Bacterium-based heavy metal biosorbents: enhanced uptake of cadmium by E. coli expressing a metallothionein fused to beta-galactosidase

We investigated the potential utility of a recombinant E. coli that expresses the human metallothionein II gene as a fusion protein with beta-galactosidase as a heavy metal biosorbent. E. coli cells expressing the metallothionein fusion demonstrated enhanced binding of Cd2+ compared to cells that lack the metallothionein. It was shown that the metallothionein fusion was capable of efficiently removing Cd2+ from solutions. Approximately 40% of the Cd2+ accumulated by the recombinant cells free in suspension was associated with the outer cell membrane, and 60% of that was present in the cytoplasm.     

Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers

Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.     

Iron-catalyzed reaction products of alpha-tocopherol with 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine (13S)-hydroperoxide

Alpha-tocopherol was reacted with 1-palmitoyl-2-[(9Z,11E)-(S)-13-hydroperoxy-9,11-octadecadienoyl]-3-sn-phosphatidylcholine (13-PLPC-OOH) in the presence of a lipid-soluble iron chelate, Fe(III) acetylacetonate, in methanol at 37 degrees C. The reaction product was isolated and identified as a mixture of 1-palmitoyl-2-[(10E)-(12S,13S)-9-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-10-octadecenoyl]-3-sn-phosphatidylcholine and 1-palmitoyl-2-[(9Z)-(12S,13S)-11-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-9-octadecenoyl]-3-sn-phosphatidylcholine (TOO-epoxyPLPC), in which the 12,13-epoxyperoxyl radicals derived from 13-PLPC-OOH attacked the 8a-position of the alpha-tocopheroxyl radical. The iron and ascorbate-catalyzed reaction of 13-PLPC-OOH with alpha-tocopherol in phosphatidylcholine (PC) liposomes was assessed by measuring the reaction products of alpha-tocopherol. When 13-PLPC-OOH and alpha-tocopherol were added in saturated dimyristoyl-PC liposomes, the products were TOO-epoxyPLPC, alpha-tocopherylquinone, and epoxy-alpha-tocopherylquinones. In 1-palmitoyl-2-linoleoyl-PC (PLPC) liposomes, alpha-tocopherol could react with both the 13-PLPC-OOH derived 12,13-epoxyperoxyl radicals and the PLPC-derived peroxyl radicals and formed the addition products together with alpha-tocopherylquinone and epoxy-alpha-tocopherylquinones. Therefore, the iron-catalyzed decomposition of phospholipid hydroperoxides primarily produces epoxyperoxyl radicals, which react with the 8a-carbon centered radical of alpha-tocopherol in liposomal systems.     

Rush and grab strategies in foraging marine endotherms: the case for haste in penguins

The speed at which air-breathing marine predators that forage by diving should swim is likely to depend on a variety of factors that differ substantially from those relevant in animals for which access to oxygen is unlimited. We used loggers attached to free-living penguins to examine the speed at which three species swam during periods searching for prey and compared this to their speeds during actual prey pursuit. All penguin species appeared to travel at similar speeds around 2 m/s during normal commuting between the surface and feeding depths, which accords closely with minimum costs of transport. However, Adélie penguins, Pygoscelis adeliae, slowed down to feed, Magellanic penguins, Spheniscus magellanicus, speeded up and king penguins, Aptenodytes patagonicus, travelled at a variety of speeds, although mean speed did not change from normal commuting. Since energy expenditure, and therefore oxygen usage, in swimming animals increases with the cube of the speed, we hypothesized that prey escape speed (a function of prey size) and prey density would prove critical in determining optimum pursuit speeds in predators. Simple models of this type help explain why it is that some penguin species apparently benefit by increasing speed to capture prey while others benefit by decreasing speed.     

A new dipeptide,O-phosphoserylethanolamine isolated from Agkistroden blomhoffi (mamushi)

A new dipeptide was isolated from several tissues of Agkistroden blomhoffi (mamushi: a venomous snake in Japan), using ion-exchange resins and thin-layer chromatography. It was identified as O-phosphoserylethanolamine by mass spectrometry and comparison with synthetic compounds using several methods. This compound was contained in several mamushi tissues including the liver, heart, brain, bile, and muscle. The concentrations of O-phosphoserylethanolamine in the liver, brain, muscle, skin, heart, and bile were 7.17+/-3.11,16.98+/-4.25,37.37+/-7.88,37.56+/-8.97,23.93+/-6.11, and 22.21+/-5.76 micromol/g, respectively.     

Mutual dependence of MDM2 and MDMX in their functional inactivation of p53

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.     

Negative regulation of platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1

SHPS-1 is a receptor-type glycoprotein that binds and activates the protein-tyrosine phosphatases SHP-1 and SHP-2, and thereby negatively modulates intracellular signaling initiated by various cell surface receptors coupled to tyrosine kinases. SHPS-1 also regulates intercellular communication in the neural and immune systems through its association with CD47 (integrin-associated protein) on adjacent cells. Furthermore, recent studies with fibroblasts derived from mice expressing an SHPS-1 mutant that lacks most of the cytoplasmic region suggested that the intact protein contributes to cytoskeletal function. Mice homozygous for this SHPS-1 mutation have now been shown to manifest thrombocytopenia. These animals did not exhibit a defect in megakaryocytopoiesis or in platelet production. However, platelets were cleared from the bloodstream more rapidly in the mutant mice than in wild-type animals. Furthermore, peritoneal macrophages from the mutant mice phagocytosed red blood cells more effectively than did those from wild-type mice; in addition, they exhibited an increase both in the rate of cell spreading and in the formation of filopodia-like structures at the cell periphery. These results indicate that SHPS-1 both contributes to the survival of circulating platelets and down-regulates the macrophage phagocytic response.