Osmotic stress–induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P2) was first identified as a nonabundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P2 via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P2 synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P2 levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P2 are 18–28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P2. After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P2 rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P2 plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P2 continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P2 and provide insight into why PtdIns(3,5)P2 levels rise in response to osmotic stress

Re-envisioning Addiction Treatment: A Six-Point Plan

This article is focused on improving the quality of addiction treatment. Based on observations that patients are leaving treatment too early and/or are continuing to use substances during their care, the authors propose six actions that could help reorient and revitalize this kind of clinical work: (1) conceptualize and treat addictive disorders within a psychiatric/mental health framework; (2) make the creation of a strong therapeutic alliance a core part of the healing process; (3) understand patients’ addictions and other problems using models based on multiple internal parts, voices, or modes; (4) make contingency management and the use of positive reinforcement systems a standard and central practice in all treatment settings; (5) envision long-term change and healing through the lens of identity theory; and (6) integrate the growing developments in recovery culture with formal treatment

Suppression of glioma progression by Egln3.

Grade IV astrocytoma or glioblastoma has a poor clinical outcome that can be linked to hypoxia, invasiveness and active vascular remodeling. It has recently been suggested that hypoxia-inducible factors, Hifs, increase glioma growth and aggressiveness [1], [2], [3]. Here, we tested the hypothesis that Egl 9 homolog 3 (Egln3), a prolyl-hydroxylase that promotes Hif degradation, suppresses tumor progression of human and rodent glioma models. Through intracranial tumorigenesis and in vitro assays, we demonstrate for the first time that Egln3 was sufficient to decrease the kinetics of tumor progression and increase survival. We also find that Klf5, a transcription factor important to vascular remodeling, was regulated by hypoxia in glioma. An analysis of the tumor vasculature revealed that elevated Egln3 normalized glioma capillary architecture, consistent with a role for Egln3 in eliciting decreases in the production of Hif-regulated, angiogenic factors. We also find that the hydroxylase-deficient mutant, Egln3(H196A) partially maintained tumor suppressive activity. These results highlight a bifurcation of Egln3 signaling and suggest that Egln3 has a non-hydroxylase-dependent function in glioma. We conclude that Egln3 is a critical determinant of glioma formation and tumor vascular functionality

Inducibly-expressed Egln3 down-regulates Hif-2α, Oct4 and Klf5 in glioma cells.

<p>(A) Total cell lysates were prepared from Hu-glioma cells grown for 6hrs under hypoxia in the presence of 0–1µg/ml of Dox and analyzed for Egln3, Hif-1α and Hif-2α expression by Western. Changes in protein expression levels of Hif-1α ℓ (y-axis scale, 0–1.2), Hif-2 _ (y-axis scale, 0–1.2), Egln3 (y-axis scale, 0–60) were quantified relative to controls (hypoxia 6hrs, -Dox). Klf5 and Oct4 mRNA expression levels were determined by RT-QPCR (far right panel) performed on Hu-glioma cells Egln3 +/− 0.25µg/mL Dox that were cultured under hypoxia for 6hrs. (B) Rt-glioma cells Egln3 +/− 1µg/ml Dox were assayed for Egln3, Hif-1α and Hif-2α expression by Western. Klf5 and Oct4 mRNA expression analysis was conducted by RT-QPCR (far right panel). Hypoxic samples are shown; data are expressed as fold changes relative to control (hypoxia 6hrs, -Dox). Mean values +/− s.d. are shown; n = 3.</p