Desensitization to norepinephrine includes refractoriness of calcium release in myocardial cells

Localization of myoplasmic free calcium was measured in fura2-loaded single rat myocardial cells to determine whether the mechanism of norepinephrine desensitization includes redistribution of calcium. Fluorescence intensities at each pixel were quantitated by use of a photon-counting, microchannel plate camera. From these images, values of calcium-dependent fluorescence intensity averages in whole cells, areas of calcium release (as zones of high intracellular calcium concentrations), and ratios of fluorescence intensity in central vs. peripheral sites were determined. Stimulation by 1 nM norepinephrine caused an increase in total free intracellular calcium and an activation of intracellular calcium release sites from subsarcolemmal pools initially and later from centrally located calcium pools. Subsequent addition of 100 nM norepinephrine failed to cause significant intracellular calcium release from centrally located pools. In contrast, forskolin exposure still released high concentrations of calcium from these central pools. These results indicate that pretreatment with even a relatively small concentration of norepinephrine causes markedly decreased subsequent intracellular calcium release from centrally located sarcoplasmic reticulum because of a refractoriness of the link between receptor activation and calcium release.     

Inhibitory effects of some esters of 2- and 3-substituted alkoxyphenylcarbamic acids on photosynthetic characteristics

The inhibitory effect of 23N-alkyl-4-piperidylesters (alkyl = ethyl-butyl) (APEA) and 8N-ethyl-2-pyrrolidinylmethylesters (EPMEA) of 2- and 3-substituted alkoxyphenylcarbamic acids (alkoxy = butoxy-heptyloxy-) on photosynthetic Hill reaction activity of spinach chloroplasts and on chlorophyll (Chl) synthesis in green algaeChlorella vulgaris was investigated. Inhibitory activities of these compounds were strongly connected with the lipophilicity of the whole molecule. A lower inhibitory activity of 2-alkoxy-substituted derivatives in relation to the corresponding 3-substituted ones was confirmed. Electron spin resonance (ESR) spectra of spinach chloroplasts demonstrated that the studied compounds affected the structure of photosystem (PS) 2 with the release of Mn²⁺ ions into interior of thylakoid membranes.     

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2- aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.      

Design and implementation of a qualitative simulation model of lambda phage infection

MOTIVATION: Molecular biology databases hold a large number of empirical facts about many different aspects of biological entities. That data is static in the sense that one cannot ask a database 'What effect has protein A on gene B?' or 'Do gene A and gene B interact, and if so, how?'. Those questions require an explicit model of the target organism. Traditionally, biochemical systems are modelled using kinetics and differential equations in a quantitative simulator. For many biological processes however, detailed quantitative information is not available, only qualitative or fuzzy statements about the nature of interactions. RESULTS: We designed and implemented a qualitative simulation model of lambda phage growth control in Escherichia coli based on the existing simulation environment QSim. Qualitative reasoning can serve as the basis for automatic transformation of contents of genomic databases into interactive modelling systems that can reason about the relations and interactions of biological entities.      

When dispersal fails: unexpected genetic separation in Gibraltar macaques (Macaca sylvanus)

Barbary macaques (Macaca sylvanus), now restricted in the wild to a few isolated forested areas of Morocco and Algeria, are present in a free‐ranging colony on Gibraltar. For many decades, the Gibraltar colony was exposed to multiple bottlenecks due to highly nonrandom removal of animals, followed by repeated introductions of animals from North Africa. Moreover, because of complete isolation, Gibraltar's several social groups of macaques provide an ideal system to study the genetic consequences of dispersal in cercopithecines in situ. Predictions of genetic consequences due to male‐biased dispersal in cercopithecines will be different for autosomal and maternally inherited genetic markers, such as the control region of the mitochondrial DNA. We used a panel of 14 highly polymorphic microsatellite loci and part of the hypervariable region I of the mitochondrial control region to estimate genetic structure between five social groups in Gibraltar. Surprisingly, for autosomal markers, both classical summary statistics and an individual‐based method using a Bayesian framework detected significant genetic structure between social groups in Gibraltar, despite much closer proximity than wild Algerian and Moroccan populations. Mitochondrial data support this finding, as a very substantial portion of the total genetic variation (70.2%) was found between social groups. Using two Bayesian approaches, we likewise identified not only a small number of male first‐generation immigrants (albeit less than expected for cercopithecines) but also unexpectedly a few females. We hypothesize that the culling of males that are more likely to disperse might slow down genetic homogenization among neighbouring groups, but may also and more perversely produce selection on certain behavioural traits. This may have important repercussions for conservation, as it could lead to evolutionary changes that are not due to inbreeding or genetic drift.     

Nomegestrol acetate and vascular reactivity: nonhuman primate experiments

Prevention of coronary artery disease has been recognized as a major benefit of estrogen replacement therapy (ERT) in postmenopausal women. However, endometrial hyperplasia induced by unopposed ERT has raised important safety concerns. Progesterone or synthetic progestins have been used in combined hormone replacement therapy (HRT) to prevent endometrial cancer risk. Therefore, a major concern has been to ensure that the vascular beneficial effects of estrogens are not opposed when combined with progestins. Nomegestrol acetate (NOMAC) is an orally active progestin widely prescribed for HRT. Its vascular effects were evaluated in two models of coronary vascular reactivity in primates: 1) the paradoxical vasoconstriction to acetylcholine (Ach) coronary infusion after 5 months of mildly atherogenic diet in ovariectomized (OVX) Cynomolgus monkeys and 2) the pharmacologically evoked coronary vasospasm in the OVX Rhesus monkey. In the first model, after 3 months of continuous oral administration in the diet at 0.1 mg/kg/day, E2 prevented the paradoxical response to Ach, alone as well as combined with 0.25 mg/kg/day NOMAC, whereas NOMAC counteracted the endometrial stimulation. In the second model, after one artificial cycle consisting of 28 days of E2 subcutaneous (s.c.) implant and of daily oral gavage with 1 mg/kg/day of NOMAC for the last 14 days, no vasospasm (0 of 11 tested animals) occurred when the complete challenge protocol, including serotonin and the thromboxane agonist U46619, was administered to OVX Rhesus monkeys. In the balanced crossover design, identical artificial cycles with medroxyprogesterone acetate (MPA) at the same dose resulted in 7 vasospasms in 12 animals. In parallel, effective progestative activity was demonstrated by a secretory pattern in endometrial sections obtained at the end of the cycle. In these two nonhuman primate cardiovascular models, NOMAC did not have the negating effects observed with MPA.     

GeneTools – application for functional annotation and statistical hypothesis testing

Background  

Modern biology has shifted from "one gene" approaches to methods for genomic-scale analysis like microarray technology, which allow simultaneous measurement of thousands of genes. This has created a need for tools facilitating interpretation of biological data in "batch" mode. However, such tools often leave the investigator with large volumes of apparently unorganized information. To meet this interpretation challenge, gene-set, or cluster testing has become a popular analytical tool. Many gene-set testing methods and software packages are now available, most of which use a variety of statistical tests to assess the genes in a set for biological information. However, the field is still evolving, and there is a great need for "integrated" solutions.     

Spontaneous contractions of dispersed vascular muscle in cell culture.

Dispersed vascular muscle cells from chick omphalomesenteric vessels maintained in primary cell culture contracted spontaneously. Six methods which produced contracting isolated muscle cells are described and compared. The combination of dispersion method and culture conditions to produce contracting muscle cells was more critical for vascular than for heart muscle. These findings of continuing pacemaker function demonstrate that functional integrity of isolated vascular muscle cells is possible to maintain. Further indication of the full functional state of the isolated vascular muscle cells was demonstrated by the sensitivity to norepinephrine at a physiological concentration (0.1 muM). Spontaneous contraction frequencies were similar to the range found in situ, and spontanious or norepinephrine-induced contractions had time courses corresponding to intact vessel contractions. This is the first report that isolated vascular muscle cells in primary cell culture retain functional characteristics found in situ and are suitable for pharmacological characterization of individual muscle cells.