Insulin signaling: inositol phosphates get into the Akt

An acute but transient response to insulin is essential for glucose homeostasis in mammals. Chakraborty et?al. (2010) uncover a new feedback mechanism regulating insulin signaling. They show that the inositol pyrophosphate IP7, which is produced in response to insulin, inhibits the Akt kinase, a primary effector of insulin signaling.     

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.     

Isolation and characterization of microsatellite loci in Santalum lanceolatum and Santalum leptocladum (Santalaceae)

? Premise of the study: Microsatellite primers were developed for the first time in the native Australian sandalwood species Santalum lanceolatum. ? Methods and Results: Using an enrichment cloning protocol, five novel polymorphic codominant loci were developed and characterized in S. lanceolatum and S. leptocladum. In addition to these, three existing microsatellite loci from other sandalwood species were successfully amplified and characterized for S. lanceolatum and S. leptocladum. Among the eight loci, allelic diversity ranged from 4 to 29. ? Conclusions: Primers will be useful for studies of clonality, genetic diversity and spatial genetic structure in wild populations. When coupled with other molecular techniques will help investigate the relationship between S. lanceolatum and S. leptocladum, species of commercial and conservation interest.     

Peptide ligands that use a novel binding site to target both TGF-β receptors

The transforming growth factor beta (TGF-β) signaling pathway plays myriad roles in development and disease. TGF-β isoforms initiate signaling by organizing their cell surface receptors TβRI and TβRII. Exploration and exploitation of the versatility of TGF-β signaling requires an enhanced understanding of structure-function relationships in this pathway. To this end, small molecule, peptide, and antibody effectors that bind key signaling components would serve as valuable probes. We focused on the extracellular domain of TβR1 (TβRI-ED) as a target for effector screening. The observation that TβRI-ED can bind to a TGF-β coreceptor (endoglin) suggests that the TβRI-ED may have multiple interaction sites. Using phage display, we identified two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the TβRI-ED (K(d)≈ 10(-5) M). Although our screen focused on TβRI-ED, the hit peptides interact with the TβRII-ED with similar affinities. The peptide ligands occupy the same binding sites on TβRI and TβRII, as demonstrated by their ability to compete with each other for receptor binding. Moreover, neither interferes with TGF-β binding. These results indicate that both TβRI and TβRII possess hot spots for protein-protein interactions that are distinct from those used by their known ligand TGF-β. To convert these compounds into high affinity probes, we exploited the observation that TβRI and TβRII exist as dimers on the cell surface; therefore, we assembled a multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the role of receptor assembly in TGF-β function.     

Spatial variation in molluscan assemblages from coralline turfs of Argentinean Patagonia

Patterns of spatial variation of molluscan communities associatedwith coralline algal turfs were evaluated over 1,000 kmof the coast of Argentinean Patagonia. A hierarchically-nestedexperimental design was used to determine the relative importanceof molluscan assemblage variation at three different spatialscales (shores, sites and cores). Hypotheses were also testedabout the potential role of habitat variables (frond density,frond length, sediment and epiphytes) for determining molluscancommunity structure. In total, 38 molluscan species were foundcomprising 16, 18 and 4 species of bivalves, gastropods andpolyplacophorans, respectively. Densities of molluscs in corallineturfs reached ca 77,000 individuals per m² and were dominatedby mussels, especially Perumytilus purpuratus. Multivariateand univariate analyses of assemblage structure consistentlyshowed that variation at scales of metres and hundreds of kilometresdominated, with sites 20–50 m apart always contributingless than 24% of the total. Significant associations betweenmolluscan community structure and both frond density and frondlength demonstrated the potential importance of habitat structurein determining community structure at local scales. Variationin molluscan assemblages at the scale of shores, however, didnot appear to correlate with latitudinal, temperature or waveexposure gradients, indicating that other processes must beoperating. The compositions of molluscan assemblages in corallineturfs on the coast of Argentina were similar to those reportedfor central Chile. Comparisons of the richness of these SouthAmerican assemblages to other parts of the world revealed somestriking biogeographical patterns that warrant further investigation.Overall, this work highlights the general importance of small-scalevariation in molluscan assemblages on rocky shores and the consistentinfluence of habitat complexity in determining the structureof diverse molluscan communities associated with mat-like habitats. (Received 10 August 2006; accepted 20 January 2007)     

AKT/PKB signaling: navigating downstream

The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Here, we review the molecular properties of Akt and the approaches used to characterize its true cellular targets. In addition, we discuss those Akt substrates that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth, proliferation, angiogenesis, metabolism, and migration.     

A five-strain probiotic combination reduces pathogen shedding and alleviates disease signs in pigs challenged with Salmonella enterica Serovar Typhimurium

Salmonella spp. infection is a major cause of gastroenteritis, with many thousands of cases reported in the European Union every year. The use of probiotics offers the potential to improve this situation. Here, we investigate the effects of oral treatment of pigs with a defined lactic acid bacteria culture mixture on both clinical and microbiological signs of Salmonella enterica serovar Typhimurium infection. Fifteen weaned pigs blocked by sex and weight were administered control milk or a mixture of five probiotic strains as either a milk fermentate or milk suspension for a total of 30 days. The mixture consisted of two strains of Lactobacillus murinus and one strain each of Lactobacillus salivarius subsp. salivarius, Lactobacillus pentosus, and Pediococcus pentosaceous. Following probiotic administration for 6 days, animals were challenged orally with serovar Typhimurium; the health of the animals and the microbiological composition of their feces were monitored for 23 days postinfection. Animals treated with probiotic showed reduced incidence, severity, and duration of diarrhea. These animals also gained weight at a greater rate than control pigs administered skim milk. Mean fecal numbers of Salmonella were significantly reduced in probiotic-treated animals at 15 days postinfection (P = 0.01). The administered probiotic bacteria improved both the clinical and microbiological outcome of Salmonella infection. These strains offer significant benefit for use in the food industry and may have potential in human applications.     

Trophic cul-de-sac, Pyrazus ebeninus, limits trophic transfer through an estuarine detritus-based food web

The importance to food‐webs of trophic cul‐de‐sacs, species that channel energy flow away from higher trophic levels, is seldom considered outside of the pelagic systems in which they were first identified. On intertidal mudflats, inputs of detritus from saltmarshes, macroalgae or microphytobenthos are generally regarded as a major structuring force underpinning food‐webs and there has been no consideration of trophic cul‐de‐sacs to date. A fully orthogonal three‐factor experiment manipulating the density of the abundant gastropod, Pyrazus ebeninus, detritus and macrobenthic predators on a Sydney mudflat revealed large deleterious effects of the gastropod, irrespective of detrital loading or the presence of predators. Two months after experimental manipulation, the standing‐stock of microphytobenthos in plots with high (44 per m²) densities of P. ebeninus was 20% less than in plots with low (4 per m²) densities. Increasing densities of P. ebeninus from low to high halved the abundance of macroinvertebrates and the average number of species. In contrast, the addition of detritus had differing effects on microphytobenthos (positively affected) and macroinvertebrates (negatively affected). Over the two‐months of our experiment, no predatory mortality of P. ebeninus was observed and high densities of P. ebeninus decreased impacts of predators on macroinvertebrate abundances. Given that the dynamics of southeast Australian mudflats are driven more by disturbance than seasonality in predators and their interactions with prey, it is likely that Pyrazus would be similarly resistant to predation and have negative effects on benthic assemblages at other times of the year, outside of our study period. Thus, in reducing microphytobenthos and the abundance and species richness of macrofauna, high abundances of the detritivore P. ebeninus may severely limit the flow of energy up the food chain to commercially‐important species. This study therefore suggests that trophic cul‐de‐sacs are not limited to the eutrophied pelagic systems in which they were first identified, but may exist in other systems as well.     

C-terminal tail phosphorylation of N-formyl peptide receptor: differential recognition of two neutrophil chemoattractant receptors by monoclonal antibodies NFPR1 and NFPR2

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni(2+)-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45-75 kDa species (peak M(r) approximately 60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of M(r) approximately 40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting approximately 60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337-346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.     

DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur

Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.