Modulation of rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein.

We used the yeast two-hybrid system to identify proteins that interact directly with Galpha(o). Mutant-activated Galpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that Galpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with Galpha(o) and Galpha(i) but not with Galpha(s) or Galpha(q). Rap1GAP interacts more avidly with the unactivated Galpha(o) as compared with the mutant (Q205L)-activated Galpha(o). When expressed in HEK-293 cells, unactivated Galpha(o) co-immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin. When unactivated Galpha(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-Galpha(o). Expression of unactivated Galpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of Galpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions.     

A dye-binding induced conformational transition of poly-(methacrylic acid) by Auramine O in aqueous solutions. II. Theoretical interpretation and discussion of the experimental results

The binding of Auramine O to poly-(methacrylic acid) (PMA) is explained using a two-state model for the polyelectrolyte and preferential binding of the dye to the hypercoiled conformational state of PMA predominantly present for the dye-free polyelectrolyte at low degrees of neutralization. Bound-dye interactions were neglected leading to a binding isotherm as given by Monod et al. to which the experimental dialysis results could be fitted. It is shown that this model predicts a conformational transition from the more extended conformational state of PMA to the hyper-coiled one upon progressive binding of the dye. The experimental results obtained by potentiometric and viscosimetric titrations as well as the fluorcsence intensity measurements of the AuO—PMA system arc consistent with the conclusions based on this model.     

Regional hemodynamic responses to hypoxia in polycythemic dogs

Polycythemia increases blood viscosity so that systemic O2 delivery (QO2) decreases and its regional distribution changes. We examined whether hypoxia, by promoting local vasodilation, further modified these effects in resting skeletal muscle and gut in anesthetized dogs after hematocrit had been raised to 65%. One group (CON, n = 7) served as normoxic controls while another (HH, n = 6) was ventilated with 9% O2--91% N2 for 30 min between periods of normoxia. Polycythemia decreased cardiac output so that QO2 to both regions decreased approximately 50% in both groups. In compensation, O2 extraction fraction increased to 65% in muscle and to 50% in gut. When QO2 was reduced further during hypoxia, blood flow increased in muscle but not in gut. Unlike previously published normocythemic studies, there was no initial hypoxic vasoconstriction in muscle. Metabolic vasodilation during hypoxia was enhanced in muscle when blood O2 reserves were first lowered by increased extraction with polycythemia alone. The increase in resting muscle blood flow during hypoxia with no change in cardiac output may have decreased O2 availability to other more vital tissues. In that sense and under these experimental conditions, polycythemia caused a maladaptive response during hypoxic hypoxia.     

NCAM deficiency in the mouse forebrain impairs innate and learned avoidance behaviours

The neural cell adhesion molecule (NCAM) has been implicated in the development and plasticity of neural circuits and the control of hippocampus‐ and amygdala‐dependent learning and behaviour. Previous studies in constitutive NCAM null mutants identified emotional behaviour deficits related to disturbances of hippocampal and amygdala functions. Here, we studied these behaviours in mice conditionally deficient in NCAM in the postmigratory forebrain neurons. We report deficits in both innate and learned avoidance behaviours, as observed in elevated plus maze and passive avoidance tasks. In contrast, general locomotor activity, trait anxiety or neophobia were unaffected by the mutation. Altered avoidance behaviour of the conditional NCAM mutants was associated with a deficit in serotonergic signalling, as indicated by their reduced responsiveness to (±)‐8‐hydroxy‐2‐(dipropylamino)‐tetralin‐induced hypo-thermia. Another serotonin‐dependent behaviour, namely intermale aggression that is massively increased in constitutively NCAM‐deficient mice, was not affected in the forebrain‐specific mutants. Our data suggest that genetically or environmentally induced changes of NCAM expression in the late postnatal and mature forebrain determine avoidance behaviour and serotonin (5‐HT)_1A receptor signalling.     

Anxiety and increased 5-HT1A receptor response in NCAM null mutant mice.

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety-like behavior of homozygous (NCAM-/-) and heterozygous (NCAM/-) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety-like behavior was reduced in both NCAM+/+ and NCAM-/- mice by systemic administration of the benzodiazepine agonist diazepam and the 5-HT1A receptor agonists buspirone and 8-OH-DPAT. However, NCAM-/- mice showed anxiolytic-like effects at lower doses of buspirone and 8-OH-DPAT than NCAM+/+ mice. Such increased response to 5-HT1A receptor stimulation suggests a functional change in the serotonergic system of NCAM-/- mice, likely involved in the control of anxiety and aggression. However, 5-HT1A receptor binding and tissue content of serotonin and its metabolite 5-hydroxyindolacetic acid were found unaltered in every brain area of NCAM-/- mice investigated, indicating that expression of 5-HT1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM-/- mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5-HT1A receptors and inwardly rectifying K+ channels as the respective effector systems.     

A dye-binding induced conformational transition of poly-(methacrylic acid) by Auramine O in aqueous solutions. I. Experimental results

Binding of auramine O to poly-(methacrylic acid) (PMA) has been established over a large range of C⁰_p/C⁰_d values using spectroscopic methods (UV absorption and visible fluorescence emission spectra), equilibrium and sedimentation dialysis, potentiometric and viscosimetric titrations. All the results show qualitative agreement with those obtained previously with the system crystal violet-PMA although the binding seems to be less strong for auramine O than for crystal violet. From dialysis experiments binding isotherms were obtained at three different degrees of neutralization α'; at α' = 0.10 the results could be fitted to a Langmuir isotherm but at α' equal to 0.40 and 0.65 deviations with respect to such an isotherm occur. The results of potentiometric and viscosimetric titrations confirm that the conformational transition which the dye-free PMA exhibits upon ionization is affected by the dye binding. The region in which the conformational transion occurs is broadened and is less sharply defined in the presence of auramine O.     

The Conservation of Saproxylic Insects in Tropical Forests: A Research Agenda

Invariably, insects are overlooked when tropical forest management issues are discussed, because there are so many species, they are taxonomically intractable and so poorly known. Often people take the view that if you look after the vegetation and vertebrates, the insects will look after themselves. This may be true for some functional groups, but for saproxylic insects, this seems unlikely. Their study deserves high priority, since they are dependent on the very resource – wood – whose removal from the ecosystem is the usual object of forest management. Given the current international effort to develop 'criteria and indicators' to monitor sustainable forest management for biodiversity values, there is a window of opportunity for sound ecological research on saproxylic insects to influence the formulation of forest policy such that their needs can be taken into account. There is already a large body of knowledge on temperate and boreal region saproxylic insects, and on the effects that logging has on them, but knowledge of the tropical forest situation lags far behind. This paper proposes a research agenda to enable the needs of saproxylic insects to be taken into account in natural forest management in the tropics. Basic questions, such as whether logging has so far had an impact on tropical saproxylic insects, and whether there are workable sampling techniques to investigate this, still remain to be addressed and deserve high priority. The links between the responses of saproxylic insects and more 'charismatic' study species need to be investigated. We also need to know whether there is a correlation between the intensity of logging and the response of saproxylic insects, and, critically, whether we would be justified in measuring some surrogate aspects of forest structure (as potential habitat for saproxylic insects) rather than the saproxylic insects themselves, and modelling this to determine likely impacts of different management regimes. We consider such an ambitious research agenda as justified given the scale of impact that forest use and management is likely to have on tropical forest insects in the future.