Quest for agonist and antagonist selectivity at muscarinic receptors in guinea-pig smooth muscles and cardiac atria.

Potencies of 11 muscarinic agonists in eliciting contraction of smooth muscle in guinea-pig ileum, trachea, urinary bladder and uterus and in inhibiting the rate of contractions of cardiac atria were compared. While acetylcholine (ACh) was the most potent agonist on the ileum, uterus and cardiac atria, cis-L(+)-dioxolane was equally as potent as ACh on the ileum and more potent on the urinary bladder and trachea. Compared to ACh, methylfurmethide, oxotremorine, acetoxybut-2-inyl-trimethylammonium and cis-L(+)-dioxolane acted weakly on the atria. Aceclidine, arecoline and acetyl-beta-methylcholine displayed selectivity for the urinary bladder and pilocarpine for the tracheal and urinary bladder smooth muscles. Oxotremorine had very low activity on the uterus. The stereoselectivity of muscarinic ACh receptors (mAChRs) for cis-L(+)-and cis-D(-)-dioxolane was low in the urinary bladder and uterus and high in the ileum and trachea. Most antagonists showed little selectivity between different organs, but S(-)-phenylcyclohexylglycoloyl choline was 6 times more active on the urinary bladder than on the ileum and AF-DX 116 was 12-30 times more active on the atria than on the smooth muscles. Among the N-alkyl derivatives of benzilylcholine, the octyl derivative as 400 times more active on the ileum than on the atria, while among the N-alkyl derivatives of QNB, the N-decyl derivative was 41 times more active on the ileum. The observed differences in the potency of various agonists and their stereoisomers on different smooth muscles cannot be explained by differences in the accessibility of receptors or in receptor reserve.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of the neuromuscular blocking drugs alcuronium, decamethonium, gallamine, pancuronium, ritebronium, tercuronium and d-tubocurarine with muscarinic acetylcholine receptors in the heart and ileum.

Neuromuscular blocking drugs have a high affinity for muscarinic acetylcholine receptors in the heart atria and ileal smooth muscle. In experiments on homogenates, alcuronium, gallamine, pancuronium, tercuronium and ritebronium inhibited the binding of the muscarinic antagonist (3H)quinuclidinyl benzilate (QNB) to rat heart atria with IC50 values of 0.15-0.53 mumol X 1(-1) and to ileal longitudinal muscles with IC50 values of 0.12-0.45 mumol X 1(-1). d-Tubocurarine and decamethonium inhibited (3H)QNB binding to these tissues with IC50 values of 6.2-8.5 mumol X 1(-1). For each neuromuscular blocking drug, the IC50 values were virtually identical for (3H)QNB displacement in the homogenates of the atria and of the ileal muscle. Alcuronium and gallamine differed from the other blocking agents in that they produced less steep (3H)QNB displacement curves both in the atria and the ileal muscle; Hill coefficients for the binding of alcuronium and gallamine to atrial and ileal homogenates were lower than unity. On isolated atria, gallamine, pancuronium, ritebronium and tercuronium antagonized the inhibition of tension development caused by the muscarinic agonist, methylfurmethide, with Kd values which were of the same order of magnitude as the IC50 values for the displacement of (3H)QNB binding to homogenates; the Kd of alcuronium was 12.5 times higher. d-Tubocurarine and decamethonium did not antagonize the effects of methylfurmethide at concentrations up to 100 mumol X 1(-1). On isolated ileal longitudinal muscle, gallamine and pancuronium antagonized the effects of methylfurmethide with Kd values that were 53 times and 100 times higher than their respective Kd values in the atria.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the Epilimnetic Bacterial Community Composition, Production, and Protist-Induced Mortality along the Longitudinal Axis of a Highly Eutrophic Reservoir.

We studied changes in the epilimnetic bacterial community composition (BCC), bacterial biomass and production, and protistan succession and bacterivory along the longitudinal axis of the canyon-shaped, highly eutrophic Sau Reservoir (NE Spain) during two sampling campaigns, in April and July 1997. Longitudinal changes in BCC from the river inflow to the dam area of the reservoir were detected by using oligonucleotide probes targeted to the kingdom Bacteria, to the alpha, beta, and gamma subclasses (ALFA, BETA, and GAMA) of the class Proteobacteria, and to the Cytophaga/Flavobacterium (CF) cluster. In general, the inflow of the organically loaded Ter river, with highly abundant allochthonous bacterial populations, induced a clearly distinguishable longitudinal succession of the structure of the microbial food web. The most dynamic changes in microbial parameters occurred at the plunge point, the mixing area of river water and the reservoir epilimnion. Changes within members of BETA and CF were the most important in determining changes in BCC, bacterial abundance and biomass. Much less relevant changes occurred within the less abundant ALFA and GAMA bacteria. From the plunge point downstream, we described a significant shift in BCC in the form of decreased proportions of BETA and CF. This shift spatially coincided with the highest values of heterotrophic nanoflagellate bacterivory (roughly doubled the bacterial production). CF numerically dominated throughout the reservoir without any marked longitudinal changes in their mean cell volume. In contrast, very large cells affiliated to BETA clearly dominated in the allochthonous bacterial biomass brought by the river. BETA showed a marked downstream trend of decreasing mean cell volume. We conclude that the observed BCC shift and the longitudinal shift in food web structure (bacteria-heterotrophic nanoflagellates-ciliates) resulted from highly complex interactions brought about by several major factors: varying hydrology, the high localized allochthonous input of organic matter brought by the river, downstream changing substrate availability, and selective protistan bacterivory.

Application of numerical modelling of osteotomy to orthopaedic practice.

During the development of an osteoarthritic hip joint, osteophytes, cysts as well as arthrosis originate on the contact boundary between the femoral head and the acetabulum in highly loaded human joints. The purpose of proximal femur osteotomy is to bring the femoral head into a new position inside the acetabulum. The corrective options of intertrochanteric osteotomy most frequently used in orthopaedic practice are valgization, varization, displacements, oblique displacements, lateralization, extension, rotation or shortening of the shaft. The present widespread tomographic measurement methods, such as computer tomography (CT), magnetic resonance imaging (MRI) and 2D or 3D reconstruction involve the simulation of surgical problems of osteotomy, as well as the simulation of the reconstruction of the function of the hip joint after the operation. The results of numerical modelling of osteotomy concerning contact problems, will be presented in the contribution.

2D and 3D finite element pre- and post-processing in orthopaedy.

The present widespread tomographic techniques based on computed tomography (CT), magnetic resonance imaging (MRI) and automatic 2D and 3D reconstructions also involve numerous new points of view for planning surgical techniques in orthopaedy. These require efficient pre- and post-processing methods. By pre-processing and post-processing is meant both the generation of a finite element mesh that numerically models human joints and the visualization of the results obtained as required by the surgeon. Since the numerical reconstructions of human joints are based on the finite element approximation of contact problems assuming linear elasticity, the main goal of pre-processing is to generate triangular (2D) and tetrahedral (3D) meshes of objects with a 'reasonable' boundary-human joints. In our paper we restrict ourselves, for the sake of simplicity, to polygonal (resp. polyhedral) objects. The idea of preprocessing is based on the modified method of Delaunay Mangulalia and on the so-called relaxation technique. After the generation of the finite element mesh and after numerical simulation of surgical problems of orthopaedy, the results obtained as stress-strain fields, temperature, etc. may be visualized in a graphical form on the computer screen as well as on other media.

Muscarinic acetylcholine receptors in the heart of rats before and after birth.

Atropine-displaceable binding of (3H)quinuclidinyl benzilate (QNB) to homogenates was used to identify the muscarinic binding sites in rat heart atria and ventricles and to investigate developmental changes in their concentration and binding properties between the 15th day of prenatal life and 3 months after birth. On the 15th day of prenatal life, muscarinic binding sites were already present in the heart. Their concentration increased steeply between the 15th and 19th days of prenatal development; in the atria, it remained high until the 1st day after birth and thereafter it diminished throughout the postnatal life, while in the ventricles the decrease started before the first postnatal day. The concentration of the binding sites was 1.8-3.0 times higher in the atria than in the ventricles at all time points investigated. Their affinity for QNB (the antagonist) was the same in the atria and ventricles and did not change during postnatal development (KD of 17.8 pmol/l at an infinitely low concentration of the binding sites). The binding of carbamoylcholine (the agonist) to muscarinic binding sites was analysed in experiments with the displacement of (3H)QNB binding, assuming the presence of high- and low-affinity binding sites for agonists. The proportion between the concentrations of the two classes of agonist binding sites is close to 1:1 both in the atria and the ventricles and does not change with age. No statistical significant differences were discovered between the affinities of the high- and low-affinity binding sites for carbamoylcholine between the atria and the ventricles and between new-born and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Methylumbelliferyl-beta-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory.

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-beta-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity beta-N-acetylglucosaminidase-like enzyme (K(m), >>100 mumol liter) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, beta-N-acetylglucosaminidase-like enzyme (K(m), <0.5 mumol liter). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r = 0.96) and Cyclidium sp. (r = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of beta-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.

Impact of ionic aluminium on extracellular phosphatases in acidified lakes.

We studied direct inhibiting effects of aluminium (Al) on extracellular phosphatases produced by the plankton of acidified lakes in the Bohemian Forest. In laboratory experiments we tested the effect of different Al concentrations (0-1000 microg l(-1)) on kinetic parameters of acid phosphatases (pH optimum approximately 5.0) at pH between 4.5 and 5.2. We observed a significant reduction of an apparent substrate affinity at Al concentrations between 300 and 1000 microg l(-1) at pH 4.5 and 4.8 (but not at 5.2). In contrast, maximum acid phosphatase activity (AcPA) remained unchanged. Such behaviour of saturation kinetics is compatible with the assumption that ionic Al acts as a competitive inhibitor of acid phosphatases. To decide whether the observed Al effects could be explained alternatively by complexation of Al with substrate, we tested statistically the best fits of data with both possible models (competitive versus complexation). Experimental results supported the competitive hypothesis rather than the complexation model suggested originally by some authors. Furthermore, we tested the Al effect within a wide range of pH from 4.0 to 6.0. For pH values < 5.2, the results of an Al-pH matrix experiment gave a more detailed picture: the higher the Al concentration, the wider the pH range in which Al could negatively affect AcPA. The ecological ramifications of this effect were evaluated in the context of field AcPA data on three strongly acidified lakes.

Morphological and compositional shifts in an experimental bacterial community influenced by protists with contrasting feeding modes.

In a two-stage continuous-flow system, we studied the impacts of different protozoan feeding modes on the morphology and taxonomic structure of mixed bacterial consortia, which were utilizing organic carbon released by a pure culture of a Rhodomonas sp. grown on inorganic medium in the first stage of the system. Two of three second stages operated in parallel were inoculated by a bacterivorous flagellate, Bodo saltans, and an algivorous ciliate, Urotricha furcata, respectively. The third vessel served as a control. In two experiments, where algal and bacterial populations grew at rates and densities typical for eutrophic waters, we compared community changes of bacteria, algae, and protozoa under quasi-steady-state conditions and during the transient stage after the protozoan inoculation. In situ hybridization with fluorescent oligonucleotide probes and cultivation-based approaches were used to tentatively analyze the bacterial community composition. Initially the cell size distribution and community structure of all cultivation vessels showed similar patterns, with a dominance of 1- to 2.5-(mu)m-long rods from the beta subdivision of the phylum Proteobacteria ((beta)-Proteobacteria). Inoculation with the ciliate increased bacterial growth in this substrate-controlled variant, seemingly via a recycling of nutrients and substrate released by grazing on algae, but without any detectable effect on the composition of bacterial assemblage. In contrast, an inoculation with the bacterivore, B. saltans, resulted in a decreased proportion of the (beta)-Proteobacteria. One part of the assemblage (<4% of total bacterial numbers), moreover, produced large grazing-resistant threadlike cells. As B. saltans ingested only cells of <3 (mu)m, this strategy yielded a refuge for (symbl)70% of total bacterial biomass from being grazed. Another consequence of the heavy predation in this variant was a shift to the numerical dominance of the (alpha)-Proteobacteria. The enhanced physiological status of the heavily grazed-upon segment of bacterial community resulted in a much higher proportion of CFU (mean, 88% of total bacterial counts) than with other variants, where CFU accounted for (symbl)30%. However, significant cultivation-dependent shifts of the bacterial community were observed toward (gamma)-Proteobacteria and members of the Cytophaga/Flavobacterium group, which demonstrated the rather poor agreement between cultivation-based approaches and oligonucleotide probing.

Stabilization of antagonist binding to cardiac muscarinic acetylcholine receptors by gallamine and other neuromuscular blocking drugs.

The effects of neuromuscular blocking drugs and muscarinic agonists and antagonists on the dissociation of [3H]quinuclidinylbenzilate ([3H]QNB) from muscarinic receptors was studied on rat atrial homogenates. In typical experiments the investigated drug was added to the homogenate equilibrated with [3H]QNB and the amount of undissociated [3H]QNB receptor complexes was measured 40 min later. The antagonists atropine and pirenzepine, agonists carbamoylcholine and methylfurmethide and neuromuscular blockers pancuronium, d-tubocurarine and decamethonium caused a concentration-dependent dissociation of [3H]QNB from the receptors, which may be explained by their competition with [3H]QNB for the same (primary) binding sites. In accordance with this, these drugs did not affect the dissociation of [3H]QNB elicited by an excess of atropine, which indicates that the kinetics of dissociation of the [3H]QNB receptor complex remained unchanged in their presence. Neuromuscular blockers alcuronium, gallamine and to a lesser degree tercuronium differed from the other drugs in that 1) their effect on [3H]QNB dissociation was biphasic, being higher at their low (10(-6) to 10(-5) M) than at their high concentrations (10(-4) to 10(-3) and that 2) at high concentrations they strongly inhibited the dissociation of [3H]QNB receptor complexes elicited by the excess of atropine. Their behavior may be rationalized by assuming that at low concentrations they bind to the primary binding site making rebinding of once dissociated [3H]QNB molecules improbable (competitive mechanism), whereas at high concentrations they also act on a secondary (allosteric) binding site stabilizing the [3H]QNB receptor complexes by slowing their off-kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive cooperativity in the binding of alcuronium and N-methylscopolamine to muscarinic acetylcholine receptors.

The effect of the neuromuscular blocker alcuronium on the binding of N-[3H]-methylscopolamine [( 3H]NMS) and l-[3H]quinuclidinylbenzilate ([3H]QNB) to muscarinic binding sites in rat heart atria, longitudinal smooth muscle of the ileum, cerebral cortex, cerebellum, and submaxillary glands was measured using filtration techniques. In the presence of 10(-5) M alcuronium, the binding of [3H]NMS (which was present at a subsaturating concentration of 2 x 10(-10) M) was increased 5.3-fold in the atria and smooth muscle and 3-fold in the cerebellum; no increase was observed in the brain cortex and salivary glands. The binding of [3H]NMS was inhibited at 10(-3) M and higher concentrations of alcuronium. The rates of [3H]NMS association to and dissociation from muscarinic binding sites in the atria were diminished by 10(-5) M alcuronium. Scatchard plots of [3H]NMS binding data obtained with and without 10(-5) M alcuronium indicated that the maximum number of binding sites was not altered by the drug, whereas the apparent Kd for [3H]NMS was diminished. In contrast to [3H] NMS, the effects of alcuronium on the binding of [3H]QNB were only inhibitory. The concentration of alcuronium required to diminish the binding of [3H]QNB by 50% (IC50) was 4-7 microM in the atria, ileal smooth muscle, and the cerebellum, 140 microM in the brain cortex, and 1200 microM in the parotid gland. The results suggest that the binding of low concentrations of alcuronium to muscarinic receptors in the heart, ileal smooth muscle, and cerebellum allosterically increases the affinity of muscarinic receptors towards [3H]NMS, although not [3H]QNB. At high concentrations, alcuronium inhibits the binding of muscarinic ligands, presumably by competition for the classical muscarinic binding site. Positive cooperativity induced by alcuronium appears to be specific for the m2 (cardiac) subtype of muscarinic receptors.

Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir.

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-microm treatment) or enhance (<5-microm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the "grazer-free" (0.8-microm-filtered) treatment, subject only to a relatively low mortality rate (approximately 17% day(-1)) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-microm filtrate), grazing mortality was approximately 200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.