Synthesis of a multivalent chelator lipid for stably tethering histidine-tagged proteins onto membranes

This protocol describes the synthesis of a lipid-like molecule carrying a head group containing two nitrilotriacetic acid moieties. This multivalent chelator lipid can be incorporated into lipid membranes, to which histidine-tagged protein can then be tethered in an oriented fashion. Possible applications of this lipid are protein tethering to solid-supported membranes, to lipid vesicles or to live cells. As compared to conventional monovalent chelator lipids, this lipid can achieve highly stable tethering of proteins by the multivalent chelator head. The eight-step synthesis described in this protocol can be completed within 4-5 weeks.     

Systemic dysregulation of CEACAM1 in melanoma patients

It was previously shown that CEACAM1 on melanoma cells strongly predicts poor outcome. Here, we show a statistically significant increase of serum CEACAM1 in 64 active melanoma patients, as compared to 48 patients with no evidence of disease and 37 healthy donors. Among active patients, higher serum CEACAM1 correlated with LDH values and with decreased survival. Multivariate analysis with neutralization of LDH showed that increased serum CEACAM1 carries a hazard ratio of 2.40. In vitro, soluble CEACAM1 was derived from CEACAM1(+), but neither from CEACAM1(?) melanoma cells nor from CEACAM1(+) lymphocytes, and directly correlated with the number of CEACAM1(+) melanoma cells. Production of soluble CEACAM1 depended on intact de novo protein synthesis and secretion machineries, but not on metalloproteinase function. An unusually high percentage of CEACAM1(+) circulating NK and T lymphocytes was demonstrated in melanoma patients. CEACAM1 inhibited killing activity in functional assays. CEACAM1 expression could not be induced on lymphocytes by serum from patients with high CEACAM1 expression. Further, expression of other NK receptors was impaired, which collectively indicate on a general abnormality. In conclusion, the systemic dysregulation of CEACAM1 in melanoma patients further denotes the role of CEACAM1 in melanoma and may provide a basis for new tumor monitoring and prognostic platforms.     

What is more important for invertebrate colonization in a stream with low-quality litter inputs: exposure time or leaf species?

The objective of this study was to evaluate the influences of detritus from the leaves of different species, and of exposure time on invertebrate colonization of leaves in a shaded Cerrado stream. We hypothesized that the exposure time is the main factor that influences the colonization of leaves by invertebrates. We used leaves of five tree species native to the Brazilian Cerrado: Protium heptaphyllum and Protium brasiliense (Burseraceae), Ocotea sp. (Lauraceae), Myrcia guyanensis (Myrtaceae), and Miconia chartacea (Melastomataceae), which are characterized by their toughness and low-nutritional quality. Litter bags, each containing leaves from one species, were placed in a headwater stream and removed after 7, 15, 30, 60, 90, and 120 days. The dominant taxon was Chironomidae, which comprised ca. 52% of all organisms and ca. 20% of the total biomass. The taxonomic richness of colonizing organisms did not vary among the leaf species. However, the density and biomass of the associated organisms varied differently among the kinds of detritus during the course of the incubation. The collector-gatherers and shredders reached higher densities in the detritus that decomposed more rapidly (Ocotea sp. and M. guyanensis), principally in the more advanced stages of colonization. The collector-filterers reached higher densities in the detritus that decomposed more slowly (P. heptaphyllum, P. brasiliense, and M. chartacea), principally in the initial stages of incubation. A cluster analysis divided the detritus samples of different leaf species according to the exposure time (initial phase: up to 7 days; intermediate phase: 7–30 days; advanced phase: 30–120 days), suggesting some succession in invertebrate colonization, with differences in taxon composition (indicator taxa analysis). These results suggest that regardless of the leaf-detritus species, exposure time was the main factor that influenced the colonization process of aquatic invertebrates.     

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.     

A garden of Notch-ly delights

Over the past decade, the Notch signaling pathway has been shown to be crucially important for normal metazoan development and to be associated with several human inherited and late onset diseases. The realization that altered Notch signaling contributes at various levels to human disease lead in May to the first meeting dedicated solely to Notch signaling in vertebrate development and disease in Madrid, Spain. Hosted by the Cantoblanco Workshops on Biology and organized by Tom Gridley, José Luis de la Pompa and Juan Carlos Izpisúa Belmonte, the meeting covered diverse aspects of this important signaling pathway.     

Two biflavonoids from Ouratea nigroviolacea

The leaves of Ouratea nigroviolacea (Ochnaceae) afforded two biflavonoids, ouratine A and B together with agathisflavone and stigmasterol. The biflavonoids were characterized as 4'-O-methylated apigeninyl-(I-6, II-8)-4'-O-methylatedapigenin and 4'-O-methylated apigeninyl-(I-6, II-8) apigenin by spectral and chemical transformation studies.     

Current perspectives on the mechanism of action of artemisinins

Artemisinin derivatives are the most recent single drugs approved and introduced for public antimalarial treatment. Although their recommended use is for treatment of Plasmodium falciparum infection, these drugs also act against other parasites, as well as against tumor cells. The mechanisms of action attributed to artemisinin include interference with parasite transport proteins, disruption of parasite mitochondrial function, modulation of host immune function and inhibition of angiogenesis. Artemisinin combination therapies are currently the preferred treatment for malaria. These combinations may prevent the induction of parasite drug resistance. However, in view of the multiple mechanisms involved, especially when additional drugs are used, the combined therapy should be carefully examined for antagonistic effects. It is now a general theory that the crucial mechanism is interference with plasmodial SERCA. Therefore, future development of resistance may be associated with overproduction or mutations of this transporter. However, a general mechanism, such as alterations in general drug transport pathways, is feasible. In this article, we review the evidence for each mechanism of action suggested.