Concanavalin A receptor(s) possibly interacts with at least two kinds of GTP-binding proteins in murine thymocytes

A part of the GTP gamma S-binding activity in murine thymocyte membranes was found to have affinity to a concanavalin A (Con A)-Sepharose column. The material was identified as Gi (inhibitory GTP-binding protein) on the basis of the molecular weight and by islet activating protein-dependent ADP-ribosylation and anti-alpha i (alpha subunit of Gi) immunoblotting. However, when the membranes prepared from Con A-stimulated thymocytes were used, no GTP gamma S-binding activity was detected in the Con A-bound fraction, suggesting that Gi physically and specifically associated with Con A acceptors dissociates upon Con A stimulation. Furthermore, another GTP gamma S-binding protein (25 kDa), which is quite similar to a novel phosphoinositide-specific phospholipase C (PI-PLC)-associated G protein in calf thymocytes (Wang, P., Toyoshima, S., & Osawa, T. (1988) J. Biochem. 103, 137-142), was detected among the Con A-Sepharose-bound proteins with the chemical cross-linking technique. When the 40 kDa and 25 kDa G proteins associated with Con A receptor(s) were isolated and their direct effects on the activity of partially purified PI-PLC as to phosphatidylinositol 4,5-bisphosphate hydrolysis were examined, the 25 kDa G protein was found to enhance the PI-PLC activity more effectively. On the other hand, pretreatment of cells with islet-activating protein completely abolished the inhibitory effect of Con A on the prostaglandin E1 and isoproterenol-induced increases of cellular cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.     

Effect of deuterium oxide on the thermodynamic quantities associated with phase transitions of phosphatidylcholine bilayer membranes

The bilayer phase transitions of three kinds of phospholipids, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dihexadecylphosphatidylcholine (DHPC), in deuterium oxide (D₂O) and hydrogen oxide (H₂O) were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The DSC measurements showed that the substitution of H₂O by D₂O affected the pretransition temperatures and the main-transition enthalpies of all PC bilayers. The temperature-pressure phase diagrams for these PC bilayer membranes in both solvents were constructed by use of the data of light-transmittance measurements. Regarding the main transition of all PC bilayer membranes, there was no appreciable difference between the transition temperatures in D₂O and H₂O under high pressure. On the other hand, the phase transitions among the gel phases including the pretransition were significantly affected by the solvent substitution. The thermodynamic quantities of phase transitions for the PC bilayer membranes were evaluated and the differences in thermodynamic properties by the water substitution were considered from the difference of interfacial-free energy per molecule in the bilayer in both solvents. It was proved that the substitution of H₂O by D₂O causes shrinkage of the molecular area of phospholipid at bilayer interface due to the difference in bond strength between deuterium and hydrogen bonds and produces the great influence on the bilayer phase with the smaller area. Further, the induction of bilayer interdigitation in D₂O turned out to need higher pressures than in H₂O.     

A calcitonin-like action of prostaglandin E1

The pharmacological effects of PGE₁ (6 and 9 days, 21,250 μg/kg per day subcutaneously) upon the growth and the bone resorption of mammals were studied using the proximal tibia and upper incisor of immature rats along with lead acetate as a time marker, and upon the serum calcium and inorganic phosphorus levels. The following results were obtained. 1. PGE₁ hardly affected the body weight or the weight of organs of the rats but apparently inhibited the longitudinal growth of proximal tibia in a dose related manner. 2. PGE₁ clearly inhibited not only the longitudinal growth (incisor growth) but also the appositional growth (dentin formation) of incisal dentin. 3. The grade of the inhibitory effect on the growth was in the order of bone growth >dentin formation >incisor growth. 4. The occurrence of osteoporosis due to a low calcium diet was inhibited by the simultaneous administration of PGE₁, the mechanism being considered to be mainly due to the inhibitory effect on the bone resorption. 5. PGE₁ lowered the level of serum calcium and the lowering effect was not observed in the thyro-parathyroidectomized rat. From the facts that the above effects were exactly the same as those of calcitonin (1), the possibility that the subcutaneous injection of PGE₁ may induce a calcitonin-like action, a part of which may dependent on the calcinonin secretion is suggested.     

Aluminum-induced 1-->3-beta-D-glucan inhibits cell-to-cell trafficking of molecules through plasmodesmata. A new mechanism of aluminum toxicity in plants

Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1-->3-beta-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.     

Identification of a stable chymase inhibitor using a pharmacophore-Based database search

In general, serine protease chymase inhibitors readily decompose in plasma. We previously found that thiazolidine-2,4-dione and thiadiazole derivatives are also unstable. Using a pharmacophore-based database search, we identified a benzo[b]thiophen-2-sulfonamide derivative as a stable chymase inhibitor. Finding a lead compound with adequate activity and stability by a pharmacophore-based approach is more efficient than modifying an unstable compound to reduce its instability without simultaneously decreasing its inhibitory activity. Our pharmacophore model of chymase inhibitors suggests that the two hydrophobic interactions in the S1 and S1' regions and the two H-bonding interactions between them play important roles in chymase inhibitors.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

A genetically explicit model of speciation by sensory drive within a continuous population in aquatic environments

Background  

The sensory drive hypothesis predicts that divergent sensory adaptation in different habitats may lead to premating isolation upon secondary contact of populations. Speciation by sensory drive has traditionally been treated as a special case of speciation as a byproduct of adaptation to divergent environments in geographically isolated populations. However, if habitats are heterogeneous, local adaptation in the sensory systems may cause the emergence of reproductively isolated species from a single unstructured population. In polychromatic fishes, visual sensitivity might become adapted to local ambient light regimes and the sensitivity might influence female preferences for male nuptial color. In this paper, we investigate the possibility of speciation by sensory drive as a byproduct of divergent visual adaptation within a single initially unstructured population. We use models based on explicit genetic mechanisms for color vision and nuptial coloration.