Synergistic stimulation of the Ca2+ influx in rat hepatocytes by glucagon and the Ca2+-linked hormones vasopressin and angiotensin II

Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin II, and the effects on the initial 45Ca2+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly increased the initial 45Ca2+ uptake rate. When glucagon was added together with vasopressin or angiotensin II--both of which when added separately increase the initial 45Ca2+ uptake rate but did not affect the cellular content of cyclic AMP--the measured initial 45Ca2+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca2+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or forskolin, suggesting that cyclic AMP augments both the resting Ca2+ and the vasopressin- or angiotensin II-stimulated influx. Measurement of the initial 45Ca2+ uptake rate as a function of the extracellular Ca2+ concentration indicated that the increase in the Ca2+ influx resulting from single or combined glucagon and vasopressin administration occurred through a homogeneous population of Ca2+ gates. These hormones were found to raise both the apparent Km for external Ca2+ and the apparent Vmax of the Ca2+ influx. The maximal increase in these two parameters was observed when the two hormones were added together. This suggests that glucagon and vasopressin synergistically stimulate the same Ca2+ gating mechanism. The dose-response curves for the action of glucagon or vasopressin applied in the presence of increasing concentrations of vasopressin or glucagon, respectively, showed that each hormone increases the maximal response to the other without affecting its ED50. It is proposed that glucagon and the Ca2+-linked hormones control the cellular concentration of two intermediates which are both necessary to allow Ca2+ entry into the cells.     

Production and purification of monoclonal antibodies to Pisum and Avena phytochrome

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Hormonal regulation of inositol 1,4,5-trisphosphate receptor in rat liver

Inositol 1,4,5-trisphosphate (IP3) is a second messenger which induces Ca2+ release from an intracellular store. We have investigated the properties of the [32P]IP3 binding sites in rat liver. Two specific [32P]IP3 receptors with KD of 2.3 and 88 nM and respective capacities of 33 fmol/mg protein and 195 fmol/mg protein have been detected in a crude membrane fraction prepared from rat liver homogenate. The pretreatment of the liver with IP3-dependent hormones increased two-fold the capacity of the high affinity site. This effect was partly reversed by dibutyryl cyclic AMP. Permeabilized hepatocytes also displayed two [32P]IP3 binding sites with KD of 1.5 and 84 nM and respective capacities of 8 and 300 fmol/10(6) cells. We have measured the [32P]IP3 binding and the IP3-induced 45Ca2+ release in the same batch of permeabilized hepatocytes. In a low Mg2+ medium, the EC50 for 45Ca2+ release was in close correlation with the KD for the low affinity site. These data suggest that an equilibrium between two states of the IP3 receptor is regulated by hormone action and the low affinity state is responsible for the intracellular Ca2+ release.     

Inhibitory effect of dihydroxyacetone onGluconobacter oxydans: Kinetic aspects and expression by mathematical equations

Summary Microbial conversion of glycerol into dihydroxyacetone (DHA) byGluconobacter oxydans was subjected to inhibition by excess substrate. Comparison of cultures containing increasing initial DHA contents (0 to 100 g l^–1) demonstrated that DHA also inhibited this fermentation process. The first effect was on bacterial growth (cellular development stopped when DHA concentration reached 67 gl^–1), and then on oxidation of glycerol (DHA synthesis only occurred when the DHA concentration in the culture medium was lower than 85 g l^–1). Productivity, specific rates and, to a lesser extent, conversion yields decreased as initial concentrations of DHA increased. The changes in the specific parameters according to increasing initial DHA contents were described by general equations. These formulae satisfactorily express the concave aspect of the curves and the reduction in biological activity when the cells were in contact with DHA concentrations of up to 96 g l^–1.Abbreviations X, S, P biomass, substrate, product concentrations - r _x,r _s,r _p rates of growth, consumption and production - ,q _s,q _p specific rates of growth, glycerol consumption and DHA production - Y _x/s, Y_p/s conversion yields of substrate into biomass and product - K _s constant of affinity of cells to the substrate - K _ip product inhibition constant - P _m threshold concentration of DHA in substrate     

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.     

Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys

The ovariectomized old cynomolgus monkey is a recognized model of human osteoporosis, and the same species can be used for the assessment of the efficacy and potential toxicity of agents intended to prevent or treat osteoporosis. Several assays have been developed that can measure the same biochemical markers of bone turnover as are used in human patients for the diagnosis and treatment follow-up of bone-related diseases, including osteoporosis. The aim of the present study was to describe the results obtained with these assays in normal control monkeys, their variations with age and sex, and their sensitivity in monitoring the bone turnover induced by ovariectomy in old skeletally mature cynomolgus monkeys. Seven old cynomolgus monkeys were bilaterally ovariectomized and 13 age-matched monkeys were sham-operated. Bone mineral density and biochemical markers were measured before and at regular intervals after surgery for up to 20 months. Total alkaline phosphatase (total ALP), bone-specific alkaline phosphatase isoenzyme (bone ALP) and osteocalcin (OC) were highly correlated to the decrease in bone mineral density (BMD) induced by ovariectomy. Deoxypyridinoline (DPD) measured by enzyme-linked immunoassay was insensitive to the bone resorption induced by ovariectomy, but cross-linked N-telopeptide (NTX-I) was higher in ovariectomized monkeys than in control monkeys. These results demonstrate that reliable biochemical parameters are available to adequately monitor and provide insight into osteoclastic bone resorption and osteoblastic bone formation, the two components of bone turnover in this animal model, and can thus be used to assess the efficacy and toxicity of potential therapeutic agents.     

Audio-Visual Perception of 3D Cinematography: An fMRI Study Using Condition-Based and Computation-Based Analyses

The use of naturalistic stimuli to probe sensory functions in the human brain is gaining increasing interest. Previous imaging studies examined brain activity associated with the processing of cinematographic material using both standard “condition-based” designs, as well as “computational” methods based on the extraction of time-varying features of the stimuli (e.g. motion). Here, we exploited both approaches to investigate the neural correlates of complex visual and auditory spatial signals in cinematography. In the first experiment, the participants watched a piece of a commercial movie presented in four blocked conditions: 3D vision with surround sounds (3D-Surround), 3D with monaural sound (3D-Mono), 2D-Surround, and 2D-Mono. In the second experiment, they watched two different segments of the movie both presented continuously in 3D-Surround. The blocked presentation served for standard condition-based analyses, while all datasets were submitted to computation-based analyses. The latter assessed where activity co-varied with visual disparity signals and the complexity of auditory multi-sources signals. The blocked analyses associated 3D viewing with the activation of the dorsal and lateral occipital cortex and superior parietal lobule, while the surround sounds activated the superior and middle temporal gyri (S/MTG). The computation-based analyses revealed the effects of absolute disparity in dorsal occipital and posterior parietal cortices and of disparity gradients in the posterior middle temporal gyrus plus the inferior frontal gyrus. The complexity of the surround sounds was associated with activity in specific sub-regions of S/MTG, even after accounting for changes of sound intensity. These results demonstrate that the processing of naturalistic audio-visual signals entails an extensive set of visual and auditory areas, and that computation-based analyses can track the contribution of complex spatial aspects characterizing such life-like stimuli.