Visual detection of carcinoembryonic antigen on surfaces.

An attempt is made to detect carcinoembryonic antigen (CEA) at clinically interesting concentrations by using a simple immunologic surface test. Antibodies to CEA are detected in a direct test at concentrations below 1 ng/ml. The sensitivity of this assay is mainly limited by diffusion to the reacting surface. CEA is detected in an inhibition test at concentrations down to 20 ng/ml. The sensitivity of this inhibition test is limited by the average equilibrium constant in solution of the antibody-antigen reaction.     

Substrate mechanics and cell spreading

Cell spreading and cell locomotion arise from forces exerted by actin microfilaments upon the substratum. Using modified protein films at fluorocarbon oil--water interfaces as substrates, we have measured some minimal mechanical properties required of these films to support cell spreading forces in vitro. For murine 3T3-L1 fibroblasts, complete cell spreading was obtained when the films exceeded surface shear moduli and surface fracture points of 15 and 5 dyne/cm, respectively. The human WI-38 fibroblast required more robust films than did its transformed counterpart (WI-38/VA 13) in order to achieve equivalent spreading. These results are of significance in understanding the metastatic capabilities of cancer cells.     

<Emphasis Type="Italic">Prorocentrum rivalis</Emphasis> sp. nov. (Dinophyceae) and its phylogenetic affinities inferred from analysis of a mixed morphological and LSU rRNA data set

A new freshwater epiphytic Prorocentrum species, Prorocentrum rivalis, from the temperate region of the Haute-Vienne, France, is described. This species is the third freshwater species identified among approximately 60 marine Prorocentrum species. This new species is described using scanning electron microscope and phylogenetic analyses by a polyphasic approach (LSU rRNA sequences combined with 9 morphological characters). The phylogenetic analysis attests that P. rivalis is close to other planktonic freshwater species and the freshwater Prorocentrum clade is evolutionarily derived from an epiphytic freshwater prorocentroid ancestor. The unique marine species in the freshwater clade results from an ecophysiological reversion. P. rivalis differs from other epiphytic taxa by its rarity, its temperate distribution and its ecophysiological needs. The phylogeny confirms also that all planktonic Prorocentrum species are evolutionarily derived from epiphytic/benthic ancestors.     

PPAR γ is highly expressed in F4/80 adipose tissue macrophages and dampens adipose-tissue inflammation

Macrophage infiltration into adipose tissue is a hallmark of obesity. We recently reported two phenotypically distinct subsets of adipose tissue macrophages (ATM) based on the surface expression of the glycoprotein F4/80 and responsiveness to treatment with a peroxisome proliferator-activated receptor (PPAR) γ agonist. Hence, we hypothesized that F4/80^hi and F4/80^lo ATM differentially express PPAR γ. This study phenotypically and functionally characterizes F4/80^hi and F4/80^lo ATM subsets during obesity. Changes in gene expression were also examined on sorted F4/80^lo and F4/80^hi ATM by quantitative real-time RT-PCR. We show that while F4/80^lo macrophages predominate in adipose tissue of lean mice, obesity causes accumulation of both F4/80^lo and F4/80^hi ATM. Moreover, accumulation of F4/80^hi ATM in adipose tissue is associated with impaired glucose tolerance. Phenotypically, F4/80^hi ATM express greater amounts of CD11c, MHC II, CD49b, and CX3CR1 and produce more TNF-α, MCP-1, and IL-10 than F4/80^lo ATM. Gene expression analyses of the sorted populations revealed that only the F4/80^lo population produced IL-4, whereas the F4/80^hi ATM expressed greater amounts of PPAR γ, δ, CD36 and toll-like receptor-4. In addition, the deficiency of PPAR γ in immune cells favors expression of M1 and impairs M2 macrophage marker expression in adipose tissue. Thus, PPAR γ is differentially expressed in F4/80^hi versus F4/80^low ATM subsets and its deficiency favors a predominance of M1 markers in WAT.     

Local temperatures inferred from plant communities suggest strong spatial buffering of climate warming across Northern Europe

Recent studies from mountainous areas of small spatial extent (<2500 km²) suggest that fine‐grained thermal variability over tens or hundreds of metres exceeds much of the climate warming expected for the coming decades. Such variability in temperature provides buffering to mitigate climate‐change impacts. Is this local spatial buffering restricted to topographically complex terrains? To answer this, we here study fine‐grained thermal variability across a 2500‐km wide latitudinal gradient in Northern Europe encompassing a large array of topographic complexities. We first combined plant community data, Ellenberg temperature indicator values, locally measured temperatures (LmT) and globally interpolated temperatures (GiT) in a modelling framework to infer biologically relevant temperature conditions from plant assemblages within <1000‐m² units (community‐inferred temperatures: CiT). We then assessed: (1) CiT range (thermal variability) within 1‐km² units; (2) the relationship between CiT range and topographically and geographically derived predictors at 1‐km resolution; and (3) whether spatial turnover in CiT is greater than spatial turnover in GiT within 100‐km² units. Ellenberg temperature indicator values in combination with plant assemblages explained 46–72% of variation in LmT and 92–96% of variation in GiT during the growing season (June, July, August). Growing‐season CiT range within 1‐km² units peaked at 60–65°N and increased with terrain roughness, averaging 1.97 °C (SD = 0.84 °C) and 2.68 °C (SD = 1.26 °C) within the flattest and roughest units respectively. Complex interactions between topography‐related variables and latitude explained 35% of variation in growing‐season CiT range when accounting for sampling effort and residual spatial autocorrelation. Spatial turnover in growing‐season CiT within 100‐km² units was, on average, 1.8 times greater (0.32 °C km^?1) than spatial turnover in growing‐season GiT (0.18 °C km^?1). We conclude that thermal variability within 1‐km² units strongly increases local spatial buffering of future climate warming across Northern Europe, even in the flattest terrains.     

Sirt2 Deacetylase Is a Novel AKT Binding Partner Critical for AKT Activation by Insulin

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr³⁰⁸ and Ser⁴⁷³, mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis. Here, we identify the cytoplasmic NAD⁺-dependent deacetylase, Sirt2, as a novel AKT interactor, required for optimal AKT activation. Pharmacological inhibition or genetic down-regulation of Sirt2 diminished AKT activation in insulin and growth factor-responsive cells, whereas Sirt2 overexpression enhanced the activation of AKT and its downstream targets. AKT was prebound with Sirt2 in serum or glucose-deprived cells, and the complex dissociated following insulin treatment. The binding was mediated by the pleckstrin homology and the kinase domains of AKT and was dependent on AMP-activated kinase. This regulation involved a novel AMP-activated kinase-dependent Sirt2 phosphorylation at Thr¹⁰¹. In cells with constitutive PI3K activation, we found that AKT also associated with a nuclear sirtuin, Sirt1; however, inhibition of PI3K resulted in dissociation from Sirt1 and increased association with Sirt2. Sirt1 and Sirt2 inhibitors additively inhibited the constitutive AKT activity in these cells. Our results suggest potential usefulness of Sirt1 and Sirt2 inhibitors in the treatment of cancer cells with up-regulated PI3K activity and of Sirt2 activators in the treatment of insulin-resistant metabolic disorders.     

Distinct activities of the related protein kinases Cdk1 and Ime2

In budding yeast, commitment to DNA replication during the normal cell cycle requires degradation of the cyclin-dependent kinase (CDK) inhibitor Sic1. The G1 cyclin-CDK complexes Cln1-Cdk1 and Cln2-Cdk1 initiate the process of Sic1 removal by directly catalyzing Sic1 phosphorylation at multiple sites. Commitment to DNA replication during meiosis also appears to require Sic1 degradation, but the G1 cyclin-CDK complexes are not involved. It has been proposed that the meiosis-specific protein kinase Ime2 functionally replaces the G1 cyclin-CDK complexes to promote Sic1 destruction. To investigate this possibility, we compared Cln2-Cdk1 and Ime2 protein kinase activities in vitro. Both enzyme preparations were capable of catalyzing phosphorylation of a GST-Sic1 fusion protein, but the phosphoisomers generated by the two activities had significantly different electrophoretic mobilities. Furthermore, mutation of consensus CDK phosphorylation sites in Sic1 affected Cln2-Cdk1- but not Ime2-dependent phosphorylation. Phosphoamino acid analysis and phosphopeptide mapping provided additional evidence that Cln2-Cdk1 and Ime2 targeted different residues within Sic1. Examination of other substrates both in vitro and in vivo also revealed differing specificities. These results indicate that Ime2 does not simply replace G1 cyclin-CDK complexes in promoting Sic1 degradation during meiosis.