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Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus. Different phosphoinositide isomers are involved in a variety of physiological and pathological processes. Here, the authors combine chiral column chromatography and mass spectrometry to measure phosphoinositide regioisomers, revealing their dynamic changes in intra- and extracellular cancer cell milieus. [ABSTRACT FROM AUTHOR]

Inflammation has a critical role in the development of insulin resistance. Recent evidence points to a contribution by the central nervous system in the modulation of peripheral inflammation through the anti-inflammatory reflex. However, the importance of this phenomenon remains elusive in type 2 diabetes pathogenesis. Here we show that rat insulin-2 promoter (Rip)-mediated deletion of Pten, a gene encoding a negative regulator of PI3K signaling, led to activation of the cholinergic anti-inflammatory pathway that is mediated by M2 activated macrophages in peripheral tissues. As such, Rip-cre+ Ptenflox/flox mice showed lower systemic inflammation and greater insulin sensitivity under basal conditions compared to littermate controls, which were abolished when the mice were treated with an acetylcholine receptor antagonist or when macrophages were depleted. After feeding with a high-fat diet, the Pten-deleted mice remained markedly insulin sensitive, which correlated with massive subcutaneous fat expansion. They also exhibited more adipogenesis with M2 macrophage infiltration, both of which were abolished after disruption of the anti-inflammatory efferent pathway by left vagotomy. In summary, we show that Pten expression in Rip+ neurons has a critical role in diabetes pathogenesis through mediating the anti-inflammatory reflex. [ABSTRACT FROM AUTHOR]

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P2 phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a-/- mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P2, PtdIns(3,4,5)P3 and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P2 metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P2 metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related mortality in the world, resulting in over a million deaths each year. Non-small cell lung cancers (NSCLCs) are characterized by a poor immunogenic response, which may be the result of immunosuppressive factors such as prostaglandin E2 (PGE2) present in the tumor environment. The effect of PGE2 in the suppression of anti-tumor immunity and its promotion of tumor survival has been established for over three decades, but with limited mechanistic understanding. We have previously reported that PGE2 activates hematopoietic progenitor kinase 1 (HPK1), a hematopoietic-specific kinase known to negatively regulate T-cell receptor signaling. Here, we report that mice genetically lacking HPK1 resist the growth of PGE2-producing Lewis lung carcinoma (LLC). The presence of tumor-infiltrating lymphocytes (TILs) and T-cell transfer into T cell-deficient mice revealed that tumor rejection is T cell mediated. Further analysis demonstrated that this may be significantly due to the ability of HPK1−/− T cells to withstand PGE2-mediated suppression of T-cell proliferation, IL-2 production, and apoptosis. We conclude that PGE2 utilizes HPK1 to suppress T cell-mediated anti-tumor responses. [ABSTRACT FROM AUTHOR]

The study evaluated biological reaction of four types of novel double network gels in muscle and subcutaneous tissues, using implantation tests according to the international guideline. The implantation tests demonstrated that, although poly (2-acrylamide-2-metyl-propane sulfonic acid)/poly ( N, N′-dimetyl acrylamide) (PAMPS/PDMAAm) gel induced a mild inflammation at 1 week, the degree of the inflammation significantly decreased into the same degree as that of the negative control at 4 and 6 weeks. This gel has a potential to be applied as artificial cartilage. In addition, Cellulose/Gelatin gel showed the same degree of inflammation as that of the negative control at 1 week, and then, showed a gradually absorbable property at 4 and 6 weeks. This gel has a potential to be applied as an absorbable implant. The PAMPS/polyacrylamide and Cellulose/PDMAAm gels induced a significant inflammation at each week. These DN gels are difficult to be applied as clinical implants in the current situation. [ABSTRACT FROM AUTHOR]

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco ( Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, 15NH was incorporated into [5−15N]glutamine and [2−15N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−15N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−15N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. [ABSTRACT FROM AUTHOR]

Pten is an important phosphatase, suppressing the phosphatidylinositol-3 kinase/Akt pathway. Here, we generated adipose-specific Pten-deficient (AdipoPten-KO) mice, using newly generated Acdc promoter-driven Cre transgenic mice. AdipoPten-KO mice showed lower body and adipose tissue weights despite hyperphagia and enhanced insulin sensitivity with induced phosphorylation of Akt in adipose tissue. AdipoPten-KO mice also showed marked hyperthermia and increased energy expenditure with induced mitochondriagenesis in adipose tissue, associated with marked reduction of p53, inactivation of Rb, phosphorylation of cyclic AMP response element binding protein (CREB) and increased expression of Ppargc1a, the gene that encodes peroxisome proliferative activated receptor gamma coactivator 1 alpha. Physiologically, adipose Pten mRNA decreased with exposure to cold and increased with obesity, which were linked to the mRNA alterations of mitochondriagenesis. Our results suggest that altered expression of adipose Pten could regulate insulin sensitivity and energy expenditure. Suppression of adipose Pten may become a beneficial strategy to treat type 2 diabetes and obesity. [ABSTRACT FROM AUTHOR]

Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription–polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15NH4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine synthesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO3- assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis. [ABSTRACT FROM AUTHOR]

Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain. [ABSTRACT FROM AUTHOR]

Transformed tobacco (Nicotiana tabacum L.) plants with varying activities of the key enzyme of ammonia assimilation, ferredoxin-glutamine-α-ketoglutarate aminotransferase (Fd-GOGAT; EC 1.4.7.1), were used to examine the roles of ammonium, glutamine (Gln) and α-ketoglutarate (α-KG) in the regulation of nitrate reductase (NR; EC 1.6.6.1) transcript abundance. In wild-type leaf discs, NR mRNA abundance was increased following feeding with NO3–, sucrose and α-KG and decreased by feeding Gln. In air, leaves with decreased GOGAT accumulated Gln and α-KG simultaneously; this was accompanied by increased NR transcripts. The inhibition of NR transcription by Gln observed in leaf-disc experiments was therefore not observed in the low-Fd-GOGAT plants that accumulate Gln in vivo. The results suggest that the negative effect of Gln on NR transcript abundance was offset by high α-KG and that the relative amounts of α-KG and Gln are more important in controlling NR gene transcription than the concentration of either metabolite alone. [ABSTRACT FROM AUTHOR]

Reports that genetic inactivation of the p110-gamma catalytic subunit of phosphoinositide-3-OH kinase (PI(3)Ks) leads to development of invasive colorectal adenocarcinomas in mice. Role of the subunit in tumorigenesis; Finding that p110-gamma can block the growth of human colon cancer cells.

The cellular localization of the enzymes involved in primary nitrogen assimilation was investigated following separation of mesophyll protoplasts and bundle-sheath cells of maize ( Zea mays L.) leaves. Determination of the enzymatic activities in the two types of cell revealed that nitrate and nitrite reductase are principally located in the mesophyll cells whereas glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) are present in both tissues with a preferential location in the bundle-sheath strands. In order to confirm the results obtained by this conventional biochemical method we have used an in-situ immunofluorescence technique to unambiguously localize GS and Fd-GOGAT at the cellular level. Thin-sectioned maize leaves treated with specific GS and Fd-GOGAT antisera followed by conjugation with fluorescein-isothiocyanate-labelled sheep anti-rabbit immunoglobulins clearly show that GS is equally distributed within the leaf whereas Fd-GOGAT is mostly present in the chloroplasts of the bundle-sheath cells. The cellular localization of nitrate reductase, nitrite reductase, GS-2 and Fd-GOGAT in maize leaf cell types strongly indicates that primary nitrogen assimilation functions in the mesophyll cells while photorespiratory nitrogen recycling is restricted to the bundle-sheath cells. [ABSTRACT FROM AUTHOR]

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco ( Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C) from a tobacco λgt11 expression library. A longer Fd-GOGAT cDNA clone (C) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco λgt10 cDNA library using C as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression. [ABSTRACT FROM AUTHOR]

The action spectrum for photoinduction of pileus formation in Favolus arcularius (Fr.) Ames. shows four maxima, at ca. 375-385, 420, 446, and 480 nm, and minima near 365, 400, 435, and 470 nm. Wavelengths above 510 nm were ineffective. The maxima coincided with four of the six maxima for primordium formation of this fungus, but the two maxima for the latter around 398 and 514 nm could not be detected. The photosensitive region for the initiation of pileus formation was localized in the apical region of the stipes. [ABSTRACT FROM AUTHOR]

We performed basic studies on a new high-yield culture system (concentrate rotary tissue-culture system) for application to adoptive immunotherapy with lymphokine-activated killer (LAK) cells. Using this system, we demonstrated that up to 2 × 10 peripheral blood mononuclear cells/ml could be cultured in interleukin-2 with a sufficient recovery rate and cytotoxicity in short-term cultures (6 days). This system can also be used to proliferate LAK cells to four times the initial cell number with sufficient cytotoxicity for 14 days of culture. Thus, this system allows activation of sufficient numbers of cells to conduct clinical trials on humans. [ABSTRACT FROM AUTHOR]

This corrects the article DOI: 10.1038/ncomms16017 [ABSTRACT FROM AUTHOR]

The presence of senescent, transformed or damaged cells can impair tissue function or lead to tumorigenesis; therefore, organisms have evolved quality control mechanisms to eliminate them. Here, we show that YAP activation induced by inactivation of the Hippo pathway specifically in damaged hepatocytes promotes their selective elimination by using in vivo mosaic analysis in mouse liver. These damaged hepatocytes migrate into the hepatic sinusoids, undergo apoptosis and are engulfed by Kupffer cells. In contrast, YAP activation in undamaged hepatocytes leads to proliferation. Cellular stresses such as ethanol that damage both liver sinusoidal endothelial cells and hepatocytes switch cell fate from proliferation to migration/apoptosis in the presence of activated YAP. This involves the activation of CDC42 and Rac that regulate cell migration. Thus, we suggest that YAP acts as a stress sensor that induces elimination of injured cells to maintain tissue and organ homeostasis. [ABSTRACT FROM AUTHOR]