PF-4708671 Activates AMPK Independently of p70S6K1 Inhibition

The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I.     

The value of spatial experience and group size for ant colonies in direct competition

Animals often search for food more efficiently with experience. However, the contribution of experience to foraging success under direct competition has rarely been examined. Here we used colonies of an individually foraging desert ant to investigate the value of spatial experience. First, we trained worker groups of equal numbers to solve either a complex or a simple maze. We then tested pairs of both groups against one another in reaching a food reward. This task required solving the same complex maze that one of the groups had been trained in, to determine which group would exploit better the food reward. The worker groups previously trained in the complex mazes reached the food reward faster and more of these workers fed on the food than those trained in simple mazes, but only in the intermediate size group. To determine the relative importance of group size versus spatial experience in exploiting food patches, we then tested smaller trained worker groups against larger untrained ones. The larger groups outcompeted the smaller ones, despite the latter's advantage of spatial experience. The contribution of spatial experience, as found here, appears to be small, and depends on group size: an advantage of a few workers of the untrained group over the trained group negates its benefits.     

Use of monoclonal antibodies to pregnanediol-3α-glucuronide for the development of a solid phase chemiluminescence immunoassay

Monoclonal antibodies to pregnanediol-3α-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specifity and affinity to pregnanediol-3α-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3α-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3α-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r=0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.     

Transglutaminase Activity in Rat Brain: Characterization, Distribution, and Changes with Age

The activity of transglutaminase was characterized in the rat brain. In adults, comparable levels of transglutaminase activity are present in all brain regions examined. The activity is present in all subcellular fractions, as studied by differential centrifugation, but the soluble fraction contains the highest specific activity. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate casein) is very low in all subcellular fractions, except in the synaptosomal fraction where its highest levels are about 40-60% of the activity assayed in the presence of casein. Furthermore, enzyme activity is present on the external surface of synaptosomes. In the soluble fraction, maximal activity can be detected between pH values of 9 and 10 when assayed in the presence of 5 mM CaCl2 (with half-maximal activity requiring 0.75 mM CaCl2) and 0.4 mM putrescine (with an apparent Km for putrescine of 0.1 mM). The activity can be partially inhibited by ZnCl2 (with an IC50 of 4.5 mM) and by AlCl3 (with an IC50 of 5.1 mM). In the cerebellum, where the full span of neuronal development can be studied after birth, the highest specific activity is observed just after birth, thereafter the activity starts to decline and by 14 days, after a reduction of about 65%, it reaches levels observed throughout life.     

Synergistic amplification of beta-amyloid- and interferon-gamma-induced microglial neurotoxic response by the senile plaque component chromogranin A

Activation of the microglial neurotoxic response by components of the senile plaque plays a critical role in the pathophysiology of Alzheimer's disease (AD). Microglia induce neurodegeneration primarily by secreting nitric oxide (NO), tumor necrosis factor- (TNF), and hydrogen peroxide. Central to the activation of microglia is the membrane receptor CD40, which is the target of costimulators such as interferon- (IFN). Chromogranin A (CGA) is a recently identified endogenous component of the neurodegenerative plaques of AD and Parkinson's disease. CGA stimulates microglial secretion of NO and TNF, resulting in both neuronal and microglial apoptosis. Using electrochemical recording from primary rat microglial cells in culture, we have shown in the present study that CGA alone induces a fast-initiating oxidative burst in microglia. We compared the potency of CGA with that of -amyloid (A) under identical conditions and found that CGA induces 5–7 times greater NO and TNF secretion. Coapplication of CGA with A or with IFN resulted in a synergistic effect on NO and TNF secretion. CD40 expression was induced by CGA and was further increased when A or IFN was added in combination. Tyrphostin A1 (TyrA1), which inhibits the CD40 cascade, exerted a dose-dependent inhibition of the CGA effect alone and in combination with IFN and A. Furthermore, CGA-induced mitochondrial depolarization, which precedes microglial apoptosis, was fully blocked in the presence of TyrA1. Our results demonstrate the involvement of CGA with other components of the senile plaque and raise the possibility that a narrowly acting agent such as TyrA1 attenuates plaque formation. Alzheimer's disease; oxidative burst; apoptosis; nitric oxide; tyrphostin A1     

Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells

Fusobacterium nucleatum is a common oral anaerobe associated with gingivitis, periodontal disease and preterm deliveries. Coaggregation among oral bacteria is considered to be a significant factor in dental plaque development. Adhesion to host cells was suggested to be important for the F. nucleatum virulence associated with oral inflammation and with preterm births. An uncharacterized fusobacterial galactose inhibitible adhesin mediates coaggregation of F. nucleatum 12230 and F. nucleatum PK1594 with the periodontal pathogen Porphyromonas gingivalis. This adhesin is also involved with the attachment of both fusobacterial strains to host cells. However, it has been suggested that additional unidentified fusobacterial adhesins are involved in F. nucleatum virulence associated with preterm births. In this study, a fluorescence-based high throughput sensitive and reproducible method was developed for measuring bacterial coaggregation and bacterial attachment to mammalian cells. Using this method we found that coaggregation of F. nucleatum 4H with P. gingivalis and its attachment to murine macrophages is less inhibitible by galactose than that of F. nucleatum PK1594. These findings suggest that F. nucleatum 4H can serve as a model organism for identifying nongalactose inhibitible F. nucleatum adhesins considered to be involved in fusobacterial attachment to mammalian cells.     

Optical Immunosensors for the Efficient Detection of Target Biomolecules

Recently, immunosensors have attracted attention because they are widely applied for the detection of various pathogens. Among the commonly used immunosensors, the optical immunosensor features prominently as an effective tool for the quantification of the amount of antibodies, antigens, or haptens in complex samples with high sensitivity and specificity. However, very few studies provide comprehensive overviews of optical immunosensors. In this review, we present various methods and applications of optical immunosensors in pathogen detection. We introduced a concise definition of optical immunosensors and the principle of using them for detection. We subsequently discuss the main categories of optical immunosensors and their application to the detection of pathogens, as well as their advantages and limitations. Recent publications from 2006 to 2015 on variously designed optical immunosensors have also been updated. We conclude the review with a brief summary and discuss future directions of optical immunosensors.