Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa

During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.     

No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine

A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105) or placebo treatment (n = 111) for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79). There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70), baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.     

Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor

The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.     

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16^INK4 and p19^ARF. These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.     

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1

Poly(ADP-ribose) polymerases or PARPs are a family of NAD⁺-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP1 activation following genotoxin treatment causes NAD⁺ depletion and cell death, whereas pharmacological PARP1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD⁺ metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin–Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1500 to 60,000 copies per cell) lowers steady-state NAD⁺ levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H₂O₂ and the alkylating agent MNNG, genotoxins that kill cells by activating PARP1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP1-mediated reactions such as PARP1 automodification and NAD⁺ consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD⁺ homeostasis and inhibition of PARP1 function.     

Farnesol concentrations required to block germ tube formation in Candida albicans in the presence and absence of serum

Concentrations of (E,E)-farnesol needed to inhibit germ tube formation were determined for Candida albicans strains A72 and SC5314 by using six different conditions known to trigger germination. For defined media, 1 to 2 microM farnesol was sufficient. However, with serum at 2 to 20%, up to 250 microM farnesol was required. Farnesol blocked germ tube formation but did not block elongation of existing germ tubes.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.