Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

Background

The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported.

Methodology/Principal Findings

Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ.

Conclusion/Significance

This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.     

Cashmirols A and B,New Lipoxygenase Inhibiting Triterpenes from Sorbus cashmiriana

Two new triterpenes, cashmirols A and B ( 1 and 2 , resp.), along with three known compounds have been isolated from the AcOEt‐soluble fraction of Sorbus cashmiriana, and their structures were elucidated by spectroscopic techniques including two‐dimensional NMR. Both compounds displayed lipoxygenase enzyme inhibitory potential.     

Billburttia, a new genus of Apiaceae (tribe Apieae) endemic to Madagascar

The genus Billburttia is described to include two new species endemic to Madagascar, B. capensoides and B. vaginoides. Both species were tentatively placed within the problematic genus Peucedanum L. (as Peucedanum sp. A and Peucedanum sp. B) based on their dorsally compressed fruits lacking prominent dorsal ribs and with winged marginal ribs. Recently, however, the African members of Peucedanum have been shown to be only distantly related to the type of the genus and have therefore been segregated into six African endemic genera. While the Malagasy species appear superficially similar to members from one of these segregate genera, viz. Notobubon, they differ markedly in their fruit anatomical characters, notably a narrower commissure, six commissural vittae, vascular tissue in the tip of the ribs and sphaerocrystals distributed in and around the epidermis. The latter two characters apparently represent apomorphies for Billburttia. The non-peucedanoid affinity of the genus, as suggested by the fruit anatomical data, was confirmed using ITS and rps16 intron sequences. Both parsimony and Bayesian analyses of these data place Billburttia within the tribe Apieae of subfamily Apioideae and not closely related to either Peucedanum (Selineae) or the African peucedanoid genera (Lefebvrea clade of Tordylieae).     

Haplophytin-A induces caspase-8-mediated apoptosis via the formation of death-inducing signaling complex in human promyelocytic leukemia HL-60 cells

Haplophytin-A (10-methoxy-2,2-dimethyl-2,6-dihydro-pyrano[3,2-c]quinolin-5-one), a novel quinoline alkaloid, was isolated from the Haplophyllum acutifolium. In this study, we investigated the effect of haplophytin-A on the apoptotic activity and the molecular mechanism of action in human promyelocytic leukemia HL-60 cells. Treatment with haplophytin-A (50 μM) induced classical features of apoptosis, such as, DNA fragmentation, DNA ladder formation, and the externalization of annexin-V-targeted phosphatidylserine residues in HL-60 cells. In addition, haplophytin-A triggered the activations of caspase-8, -9, and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP) in HL-60 cells. In addition, haplophytin-A caused the loss of mitochondrial membrane potential (ΔΨ_m) and the release of cytochrome c and Smac/DIABLO to the cytosol, and modulated the expression levels of Bcl-2 family proteins. We further demonstrated that knockdown of caspase-8 using its siRNA inhibited the mitochondrial translocation of tBid, the activations of caspase-9 and caspase-3, and subsequent DNA fragmentation by haplophytin-A. Furthermore, haplophytin-A-induced the formation of death-inducing signaling complex (DISC) and then activated caspase-8 in HL-60 cells. During haplophytin-A-induced apoptosis, caspase-8-stimulated tBid provide a link between the death receptor-mediated extrinsic pathway and the mitochondria- mediated intrinsic pathway. Taken together, these results suggest that the novel compound haplophytin-A play therapeutical role for leukemia via the potent apoptotic activity through the extrinsic pathway, involving the intrinsic pathway.     

Urocortin 1 administered into the hypothalamic supraoptic nucleus affects open-field behaviour in rats

The presence of both Urocortin 1 (Ucn1) and corticotropin-releasing factor 2 receptors (CRF₂R) in the hypothalamic supraoptic nucleus (SON) suggests that endogenous Ucn1 released within this brain area acts as a local signal that might be involved in the regulation of not only endocrine but also behavioural stress responses. To test this hypothesis, we monitored the effects induced by the administration of a range of doses of synthetic Ucn1 (0.001–1.0 μg) bilaterally into the SON of rats in the open field test (OFT). Ucn1 administration produced an inverted U-shaped dose–response curve on OFT behaviour, in particular the dose of 0.01 μg of Ucn1 significantly increased the number of rearing and grooming episodes without affecting locomotion. In addition, this dosage augmented also the latency to visit the centre of the open field. Pre-treatment with the CRF₂R antagonist, astressin-2B (0.1 μg) normalized Ucn1 treatment-induced effects. These results suggest that Ucn1 released within the SON area interacts with CRF₂R to control the state of arousal.     

Hypothalamic-Specific Manipulation of Fto,the Ortholog of the Human Obesity Gene FTO,Affects Food Intake in Rats

Sequence variants in the first intron of FTO are strongly associated with human obesity and human carriers of the risk alleles show evidence for increased appetite and food intake. Mice globally lacking Fto display a complex phenotype characterised by both increased energy expenditure and increased food intake. The site of action of FTO on energy balance is unclear. Fasting reduces levels of Fto mRNA in the arcuate nucleus (ARC) of the hypothalamus, a site where Fto expression is particularly high. In this study, we have extended this nutritional link by demonstrating that consumption of a high fat diet (45%) results in a 2.5 fold increase in Arc Fto expression. We have further explored the role of hypothalamic Fto in the control of food intake by using stereotactic injections coupled with AAV technology to bi-directionally modulate Fto expression. An over expression of Fto protein by 2.5-fold in the ARC results in a 14% decrease in average daily food intake in the first week. In contrast, knocking down Arc Fto expression by 40% increases food intake by 16%. mRNA levels of Agrp, Pomc and Npy, ARC-expressed genes classically associated with the control of food intake, were not affected by the manipulation of Fto expression. However, over expression of Fto resulted in a 4-fold increase in the mRNA levels of Stat3, a signalling molecule critical for leptin receptor signalling, suggesting a possible candidate for the mediation of Fto''s actions. These data provide further support for the notion that FTO itself can influence key components of energy balance, and is therefore a strong candidate for the mediation of the robust association between FTO intronic variants and adiposity. Importantly, this provide the first indication that selective alteration of FTO levels in the hypothalamus can influence food intake, a finding consistent with the reported effects of FTO alleles on appetite and food intake in man.     

Syntheses and antifilarial profile of 7-chloro-4-(substituted amino) quinolines: a new class of antifilarial agents

The syntheses of 7-chloro-4-(substituted amino) quinolines (2-22) and their antifilarial activities are delineated. Some of the screened compounds have shown promising filarial response and sterilization effect on female Acanthocheilonema viteae in rodents.     

Over-expression and production of plant allergens by molecular farming strategies

Recombinant allergens have become a valuable tool for diagnosis and may also be used for therapy in the near future. To supply the required large amounts of functional recombinant proteins on a cost-effective basis, the production of allergens in plants by molecular farming is an alternative to microbial expression systems. Especially as post-translational modifications of the allergens, e.g., phosphorylation and glycosylation, may be important for recognition by the human immune system, the plant-based production of recombinant allergens enables the correct folding, glycosylation, and other modifications of the recombinant allergen. An introduction to the methods for plant transformation via the tumor-inducing bacterium, Agrobacterium tumefaciens, is given in this paper.     

Gut integrity and duodenal enteropathogen burden in undernourished children with environmental enteric dysfunction

Environmental enteric dysfunction (EED) is a subclinical condition of intestinal inflammation, barrier dysfunction and malabsorption associated with growth faltering in children living in poverty. This study explores association of altered duodenal permeability (lactulose, rhamnose and their ratio) with higher burden of enteropathogen in the duodenal aspirate, altered histopathological findings and higher morbidity (diarrhea) that is collectively associated with linear growth faltering in children living in EED endemic setting. In a longitudinal birth cohort, 51 controls (WHZ > 0, HAZ > −1.0) and 63 cases (WHZ< -2.0, refractory to nutritional intervention) were recruited. Anthropometry and morbidity were recorded on monthly bases up to 24 months of age. Dual sugar assay of urine collected after oral administration of lactulose and rhamnose was assessed in 96 children from both the groups. Duodenal histopathology (n = 63) and enteropathogen analysis of aspirate via Taqman array card (n = 60) was assessed in only cases. Giardia was the most frequent pathogen and was associated with raised L:R ratio (p = 0.068). Gastric microscopy was more sensitive than duodenal aspirate in H. pylori detection. Microscopically confirmed H. pylori negatively correlated with HAZ at 24 months (r = −0.313, p = 0.013). Regarding histopathological parameters, goblet cell reduction significantly correlated with decline in dual sugar excretion (p< 0.05). Between cases and controls, there were no significant differences in the median (25^th, 75^th percentile) of urinary concentrations (μg/ml) of lactulose [27.0 (11.50, 59.50) for cases vs. 38.0 (12.0, 61.0) for controls], rhamnose [66.0 (28.0, 178.0) vs. 86.5 (29.5, 190.5)] and L:R ratio [0.47 (0.24, 0.90) vs. 0.51 (0.31, 0.71)] respectively. In multivariable regression model, 31% of variability in HAZ at 24 months of age among cases and controls was explained by final model including dual sugars. In conclusion, enteropathogen burden is associated with altered histopathological features and intestinal permeability. In cases and controls living in settings of endemic enteropathy, intestinal permeability test may predict linear growth. However, for adoption as a screening tool for EED, further validation is required due to its complex intestinal pathophysiology.