Membrane interaction of erythroid spectrin: surface-density-dependent high-affinity binding to phosphatidylethanolamine

Density-dependent spectrin binding to dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine (DMPC/DMPE) small uni-lamellar vesicles (SUVs) has been directly evaluated in this work from the increase in the extent of quenching of the tryptophan fluorescence of spectrin at two different temperatures, above and below the main phase transition temperatures (Tm). Results from the binding studies of spectrin to phospholipid SUVs indicated that the binding dissociation constant Kd, increased from 45 +/- 7 nM in pure DMPC SUVs to 219 +/- 20 nM in DMPC/DMPE (50:50) SUVs, both in the gel and liquid crystalline phase. However, in pure DMPE SUVs the Kd decreased drastically to 0.7 +/- 0.2 nM in the gel phase at 18 degrees C and to 2.6 +/- 0.7 nM in the fluid phase at 55 degrees C indicating a high affinity binding of spectrin for the bilayer-forming DMPE. The maximum extent of phospholipid-induced quenching and the number of spectrin molecules associated with one SUV particle, evaluated in the present work, led to a model in DMPC/DMPE bilayer membranes indicating the PE-binding site of spectrin to localize at one of the terminal domains of the dimeric spectrin. A direct evidence of the localization of the PE-binding site at one of the terminal ends of the spectrin dimer also came from electron microscopic observation in fluid membranes made of bovine brain PE.     

CXCR4-tropic HIV-1 envelope glycoprotein functions as a viral chemokine in unstimulated primary CD4+ T lymphocytes

Interaction of HIV-1 envelope glycoprotein gp120 with the chemokine receptor CXCR4 triggers not only viral entry but also an array of signal transduction cascades. Whether gp120 induces an incomplete or aberrant set of signals, or whether it can function as a full CXCR4 agonist, remains unclear. We report that, in unstimulated human primary CD4(+) T cells, the spectrum of signaling responses induced by gp120 through CXCR4 paralleled that induced by the natural ligand stromal cell-derived factor 1/CXCL12. gp120 activated heterotrimeric G proteins and the major G protein-dependent pathways, including calcium mobilization, phosphoinositide-3 kinase, and Erk-1/2 MAPK activation. Interestingly, gp120 caused rapid actin cytoskeleton rearrangements and profuse membrane ruffling, as evidenced by dynamic confocal imaging. This coordinated set of events resulted in a bona fide chemotactic response. Inactivated HIV-1 virions that harbored conformationally intact envelope glycoproteins also caused actin polymerization and chemotaxis, while similar virions devoid of envelope glycoproteins did not. Thus gp120, in monomeric as well as oligomeric, virion-associated form, elicited a complex cellular response that mimicked the effects of a chemokine. HIV-1 has therefore the capacity to dysregulate the vast CD4(+) T cell population that expresses CXCR4. In addition, HIV-1 may exploit its chemotactic properties to retain potential target cells and locally perturb their cytoskeleton, thereby facilitating viral transmission.     

Production of biodegradable plastics from activated sludge generated from a food processing industrial wastewater treatment plant

Most of the excess sludge from a wastewater treatment plant (60%) is disposed by landfill. As a resource utilization of excess sludge, the production of biodegradable plastics using the sludge has been proposed. Storage polymers in bacterial cells can be extracted and used as biodegradable plastics. However, widespread applications have been limited by high production cost. In the present study, activated sludge bacteria in a conventional wastewater treatment system were induced, by controlling the carbon: nitrogen ratio to accumulate storage polymers. Polymer yield increased to a maximum 33% of biomass (w/w) when the C/N ratio was increased from 24 to 144, where as specific growth yield decreased with increasing C/N ratio. The conditions which are required for the maximum polymer accumulation were optimized and are discussed.     

Structural determinants of proton blockage in aquaporins

Aquaporins are an important class of membrane channels selective for water and linear polyols but impermeable to ions, including protons. Recent computational studies have revealed that the relay of protons through the water-conduction pathway of aquaporin channels is opposed by a substantial free energy barrier peaking at the signature NPA motifs. Here, free-energy simulations and continuum electrostatic calculations are combined to examine the nature and the magnitude of the contribution of specific structural elements to proton blockage in the bacterial glycerol uptake facilitator, GlpF. Potential of mean-force profiles for both hop and turn steps of structural diffusion in the narrow pore are obtained for artificial variants of the GlpF channel in which coulombic interactions between the pore contents and conserved residues Asn68 and Asn203 at the NPA signature motifs, Arg206 at the selectivity filter, and the peptidic backbone of the two half-helices M3 and M7, which are arranged in head-to-head fashion around the NPA motifs, are turned off selectively. A comparison of these results with electrostatic energy profiles for the translocation of a probe cation throughout the water permeation pathway indicates that the free-energy profile for proton movement inside the narrow pore is dominated by static effects arising from the distribution of charged and polar groups of the channel, whereas dielectric effects contribute primarily to opposing the access of H+ to the pore mouths (desolvation penalty). The single most effective way to abolish the free-energy gradients opposing the movement of H+ around the NPA motif is to turn off the dipole moments of helices M3 and M7. Mutation of either of the two NPA Asn residues to Asp compensates for charge-dipole and dipole-dipole effects opposing the hop and turn steps of structural diffusion, respectively, and dramatically reduces the free energy barrier of proton translocation, suggesting that these single mutants could leak protons.     

Activation of a Plasmodium falciparum cdc2-related kinase by heterologous p25 and cyclin H. Functional characterization of a P. falciparum cyclin homologue

Several Plasmodium falciparum genes encoding cdc2-related protein kinases have been identified, but the modalities of their regulation remains largely unexplored. In the present study, we investigated the regulation in vitro of PfPK5, a putative homologue of Cdk1 (cdc2) in P. falciparum. We show that (i) PfPK5 is efficiently activated by heterologous (human) cyclin H and p25, a cyclin-like molecule that specifically activates human Cdk5; (ii) the activated enzyme can be inhibited by chemical Cdk inhibitors; (iii) Pfmrk, a putative P. falciparum homologue of the Cdk-activating kinase, does neither activate nor phosphorylate PfPK5; and (iv) PfPK5 is able to autophosphorylate in the presence of a cyclin. Taken together, these results suggest that the regulation of Plasmodium Cdks may differ in important aspects from that of their human counterparts. Furthermore, we cloned an open reading frame encoding a novel P. falciparum protein possessing maximal homology to cyclin H from various organisms, and we show that this protein, called Pfcyc-1, is able to activate recombinant PfPK5 in vitro with an efficiency similar to that of human cyclin H and p25. This work opens the way to the development of screening procedures aimed at identifying compounds that specifically target the parasite Cdks.     

Amphotericin B both inhibits and enhances T-cell proliferation: inhibitory effect is mediated through H(2)O(2) production via cyclooxygenase pathway by macrophages

Amphotericin B (AmB) has been shown to have both immunosuppressive and -enhancing effects, making its precise nature of action enigmatic. In the present study, we found that AmB inhibited concanavalin A (Con A)-induced T cell proliferation if added within first 30 min of stimulation, after which inhibition began to diminish rapidly. However, AmB did not inhibit T-cell proliferation induced by a combination of PMA and ionomycin. AmB inhibition of Con A-induced proliferation was completely overcome by cyclooxygenase inhibitor ibuprofen ([alpha-methyl-4-(isobutyl)phenylacetic acid]) and H(2)O(2) scavenger catalase. In fact, in the presence of ibuprofen and catalase, AmB enhanced, instead of suppressing, Con A-induced proliferation in a dose-dependent way. The effect of catalase was limited to the removal of extracellular H(2)O(2) only, as the enzyme did not enter the cells. AmB stimulated H(2)O(2) production by macrophages, but not by a lymphocyte population, which was inhibited by ibuprofen. Our T-cell preparation contained about 3% macrophages, and AmB inhibition of proliferation was further pronounced by increasing the macrophage number by as little as 1%. Finally, AmB inhibition of Con A-induced T-cell proliferation was completely overcome by 2-mercaptoethanol. On the basis of these results, we suggest that AmB stimulates H(2)O(2) production by macrophages through the activation of the cyclooxygenase pathway of arachidonate metabolism. H(2)O(2) then inhibits Con A-induced T-cell proliferation by interfering with an early step of the T-cell receptor signaling pathway through the oxidative modification of some signaling proteins. Our results also show that AmB enhances T-cell proliferation, which can be seen only after blocking its inhibitory effect.     

Diacylglycerol mediates the T-cell receptor-driven Ca(2+) influx in T cells by a novel mechanism independent of protein kinase C activation

The mechanism of Ca(2+) influx in nonexcitable cells is not known yet. According to the capacitative hypothesis, Ca(2+) influx is triggered by IP(3)-mediated Ca(2+) release from the intracellular Ca(2+) stores. Conversely, many workers have reported a lack of association between release and influx. In this work, the role of diacylglycerol (DAG) as the mediator of T-cell receptor (TCR)-driven Ca(2+) influx in T cells was investigated. Stimulation of mouse splenic T cells with naturally occurring DAG caused Ca(2+) entry in a dose- and time-dependent manner. Such stimulation was blocked by Ni(2+), a divalent cation known to block Ca(2+) channels. Inhibition of protein kinase C (PKC) by calphostin C did not inhibit, but slightly enhanced, the DAG-stimulated Ca(2+) entry. However, inhibition of DAG metabolism by DAG kinase and lipase inhibitors enhanced the DAG-stimulated Ca(2+) entry. DAG lipase and kinase inhibitors also enhanced the Ca(2+) entry in T cells stimulated through TCR/CD3 complex with anti-CD3 antibody. Calphostin C did not affect the anti-CD3-stimulated Ca(2+) entry. These results showed that TCR-driven Ca(2+) influx in T cells is mediated by DAG through a novel mechanism(s) independent of PKC activation.