Effects of ATP and adenosine addition on activity of oxoglutarate dehydrogenase and the concentration of cytoplasmic free Ca2+ in rat hepatocytes

Addition of ATP (100 microM) to hepatocytes from starved rats incubated with 5 mM [1-14C]glutamine caused a stimulation of glucose formation; the magnitude of the concomitant increases in 14CO2 production and glutamine consumption indicate that flux from glutamine to glucose was increased. ATP also caused a simultaneous decrease in the cell content of oxoglutarate; together with the increased flux this is consistent with an activation of oxoglutarate dehydrogenase. In corroboration of this, a stimulation by ATP of gluconeogenesis and a decrease in oxoglutarate was also observed with 5 mM proline as substrate. ATP caused an increase in hepatocyte cytoplasmic free Ca2+ concentration, [Ca2+]c, as indicated by the increase in the fluorescence of cytoplasmically trapped quin2, from a resting value of about 0.2 microM to greater than 1 microM. The mechanism of oxoglutarate dehydrogenase activation may be via an increase in mitochondrial Ca2+ content as a consequence of the increase in [Ca2+]c. The effects of 100 microM adenosine were also investigated. An increase in flux from glutamine to glucose was observed together with a decrease in the cell oxoglutarate, thus indicating that adenosine addition to hepatocytes could also activate oxoglutarate dehydrogenase. The activation by adenosine was less than that produced by ATP. Adenosine caused a small apparent increase in [Ca2+]c to 0.3-0.4 microM; it remains to be established if this effect, which is small relative to that of ATP, is sufficient to elicit the activation of oxoglutarate dehydrogenase: alternative mechanisms may exist.     

Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells

Pasteurella multocida toxin, both native and recombinant, is an extremely potent mitogen for Swiss 3T3 cells and acts to enhance the formation of total inositol phosphates (Rozengurt, E., Higgins, T., Changer, N., Lax, A.J., and Staddon, J.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 123-127). P. multocida toxin also stimulates diacylglycerol production and activates protein kinase C (Staddon, J.M., Chanter, N., Lax, A.J., Higgins, T.E., and Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848). Here we analyze, by [3H]inositol labeling and high performance liquid chromatography, the inositol phosphates in recombinant P. multocida toxin-treated cells. Recombinant P. multocida toxin stimulated increases in [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and its metabolic products, including Ins(1,3,4,5)P4, Ins(1,3,4)P3, Ins(1,4)P2, Ins(4/5)P, and Ins(1/3)P. The profile of the increase in the cellular content of these distinct inositol phosphates was very similar to that elicited by bombesin. Furthermore, recombinant P. multocida toxin, like bombesin, mobilizes an intracellular pool of Ca2+. Recombinant P. multocida toxin pretreatment greatly reduces the Ca2(+)-mobilizing action of bombesin, consistent with Ca2+ mobilization from a common pool by the two agents. The enhancement of inositol phosphates and mobilization of Ca2+ by recombinant P. multocida toxin were blocked by the lysosomotrophic agents methylamine, ammonium chloride, and chloroquine and occurred after a dose-dependent lag period. The stimulation of inositol phosphate production by recombinant P. multocida toxin persisted after removal of extracellular toxin, in contrast to the reversibility of the action of bombesin. Recombinant P. multocida toxin, unlike bombesin and guanosine 5'-O-(gamma-thiotriphosphate), did not cause the release of inositol phosphates in permeabilized cells. These data demonstrate that recombinant P. multocida toxin, acting intracellularly, stimulates the phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate.     

Effects of mycorrhizal colonization and elevated atmospheric carbon dioxide on carbon fixation and below-ground carbon partitioning in Plantago lanceolata

Plantago lanceolata with or without the mycorrhizal fungus Glomus mosseae were grown over a 100 d period under ambient (38050 mol mol^-1) and elevated (600150 mol mol^-1) atmospheric CO2 conditions. To achieve similar growth, non-mycorrhizal plants received phosphorus in solution whereas mycorrhizal plants were supplied with bonemeal. Measures of plant growth, photosynthesis and carbon input to the soil were obtained. Elevated CO2 stimulated plant growth to the same extent in mycorrhizal and non0mycorrhizal plants, but had no effect on the partitioning of carbon between shoots and roots or on shoot tissue phosphorus concentration. Mycorrhizal colonization was low, but unaffected by CO2 treatment. Net photosynthesis was stimulated both by mycorrhizal colonization and elevated CO2, and there was a more than additive effect of the two treatments on net photosynthesis. Colonization by mycorrhizal fungi inhibited acclimation, in terms of net carbon assimilation, or plants to elevated CO2. ¹³C natural abundance techniques were used to measure carbon input into the soil, although the results were not conclusive. Direct measurements of below-ground root biomass showed that elevated CO2 did stimulate carbon flow below-ground and this was higher in mycorrhizal than non-mycorrhizal plants. For the four treatment combinations, the observed relative differences in amount of below-ground carbon were compared with those expected from the differences in net photosynthesis. A considerable amount of the extra carbon fixed both as a result of mycorrhizal colonization and growth in elevated CO2 did not reveal itself as increased plant biomass. As there was no evidence for a substantial increase in soil organic matter, most of this extra carbon must have been respired by the mycorrhizal fungus and the roots or by the plants as dark-respiration. The need for detailed studies in this area is emphasized.     

Carbon isotopes in functional soil ecology

Soil is an integral part of terrestrial ecosystems. Many soil ecologists interested in soil ecosystem functioning rely, to some degree, on stable isotope methodologies. The study of the natural abundance of carbon isotopes, especially (13)C but also (14)C, in the environment and the use of stable carbon isotope tracers have proved very useful in investigating the soil carbon cycle and soil trophic relationships. Recent methodological and technical advances have greatly extended the possibilities for the application of stable carbon isotopes to terrestrial ecology and have vastly improved our knowledge of belowground ecosystem functioning and will continue to do so. A better understanding of soil processes is invaluable in predicting the future impacts of global environmental change on terrestrial ecosystems.     

The speed of soil carbon throughput in an upland grassland is increased by liming

In situ (13)C pulse labelling was used to measure the temporal and spatial carbon flow through an upland grassland. The label was delivered as (13)C-CO(2) to vegetation in three replicate plots in each of two treatments: control and lime addition. Harvests occurred over a two month period and samples were taken along transects away from the label delivery area. The (13)C concentration of shoot, root, bulk soil, and soil-respired CO(2) was measured. There was no difference in the biomass and (13)C concentration of shoot and root material for the control and lime treatments meaning that the amount of (13)C-CO(2) assimilated by the vegetation and translocated below ground was the same in both treatments. The (13)C concentration of the bulk soil was lower in the lime treatment than in the control and, conversely, the (13)C concentration of the soil-respired CO(2) was higher in the lime. Unlike the difference in bulk soil (13)C concentration between treatments, the difference in the (13)C concentration of the soil-respired CO(2) was obvious only at the delivery site and primarily within 1 d after labelling. An observed increase in the abundance of mycorrhizal fungi in the lime treatment was a possible cause for this faster carbon throughput. The potential key role of mycorrhizas in the soil carbon cycle is discussed. The importance of a better understanding of soil processes, especially biological ones, in relation to the global carbon cycle and environmental change is highlighted.     

Occludin phosphorylation: identification of an occludin kinase in brain and cell extracts as CK2

In epithelial and endothelial cells, tight junctions limit paracellular flux of ions, proteins and other macromolecules. However, mechanisms regulating tight junction function are not clear. Occludin, a tight junction protein, undergoes phosphorylation changes in several situations but little is known about occludin kinases. A recombinant C-terminal fragment of occludin is a substrate for a kinase in crude extracts of brain. This activity was purified about 10000-fold and identified as CK2 (casein kinase 2) by peptide mass fingerprinting, immunoblotting and mutation of CK2 sites within the occludin sequence. CK2 is therefore a candidate kinase for regulation of occludin phosphorylation in vivo.     

Habituation, memory and the brain: the dynamics of interval timing

Memory decay is rapid at first and slower later-a feature that accounts for Jost's memory law: that old memories gain on newer ones with lapse of time. The rate-sensitive property of habituation-that recovery after spaced stimuli may be slower than after massed-provides a clue to the dynamics of memory decay. Rate-sensitive habituation can be modeled by a cascade of thresholded integrator units that have a counterpart in human brain areas identified by magnetic source imaging (MSI). The memory trace component of the multiple-time-scale model for habituation can provide a 'clock' that has the properties necessary to account for both static and dynamic properties of interval timing: static proportional and Weber-law timing as well as dynamic tracking of progressive, 'impulse' and periodic interval sequences.     

Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.     

Consciousness and Theoretical Behaviorism

There are three domains of experience that concern studentsof behavior: Domain 1. The domain of felt experience, the phenomenologicaldomain. Domain 2. The domain of physiology, the real-time functioningof the brain. Domain 3. The domain of behavioral data, "intersubjectivelyverifiable" reports and judgments by experimental subjects.Consciousness has meanings in each of these domains. Domain1 consciousness is beyond the reach of science as public knowledge.Empathy and plausible inference may tell us that our spouse,or our dog, is as conscious as we are. Science cannot. Researchin Domains 2 and 3 permits us to infer similarities and differencesbetween human and non-human psychology. Unfortunately, thesewill never permit us to know ‘what it is like’ tobe another creature. An example from the study of motion perceptionillustrates the point that the fruitless attempt to answer thisquestion can actually impede the objective study of behavioralprocesses we share with non-human animals.     

Dispersal of fig seeds in the Cook Islands: introduced frugivores are no substitutes for natives

Across the Pacific, island vegetation is altering in response to changes in seed disperser assemblages brought about by extinctions and introductions of birds and other animals. On the Cook Islands in the South Pacific, the Pacific Banyan (Ficus prolixa, Moraceae) is undergoing little if any recruitment, possibly linked to a lack of dispersal agents. On Rarotonga, where F. prolixa is found in semi-urban and agricultural environments, there is no recent recruitment in contrast to the situation on Atiu where the tree is common in native forest. We examined the quality and quantity of seed dispersal offered to F. prolixa by the available frugivores on these islands, comparing the effectiveness of extant native and introduced species. The native Cook Islands fauna, particularly birds and bats, appear to be the most effective seed dispersers of F. prolixa, both in terms of quantity and quality. Whilst these are relatively numerous on Atiu, they rarely visit F. prolixa on Rarotonga. The native Chocolate hermit crab Coenobita cavipes is a previously unreported additional native seed disperser, conferring low quantity, but high quality dispersal. Introduced birds and mammals are the most numerous F. prolixa frugivores on Rarotonga and in non-forest environments on Atiu, but they act mainly as seed predators. Consequently, the losses and rarity of remaining native frugivores have not been compensated for by introduced species on Rarotonga which may be contributing to the absence of recruitment there.