Arabidopsis thaliana ASN2 encoding asparagine synthetase is involved in the control of nitrogen assimilation and export during vegetative growth

We investigated the function of ASN2, one of the three genes encoding asparagine synthetase (EC 6.3.5.4), which is the most highly expressed in vegetative leaves of Arabidopsis thaliana. Expression of ASN2 and parallel higher asparagine content in darkness suggest that leaf metabolism involves ASN2 for asparagine synthesis. In asn2‐1 knockout and asn2‐2 knockdown lines, ASN2 disruption caused a defective growth phenotype and ammonium accumulation. The asn2 mutant leaves displayed a depleted asparagine and an accumulation of alanine, GABA, pyruvate and fumarate, indicating an alanine formation from pyruvate through the GABA shunt to consume excess ammonium in the absence of asparagine synthesis. By contrast, asparagine did not contribute to photorespiratory nitrogen recycle as photosynthetic net CO₂ assimilation was not significantly different between lines under both 21 and 2% O₂. ASN2 was found in phloem companion cells by in situ hybridization and immunolocalization. Moreover, lack of asparagine in asn2 phloem sap and lowered ¹⁵N flux to sinks, accompanied by the delayed yellowing (senescence) of asn2 leaves, in the absence of asparagine support a specific role of asparagine in phloem loading and nitrogen reallocation. We conclude that ASN2 is essential for nitrogen assimilation, distribution and remobilization (via the phloem) within the plant.     

Downregulation of FIP200 induces apoptosis of glioblastoma cells and microvascular endothelial cells by enhancing Pyk2 activity

The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.     

BMPR2 expression is suppressed by signaling through the estrogen receptor

Background

Low endogenous testosterone levels have been shown to be a risk factor for the development of cardiovascular disease and cardiovascular benefits associated with testosterone replacement therapy are being advocated; however, the effects of endogenous testosterone levels on acute coronary vasomotor responses to androgen administration are not clear. The objective of this study was to compare the effects of acute androgen administration on in vivo coronary conductance and in vitro coronary microvascular diameter in intact and castrated male swine.

Methods

Pigs received intracoronary infusions of physiologic levels (1?C100 nM) of testosterone, the metabolite 5??-dihydrotestosterone, and the epimer epitestosterone while left anterior descending coronary blood flow and mean arterial pressure were continuously monitored. Following sacrifice, coronary arterioles were isolated, cannulated, and exposed to physiologic concentrations (1?C100 nM) of testosterone, 5??-dihydrotestosterone, and epitestosterone. To evaluate effects of the androgen receptor on acute androgen dilation responses, real-time PCR and immunohistochemistry for androgen receptor were performed on conduit and resistance coronary vessels.

Results

In vivo, testosterone and 5??-dihydrotestosterone produced greater increases in coronary conductance in the intact compared to the castrated males. In vitro, percent maximal dilation of microvessels was similar between intact and castrated males for testosterone and 5??-dihydrotestosterone. In both studies epitestosterone produced significant increases in conductance and microvessel diameter from baseline in the intact males. Androgen receptor mRNA expression and immunohistochemical staining were similar in intact and castrated males.

Conclusions

Acute coronary vascular responses to exogenous androgen administration are increased by endogenous testosterone, an effect unrelated to changes in androgen receptor expression.     

Tetratricopeptide Repeat Domain 9A Negatively Regulates Estrogen Receptor Alpha Activity

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.     

1H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

NSD3s, the proline-tryptophan-tryptophan-proline (PWWP) domain-containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using ¹H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein-dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.     

A non‐canonical role of the p97 complex in RIG‐I antiviral signaling

RIG‐I is a well‐studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4‐Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG‐I. The p97 complex is able to directly bind both non‐ubiquitinated RIG‐I and the E3 ligase RNF125, promoting K48‐linked ubiquitination of RIG‐I at residue K181. Viral infection significantly strengthens the interaction between RIG‐I and the p97 complex by a conformational change of RIG‐I that exposes the CARDs and through K63‐linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG‐I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.     

The Cytoplasmic pH Influences Hyphal Tip Growth and Cytoskeleton-Related Organization

The regulatory role of protons in hyphal tip growth was investigated by using membrane-permeant weak acids to acidify cytoplasm of the oomycete Saprolegnia ferax. Acetic acid decreased cytoplasmic pH from approximately pH 7.2 to 6.8, as shown by SNARF-1 measurements of cytoplasmic pH. Inhibition of growth in a dose-dependent manner by acetic, propionic, and isobutyric acid was accompanied by changes in positioning and morphology of mitochondria and nuclei, condensation of chromatin, disruptions in peripheral actin, and increases in hyphal diameter. These cellular alterations were fully reversible, and during recovery, major cytoplasmic movements and extensive apical vacuolations were observed. The results are consistent with proton regulation of the cytoskeleton, nuclear matrix, and/or chromosomes. However, a macroscopic cytoplasmic gradient of H+ in hyphae was not revealed by SNARF-1, indicating that if such a H+ gradient were required, it must occur at a finer level than we detected.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Urokinase-type Plasminogen Activator Receptor (uPAR) Ligation Induces a Raft-localized Integrin Signaling Switch That Mediates the Hypermotile Phenotype of Fibrotic Fibroblasts

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.     

Mapping quantitative trait loci (QTLs) for fatty acid composition in an interspecific cross of oil palm

Background  

Marker Assisted Selection (MAS) is well suited to a perennial crop like oil palm, in which the economic products are not produced until several years after planting. The use of DNA markers for selection in such crops can greatly reduce the number of breeding cycles needed. With the use of DNA markers, informed decisions can be made at the nursery stage, regarding which individuals should be retained as breeding stock, which are satisfactory for agricultural production, and which should be culled. The trait associated with oil quality, measured in terms of its fatty acid composition, is an important agronomic trait that can eventually be tracked using molecular markers. This will speed up the production of new and improved oil palm planting materials.