Amino acid starvation inhibits autophagy in lipid droplet-deficient cells through mitochondrial dysfunction.

Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions, they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast. It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy.

Increased fatty acid synthesis inhibits nitrogen starvation-induced autophagy in lipid droplet-deficient yeast.

Macroautophagy is a degradative pathway whereby cells encapsulate and degrade cytoplasmic material within endogenously-built membranes. Previous studies have suggested that autophagosome membranes originate from lipid droplets. However, it was recently shown that rapamycin could induce autophagy in cells lacking these organelles. Here we show that lipid droplet-deprived cells are unable to perform autophagy in response to nitrogen-starvation because of an accelerated lipid synthesis that is not observed with rapamycin. Using cerulenin, a potent inhibitor of fatty acid synthase, and exogenous addition of palmitic acid we could restore nitrogen-starvation induced autophagy in the absence of lipid droplets.

Canthin-6-one displays antiproliferative activity and causes accumulation of cancer cells in the G2/M phase.

Canthinones are natural substances with a wide range of biological activities, including antipyretic, antiparasitic, and antimicrobial. Antiproliferative and/or cytotoxic effects of canthinones on cancer cells have also been described, although their mechanism of action remains ill defined. To gain better insight into this mechanism, the antiproliferative effect of a commercially available canthin-6-one (1) was examined dose-dependently on six cancer cell lines (human prostate, PC-3; human colon, HT-29; human lymphocyte, Jurkat; human cervix, HeLa; rat glioma, C6; and mouse embryonic fibroblasts, NIH-3T3). Cytotoxic effects of 1 were investigated on the same cancer cell lines by procaspase-3 cleavage and on normal human skin fibroblasts. Strong antiproliferative effects of the compound were observed in all cell lines, whereas cytotoxic effects were very dependent on cell type. A better definition of the mechanism of action of 1 was obtained on PC-3 cells, by showing that it decreases BrdU incorporation into DNA by 60% to 80% and mitotic spindle formation by 70% and that it causes a 2-fold accumulation of cells in the G2/M phase of the cell cycle. Together, the data suggest that the primary effect of canthin-6-one (1) is antiproliferative, possibly by interfering with the G2/M transition. Proapoptotic effects might result from this disturbance of the cell cycle.

Activation of rhodopsin gene transcription in cultured retinal precursors of chicken embryo: role of Ca(2+) signaling and hyperpolarization-activated cation channels.

This study reports that the spontaneous 50-fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13-day chicken embryo, relies on a Ca(2+)-dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant-negative form of the cAMP-responsive element-binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca(2+)/calmodulin-dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca(2+) rather than cAMP. This was confirmed by the inhibitory effect of general and T-type selective Ca(2+) channel blockers. Oscillations in Ca(2+) fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization-activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage-gated Ca(2+)-channels, activation of Ca(2+)/calmodulin-dependent kinase II and phosphorylation of cAMP-responsive element-binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+-dependent mechanism whereby hyperpolarization-activated cyclic nucleotide-gated channels (HCN) activate T-type voltage-dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin-dependent kinase II (CaMKII) to phosphorylate the cAMP-responsive element-binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo.

The MFS-type efflux pump Flr1 induced by Yap1 promotes canthin-6-one resistance in yeast.

Screening for suppressors of canthin-6-one toxicity in yeast identified Yap1, a transcription factor involved in cell response to a broad range of injuries. Although canthin-6-one did not promote a significant oxidative stress, overexpression of YAP1 gene clearly increased resistance to this drug. We demonstrated that Yap1-mediated resistance involves the plasma membrane major-facilitator-superfamily efflux pump Flr1 but not the vacuolar ATP-binding-cassette transporter Ycf1. FLR1 overexpression was sufficient to reduce sensitivity to the drug, but strictly dependent on a functional YAP1 gene.

Cyclic AMP-dependent regulation of tyrosine hydroxylase mRNA and immunofluorescence levels in rat retinal precursor cells.

Stimulation of tyrosine hydroxylase (TH) gene transcription by cyclic AMP (cAMP) has been clearly established in adrenal medula cells and neural-crest-derived cell lines but information on this mechanism is still lacking in dopaminergic neurons. Because they are easily amenable to in vitro experiments, dopaminergic amacrine cells of the retina might constitute a valuable model system to study this mechanism. We have used real-time reverse transcription with the polymerase chain reaction to quantify TH mRNA levels in the rat retina during post-natal development and in retinal precursor cells obtained from neonatal rats and cultured for 3 days in serum-free medium. Whereas the TH mRNA concentration remains consistantly low in control cultures, treatment with cAMP-increasing agents (forskolin, membrane depolarization, phosphodiesterase inhibitors) is sufficient to raise it to the level observed in adult retina (15-fold increase). Treatment of the cultured cells can be delayed by up to 2 days with identical results at the TH mRNA level, thus ruling out a survival-promoting effect of cAMP. TH immunofluorescence has confirmed cAMP-dependent regulation of TH expression at the protein level and indicates that the frequency of TH-positive cells in the cultures is similar to that observed in the adult retina. Selective phosphodiesterase inhibitors suggest that PDE4 is the major subtype involved in the dopaminergic amacrine cell response. Our data clearly establish the cAMP-dependent regulation of TH mRNA and immunofluorescence levels in retinal precursor cells. The possible role of this regulation mechanism in the developmental activation of TH gene expression is discussed.

Accuracy and trust of self-testing for bacterial vaginosis.

PURPOSE: Two point-of-care tests are available to detect bacterial vaginosis (BV), a common vaginal condition. This study aimed to (1) compare the accuracy of two self-performed BV tests with clinician-performed BV tests and with clinical diagnosis of BV; and (2) compare trust of results for self-performed BV testing with clinician-performed BV testing. METHODS: Participants (14-22 years old) in a study assessing self-testing for Trichomonas vaginalis were also asked to perform a self-test for BV (using a pH or sialidase test). Results were compared with clinician-performed tests and with clinical diagnosis of BV (defined by modified Amsel criteria). A two-item subscale from a larger acceptability scale was used to assess trust at baseline, after testing, and after discussion of results. RESULTS: All 131 women performed self-BV testing correctly. Agreement between self- and clinician-performed tests was good (κ: .5-.7) Compared with clinical diagnosis of BV, self-pH was 73% sensitive and 67% specific, and self-sialidase was 40% sensitive and 90% specific. Trust in self-performed BV testing was lower than trust in clinician-performed BV testing at baseline, but increased after testing and discussion of results. CONCLUSIONS: Young women can perform self-tests for BV with reasonable accuracy, which could increase testing when pelvic examinations are not feasible. Trust in self-testing increased after experience and after discussion of test results. Although the pH test is available over the counter, young women may continue to rely on clinicians for testing.

Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo.

The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation.

Photoreceptor-specific expression, light-dependent localization, and transcriptional targets of the zinc-finger protein Yin Yang 1 in the chicken retina.

The zinc-finger transcription factor Yin Yang 1 (YY1) is a multifunctional protein that plays a critical role in embryonic development. Although it has been shown to play a role in eye development, its expression in the retina was not previously described. Here, we investigated YY1 expression in chicken tissues and we identified the neural retina as one of the tissues with highest YY1 protein levels. Immunohistochemical detection of YY1 in the retina revealed a clear-cut photoreceptor specificity and day/night differences in the cytoplasmic localization of the protein. YY1 was also present at high concentration in the nuclei of some photoreceptors. Gel-shift assays indicated YY1 bound to regulatory regions of several genes specifically expressed in photoreceptors. One of these genes, hydroxyindole-O-methyltransferase (EC 2.1.1.4), encodes the last enzyme of the melatonin synthesis pathway. Although over-expression of chicken YY1 was not sufficient to activate the chicken hydroxyindole-O-methyltransferase promoter in HEK293 cells, the YY1-binding site contained in this promoter was clearly required for full transcriptional activity in chicken embryonic retinal cells. These results suggest a role of YY1 in regulating the melatoninergic function of retinal photoreceptors.

Melatoninergic differentiation of retinal photoreceptors: activation of the chicken hydroxyindole-O-methyltransferase promoter requires a homeodomain-binding element that interacts with Otx2.

The gene encoding the last enzyme of the melatonin-synthesis pathway, hydroxyindole-O-methyltransferase (HIOMT), is selectively expressed in retinal photoreceptors and pineal cells. Here, we analysed the promoter of the chicken HIOMT gene and we found that a homeodomain-binding element located in the proximal region of this promoter was essential for its activation in primary cultures of embryonic chicken retinal cells. This homeodomain-regulatory element interacted with a protein expressed in the chicken retina and pineal gland, which was recognized by an anti-Otx2 antiserum. Recombinant Otx2 expressed in vitro was able to bind this DNA element and to directly transactivate the chicken HIOMT promoter. This promoter was also transactivated by another member of the Otx family, Otx5, but the amplitude of stimulation was lower than with Otx2. The spatio-temporal pattern of Otx2 expression was compatible with a possible role of this transcription factor in HIOMT gene activation. In adult chicken, Otx2 mRNA was found to be present in those two tissues that express HIOMT: the retina and the pineal gland. During development, a burst of Otx2 mRNA closely matched the timing of HIOMT gene activation in these two tissues. In the pineal, Otx2 immunolabelling was specifically localized in the nuclei of photoreceptor cells. In the neural retina, Otx2 immunoreactivity brightly decorated the photoreceptor nuclei and extended more faintly to the outer half of the inner nuclear layer. Together, the data support a role of Otx2 in the onset of HIOMT expression in developing chicken photoreceptors.

Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursor cells.

PURPOSE: Photoreceptor differentiation involves the activation of two specific sets of genes; those encoding the proteins of the phototransduction cascade and those encoding the enzymes of the melatonin synthesis pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT). The purpose of the present study was to examine the conditions of AANAT and HIOMT gene activation, relative to that of selected phototransduction markers (alpha-transducin and opsins), in both in vivo and in vitro differentiating photoreceptors of the chicken retina. METHODS: Neural retina RNA was obtained between embryonic day 7 (E7) and posthatch day 8 (P8) and analyzed on northern blots with cDNA probes to AANAT, HIOMT, visinin, alpha-transducin, rhodopsin, and the four cone opsins. Cell cultures were prepared from E7 chicken neural retina and incubated for two to four days in vitro, either in basal medium or in serum-supplemented medium or in medium containing an insulin-based supplement. RNA from the cultured cells was analyzed on northern blots as above. Real time RT-PCR was used to confirm in vitro changes in HIOMT and red opsin mRNA levels. The cultured cells were transfected with promoter-reporter plasmids for direct analysis of HIOMT promoter regulation by the dual luciferase method. RESULTS: The different mRNAs composing the photoreceptor phenotype appeared at E7 (visinin), E10 (alpha-transducin), E14 (HIOMT), E15 (rhodopsin, red opsin, and green opsin), E16 (AANAT), E17 (blue opsin), and E18 (violet opsin). In the early differentiating cones of the central retina, HIOMT mRNA appeared two days earlier than red opsin and green opsin mRNAs (E12 rather than E14). In cultured embryonic neural retina cells, basal medium was sufficient to activate alpha-transducin gene transcription, an insulin-based supplement was sufficient to activate HIOMT gene transcription, whereas serum was required for red opsin gene transcription after two days in vitro. All serum batches were able to activate red opsin gene transcription, whereas some of them failed to activate HIOMT gene transcription. Activation of the HIOMT gene promoter by an insulin-based supplement and by serum was confirmed after transfection of chicken embryonic neural retina cells with promoter-reporter plasmids. CONCLUSIONS: Activation of the melatonin synthesis genes in vivo takes place in a time window very close to that of early opsins. However, a 24-48 h lead of HIOMT gene expression over early opsins was clearly observed. Our in vitro experiments indicate that different exogenous signals are required to activate the different genes encoding photoreceptor specific functions. Significantly, marker genes for light sensitivity (red opsin) and for melatonin synthesis (HIOMT) appear to be activated in response to different signals.