A comparison of the antimuscarinic effects of pirenzepine and N-methylatropine on ganglionic and vascular muscarinic receptors in the rat

The antimuscarinic properties of pirenzepine and N-methylatropine were evaluated in two intact preparations by measuring A) the inhibition of increase in mean arterial pressure evoked by McN-A-343 in pithed rats through activation of ganglionic muscarinic receptors and B) the inhibition of fall in arterial pressure evoked by methacholine in anaesthetized rats through activation of vascular muscarinic receptors. To characterize the antimuscarinic potencies of pirenzepine and N-methylatropine, for both antagonists doses were calculated that produce a 10-fold shift to the right of the dose-response curves for A) the pressor response to McN-A-343 (i.v. administration) in pithed rats (D10-p.r.) and B) for the depressor effect to methacholine (i.v. administration) in anaesthetized rats (D10-an.r.), respectively. Whereas N-methylatropine was virtually equieffective in blocking both muscarinic responses (D10-an.r./D10-p.r. approximately equal to 1), pirenzepine, however, was considerably more potent at ganglionic than at vascular muscarinic receptors (D10-an.r./D10-p.r. approximately equal to 16). These data confirm the existence of excitatory ganglionic muscarinic receptors with high affinity for pirenzepine (M1) and provide evidence for the presence of M2 receptors - receptors which show a low sensitivity to pirenzepine - on vascular smooth muscle cells. To further characterize the anticholinergic properties of pirenzepine, its effect on the pressor response to DMPP, a nicotinic ganglionic stimulant, was investigated in pithed rats. A high dose of pirenzepine (1.13 mumol/kg), given i.v., did not affect nicotinic ganglionic transmission.     

Hepatocystis in populations of baboons (Papio hamadryas s.l.) of Tanzania and Ethiopia

Bloodsmears from 119 yellow baboons (Papio hamadryas cynocephalus) in six groups in Mikumi National Park, Tanzania, and from 189 olive (P.h. anubis) hamadryas (P.h. hamadryas) and hybrid baboons in three groups in Awash National park, Ethiopia showed differing levels of infection with Hepatocystis simiae. 0% to 42% in the Tanzanian groups were carriers of the parasite but none of the Ethiopian baboons was positive. The remarkable absence of H. simiae in the Ethiopian baboons is believed to be related to environmental factors that exclude transmission in the absence of the Culicoides vector.     

A model for analyzing growth kinetics of a slowly growing Mycobacterium sp

This report describes a simple method for quantifying viable mycobacteria and for determining generation time. We used statistical models and computer analysis of growth curves generated for the slowly growing mycobacterium Mycobacterium paratuberculosis under controlled conditions to derive a mathematical formula relating the dependent variable, growth, to the independent variables, log10 number of organisms in the inoculum (inoculum size) and incubation time. Growth was measured by a radiometric method which detects 14CO2 release during metabolism of a 14C-labeled substrate. The radiometric method allowed for early detection of growth and detected as few as three viable bacteria. The coefficient of variation between culture vials inoculated with the same number of M. paratuberculosis was 0.083. Radiometric measurements were highly correlated to spectrophotometric and plate count methods for measuring growth (r = 0.962 and 0.992, respectively). The proportion of the total variability explained by the model in a goodness of fit test was 0.9994. Application of the model to broth cultures provided accurate estimates of the number of M. paratuberculosis (standard error = 0.21, log10 scale) and the growth rate (coefficient of variation, 0.03). Generation time was observed to be dependent upon the number of organisms in the inoculum. The model accurately described all phases of growth of M. paratuberculosis and can likely be applied to other slowly growing microorganisms.     

Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.     

Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.     

The adsorptive immobilization of phospholipids D mediated by calcium ions

Immobilization of phospholipase D from cabbage was studied with the aim of stabilizing the enzyme for its use in synthesis of phospholipids. It was shown that phospholipase D can be immobilized by adsorption to polymeric carriers containing long chain anchor groups such as octadecyl, octyl, or other alkyl residues. Starting from the crude enzyme, phospholipase D activity is preferentially bound (up to 100%) in competition with contaminating proteins. A prerequisite of high binding rates is the presence of calcium ions, which play a mediating role in the adsorption process. The maximum activity of the carrier-enzyme complexes depends upon the calcium concentration in the immobilization process and the carrier material (>/=10mM CaCl(2) with octadecyl-Si40, >/=40 mM CaCl(2) with octyl-sepharose and butyl-fractogel). Immobilization of phospholipase D to octyl-sepharose was shown to result in a distinctly increased storage stability and an enlarged pH-optimum range for the catalytic activity. Operational stability of different phospholipase D-carrier complexes was compared. (c) 1993 Wiley & Sons, Inc.     

The design and pharmacology of novel selective muscarinic agonists and antagonists

The muscarinic pharmacology of C1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent M1-selective agonist known today (pEC50: M1/rabbit vas deferens = 7.83; M2/guinea-pig atria = 6.35; M3/guinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)-4-F-MePyMcN was a potent partial agonist (pEC50 = 7.22; apparent efficacy = 0.83). These results indicate that (S)-4-F-MePyMcN might be a useful tool to study M1 receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7.86/atria; pKi = 7.8/rat heart) with lower affinities for the M1 (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6.92/guinea-pig ileum; pKi = 6.7/rat pancreas) and M4 receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal H1 receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging.     

Hepatocystis Parasitemia in Wild Kenya Vervet Monkeys (Cercopithecus aethiops)

Blood smears of 159 vervet monkeys from three sites in Kenya were stained with Giemsa and examined for Hepatocystis parasites. The populations differ in incidence of parasitemia, ranging from 0–62% affected individuals. These differences are probably due to altitude and local environmental conditions.     

Ghrelin and NUCB2/nesfatin-1 are expressed in the same gastric cell and differentially correlated with body mass index in obese subjects

The orexigenic peptide ghrelin and the anorexigenic peptide nesfatin-1 are expressed by the same endocrine cell of the rat stomach, the X/A-like cell. However, data in humans are lacking, especially under conditions of obesity. We collected gastric tissue of obese patients undergoing sleeve gastrectomy and investigated the expression of nesfatin-1 and ghrelin in the gastric oxyntic mucosa by immunofluorescence. Nesfatin-1 immunoreactivity was detected in the human oxyntic mucosa in cells with an endocrine phenotype. A major portion of nesfatin-1 immunoreactive cells (78 %) co-localized with ghrelin indicating the occurrence in human X/A-like cells. In patients with very high body mass index (BMI 55–65 kg/m²), the number of nesfatin-1 immunoreactive cells/low-power field was significantly higher than in obese patients with lower BMI (40–50 kg/m², 118 ± 10 vs. 82 ± 11, p < 0.05). On the other hand, the number of ghrelin immunoreactive cells was significantly reduced in obese patients with higher compared to lower BMI (96 ± 12 vs. 204 ± 21, p < 0.01). Also the ghrelin-acylating enzyme ghrelin-O-acyltransferase decreased with increasing BMI. In conclusion, nesfatin-1 immunoreactivity is also co-localized with ghrelin in human gastric X/A-like cells giving rise to a dual role of this cell type with differential effects on stimulation and inhibition of appetite dependent on the peptide released. The expression of these two peptides is differentially regulated under obese conditions with an increase of nesfatin-1 and a decrease of ghrelin immunoreactivity with rising BMI pointing towards an adaptive change of expression that may counteract further body weight increase.