quantitative structure-activity relationship study. The analysis has helped to ascertain the role of different substituents in explaining the observed binding potencies of these analogues. In the first category of compounds, three sites R1, R2, and X were varied and from the quantitative structure-activity relationship, it emerged that X- and R1-substituents having respectively, the high values of field and resonance effects may lead to more potent alpha1a-antagonists. The substituent of R2, being either CH3 or C2H5, does not add to improve the activity and thus the site, at present, becomes redundant. This site may, however, be explored for some additional substituents in future. In the second series of compounds, the phenyl ring, linked to a piperazine moiety at the end of a side chain, was substituted with various groups onto different positions. From derived significant correlations, it appeared the less polar and/or bulky substituents at the meta- and para-positions and a more hydrophobic substituent at the para-position are advantageous.