Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid ��-Spectrin in a Calcium- and Calmodulin-dependent Manner

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph^1J/sph^1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G₃ peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K_d = 0.30 μm). EF-domains bearing the sph^1J mutation are inactive. Binding of protein 4.2 to band 3 (K_d = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca²⁺ (but not Mg²⁺) by three to five times. Calmodulin also binds to the EF-domain (K_d = 17 μm), and Ca²⁺-calmodulin blocks Ca²⁺-dependent binding of protein 4.2 but not Ca²⁺-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

Zinc-induced Neurotoxicity Mediated by Transient Receptor Potential Melastatin 7 Channels

Transient receptor potential melastatin 7 (TRPM7) channels are novel Ca²⁺-permeable non-selective cation channels ubiquitously expressed. Activation of TRPM7 channels has been shown to be involved in cellular Mg²⁺ homeostasis, diseases caused by abnormal magnesium absorption, and in Ca²⁺-mediated neuronal injury under ischemic conditions. Here we show strong evidence suggesting that TRPM7 channels also play an important role in cellular Zn²⁺ homeostasis and in Zn²⁺-mediated neuronal injury. Using a combination of fluorescent Zn²⁺ imaging, small interfering RNA, pharmacological analysis, and cell injury assays, we show that activation of TRPM7 channels augmented Zn²⁺-induced injury of cultured mouse cortical neurons. The Zn²⁺-mediated neurotoxicity was inhibited by nonspecific TRPM7 blockers Gd³⁺ or 2-aminoethoxydiphenyl borate, and by knockdown of TRPM7 channels with small interfering RNA. In addition, Zn²⁺-mediated neuronal injury under oxygen-glucose deprivation conditions was also diminished by silencing TRPM7. Furthermore, we show that overexpression of TRPM7 channels in HEK293 cells increased intracellular Zn²⁺ accumulation and Zn²⁺-induced cell injury, while silencing TRPM7 by small interfering RNA attenuated the Zn²⁺-mediated cell toxicity. Thus, TRPM7 channels may represent a novel target for neurological disorders where Zn²⁺ toxicity plays an important role.     

Regulation of phosphate starvation responses in higher plants

Background

Phosphorus (P) is often a limiting mineral nutrient for plant growth. Many soils worldwide are deficient in soluble inorganic phosphate (P_i), the form of P most readily absorbed and utilized by plants. A network of elaborate developmental and biochemical adaptations has evolved in plants to enhance P_i acquisition and avoid starvation.

Scope

Controlling the deployment of adaptations used by plants to avoid P_i starvation requires a sophisticated sensing and regulatory system that can integrate external and internal information regarding P_i availability. In this review, the current knowledge of the regulatory mechanisms that control P_i starvation responses and the local and long-distance signals that may trigger P_i starvation responses are discussed. Uncharacterized mutants that have P_i-related phenotypes and their potential to give us additional insights into regulatory pathways and P_i starvation-induced signalling are also highlighted and assessed.

Conclusions

An impressive list of factors that regulate P_i starvation responses is now available, as is a good deal of knowledge regarding the local and long-distance signals that allow a plant to sense and respond to P_i availability. However, we are only beginning to understand how these factors and signals are integrated with one another in a regulatory web able to control the range of responses demonstrated by plants grown in low P_i environments. Much more knowledge is needed in this agronomically important area before real gains can be made in improving P_i acquisition in crop plants.     

Hepatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations

Background  

Glutaminase predominates in periportal hepatocytes and it has been proposed that it determines the glutamine-derived nitrogen flow through the urea cycle. Glutamine-derived urea production should, thus, be considerably faster in periportal hepatocytes. This postulate, based on indirect observations, has not yet been unequivocally demonstrated, making a direct investigation of ureogenesis from glutamine highly desirable.     

Substance P binding sites in the nucleus tractus solitarius of the cat

Substance P binding sites in the nucleus tractus solitarius were visualized with receptor autoradiography using Bolton-Hunter [¹²⁵I]substance P. Substance P binding sites were found to have distinct patterns within the cat nucleus tractus solitarius. The majority of substance P binding sites were present in the medial, intermediate and the peripheral rim of the parvocellular subdivisions. Lower amounts of substance P binding sites were present in the commissural, ventrolateral, interstitial and dorsolateral subdivisions. No substance P binding sites were present in the central region of the parvocellular subdivision or the solitary tract. The localization of substance P binding sites in the nucleus tractus solitarius is very similar to the patterns of substance P immunoreactive fibers previously described for this region. Results of this study add further support for a functional role of substance P in synaptic circuits of the nucleus tractus solitarius.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

Morphological variation of Echinococcus granulosus protoscoleces from hydatid cysts of human and various domestic animals in Jordan

, and 1988. Morphological variation of Echinococcus granulosus protoscoleces from hydatid cysts of human and various domestic animals in Jordan. International Journal for Parasitology 18: 1111–1114. Rostellar hook morphology of protoscoleces was employed to study the possible existence of Echinococcus granulosus strains in humans and various domestic animals in Jordan. A distinct form in the donkey was evident as the protoscoleces from this host did not share any characteristics with those from the other hosts examined. Sheep, goats and cattle appeared to be affected by another form since the protoscoleces from their hydatid cysts shared six out of nine characteristics studied. Protoscoleces of camel and human cysts shared seven out of nine characteristics studied and they were different in six characteristics from protoscoleces from other hosts. Differences observed among the three forms may reflect strain variation of E. granulosus in this country.