Systems analysis of digoxin kinetics and inotropic response in the rat heart: effects of calcium and KB-R7943

To gain more insight into the mechanistic processes controlling the kinetics of inotropic response of digoxin in the perfused whole heart, an integrated kinetic model was developed incorporating digoxin uptake, receptor binding (Na(+)-K(+)-ATPase inhibition), and cellular events linking receptor occupation and response. The model was applied to data obtained in the single-pass Langendorff-perfused rat heart for external [Ca(2+)] of 0.5 and 1.5 mM under control conditions and in the presence of the reverse-mode Na(+)/Ca(2+) exchange inhibitor KB-R7943 (0.1 microM) in perfusate. Outflow concentration and left ventricular developed pressure data measured for three consecutive doses (15, 30, and 45 microg) in each heart were analyzed simultaneously. While disposition kinetics of digoxin was determined by interaction with a heterogeneous receptor population consisting of a high-affinity/low-capacity and a low-affinity/high- capacity binding site, response generation was >80% mediated by binding to the high-affinity receptor. Digoxin sensitivity increased at lower external [Ca(2+)] due to higher stimulus amplification. Coadministration of KB-R7943 significantly reduced the positive inotropic effect of digoxin at higher doses (30 and 45 microg) and led to a saturated and delayed receptor occupancy-response relationship in the cellular effectuation model. The results provide further evidence for the functional heterogeneity of the Na(+)-K(+)-ATPase and suggest that in the presence of KB-R7943 a reduction of the Ca(2+) influx rate via the reverse mode Na(+)/Ca(2+) exchanger might become the limiting factor in digoxin response generation.     

Isolation and Characterization of a Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">morrisoni</Emphasis> PG-14 Encoding δ-Endotoxin Cry1Ac

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.     

Signaling from Nucleotide Receptors to Protein Kinase Cascades in Astrocytes

In the CNS, extracellular ATP can function as an excitatory neurotransmitter as well as a trophic factor. These short-term and long-term actions are mediated by nucleotide receptors. Extracellular ATP can also act as a co-mitogen in conjunction with polypeptide growth factors such as basic fibroblast growth factor (FGF2). Cellular proliferation, differentiation and survival are regulated by signaling cascades composed of protein kinases, including extracellular signal regulated protein kinase (ERK) and protein kinase B (also called Akt). Here we summarize recent studies on nucleotide receptor signaling to ERK and Akt in astrocytes and the role of protein kinase cascades in mediating the trophic actions of extracellular ATP, alone or together with FGF2. Because extracellular ATP and FGF2 contribute to the hyperplastic and hypertrophic response of astrocytes to CNS injuries, an understanding of their protein kinase signaling mechanisms may lead to novel therapeutic approaches for neurological conditions that involve gliosis and the generation of reactive astrocytes, such as trauma, stroke, seizure and neurodegenerative and demyelinating disorders.Special issue dedicated to Lawrence F. Eng.     

Application of monoclonal antibody, specific for intracellular Orientia tsutsugamushi, to immunofluorescent antibody test for determining antibiotic susceptibility

The simple quantification of viable intracellular bacteria is important for the study of an obligate intracellular bacterium, Orientia tsutsugamushi. We applied a novel monoclonal antibody (M686-13)--specific for intracellular Orientia--to an immunofluorescent antibody (IFA) test for determining antibiotic susceptibility of O. tsutsugamushi. M686-13 did not react with Orientia that was inhibited by doxycycline, although bacterial particles still remained in the cells. This preferential staining of proliferating bacteria made the IFA test rapid and precise. Using this method, we could successfully measure the minimal inhibitory concentration (MIC) of a Korean strain of O. tsutsugamushi to doxycycline and clindamycin. This method may be used in other procedures to evaluate the growth of Orientia.     

Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin-dependent endocytosis and not by caveolae-mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule-disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin-mediated endocytosis is likely to be a major route of RV infectious entry.     

Involvement of acetaldehyde in seed deterioration of some recalcitrant woody species through the acceleration of aerobic respiration

The rate of acetaldehyde (Ald) evolution in the deterioration of recalcitrant woody seeds was investigated. Four plant species, Ligustrum japonicum, Quercus serrata, Quercus myrsinaefolia and Camellia japonica, were used for the experiments. Similar to orthodox seeds, all of the recalcitrant seeds used contained Ald in addition to methanol and ethanol, although the amount of Ald in Camellia, a typical oil seed, was very small. These volatiles were accumulated in a container in which Ligustrum and Q. serrata seeds were stored for a short period. Moreover, all of the seeds that had been previously exposed to Ald for only 6 d at 3 or 13 degrees C lost their vigor rapidly in proportion to the concentration of Ald. The occasional removal by decompression of Ald accumulated in the container prolonged the life span of Q. serrata seeds from 4 to 6 months. These findings suggest that a short life span of the hydrated recalcitrant seeds may involve Ald synthesis as in the orthodox seeds. However, the action mechanism of Ald in Ligustrum and Quercus seeds in which storage substances were polysaccharides seems to differ slightly from that in orthodox seeds, because their aerobic respiration was significantly stimulated by exposure to exogenously applied Ald. It was, therefore, thought that the rapid deterioration of some recalcitrant seeds in woody species may result from a decline in vigor, not only due to the denaturation of functional proteins by Ald as in the orthodox seeds but also due to the rapid consumption of direct substrates for the Ald-stimulated aerobic respiration and related co-enzymes within seeds. In contrast, in the oil-bearing Camellia seeds, Ald was slightly produced and their aerobic respiration was not enhanced by Ald, although they were very sensitive to Ald. Desiccation storage of Camellia seeds caused the deterioration of their outer part, which was accelerated by exogenously applied Ald, which suggests that in Camellia Ald acts only to denature the functional proteins as in orthodox seeds. Thus, the short longevity of these woody recalcitrant seeds is discussed in relation to the actions of Ald produced endogenously.     

Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.     

Characterisation and transferability of apple SSRs to two European pear F<Subscript>1</Subscript> populations

European pear (Pyrus communis L.) is among the important fruit species for which only few genetic studies have been carried out. Available evidence indicates that simple sequence repeats (SSR) are very useful as molecular markers because they are codominant, highly polymorphic, abundant and reproducible. The present paper reports more than 100 apple SSR markers in two populations of European pear; a total of 41 SSR markers were then positioned on a genetic linkage map of the cross Passe Crassane × Harrow Sweet and 31 in the map Abbè Fétel × Max Red Bartlett. Syntenic relationships between pear and apple maps have been considered for the chromosomes carrying two or more SSR markers. The alignment among the two maps supports the colinearity of the two genomes with respect both to identification and to orientation of the linkage groups.     

Hyaluronic acid inhibits adhesion of hepatic stellate cells in spite of its stimulation of DNA synthesis

Unbalanced accumulation of fibers in extracellular matrix (ECM) results from attachment and activation of hepatic stellate cells (HSCs) during chronic liver diseases, in which the content of hyaluronic acid (HA), a glycosaminoglycan, in ECM changes. No information is available on the effect of HA on adhesion and activation of HSCs although that of collagen (Col) on HSCs was extensively studied. This study investigated the effects of HA with or without Col on adhesion of HSCs or the rate of DNA synthesis. Attachment of primary cultured HSCs was microscopically monitored in the plate simultaneously coated with HA or other ECM components. HA inhibited adhesion of quiescent HSCs at least up to 7 days after seeding, whereas HSCs were adherent to plastic or type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV) or fibronectin. Both microscopy and alpha-smooth muscle actin immunocytochemistry revealed that the number of HSCs, which had been re-seeded after 15 days of culture, attached to HA-coated area was remarkably lower compared to that of HSCs on Col-I or plastic. Incorporation of HA into Col-I prevented adhesion of activated HSCs to matrix film. The number of HSCs adherent to HA at early times after seeding was minimal and significantly lower than that of the cells adherent to plastic. In contrast, either Col-I or Col-IV increased the number of adherent cells. Attachment of HSCs to plastic was inhibited by soluble HA in culture medium. CD44, the cell surface receptor to which HA binds, was immunochemically detected in HSCs. Adhesion of HSCs to plastic, HA or Col-I was not changed by anti-CD44 antibody. Either HA or Col increased the basal or platelet-derived growth factor-inducible rate of thymidine incorporation into DNA in HSCs. In conclusion, HA inhibits adhesion of quiescent or activated HSCs in spite of its stimulation of DNA synthesis, whereas Col increases HSC attachment and DNA synthesis, and inhibition of HSC adhesion by HA does not involve CD44.