Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.

Oral supplementation of inorganic pyrophosphate in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi ) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4 P2 O7 is absorbed in humans. Here, we report that gelatin-encapsulated Na2 H2 P2 O7  has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K2 H2 P2 O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6-/- mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.

NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir.

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.

Mycobacterial Populations Partly Change the Proportions of the Cells Undergoing Asymmetric/Symmetric Divisions in Response to Glycerol Levels in Growth Medium.

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister-daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.

ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions.

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

Moringa oleifera modulates cholinergic and purinergic enzymes activity in BV-2 microglial cells.

Microglia are immune cells that are resident in central nervous system. Activation of microglial cells are detrimental to the survival of neurons. Thus, prevention of microglia activation and/or protection against microglia activation could be potential therapeutic strategy towards the management of inflammation-mediated neurodegenerative diseases. Moringa oleifera is widely consumed as food and used in folklore medicine for treating several diseases. This study was convened to investigate the effect of aqueous extract of Moringa oleifera on cell viability, cholinergic and purinergic enzymes in BV-2 microglial cultured cell. Aqueous extract of Moringa oleifera was prepared, lyophilized and reconstituted in 0.5% dimethylsulphoxide (DMSO). Cells were treated with Moringa oleifera extracts (0.1-100 µg/mL) and assessed for cell viability and nitric oxide production. Furthermore, the effect of Moringa oleifera on enzymes of cholinergic (acetylcholinesterase) and purinergic (nucleoside triphosphate diphosphohydrolase; NTPDase, 5' nucleotidase and adenosine deaminase; ADA) systems in BV-2 microglial cells were determined. Incubation of BV-2 microglia cell with M. oleifera extract maintained cell viability, modulated cholinergic and purinergic enzymes activity. The phenolic compounds found in M. oleifera extracts, include chlorogenic acid, rutin; quercetin pentoside, kaempferol derivative and quercetin derivative. Thus, this study suggest that the potential therapeutic effect of the phenolic compounds found in M. oleifera may have been responsible for the maintenance of cell viability in BV-2 microglia cells and modulation of cholinergic as well as purinergic enzymes activity.

Defense of pyrethrum flowers: repelling herbivores and recruiting carnivores by producing aphid alarm pheromone.

(E)-ß-Farnesene (EßF) is the predominant constituent of the alarm pheromone of most aphid pest species. Moreover, natural enemies of aphids use EßF to locate their aphid prey. Some plant species emit EßF, potentially as a defense against aphids, but field demonstrations are lacking. Here, we present field and laboratory studies of flower defense showing that ladybird beetles are predominantly attracted to young stage-2 pyrethrum flowers that emitted the highest and purest levels of EßF. By contrast, aphids were repelled by EßF emitted by S2 pyrethrum flowers. Although peach aphids can adapt to pyrethrum plants in the laboratory, aphids were not recorded in the field. Pyrethrum's (E)-ß-farnesene synthase (EbFS) gene is strongly expressed in inner cortex tissue surrounding the vascular system of the aphid-preferred flower receptacle and peduncle, leading to elongated cells filled with EßF. Aphids that probe these tissues during settlement encounter and ingest plant EßF, as evidenced by the release in honeydew. These EßF concentrations in honeydew induce aphid alarm responses, suggesting an extra layer of this defense. Collectively, our data elucidate a defensive mimicry in pyrethrum flowers: the developmentally regulated and tissue-specific EßF accumulation and emission both prevents attack by aphids and recruits aphid predators as bodyguards.

The role of ENPP1/PC-1 in osteoinduction by calcium phosphate ceramics.

In the past decade, calcium phosphate (CaP) ceramics have emerged as alternatives to autologous bone grafts for the treatment of large, critical-sized bone defects. In order to be effective in the regeneration of such defects, ceramics must show osteoinductive behaviour, defined as the ability to induce de novo heterotopic bone formation. While a set of osteoinductive CaP ceramics has been developed, the exact processes underlying osteoinduction, and the role of the physical and chemical properties of the ceramics, remain largely unknown. Previous studies have focused on the role of the transcriptome to shed light on the mechanism of osteoinduction at the mRNA level. To complement these studies, a proteomic analysis was performed to study the behaviour of hMSCs on osteoinductive and non-osteoinductive CaPs. The results of this analysis suggest that plasma cell glycoprotein 1 (PC-1), encoded by the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, plays a key role in the process of osteoinduction by CaP ceramics. Validation experiments have confirmed that indeed, the mRNA expression of ENPP1 and the production of PC-1 are higher on osteoinductive than on non-osteoinductive CaP ceramics, a trend that was also observed for other osteogenic markers such as bone morphogenetic protein 2 (BMP2) and osteopontin (OPN), but not for alkaline phosphatase (ALP). Our results also showed that the expression of PC-1 is restricted to those cells which are in direct contact with the CaP ceramic surface, plausibly due to the localised depletion of calcium and inorganic phosphate ions from the supersaturated cell culture medium as CaP crystallises on the ceramic surface. Replicating the surface of the osteoinductive ceramic in polystyrene resulted in a significant decrease in ENPP1 expression, suggesting that surface structural properties alone are not sufficient to induce ENPP1 expression. Finally, knocking down ENPP1 expression in hMSCs resulted in increased BMP2 expression, both at the mRNA and protein level, suggesting that ENPP1 is a negative regulator of BMP-2 signalling. Taken together, this study shows, for the first time, that ENPP1/PC-1 plays an important role in CaP-induced osteogenic differentiation of hMSCs and thus possibly osteoinduction by CaP ceramics. Furthermore, we have identified a crucial role for the interfacial (chemical) events occurring on the CaP ceramic surface in the process of osteoinduction. This knowledge can contribute to the development of new bone graft substitutes, with improved osteoinductive potential.

Inhibition of Tissue-Nonspecific Alkaline Phosphatase Attenuates Ectopic Mineralization in the Abcc6-/- Mouse Model of PXE but Not in the Enpp1 Mutant Mouse Models of GACI.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.

Pseudoxanthoma Elasticum as a Paradigm of Heritable Ectopic Mineralization Disorders: Pathomechanisms and Treatment Development.

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.

The Basophil Activation Test Can Be of Value for Diagnosing Immediate Allergic Reactions to Omeprazole.

BACKGROUND: Proton pump inhibitors (PPIs) are commonly used to treat gastrointestinal diseases. Incidence of hypersensitivity reactions to PPIs has risen, likely because of increased consumption. Their diagnosis is difficult, with skin tests (STs) presenting low sensitivity, making it necessary to perform drug provocation tests (DPTs). The value of in vitro tests for the diagnosis of immediate reaction to PPI is unclear. OBJECTIVE: To analyze the diagnostic value of the basophil activation test (BAT) in a group of patients diagnosed with immediate allergy to omeprazole. METHODS: The study included 42 patients with confirmed immediate allergic reactions to omeprazole confirmed by positive ST results or DPT results and 22 age- and sex-matched subjects tolerant to PPIs. BAT was performed with omeprazole, pantoprazole, and lansoprazole using CD63 and CD203c as activation markers. RESULTS: ST sensitivity was 66.7% with a specificity of 100%. BAT using CD63 with a stimulation index of more than 2 as positive revealed a sensitivity of 73.8%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 66.7%. BAT was positive in 57.1% of patients with negative ST result, and thus by combining ST and BAT we can correctly diagnose 85.7% of patients with immediate allergy to omeprazole. CONCLUSION: BAT represents a complementary tool for inclusion in the allergological workup for patients allergic to omeprazole. When combined with ST, it can be of value to guide the clinician as to whether to perform a DPT.

Targeted Pth4-expressing cell ablation impairs skeletal mineralization in zebrafish.

Skeletal development and mineralization are essential processes driven by the coordinated action of neural signals, circulating molecules and local factors. Our previous studies revealed that the novel neuropeptide Pth4, synthesized by hypothalamic cells, was involved in bone metabolism via phosphate regulation in adult zebrafish. Here, we investigate the role of pth4 during skeletal development using single-cell resolution, two-photon laser ablation of Pth4:eGFP-expressing cells and confocal imaging in vivo. Using a stable transgenic Pth4:eGFP zebrafish line, we identify Pth4:eGFP-expressing cells as post-mitotic neurons. After targeted ablation of eGFP-expressing cells in the hypothalamus, the experimental larvae exhibited impaired mineralization of the craniofacial bones whereas cartilage development was normal. In addition to a decrease in pth4 transcript levels, we noted altered expression of phex and entpd5, genes associated with phosphate homeostasis and mineralization, as well as a delay in the expression of osteoblast differentiation markers such as sp7 and sparc. Taken together, these results suggest that Pth4-expressing hypothalamic neurons participate in the regulation of bone metabolism, possibly through regulating phosphate balance during zebrafish development.

Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349.

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5'-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5'-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.

Alkaloid extracts from Jimson weed (Datura stramonium L.) modulate purinergic enzymes in rat brain.

Although some findings have reported the medicinal properties of Jimson weed (Datura stramonium L.), there exist some serious neurological effects such as hallucination, loss of memory and anxiety, which has been reported in folklore. Consequently, the modulatory effect of alkaloid extracts from leaf and fruit of Jimson weed on critical enzymes of the purinergic [ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-NTDase), alkaline phosphatase (ALP) and Na+/K+ ATPase] system of neurotransmission was the focus of this study. Alkaloid extracts were prepared by solvent extraction method and their interaction with the activities of these enzymes were assessed (in vitro) in rat brain tissue homogenate and in vivo in rats administered 100 and 200mg/kg body weight (p.o) of the extracts for thirty days, while administration of single dose (1mg/kg body weight; i.p.) of scopolamine served as the positive control. The extracts were also investigated for their Fe2+ and Cu2+ chelating abilities and GC-MS characterization of the extracts was also carried out. The results revealed that the extracts inhibited activates of E-NTPDase, E-NTDase and ALP in a concentration dependent manner, while stimulating the activity of Na+/K+ ATPase (in vitro). Both extracts also exhibited Fe2+ and Cu2+ chelating abilities. Considering the EC50 values, the fruit extract had significantly higher (P<0.05) modulatory effect on the enzymes' activity as well as metal chelating abilities, compared to the leaf extract; however, there was no significant difference (P>0.05) in both extracts' inhibitory effects on E-NTDase. The in vivo study revealed reduction in the activities of ENTPDase, E-NTDase, and Na+/K+ ATPase in the extract-administered rat groups compared to the control group, while an elevation in ALP activity was observed in the extract-administered rat groups compared to the control group. GC-MS characterization revealed the presence of atropine, scopolamine, amphetamine, 3-methyoxyamphetamine, 3-ethoxyamhetamine cathine, spermine, phenlyephirine and 3-piperidinemethanol, among others in the extracts. Hence, alterations of activities of critical enzymes of purinergic signaling (in vitro and in vivo) by alkaloid extracts from leaf and fruit of Jimson weed suggest one of the mechanisms behind its neurological effects as reported in folklore.

Regulation of melanocortin 1 receptor in allergic rhinitis in vitro and in vivo.

BACKGROUND: α-melanocyte-stimulating hormone (α-MSH) was shown to inhibit allergic airway inflammation and exert suppressive effects on human basophils. OBJECTIVE: This study aims to extend our current knowledge on the melanocortin 1 receptor (MC1R) expression in nasal tissue of patients with allergic rhinitis (AR) and functional effects of α-MSH in human basophils especially from patients with allergic rhinitis. METHODS: MC1R expression before and after nasal allergen provocation was studied in nasal mucosal tissue of AR patients and in a mouse model of allergic airway inflammation using immunofluorescence. In vitro regulation of the MC1R and CD203c surface expression on whole-blood basophils of patients with AR and controls was assessed with flow cytometry. Functional effects of α-MSH on isolated basophils were analysed regarding apoptosis with flow cytometry and chemotaxis using a Boyden chamber assay. RESULTS: We detected an accumulation of MC1R-positive basophils in nasal mucosa tissue of patients with AR 24 h after nasal allergen provocation. Such accumulation was not present in mucosa sections from healthy controls. In mice with allergic airway inflammation, we found a clear accumulation of MC1R-positive basophils in the nasal tissue compared to control mice. MC1R expression was inducible in AR patients and controls by stimulation with anti-IgE. α-MSH inhibited anti-IgE and grass pollen induced upregulation of CD203c, but had no effect on chemotaxis or apoptosis of basophils in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: MC1R-positive basophils accumulate in the nasal mucosa of patients with AR after nasal allergen provocation. Since α-MSH suppresses proinflammatory effector functions in human basophils via the MC1R, it constitutes an interesting novel target for modulating the allergic inflammatory response.

1,25-Dihydroxyvitamin D3 and extracellular calcium promote mineral deposition via NPP1 activity in a mature osteoblast cell line MLO-A5.

While vitamin D supplementation is common, the anabolic mechanisms that improve bone status are poorly understood. Under standard mineralising conditions including media ionised calcium of 1.1 mM, 1,25-dihydroxyvitamin D3 (1,25D) enhanced differentiation and mineral deposition by the mature osteoblast/pre-osteocyte cell line, MLO-A5. This effect was markedly increased with a higher ionised calcium level (1.5 mM). Gene expression analyses revealed that 1,25D-induced mineral deposition was associated with induction of Enpp1 mRNA, coding for nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and NPP1 protein levels. Since MLO-A5 cells express abundant alkaline phosphatase that was not further modified by 1,25D treatment or exposure to increased calcium, this finding suggested that the NPP1 production of pyrophosphate (PPi) may provide alkaline phosphatase with substrate for the generation of inorganic phosphate (Pi). Consistent with this, co-treatment with Enpp1 siRNA or a NPP1 inhibitor, PPADS, abrogated 1,25D-induced mineral deposition. These data demonstrate that 1,25D stimulates osteoblast differentiation and mineral deposition, and interacts with the extracellular calcium concentration. 1,25D regulates Enpp1 expression, which presumably, in the context of adequate tissue non-specific alkaline phosphatase activity, provides Pi to stimulate mineralisation. Our findings suggest a mechanism by which vitamin D with adequate dietary calcium can improve bone mineral status.

Allergen-specific basophil reactivity exhibits daily variations in seasonal allergic rhinitis.

It remains poorly understood how symptoms in allergic rhinitis are most severe during overnight or early in the morning. The circadian clock consisting of a network of several 'clock genes' including Clock drives daily rhythms in physiology. This study showed that allergen-induced surface CD203c expression on basophils in seasonal allergic rhinitis caused by Japanese cedar pollen exhibited a time-of-day-dependent variation associated with temporal variations in canonical circadian clock gene expression. We also found that bone-marrow-derived basophils (BM basophils) generated from wild-type mice exhibited a time-of-day-dependent variation in IgE-mediated IL-4 and histamine production, which was not observed in BM basophils generated from Clock-mutated mice. Therefore, allergen-specific basophil reactivity shows daily variations depending on the circadian clock activity in basophils, which could partly explain temporal symptomatic variations in allergic rhinitis. Additionally, circadian variations in CD203c expression should be considered for interpretation of this biomarker in clinical research.

Hypoglycemic effects and mechanisms of electroacupuncture on insulin resistance.

The aim of this study was to investigate effects and mechanisms of electroacupuncture (EA) on blood glucose and insulin sensitivity in mice fed a high-fat diet. Both wild-type (WT) and adipose ectonucleotide pyrophosphate phosphodiesterase (ENPP1) transgenic (TG) mice were fed a high-fat diet for 12 wk; for each mouse, an intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed with or without EA at abdomen or auricular areas. A high-fat diet-induced insulin resistance in both WT and TG mice. In the WT mice, EA at 3 Hz and 15 Hz, but not at 1 Hz or 100 Hz, via CV4+CV12 significantly reduced postprandial glucose levels; EA at 3 Hz was most potent. The glucose level was reduced by 61.7% at 60 min and 74.5% at 120 min with EA at 3 Hz (all P < 0.001 vs. control). Similar hypoglycemic effect was noted in the TG mice. On the contrary, EA at auricular points increased postprandial glucose level (P < 0.03). 4). EA at 3 Hz via CV4+CV12 significantly enhanced the decrease of blood glucose after insulin injection, suggesting improvement of insulin sensitivity. Plasma free fatty acid was significantly suppressed by 42.5% at 15 min and 50.8% at 30 min with EA (P < 0.01) in both WT and TG mice. EA improves glucose tolerance in both WT and TG mice fed a high-fat diet, and the effect is associated with stimulation parameters and acupoints and is probably attributed to the reduction of free fatty acid.

Mutant Enpp1asj mice as a model for generalized arterial calcification of infancy.

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.