Fitness components versus total demographic effects: evaluating herbivore impacts on a perennial herb

I examined the long-term effects of cryptic mollusk herbivory on seven fitness components in the perennial herb Lathyrus vernus and also calculated measures of total fitness effects. Natural correlations and experimental exclusion of mollusks showed that herbivory is associated with an increased probability of dying or staying dormant, poorer growth, and a lower probability of flowering. The average yearly reduction in population growth rate (lambda) caused by mollusks in the experiment was 0.14. The largest contribution to this decrease in total fitness occurred through a decreased survival of established plants. In contrast, seedling emergence and probability of flowering were the fitness components that were most affected in terms of relative change. The more important a life-cycle transition was for population growth rate in terms of its elasticity, the less it was affected by herbivore damage. These results suggest that simple analyses of the magnitudes of effects on individual components of plant performance are poor predictors of the magnitude of total fitness effects and tolerance to herbivory. This is because total fitness is differently sensitive to different phases of the life cycle and because plants strive to maintain the functions most important to fitness.     

Computational modelling of the open-state Kv 1.5 ion channel block by bupivacaine

Binding of R(+)-bupivacaine to open-state homology models of the mammalian K(v)1.5 membrane ion channel is studied using automated docking and molecular dynamics (MD) methods. Homology models of K(v)1.5 are built using the 3D structures of the KcsA and MthK channels as a template. The packing of transmembrane (TM) alpha-helices in the KcsA structure corresponds to a closed channel state. Opening of the channel may be reached by a conformational transition yielding a bent structure of the internal S6 helices. Our first model of the K(v) open state involves a PVP-type of bending hinge in the internal helices, while the second model corresponds to a Gly-type of bending hinge as found in the MthK channel. Ligand binding to these models is probed using the common local anaesthetic bupivacaine, where blocker binding from the intracellular side of the channel is considered. Conformational properties and partial atomic charges of bupivacaine are determined from quantum mechanical HF/6-31G* calculations with inclusion of solvent effects. The automated docking and MD calculations for the PVP-bend model predict that bupivacaine could bind either in the central cavity or in the PVP region of the channel pore. Linear interaction energy (LIE) estimates of the binding free energies for bupivacaine predict strongest binding to the PVP region. Surprisingly, no binding is predicted for the Gly-bend model. These results are discussed in light of electrophysiological data which show that the K(v)1.5 channel is unable to close when bupivacaine is bound.     

Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate

High-resolution solid-state NMR methods have been used to analyze the conformation of the chromophore in the late photointermediate metarhodopsin-I, from observation of (13)C nuclei introduced into the beta-ionone ring (at the C16, C17, and C18 methyl groups) and into the adjoining segment of the polyene chain (at C8). Bovine rhodopsin in its native membrane was also regenerated with retinal that was (13)C-labeled close to the 11-Z bond (C20 methyl group) to provide a reporter for optimizing and quantifying the photoconversion to metarhodopsin-I. Indirect photoconversion via the primary intermediate, bathorhodopin, was adopted as the preferred method since approximately 44% conversion to the metarhodopsin-I component could be achieved, with only low levels (approximately 18%) of ground-state rhodopsin remaining. The additional photoproduct, isorhodopsin, was resolved in (13)C spectra from C8 in the chain, at levels of approximately 38%, and was shown using rotational resonance NMR to adopt the 6-s-cis conformation between the ring and the polyene chain. The C8 resonance was not shifted in the metarhodopsin-I spectral component but was strongly broadened, revealing that the local conformation had become less well defined in this segment of the chain. This line broadening slowed rotational resonance exchange with the C17 and C18 ring methyl groups but was accounted for to show that, despite the chain being more relaxed in metarhodopsin-I, its average conformation with respect to the ring was similar to that in the ground state protein. Conformational restraints are also retained for the C16 and C17 methyl groups on photoactivation, which, together with the largely preserved conformation in the chain, argues convincingly that the ring remains with strong contacts in its binding pocket prior to activation of the receptor. Previous conclusions based on photocrosslinking studies are considered in view of the current findings.     

Near-critical phenomena in intracellular metabolite pools

The supply and consumption of metabolites in living cells are catalyzed by enzymes. Here we consider two of the simplest schemes where one substrate is eliminated through Michaelis-Menten kinetics, and where two types of substrates are joined together by an enzyme. It is demonstrated how steady-state substrate concentrations can change ultrasensitively in response to changes in their supply rates and how this is coupled to slow relaxation back to steady state after a perturbation. In the one-substrate system, such near-critical behavior occurs when the supply rate approaches the maximal elimination rate, and in the two-substrate system it occurs when the rates of substrate supply are almost balanced. As systems that operate near criticality tend to display large random fluctuations, we also carried out a stochastic analysis using analytical approximations of master equations and compared the results with molecular-level Monte Carlo simulations. It was found that the significance of random fluctuations was directly coupled to the steady-state sensitivity and that the two substrates can fluctuate greatly because they are anticorrelated in such a way that the product formation rate displays only small variation. Basic relations are highlighted and biological implications are discussed.     

Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.     

Protein kinase D: a family affair

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.     

The small GTPase Rap1 is activated by turbulence and is involved in integrin [alpha]IIb[beta]3-mediated cell adhesion in human megakaryocytes

The small GTPase Rap1, which is activated by a large variety of stimuli, functions in the control of integrin-mediated cell adhesion. Here we show that in human megakaryocytes and several other commonly used hematopoietic cell lines such as K562, Jurkat, and THP-1, stress induced by gentle tumbling of the samples resulted in rapid and strong activation of Rap1. This turbulence-induced activation could not be blocked by inhibitors previously shown to affect Rap1 activation in human platelets, such as the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and various protein kinase C inhibitors. Also inhibition of actin cytoskeleton dynamics did not influence this activation of Rap1, suggesting that this activation is mediated by cell surface receptors. Human platelets, however, were refractory to turbulence-induced activation of Rap1. To determine the consequences of Rap1 activation we measured adhesion of megakaryocytes to fibrinogen, which is mediated by the integrin alphaIIbbeta3, in the presence of inhibitors of Rap1 signaling. Introduction of both Rap1GAP and RalGDS-RBD in the megakaryoblastic cell line DAMI strongly reduced basal adhesion to immobilized fibrinogen. This inhibition was partially rescued by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate but not by alpha-thrombin. From these results we conclude that in megakaryocytes turbulence induces Rap1 activation that controls alphaIIbbeta3-mediated cell adhesion.