PKC isoforms interact with and phosphorylate DNMT1

Background

DNA methyltransferase 1 (DNMT1) has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC) may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization.

Results

Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity.

Conclusions

Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.     

Archipelago-wide island restoration in the Galápagos Islands: reducing costs of invasive mammal eradication programs and reinvasion risk

Invasive alien mammals are the major driver of biodiversity loss and ecosystem degradation on islands. Over the past three decades, invasive mammal eradication from islands has become one of society's most powerful tools for preventing extinction of insular endemics and restoring insular ecosystems. As practitioners tackle larger islands for restoration, three factors will heavily influence success and outcomes: the degree of local support, the ability to mitigate for non-target impacts, and the ability to eradicate non-native species more cost-effectively. Investments in removing invasive species, however, must be weighed against the risk of reintroduction. One way to reduce reintroduction risks is to eradicate the target invasive species from an entire archipelago, and thus eliminate readily available sources. We illustrate the costs and benefits of this approach with the efforts to remove invasive goats from the Galápagos Islands. Project Isabela, the world's largest island restoration effort to date, removed >140,000 goats from >500,000 ha for a cost of US$10.5 million. Leveraging the capacity built during Project Isabela, and given that goat reintroductions have been common over the past decade, we implemented an archipelago-wide goat eradication strategy. Feral goats remain on three islands in the archipelago, and removal efforts are underway. Efforts on the Galápagos Islands demonstrate that for some species, island size is no longer the limiting factor with respect to eradication. Rather, bureaucratic processes, financing, political will, and stakeholder approval appear to be the new challenges. Eradication efforts have delivered a suite of biodiversity benefits that are in the process of revealing themselves. The costs of rectifying intentional reintroductions are high in terms of financial and human resources. Reducing the archipelago-wide goat density to low levels is a technical approach to reducing reintroduction risk in the short-term, and is being complemented with a longer-term social approach focused on education and governance.     

Uncovering the secretes of mesenchymal stem cells

Mesenchymal stem cells (MSC) show great promise in a wide array of therapeutic applications due mainly to their capacity to suppress immune and inflammatory reactions and instigate normal tissue repair processes. The secretion of bioactive factors is thought to play a predominant role in the mechanisms of action for these clinically relevant functions. As such, a large body of MSC research has focussed on characterization of the MSC secretome; including both soluble factors and factors released in extracellular vesicles (e.g., exosomes and microvesicles). This review provides an overview of our current knowledge of the MSC secretome in the context of determining the clinical relevance of these cells. In addition, the review summarizes various approaches that have been utilized to identify proteins secreted by MSC and discusses the advantages and limitations of different proteomic methods. Finally, we discuss issues that must be addressed before the clinical relevance of research into the MSC secretome can be realized.     

Phosphorylation of human DNMT1: implication of cyclin-dependent kinases

DNA methylation plays a central role in the epigenetic regulation of gene expression during development and progression of cancer diseases. The inheritance of specific DNA methylation patterns are acquired in the early embryo and are specifically maintained after cellular replication via the DNA methyltransferase 1 (DNMT1). Recent studies have suggested that the enzymatic activity of DNMT1 is possibly modulated by phosphorylation of serine/threonine residues located in the N-terminal domain of the enzyme. In the present work, we report that cyclin-dependent kinases (CDKs) 1, 2 and 5 can phosphorylate Ser154 of human DNMT1 in vitro. Further evidence of phosphorylation of endogenous DNMT1 at position 154 by CDKs is also found in 293 cells treated with roscovitine, a specific inhibitor of CDK1, 2 and 5. To determine the importance of Ser154 phosphorylation, a mutant of DNMT1 encoding a single-point mutation at position 154 (S154A) was generated. This mutation induced a severe loss of enzymatic activity when compared to wild type DNMT1. Moreover, after treatment with 5-Aza-2′-Deoxycytidine (5-aza-dC), a faster decline in DNMT1 protein level was observed for HEK-293 cells expressing DNMT1(S154A) as compared to cells expressing wild type DNMT1. Our data suggest that phosphorylation of DNMT1 at Ser154 by CDKs is important for enzymatic activity and protein stability of DNMT1. Considering that tumour-associated cell cycle defects are often mediated by alterations in CDK activity, our results suggest that dysregulation of cell cycle via CDKs could induce abnormal phosphorylation of DNMT1 and lead to DNA hypermethylation often observed in cancer cells.     

Barriers and facilitators to implement shared decision making in multidisciplinary sciatica care: a qualitative study

Background

Venous thromboembolism (VTE) is a common preventable cause of mortality in hospitalized medical patients. Despite rigorous randomized trials generating strong recommendations for anticoagulant use to prevent VTE, nearly 40% of medical patients receive inappropriate thromboprophylaxis. Knowledge-translation strategies are needed to bridge this gap.

Methods

We conducted a 16-week pilot cluster randomized controlled trial (RCT) to determine the proportion of medical patients that were appropriately managed for thromboprophylaxis (according to the American College of Chest Physician guidelines) within 24 hours of admission, through the use of a multicomponent knowledge-translation intervention. Our primary goal was to determine the feasibility of conducting this study on a larger scale. The intervention comprised clinician education, a paper-based VTE risk assessment algorithm, printed physicians’ orders, and audit and feedback sessions. Medical wards at six hospitals (representing clusters) in Ontario, Canada were included; three were randomized to the multicomponent intervention and three to usual care (i.e., no active strategies for thromboprophylaxis in place). Blinding was not used.

Results

A total of 2,611 patients (1,154 in the intervention and 1,457 in the control group) were eligible and included in the analysis. This multicomponent intervention did not lead to a significant difference in appropriate VTE prophylaxis rates between intervention and control hospitals (appropriate management rate odds ratio = 0.80; 95% confidence interval: 0.50, 1.28; p = 0.36; intra-class correlation coefficient: 0.022), and thus was not considered feasible. Major barriers to effective knowledge translation were poor attendance by clinical staff at education and feedback sessions, difficulty locating preprinted orders, and lack of involvement by clinical and administrative leaders. We identified several factors that may increase uptake of a VTE prophylaxis strategy, including local champions, support from clinical and administrative leaders, mandatory use, and a simple, clinically relevant risk assessment tool.

Conclusions

Hospitals allocated to our multicomponent intervention did not have a higher rate of medical inpatients appropriately managed for thromboprophylaxis than did hospitals that were not allocated to this strategy.     

Cep164, a novel centriole appendage protein required for primary cilium formation

Primary cilia (PC) function as microtubule-based sensory antennae projecting from the surface of many eukaryotic cells. They play important roles in mechano- and chemosensory perception and their dysfunction is implicated in developmental disorders and severe diseases. The basal body that functions in PC assembly is derived from the mature centriole, a component of the centrosome. Through a small interfering RNA screen we found several centrosomal proteins (Ceps) to be involved in PC formation. One newly identified protein, Cep164, was indispensable for PC formation and hence characterized in detail. By immunogold electron microscopy, Cep164 could be localized to the distal appendages of mature centrioles. In contrast to ninein and Cep170, two components of subdistal appendages, Cep164 persisted at centrioles throughout mitosis. Moreover, the localizations of Cep164 and ninein/Cep170 were mutually independent during interphase. These data implicate distal appendages in PC formation and identify Cep164 as an excellent marker for these structures.     

Mechanism of insulin-stimulated clearance of plasma nonesterified fatty acids in humans

Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is independent of change in plasma NEFA appearance is currently unknown. Nine nondiabetic men (age: 28+/-3 yr, body mass index: 27.2+/-1.7 kg/m2) underwent euglycemic clamps to maintain low (LINS) vs. high (HINS) physiological insulin levels for 6 h. An intravenous infusion of heparin+Intralipid (HI) was performed during 4 of the 6 h of the clamps (in the last 4 h at LINS and in the first 4 h at HINS), whereas saline infusion (SAL) was administered in the remaining 2 h to modulate plasma NEFA levels independently of plasma insulin levels. Four experimental conditions were obtained in each individual: LINS with saline (LINS/SAL) and with HI infusion (LINS/HI) and HINS with saline (HINS/SAL) and with HI infusion (HINS/HI). Plasma palmitate appearance during HINS/SAL was lower than during the three other experimental conditions (P<0.05). In contrast, plasma linoleate appearance, as expected, was increased by HI independently of insulin level (P<0.02). Plasma palmitate clearance during HINS/SAL was higher than LINS/SAL and LINS/HI (P<0.008), and this increase was blunted during HINS/HI. We observed a linear decrease in plasma palmitate clearance with increasing plasma NEFA appearance independent of insulin levels. Plasma NEFA levels increased exponentially with increase in plasma NEFA appearance. We conclude that insulin stimulates plasma NEFA clearance by reducing the endogenous appearance rate of NEFA. The relationship between plasma NEFA level and appearance rate is nonlinear.     

Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats

Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase 2 (PON2), a member of a powerful antioxidant family in human and rat intestine. Biochemical and ultrastructural experiments all showed a substantial expression of PON2 in human and rat intestine. Western blot analysis disclosed higher levels of PON2 in the jejunum than in the duodenum, ileum, and colon. Cell fractionation revealed a predominant PON2 association with microsomes and lysosomes in the human jejunum, which differed from that in rats. PON2 was detected in the intestine as early as week 15 of gestation and was significantly increased by week 20. Iron ascorbate-mediated lipid peroxidation induced a marked decrease in PON2 expression in intestinal specimens coincidental to an abundant rise in malondialdehyde (MDA). On the other hand, preincubation with potent antioxidants, such as butylated hydroxytoluene, Trolox, and N-acetylcysteine, prevented iron-ascorbate-generating PON2 reduction in parallel with MDA suppression. Finally, the preincubation of permeabilized Caco-2 cells with purified PON2 led to a protection against iron-ascorbate-induced lipid peroxidation. These observations demonstrate that the human intestine is preferentially endowed with a marked PON2 expression compared with the rat intestine and this expression shows a developmental and intracellular pattern of distribution. Furthermore, our observations suggest PON2 protective effects against prooxidant stimuli in the small intestine.