Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission

The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5′-phosphate [PLP]), in complex with artesunate at 2.4-Å resolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6. In humans, PLP biosynthesis is catalyzed by pyridoxal kinase (PDXK), a member of the ribokinase superfamily. PDXK utilizes inactive forms of vitamin B6 (pyridoxal [PL], pyridoxine, and pyridoxamine) and ATP as substrates, producing PLP along with the byproduct ADP. The corresponding reaction proceeds via a random substrate addition reaction mechanism (1) in which PLP biosynthesis takes place by transferring the γ-phosphate of ATP to the 5′-OH group of the B6 vitamers, in a process assisted by divalent metal ions such as Zn²⁺ and Mg²⁺ (2) (Fig. 1A). PLP serves as the essential active site component for more than 160 distinct human enzymatic activities (3) catalyzing crucial cellular processes such as detoxification reactions and multiple metabolic processes including amino acid, carbohydrate, and lipid metabolism. PLP-dependent enzymes also participate in neurotransmitter biosynthesis including the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine (4–6), which are synthesized by glutamic acid decarboxylase (GAD) and serine hydroxymethyl transferase (SHMT), respectively. Vitamin B6 deficiency has been implicated in multiple neurological, psychiatric, and internal disorders possibly including even diabetes, cancer, and autism (3), thus underpinning the importance of a finely tuned PLP biosynthesis.Open in a separate windowFig. 1.Biochemical basis of PDXK inhibition by artemisinins. (A) Schematic representation of the reaction catalyzed by PDXK. (B) Michaelis–Menten curve derived for the enzymatic activity of recombinantly purified PDXK. (C and D) Chemical structures of artemisinin (C) and artesunate (D). (E) Enzymatic activity of wild-type PDXK (WT-PDXK) in the absence and presence of artemisinin derivatives at a concentration of 1.5 mM (artesunate) and 156 µM (artemisinin), respectively. Data are presented as mean ± SEM (P values are *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) (one-way ANOVA test). (F) Inhibition curves of PDXK by artemisinin and artesunate used to derive the corresponding IC₅₀ values.Recently, PDXK was identified as one of the mammalian targets of the antimalarial drug artemisinin (7). Artemisinin-containing plant extracts have been used in traditional Chinese medicine for the treatment of malaria (8). Chemically, these small molecules are sesquiterpene lactones with an unusual endoperoxide bridge. Artemisinin and its semisynthetic derivatives artemether and artesunate (collectively referred to as artemisinins), in combination with quinones such as mefloquine and lumefantrine, nowadays represent the standard drug combinations used to treat malaria caused by Plasmodium falciparum (9). In addition to their antiprotozoan activities, these drugs have also been pharmacologically observed to regulate the activities of a variety of mammalian cellular processes, some of which are deregulated in various types of cancer (10, 11). Recently, it was discovered that artemisinins also modulate the differentiation of pancreatic Tα cells by inducing the transdifferentiation of glucagon-producing Tα cells into insulin-secreting Tβ cells, thus suggesting an antidiabetic activity of artemisinins (7). However, two subsequent studies contradicted this observation, thus questioning the potential clinical application of these compounds in the treatment of diabetes (12, 13).Until recently, in the absence of a single protein crystal structure in complex with artemisinins (neither a plasmodial nor a mammalian protein), the detailed framework describing the target recognition by these small molecules remained enigmatic. The first molecular insights into artemisinin recognition by a target protein were derived by us from crystal structures of the C-terminal domain of the moonlighting protein gephyrin (GephE) in complex with two artemisinin derivatives, artesunate and artemether (14). Gephyrin is the principal scaffolding protein at inhibitory postsynaptic specializations and also catalyzes the final two steps of the evolutionarily conserved molybdenum cofactor (Moco) biosynthesis (15–17). Structures of the GephE–artemisinin complexes demonstrated that artemisinins specifically target the universal receptor binding pocket of this moonlighting protein, without altering its enzymatic activity, thus inhibiting critical interactions of gephyrin with GABA type A receptors (GABA_ARs) and glycine receptors (GlyRs). As an important functional consequence, artemisinins modulate inhibitory neurotransmission in a gephyrin-dependent manner. In addition to gephyrin, various proteins were identified as putative targets of artemisinins in pancreatic cells, including the central metabolic enzyme PDXK (7), yet the molecular mechanisms underlying the modulation of these targets by artemisinins remained unknown.Here, we determined the 2.4-Å resolution crystal structure of mouse pyridoxal kinase (mPDXK) in complex with artesunate, a succinate derivative of artemisinin. The artesunate binding site partially overlaps with the substrate (PL)/product (PLP) binding site, thus suggesting a drug-induced inhibitory effect. Enzymatic activity assays in vitro indeed revealed a significant inhibition of PLP production in the presence of artemisinins with K_i values in the high micromolar range. Electrophysiological recordings and measurements of GABA biosynthesis suggest that artemisinins exert their effect by down-regulating the activity of PLP-dependent enzymes such as GAD. Taken together, our data define the molecular basis for the inhibition of PDXK by artemisinins and their consequences at the presynaptic terminals of inhibitory postsynapses and extend our current understanding of the artemisinin-induced modulation of inhibitory neurotransmission beyond gephyrin.     

Myogenic properties of human mesenchymal stem cells derived from three different sources

Mesenchymal stem cells (MSCs) of mammals have been isolated from many tissues and are characterized by their aptitude to differentiate into bone, cartilage, and fat. Differentiation into cells of other lineages like skeletal muscle, tendon/ligament, nervous tissue, and epithelium has been attained with MSCs derived from some tissues. Whether such abilities are shared by MSCs of all tissues is unknown. We therefore compared for three human donors the myogenic properties of MSCs from adipose tissue (AT), bone marrow (BM), and synovial membrane (SM). Our data show that human MSCs derived from the three tissues differ in phenotype, proliferation capacity, and differentiation potential. The division rate of AT-derived MSCs (AT-MSCs) was distinctly higher than that of MSCs from the other two tissue sources. In addition, clear donor-specific differences in the long-term maintenance of MSC proliferation ability were observed. Although similar in their in vitro fusogenic capacity with murine myoblasts, MSCs of the three sources contributed to a different extent to skeletal muscle regeneration in vivo. Transplanting human AT-, BM-, or SM-MSCs previously transduced with a lentiviral vector encoding β-galactosidase into cardiotoxin-damaged tibialis anterior muscles (TAMs) of immunodeficient mice revealed that at 30 days after treatment the frequency of hybrid myofibers was highest in the TAMs treated with AT-MSCs. Our finding of human-specific β-spectrin and dystrophin in hybrid myofibers containing human nuclei argues for myogenic programming of MSCs in regenerating murine skeletal muscle. For the further development of MSC-based treatments of myopathies, AT-MSCs appear to be the best choice in view of their efficient contribution to myoregeneration, their high ex vivo expansion potential, and because their harvesting is less demanding than that of BM- or SM-MSCs.     

Babesia: A world emerging

Babesia are tick-transmitted hemoprotozooans that infect mammals and birds, and which are acknowledged for their major impact on farm and pet animal health and associated economic costs worldwide. Additionally, Babesia infections of wildlife can be fatal if associated with stressful management practices; and human babesiosis, also transmitted by blood transfusion, is an increasing public-health concern. Due to the huge diversity of species reported to serve as Babesia hosts, all vertebrates might be potential carriers, as long as they are adequate hosts for Babesia-vector ticks. We here provide a comprehensive overview of the most relevant Babesia species, and a discussion of the classical taxonomic criteria. Babesia, Cytauxzoon and Theileria parasites are closely related and collectively referred to as piroplasmids. A possible scenario for the history of piroplasmids is presented in the context of recent findings, and its implications for future research avenues are outlined. Phylogenetic trees of all available 18S rRNA and hsp70 genes were generated, based on which we present a thoroughly revised molecular classification, comprising five monophyletic Babesia lineages, one Cytauxzoon clade, and one Theileria clade. Updated 18S rRNA and beta-tubulin gene trees of the B. microti isolates agree with those previously reported. To reconcile estimates of the origin of piroplasmids and ticks (～300 Ma, respectively), and mammalian radiation (60 Ma), we hypothesize that the dixenous piroplasmid life cycle evolved with the origin of ticks. Thus, the observed time gap between tick origin and mammalian radiation indicates the existence of hitherto unknown piroplasmid lineages and/or species in extant vertebrate taxa, including reptiles and possibly amphibians. The development and current status of the molecular taxonomy of Babesia, with emphasis on human-infecting species, is discussed. Finally, recent results from population genetic studies of Babesia parasites, and their implications for the development of pathogenicity, drug resistance and vaccines, are summarized.     

Manipulations during forced wakefulness have differential impact on sleep architecture, EEG power spectrum, and Fos induction

We propose a hypothesis suggesting that the most prominent experiences occurring during wakefulness activate specific clusters of neurons related to such experiences. These neurons could possibly then evoke the release of various types of sleep-inducing molecules, thereby causing different patterns of sleep architecture. In this study, we therefore sought to determine whether manipulations of behavior during wakefulness, such as forced wakefulness induced by gentle handling, forced wakefulness associated with a stressful condition such as immobilization, or forced wakefulness associated with excess intake of palatable food, could result in a variation of Fos immunoreactivity in selective brain structures and could also result in different sleep and EEG power density patterns. The results showed that the sleep-wake cycle of rats after all the experimental manipulations was different not only with respect to the control group but also among themselves. Additionally, power spectrum analysis showed an increase of 0.25–4.0 Hz in all experimental manipulations, whereas the 4.25–8.0 Hz increase occurred only in the situation of forced wakefulness plus stress. The Fos induction showed activation of cell clusters in cortical areas and telencephalic centers, in several hypothalamic nuclei, in monoaminergic cell groups, and in brain stem nuclei. The density of Fos-immunoreactive neurons varied in relation to the different paradigms of forced wakefulness. These results suggest that activation of cell clusters in the brain are related to the type of manipulation imposed on the rat during wakefulness and that such variation in cell activation prior to sleep may be associated with sleep architecture and EEG power.     

Cognitive functioning in bipolar patients receiving lamotrigine: preliminary results

Despite the increasing use of lamotrigine (LTG) in bipolar disorder, little is known about its impact on cognition in bipolar patients. Therefore, we have evaluated 33 bipolar I and II patients on cognitive measures (verbal memory, attention, executive functions) while receiving either LTG (n = 15) or another anticonvulsant (carbamazepine or valproate; n = 18). Patients receiving LTG were generally diagnosed as having bipolar II disorder, had experienced more depressive episodes but a lesser number of hospitalizations, and had better performance than the patients receiving carbamazepine or valproate on the verbal fluency task. A moderate effect size also suggests that both groups may differ on the immediate verbal memory test (California Verbal Learning Test). These preliminary results suggest a safer neurocognitive profile of LTG on bipolar patients, as compared with other anticonvulsants.     

Differences in radiological patterns, tumour characteristics and diagnostic precision between digital mammography and screen-film mammography in four breast cancer screening programmes in Spain

Objectives

To compare tumour characteristics between cancers detected with screen-film mammography (SFM) and digital mammography (DM) and to evaluate changes in positive predictive values (PPVs) for further assessments, for invasive procedures and for distinct radiological patterns in recalled women.

Methods

242,838 screening mammograms (171,191 SFM and 71,647 DM) from 103,613 women aged 45–69 years, performed in four population-based breast cancer screening programmes in Spain, were included. The tumour characteristics and PPVs of each group were compared. Radiological patterns (masses, calcifications, distortions and asymmetries) among recalled women were described and PPVs were evaluated.

Results

The percentages of ductal carcinoma in situ (DCIS) were higher in DM than in SFM both in the first [18.5% vs. 15.8%(p?=?0.580)] and in successive screenings [23.2% vs. 15.7%(p?=?0.115)]. PPVs for masses, asymmetries and calcifications were higher in DM, being statistically significant in masses (5.3% vs. 3.9%; proportion ratio: 1.37 95%CI: 1.08–1.72). Among cancers detected by calcifications, the percentage of DCIS was higher in DM (60.3% vs. 46.4%, p?=?0.060).

Conclusions

PPVs were higher when DM was used, both for further assessments and for invasive procedures, with similar cancer detection rates and no statistically significant differences in tumour characteristics. The greatest improvements in PPVs were found for masses.     

Expression of rat renal cortical OAT1 and OAT3 in response to acute biliary obstruction

Renal function in the course of obstructive jaundice has been the subject of great interest; however, little is known about the expression of renal organic anion transporters. The objective of this work was to study, in rats with acute extrahepatic cholestasis, the cortical renal expression of the organic anion transporter 1 (OAT1) and the organic anion transporter 3 (OAT3), in association with the pharmacokinetics and renal excretion of furosemide (FS). Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. All studies were carried out 21 hours after surgery. Rats were anesthetized and the pharmacokinetic parameters of FS and the renal elimination of FS were determined. Afterwards, the kidneys were excised and processed for immunoblot (basolateral membrane and renal homogenates) or immunocytochemical (light microscopic and confocal immunofluorescence microscopic analysis) techniques. The systemic and renal clearance of FS as well as the excreted and secreted load of FS increased in BDL rats. In kidneys from BDL rats, immunoblotting showed a significant increase in the abundance of both OAT1 and OAT3 in homogenates from renal cortex. In basolateral membranes from kidney cortex of BDL rats, OATI abundance was also increased and OAT3 abundance was not modified. Immunocytochemical techniques confirmed these results. In conclusion, acute obstructive jaundice is associated with an upregulation of OAT1 and OAT3, which might explain, at least in part, the increased systemic and renal elimination of FS.