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Pantothenate kinase (PanK) phosphorylates pantothenic acid (vitamin B(5)) and controls the overall rate of coenzyme A (CoA) biosynthesis. Pank1 gene deletion in mice results in a metabolic phenotype where fatty acid oxidation and gluconeogenesis are impaired in the fasted state, leading to mild hypoglycemia. Inactivating mutations in the human PANK2 gene lead to childhood neurodegeneration, but Pank2 gene inactivation in mice does not elicit a phenotype indicative of the neuromuscular symptoms or brain iron accumulation that accompany the human disease. Pank1/Pank2 double knockout (dKO) mice were derived to determine if the mild phenotypes of the single knockout mice are due to the ability of the two isoforms to compensate for each other in CoA biosynthesis. Postnatal development was severely affected in the dKO mice. The dKO pups developed progressively severe hypoglycemia and hyperketonemia by postnatal day 10 leading to death by day 17. Hyperketonemia arose from impaired whole-body ketone utilization illustrating the requirement for CoA in energy generation from ketones. dKO pups had reduced CoA and decreased fatty acid oxidation coupled with triglyceride accumulation in liver. dKO hepatocytes could not maintain the NADH levels compared to wild-type hepatocytes. These results revealed an important link between CoA and NADH levels, which was reflected by deficiencies in hepatic oleate synthesis and gluconeogenesis. The data indicate that PanK1 and PanK2 can compensate for each other to supply tissue CoA, but PanK1 is more important to CoA levels in liver whereas PanK2 contributes more to CoA synthesis in the brain.

Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture--a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics.

Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

Sphingolipids are mediators of inflammation; changes in their cellular concentration modulate specific cellular functions. Investigations of sphingosine kinases (SphK) and sphingosine 1 phosphate (S1P) in TNFα driven murine models of rheumatoid arthritis (RA), identified SphK/S1P as important intermediaries in TNFα mediated synovial proinflammatory pathways. Fibroblast-like synoviocytes (FLS) are key contributors to RA pathogenesis and express both SphK 1 and 2. To pinpoint the mechanisms of SphK effects in the inflammatory response of murine FLS in vitro, we derived SphK1 null (SphK1-/-) FLS and SphK1 wild-type (SphK1+/+) FLS from the knee joints of B6 mice. Significantly less MMP1a and IL-6 were produced by mTNFα-stimulated SphK1-/- FLS versus SphK1+/+ FLS. Trends toward less PGE2 as well as activated, ERK 1/2 and STAT3 were present in SphK1-/- FLS versus SphK1+/+ FLS. Thus genetic inhibition of SphK1 activity resulted in decreased expression of inflammatory mediators and decreased activation of inflammatory pathways in TNFα stimulated murine FLS. This decreased inflammatory phenotype in FLS lacking SphK1 activity is consistent with the attenuated TNF-α-driven arthritis in vivo in SphK1 deficient mice and adds to the understanding of the mechanistic role of SpK1/S1P in rheumatoid arthritis. Thus, specific therapeutic can be targeted with SphK inhibitors in rheumatoid arthritis.

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5-13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations., Competing Interests: The authors have declared that no competing interests exist.

The roles of sphingosine kinases SK1 and SK2 in ischemia-reperfusion injury have not been fully elucidated since studies have found beneficial effects of SK1 while others showed no role in this injury. To help resolve this, we used SK1 or SK2 knockout mice and confirmed that renal ischemia-reperfusion injury induced SK1, but not SK2, in the kidneys. Furthermore, knockout or pharmacological inhibition of SK1 increased injury after renal ischemia-reperfusion injury. In contrast, lack of SK2 conferred renal protection following injury. In addition, we used lentiviral gene delivery to selectively express enhanced green fluorescent protein (EGFP) or human SK1 coexpressed with EGFP (EGFP-huSK1) in the kidney. Mice with kidney-specific overexpression of EGFP-huSK1 had significantly improved renal function with lower plasma creatinine, renal necrosis, apoptosis, and inflammation. Moreover, EGFP-huSK1 overexpression in cultured human proximal tubule (HK-2) cells protected against peroxide-induced necrosis. Selective overexpression of EGFP-huSK1 led to increased HSP27 mRNA and protein expression in vivo and in vitro. Functional protection as well as induction of HSP27 with EGFP-huSK1 overexpression in vivo was blocked with sphingosine-1-phosphate-1 receptor(1) (S1P(1)) antagonism. Thus, our findings suggest that SK1 is renoprotective by S1P(1) activation and perhaps HSP27 induction. Kidney-specific expression of SK1 through lentiviral delivery may be a viable therapeutic option to attenuate renal ischemia-reperfusion injury.

Sphingosine kinases (SK) regulate the balance between proapoptotic ceramides and mitogenic sphingosine-1-phosphate (S1P); however, the functions of the two isoenzymes (SK1 and SK2) in tumor cells are not well defined. Therefore, RNA interference was used to assess the individual roles of SK1 and SK2 in tumor cell sphingolipid metabolism, proliferation, and migration/invasion. Treatment of A498, Caki-1, or MDA-MB-231 cells with siRNAs specific for SK1 or SK2 effectively suppressed the expression of the target mRNA and protein. Ablation of SK1 did not affect mRNA or protein levels of SK2 and reduced intracellular levels of S1P while elevating ceramide levels. In contrast, ablation of SK2 elevated mRNA, protein, and activity levels of SK1 and increased cellular S1P levels. Interestingly, cell proliferation and migration/invasion were suppressed more by SK2-selective ablation than by SK1-selective ablation, showing that the increased S1P does not rescue these phenotypes. Similarly, exogenous S1P did not rescue the cells from the antiproliferative or antimigratory effects of the siRNAs. Consistent with these results, differential effects of SK1- and SK2-selective siRNAs on signaling proteins, including p53, p21, ERK1, ERK2, FAK, and VCAM1, indicate that SK1 and SK2 have only partially overlapping functions in tumor cells. Overall, these data indicate that loss of SK2 has stronger anticancer effects than does suppression of SK1. Consequently, selective inhibitors of SK2 may provide optimal targeting of this pathway in cancer chemotherapy., (Mol Cancer Res; 9(11); 1509-19. ©2011 AACR.)

The shortage of geranylgeranyl-pyrophosphate (GGPP) was associated to an increased IL-1β release in the autoinflammatory syndrome mevalonate kinase deficiency (MKD), a rare inherited disease that has no specific therapy. Farnesyltransferase inhibitors (FTIs) act at the end of mevalonate pathway. Two FTIs, tipifarnib (Tip) and lonafarnib (Lon), were therefore evaluated as possible therapeutical choices for the treatment of MKD. FTIs could lead to a redirection of the limited available number of mevalonate intermediates preferentially to GGPP synthesis, eventually preventing the uncontrolled inflammatory response. The effect of Tip and Lon on intracellular cholesterol level (ICL) and on proinflammatory cytokines secretion was evaluated in a cellular model of MKD, chemically obtained treating RAW 264.7 cells with lovastatin (Lova) and alendronate (Ald). The combination of FTIs with the isoprenoid geraniol (GOH) was also tested both in this model and in monocytes isolated from MKD patients. Tip and Lon proved to revert the ICL lowering and to significantly reduce the lipopolysaccharide-induced cytokines secretion in Ald-Lova -RAW 264.7 cells. This anti-inflammatory effect was amplified combining the use of GOH with FTIs. The effect of GOH and Tip was successfully replicated in MKD patients' monocytes. Tip and Lon showed a dramatic anti-inflammatory effect in monocytes where mevalonate pathway was chemically or genetically impaired.

Mutations in DGUOK result in mitochondrial DNA (mtDNA) depletion and may present as neonatal liver failure. Neonatal hemochromatosis (NH(1)) is a liver disorder of uncertain and varied etiology characterized by hepatic and non-reticuloendothelial siderosis. To date, deoxyguanosine kinase (dGK(2)) deficiency has not been formally recognized in cases of NH. We report an African American female neonate with clinical and autopsy findings consistent with NH, and mtDNA depletion due to a homozygous mutation in DGUOK. This report highlights hepatocerebral mtDNA depletion in the differential of neonatal tyrosinemia, advocates considering dGK deficiency in cases of NH, and posits mitochondrial oxidative processes in the pathogenesis of NH., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: The goal of this study was to describe the spectrum of clinical signs of mevalonate kinase deficiency (MKD)., Methods: This was a retrospective French and Belgian study of patients identified on the basis of MKD gene mutations., Results: Fifty patients from 38 different families were identified, including 1 asymptomatic patient. Symptoms began during the first 6 months of life in 30 patients (60%) and before the age of 5 years in 46 patients (92%). Symptoms consisted of febrile diarrhea and/or rash in 23 of 35 patients (66%). Febrile attacks were mostly associated with lymphadenopathy (71%), diarrhea (69%), joint pain (67%), skin lesions (67%), abdominal pain (63%), and splenomegaly (63%). In addition to febrile attacks, 27 patients presented with inflammatory bowel disease, erosive polyarthritis, Sjögren syndrome, and other chronic neurologic, renal, pulmonary, endocrine, cutaneous, hematologic, or ocular symptoms. Recurrent and/or severe infections were observed in 13 patients, hypogammaglobulinemia in 3 patients, and renal angiomyolipoma in 3 patients. Twenty-nine genomic mutations were identified; the p.Val377Ile mutation was the most frequently found (29 of 38 families). Three patients died of causes related to MKD. The disease remained highly active in 17 of the 31 surviving symptomatic patients followed up for >5 years, whereas disease activity decreased over time in the other 14 patients. Interleukin 1 antagonists were the most effective biological agents tested, leading to complete or partial remission in 9 of 11 patients., Conclusion: MKD is not only an autoinflammatory syndrome but also a multisystemic inflammatory disorder, a possible immunodeficiency disorder, and a condition that predisposes patients to the development of renal angiomyolipoma., (Copyright © 2011 by the American Academy of Pediatrics.)

Improving the accessibility and functions of therapeutic and diagnostic glycoproteins is one of the major goals of glycobiotechnology. Here we present that stable knock-down of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the sialic acid biosynthetic pathway, dramatically increases incorporation of N-acetylmannosamine analogues into glycoproteins of HEK293 cells. By means of these GNE-deficient cells highly sialylated glycoproteins can efficiently be decorated with reactive functional groups, which can be employed in bioorthogonal functionalization strategies for fluorescence labelling or biotinylation.

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The presence of aggregates of abnormally expanded polyglutamine (polyQ)-containing proteins are a pathological hallmark of a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia-3 (SCA3). Previous studies in cellular, Drosophila, and mouse models of HD and SCA have shown that neurodegeneration can be prevented by manipulations that inhibit polyQ aggregation. We have shown that the UL97 kinase of the human cytomegalovirus (HCMV) prevents aggregation of the pp71 and pp65 viral tegument proteins. To explore whether UL97 may act as a general antiaggregation factor, we examined whether UL97 prevents aggregation of cellular non-polyQ and polyQ proteins. We report that UL97 prevents the deposition of aggregates of two non-polyQ proteins: a protein chimera (GFP170*) composed of the green fluorescent protein and a fragment of the Golgi Complex protein (GCP-170) and a chimera composed of the red fluorescent protein (RFP) fused to the Werner syndrome protein (WRN), a RecQ helicase and exonuclease involved in DNA repair. Furthermore, we show that UL97 inhibits aggregate deposition in cellular models of HD and SCA3. UL97 prevents the deposition of aggregates of the mutant huntingtin exon 1 containing 82 glutamine repeats (HttExon1-Q82) or full length ataxin-3 containing a 72 polyQ track (AT3-72Q). The kinase activity of UL97 appears critical, as the kinase-dead UL97 mutant (K335M) fails to prevent aggregate formation. We further show that UL97 disrupts nuclear PML bodies and decreases p53-mediated transcription. The universality of the antiaggregation effect of UL97 suggests that UL97 targets a key cellular factor that regulates cellular aggregation mechanisms. Our results identify UL97 as a novel means to modulate polyQ aggregation and suggest that UL97 can serve as a novel tool to probe the cellular mechanisms that contribute to the formation of aggregates in polyglutamine disorders., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Purines and pyrimidines, regarded for a long time only as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, gained increasing attention since genetically determined aberrations in their metabolism were associated clinically with various degrees of mental retardation and/or unexpected and often devastating neurological dysfunction. In most instances the molecular mechanisms underlying neurological symptoms remain undefined. This suggests that nucleotides and nucleosides play fundamental but still unknown roles in the development and function of several organs, in particular central nervous system. Alterations of purine and pyrimidine metabolism affecting brain function are spread along both synthesis (PRPS, ADSL, ATIC, HPRT, UMPS, dGK, TK), and breakdown pathways (5NT, ADA, PNP, GCH, DPD, DHPA, TP, UP), sometimes also involving pyridine metabolism. Explanations for the pathogenesis of disorders may include both cellular and mitochondrial damage: e.g. deficiency of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase are associated to the most severe pathologies, the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to impairment of mitochondrial functions. This review gathers the presently known inborn errors of purine and pyrimidine metabolism that manifest neurological syndromes, reporting and commenting on the available hypothesis on the possible link between specific enzymatic alterations and brain damage. Such connection is often not obvious, and though investigated for many years, the molecular basis of most dysfunctions of central nervous system associated to purine and pyrimidine metabolism disorders are still unexplained.

D-glyceric aciduria is a rare inborn error of serine and fructose metabolism that was first described in 1974. Most affected individuals have presented with neurological symptoms. The molecular basis of D-glyceric aciduria is largely unknown; possible causes that have been discussed are deficiencies of D-glycerate dehydrogenase, triokinase, and D-glycerate kinase. In 1989, van Schaftingen has reported decreased D-glycerate kinase activity in the liver of a single patient with D-glyceric aciduria. However, this analysis has not been performed in other affected individuals, and the underlying defect has remained unknown on the gene level until now. We report three patients with deficiency of D-glycerate kinase. They are of Serbian, Mexican, and Turkish origin and include the patient initially reported in 1974. All had homozygous mutations in exon 5 of the GLYCTK gene encoding D-glycerate kinase: c.1448delT (p.Phe483SerfsX2), c.1478T>G (p.Phe493Cys), or c.1558delC (p.Leu520CysfsX108). Transient overexpression of the variant GLYCTK genes in HEK293 cells clearly showed loss of enzyme activity and immunoreactivity when compared to the reference enzyme. Our work has revealed mutations in the GLYCTK gene as the cause of D-glycerate kinase deficiency and D-glyceric aciduria and provides a noninvasive approach for further diagnostic workup and research., (© 2010 Wiley-Liss, Inc.)

Ceramide kinase (CerK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P). We previously revealed that CerK is involved in the activation of mast cells. In this study, we performed an advanced investigation into the role of CerK on the activation of mast cells using CERK-/- mice. Although CERK-/- mice were less prone to exhibiting a passive cutaneous anaphylactic shock (PCA)-reaction compared to wild type (WT) mice, the differences were not significant. In bone marrow-derived mast cells (BMMC) activated by cross-linking antigen (Ag)/IgE, not high, but low concentrations of Ag had a reduced effect on degranulation in BMMC from CERK-/- mice compared to effects on BMMC from WT mice. Similarly, when the BMMCs were activated with calcium ionophore to focus on the downstream signaling of Ca(2+)-elevation, only a low concentration of ionophore had a reduced effect on degranulation in the BMMC from CERK-/- mice compared to the effect on BMMC from WT mice. Furthermore, the CerK inhibitor K1 reduced the differences in degranulation observed between the BMMC from CERK-/- and WT mice in a dose-dependent manner, demonstrating a contribution for CerK and its product C1P in degranulation. Although CerK is not essential for activation of mast cells, especially a potent and acute activation such as a PCA reaction, CerK might act as an modulator for mild and chronic activation of mast cells, thus increasing sensitivity to cytoplasmic Ca(2+)., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mevalonate kinase deficiency (MKD) is an autoinflammatory disorder caused by mutations in the MVK gene resulting in decreased activity of the enzyme mevalonate kinase (MK). Although MK is required for biosynthesis of all isoprenoids, in MKD, in particular, the timely synthesis of geranylgeranyl pyrophosphate appears to be compromised. Because small guanosine triphosphatases (GTPases) depend on geranylgeranylation for their proper signaling function, we studied the effect of MK deficiency on geranylgeranylation and activation of the two small GTPases, RhoA and Rac1. We demonstrate that both geranylgeranylation and activation of the two GTPases are more easily disturbed in MKD cells than in control cells when the flux though the isoprenoid biosynthesis pathway is suppressed by low concentrations of simvastatin. The limited capacity of geranylgeranylation in MKD cells readily leads to markedly increased levels of nonisoprenylated and activated GTPases, which will affect proper signaling by these GTPases.

Sphingosine kinase 1 (SphK1) is an enzyme that converts sphingosine to bioactive sphingosine-1-phosphate. Recent in vitro data suggest a potential role of SphK1 in TNF-alpha-mediated inflammation. Our aims in this study were to determine the in vivo significance of SphK1 in TNF-alpha-mediated chronic inflammation and to define which pathogenic mechanisms induced by TNF-alpha are SphK1 dependent. To pursue these aims, we studied the effect of SphK1 deficiency in an in vivo model of TNF-alpha-induced chronic inflammatory arthritis. Transgenic hTNF-alpha mice, which develop spontaneous inflammatory erosive arthritis beginning at 14-16 wk, were crossed with SphK1 null mice (SphK1(-/-)), on the C57BL6 genetic background. Beginning at 4 mo of age, hTNF/SphK1(-/-) mice had significantly less severe clinically evident paw swelling and deformity, less synovial and periarticular inflammation, and markedly decreased bone erosions as measured quantitatively through micro-CT images. Mechanistically, the mice lacking SphK1 had less articular cyclooxygenase 2 protein and fewer synovial Th17 cells than did hTNF/SphK1(+/+) littermates. Microarray analysis and real-time RT-PCR of the ankle synovial tissue demonstrated that hTNF/SphK1(-/-) mice had increased transcript levels of suppressor of cytokine signaling 3 compared with hTNF/SphK1(+/+) mice, likely also contributing to the decreased inflammation in the SphK1-deficient mice. Finally, significantly fewer mature osteoclasts were detected in the ankle joints of hTNF/SphK1(-/-) mice compared with hTNF/SphK1(+/+) mice. These data indicate that SphK1 plays a key role in hTNF-alpha-induced inflammatory arthritis via impacting synovial inflammation and osteoclast number.

Two temperature-sensitive Saccharomyces cerevisiae mutants, sec59-1 and dpm1-6, impaired, respectively, in dolichol kinase (Sec59p) and dolichyl phosphate mannose (DolPMan) synthase (Dpm1p), have an aberrant cell wall structure and composition. We tested their sensitivity to four classes of antifungal drugs used in clinical practice: 5-fluorocytosine, amphotericin B, caspofungin and itraconasole. The strains were resistant to itraconazole and sensitive to the other drugs used. The minimal inhibitory concentration (MIC) of caspofungin and amphotericin B was two-fold lower for sec59-1 and dpm1-6 than for the respective wild-type strains. The sensitivity of both mutants could be brought back to the wild-type level by a multicopy suppressor of the thermosensitive phenotype, the RER2 gene, encoding cis-prenyltransferase involved in dolichol biosynthesis. Biochemical analysis revealed slight changes of the cell wall composition, different in the mutants as compared to the wild-type in response to the drugs. Our data strongly support a relationship between dolichol phosphate level, protein glycosylation and antifungal sensitivity., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

Sphingosine kinase (SPHK) catalyzes sphingosine 1-phosphate production, promoting cell survival and reducing apoptosis in isolated rat pancreatic islets. Glucose, the primary islet beta-cell growth factor and insulin secretagogue, increased islet SPHK activity by 3- to 5-fold following acute (1 h) or prolonged (7 days) stimulation. Prolonged stimulation of islets with glucose induced SPHK1a and SPHK2 mRNA levels; there were no changes in SPHK protein expression. To isolate the metabolic effects of glucose on SPHK activation, islets were stimulated with glucose analogs or metabolites. 2-deoxy-D-glucose (2-DG), an analog phosphorylated by glucokinase but not an effective energy source, activated SPHK similarly to glucose. In contrast, 3-o-methylglucose (3-oMeG), which is transported but neither phosphorylated nor metabolized, did not increase islet SPHK activity. Glyceraldehyde and alpha-ketoisocaproic acid (KIC), metabolites that stimulate glycolysis and the citric acid cycle, respectively, did not activate islet SPHK. Moreover, inorganic phosphate blocked glucose-induced SPHK activation. A role for SPHK activity in beta-cell growth was confirmed when small interfering (si)SPHK2 RNA transfection reduced rat insulinoma INS-1e cell SPHK levels and activity and cell growth. Glucose induced an early and sustained increase in islet SPHK activity that was dependent on glucose phosphorylation, but independent of ATP generation or new protein biosynthesis. Glucose-supported beta-cell growth appears to be in part mediated by SPHK activity.

The localized control of second messenger levels sculpts dynamic and persistent changes in cell physiology and structure. Inositol trisphosphate [Ins(1,4,5)P(3)] 3-kinases (ITPKs) phosphorylate the intracellular second messenger Ins(1,4,5)P(3). These enzymes terminate the signal to release Ca(2+) from the endoplasmic reticulum and produce the messenger inositol tetrakisphosphate [Ins(1,3,4,5)P(4)]. Independent of their enzymatic activity, ITPKs regulate the microstructure of the actin cytoskeleton. The immune phenotypes of ITPK knockout mice raise new questions about how ITPKs control inositol phosphate lifetimes within spatial and temporal domains during lymphocyte maturation. The intense concentration of ITPK on actin inside the dendritic spines of pyramidal neurons suggests a role in signal integration and structural plasticity in the dendrite, and mice lacking neuronal ITPK exhibit memory deficits. Thus, the molecular and anatomical features of ITPKs allow them to regulate the spatiotemporal properties of intracellular signals, leading to the formation of persistent molecular memories.

Cryptococcus neoformans is a fungal pathogen causing pulmonary infection and a life-threatening meningoencephalitis in human hosts. The fungus infects the host through inhalation, and thus, the host response in the lung environment is crucial for containment or dissemination of C. neoformans to other organs. In the lung, alveolar macrophages (AMs) are key players in the host lung immune response, and upon phagocytosis, they can kill C. neoformans by evoking an effective immune response through a variety of signaling molecules. On the other hand, under conditions not yet fully defined, the fungus is able to survive and proliferate within macrophages. Since the host sphingosine kinase 1 (SK1) regulates many signaling functions of immune cells, particularly in macrophages, in this study we determined the role of SK1 in the host response to C. neoformans infection. Using wild-type (SK1/2(+/+)) and SK1-deficient (SK1(-/-)) mice, we found that SK1 is dispensable during infection with a facultative intracellular wild-type C. neoformans strain. However, SK1 is required to form a host lung granuloma and to prevent brain infection by a C. neoformans mutant strain lacking the cell wall-associated glycosphingolipid glucosylceramide (Delta gcs1), previously characterized as a mutant able to replicate only intracellularly. Specifically, in contrast to those from SK1/2(+/+) mice, lungs from SK1(-/-) mice have no collagen deposition upon infection with C. neoformans Delta gcs1, and AMs from these mice contain significantly more C. neoformans cells than AMs from SK1/2(+/+) mice, suggesting that under conditions in which C. neoformans is more internalized by AMs, SK1 may become important to control C. neoformans infection. Indeed, when we induced immunosuppression, a host condition in which wild-type C. neoformans cells are increasingly found intracellularly, SK1(-/-) survived significantly less than SK1/2(+/+) mice infected with a facultative intracellular wild-type strain, suggesting that SK1 has an important role in controlling C. neoformans infection under conditions in which the fungus is predominantly found intracellularly.

Sphingosine kinases (SKs) 1 and 2 produce high concentrations of sphingosine 1-phosphate (S1P) in blood and lymph. In contrast, S1P concentrations in lymphoid tissues are kept low by the S1P-degrading activity of the S1P-lyase. These differences in S1P concentrations drive lymphocyte circulation. Inhibition of the S1P-lyase prevents lymphocyte egress and causes lymphopenia because of increased S1P levels in lymphoid tissues. In this study, we investigated the source of this accumulating S1P in lymphoid tissues by using SK2-deficient (SK2(-/-)) mice. In contrast to wild-type mice, SK2(-/-) mice exhibited attenuated lymphopenia after S1P-lyase inhibition by 4-deoxypyridoxine (DOP). Consistently, S1P concentrations were only modestly increased in lymphoid tissues of SK2(-/-) mice compared with a significantly higher increase in wild-type mice after DOP treatment. Low S1P concentrations in lymphoid tissues of DOP-treated SK2(-/-) mice were accompanied by higher S1P concentrations in blood, suggesting that SK2(-/-) mice display defective S1P transport from blood into lymphoid tissues. To investigate this potential new role of SK2, RBCs loaded with traceable C17-S1P were transfused into wild-type and SK2(-/-) mice, resulting in much higher C17-S1P concentrations in blood of SK2(-/-) mice compared with wild-type mice 2 h after transfusion. Moreover, cocultures of RBCs with mouse splenocytes and endothelial cells demonstrated that SK2 regulated cellular uptake of S1P from RBCs. Collectively, our data suggest that S1P in lymphoid tissues derives from blood and point to an essential role of SK2 in S1P transport.

Mevalonate kinase deficiency (MKD) is a rare inborn error of metabolism caused by mutations in the mevalonate kinase (MVK) gene. The clinical phenotype is variable, ranging from the hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) to mevalonic aciduria (MA), a severe metabolic disease. We report here for the first time (to our knowledge) the case of a patient with MKD and congenital dyserythropoietic anemia. Clinical and laboratory characteristics of inflammatory attacks were compatible with HIDS, but mild dysmorphic features and elevated urinary mevalonic acid levels in the absence of an inflammatory attack suggested an intermediate phenotype between HIDS and MA. Genomic sequencing of the MVK gene revealed compound heterozygosity for a missense mutation previously described in MA (V310M) and a novel missense mutation (Y116H). By contrast, sequencing of the novel CDAII (SEC23B) gene revealed no mutations, suggesting that the bone marrow abnormalities were causally related to the MKD. Treatment with corticosteroids and colchicine directed at controlling the autoinflammatory disease resulted in improvement of the anemia.

Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/10(6) cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d(18:1/16:0) with a minority of d(18:1/24:1) and d(18:1/24:0). The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60 (degrees) C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d(18:1/18:0) C1P, as well as a significant portion of d(18:1/16:0), d(18:1/24:1), and d(18:1/24:0). In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT.

Deoxyguanosine kinase (DGUOK) catalyzes the first step of the mitochondrial deoxypurine salvage pathway, the phosphorylation of purine deoxyribonucleosides. Mutations in the DGUOK gene have been linked to inherited mtDNA depletion syndromes, neonatal liver failure, nystagmus, and hypotonia. Previously, we reported the first case of a heterozygous unclassified c.592-4_c.592-3delTT alteration in a patient with DGUOK deficiency without the demonstration of its pathogenicity (Dimmock et al., 2008). This alteration was predicted to cause aberrant splicing based upon two computer algorithms. We now report a homozygous c.592-4_c.592-3delTT mutation found in two affected siblings of asymptomatic consanguineous parents. The proband presented with symptoms of idiopathic hepatitis, liver dysfunction, nystagmus, and retinal blindness. This individual died at 6months of age due to liver failure. This individual's affected sibling presented similarly and has remarkable elevations of tyrosine, methionine, and alanine. Many organic acids were elevated in urine, including lactic acid, Krebs cycle intermediates, and para-hydroxy compounds; ketone bodies were also present. RNA studies support aberrant splicing. Sequencing of cDNA detected exon 5 skipping in the two affected siblings, but not in the normal control. These results indicate that the homozygous c.592-4_c.592-3delTT is deleterious and responsible for the DGUOK deficiency. The parents were subsequently confirmed to be carriers of this mutation. In summary, we have demonstrated that c.592-4_c.592-3delTT is a pathogenic splice acceptor site mutation leading to DGUOK deficiency.

Due to their high glucose permeability, insulin-secreting pancreatic beta-cells likely undergo strong intracellular protein glycation at high glucose concentrations. They may, however, be partly protected from the glucotoxic alterations of their survival and function by fructosamine-3-kinase (FN3K), a ubiquitous enzyme that initiates deglycation of intracellular proteins. To test that hypothesis, we cultured pancreatic islets from Fn3k-knockout (Fn3k(-/-)) mice and their wild-type (WT) littermates for 1-3 wk in the presence of 10 or 30 mmol/l glucose (G10 or G30, respectively) and measured protein glycation, apoptosis, preproinsulin gene expression, and Ca(2+) and insulin secretory responses to acute glucose stimulation. The more potent glycating agent d-ribose (25 mmol/l) was used as positive control for protein glycation. In WT islets, a 1-wk culture in G30 significantly increased the amount of soluble intracellular protein-bound fructose-epsilon-lysines and the glucose sensitivity of beta-cells for changes in Ca(2+) and insulin secretion, whereas it decreased the islet insulin content. After 3 wk, culture in G30 also strongly decreased beta-cell glucose responsiveness and preproinsulin mRNA levels, whereas it increased islet cell apoptosis. Although protein-bound fructose-epsilon-lysines were more abundant in Fn3k(-/-) vs. WT islets, islet cell survival and function and their glucotoxic alterations were almost identical in both types of islets, except for a lower level of apoptosis in Fn3k(-/-) islets cultured for 3 wk in G30. In comparison, d-ribose (1 wk) similarly decreased preproinsulin expression and beta-cell glucose responsiveness in both types of islets, whereas it increased apoptosis to a larger extent in Fn3k(-/-) vs. WT islets. We conclude that, despite its ability to reduce the glycation of intracellular islet proteins, FN3K is neither required for the maintenance of beta-cell survival and function under control conditions nor involved in protection against beta-cell glucotoxicity. The latter, therefore, occurs independently from the associated increase in the level of intracellular fructose-epsilon-lysines.

Bioactive sphingolipids play key roles in the regulation of several fundamental biological processes such as proliferation, apoptosis and transformation. The recent development of genetically engineered mouse (GEM) models has enabled the study of functional roles of sphingolipids in normal development and disease. In this chapter, we review the phenotypes of GEM models (knockout mice) that lack sphingolipid metabolism-related enzymes, discuss what we have learned from animal models and describe future directions of animal models in sphingolipid research.

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Rationale: Little is known about the contribution of bone marrow-derived progenitor cells (BMPCs) in the regulation endothelial barrier function as defined by microvascular permeability alterations at the level of adherens junctions (AJs)., Objective: We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS)., Methods and Results: We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1-null mice (SPHK1(-/-)), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier., Conclusions: These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.

Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C- and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for N-glycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Crosslinking of IgE receptors by antigen initiates Ca2+ mobilization in mast cells by activating phospholipase-C gamma-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. The resulting inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum (ER) activates store-operated Ca2+ entry, which is necessary for exocytotic release of inflammatory mediators. To investigate roles for PtdIns(4,5)P2-synthesizing isozymes of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIP5K-I) in mast cell signaling, we compared the ectopic expression of wild-type and catalytically inactive PIP5K-I beta in RBL-2H3 mast cells. Surprisingly, both antigen and thapsigargin-stimulated Ca2+ influx were reduced by overexpression of active PIP5K-I beta, whereas antigen-stimulated Ca2+ release from ER stores was unaffected. Consistent with these results, Ca2+ entry stimulated by antigen or thapsigargin was enhanced by expression of a plasma-membrane-associated inositol polyphosphate 5'-phosphatase, whereas antigen-stimulated Ca2+ release from stores was reduced. To investigate the role of PIP5K-I gamma in antigen-stimulated Ca2+ mobilization, we used bone-marrow-derived mast cells from PIP5K-I gamma(-/-) mice. Antigen-stimulated Ca2+ release from ER stores was substantially reduced in the absence of PIP5K-I gamma, but thapsigargin-mediated Ca2+ entry was unaffected. In summary, PIP5K-I gamma positively regulates antigen-stimulated Ca2+ release from ER stores, whereas PIP5K-I beta negatively regulates store-operated Ca2+ entry, suggesting that these different PIP5K-I isoforms synthesize functionally distinct pools of PtdIns(4,5)P2 at the plasma membrane.

Phosphoinositide (PI) lipids are intracellular membrane signaling intermediates and effectors produced by localized PI kinase and phosphatase activities. Although many signaling roles of PI kinases have been identified in cultured cell lines, transgenic animal studies have produced unexpected insight into the in vivo functions of specific PI 3- and 5-kinases, but no mammalian PI 4-kinase (PI4K) knockout has previously been reported. Prior studies using cultured cells implicated the PI4K2alpha isozyme in diverse functions, including receptor signaling, ion channel regulation, endosomal trafficking, and regulated secretion. We now show that despite these important functions, mice lacking PI4K2alpha kinase activity initially appear normal. However, adult Pi4k2a(GT/GT) animals develop a progressive neurological disease characterized by tremor, limb weakness, urinary incontinence, and premature mortality. Histological analysis of aged Pi4k2a(GT/GT) animals revealed lipofuscin-like deposition and gliosis in the cerebellum, and loss of Purkinje cells. Peripheral nerves are essentially normal, but massive axonal degeneration was found in the spinal cord in both ascending and descending tracts. These results reveal a previously undescribed role for aberrant PI signaling in neurological disease that resembles autosomal recessive hereditary spastic paraplegia.

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

Unlabelled: We report on two brothers with hyperimmunoglobulinemia D (patient 1: serum immunoglobulin D [IgD] concentration initially 61 IU/ml, later on 340 IU/ml; patient 2: serum IgD concentration 144 IU/ml; normal <100 IU/ml, 97th centile) and periodic fever syndrome (HIDS). Both are compound heterozygous for the mevalonate kinase (MVK) mutations V377I and I268T. They developed significant B cell cytopenia (7%, 129/microl and 11%, 132/microl, respectively; normal ranges 12-22%, 300-500/microl) with hypogammaglobulinemia (IgG 5.48 g/l and IgG 5.22 g/l, respectively; normal range IgG 6-13 g/l). Furthermore, the clinical spectrum shows an interesting atypical autoinflammatory symptomatology. The therapy consisted of prednisone, azathioprine, and intravenous immunoglobulins (IVIG), which results in reduced incidence and severity of febrile attacks., Conclusion: The pathogenesis and clinical presentation of HIDS is still not fully understood and show a great variability. To our knowledge, severe B cell cytopenia in children with HIDS has not been reported before. Furthermore, the therapy of febrile episodes is still performed on an individual basis in affected patients.

Yeast cells lacking the mitochondrial NADH kinase encoded by POS5 display increased sensitivity to hydrogen peroxide, a slow-growth phenotype, reduced mitochondrial function and increased levels of mitochondrial protein oxidation and mtDNA mutations. Here we examined gene expression in pos5Delta cells, comparing these data to those from cells containing deletions of superoxide dismutase-encoding genes SOD1 or SOD2. Surprisingly, stress-response genes were down-regulated in pos5Delta, sod1Delta and sod2Delta cells, implying that cells infer stress levels from mitochondrial activity rather than sensing reactive oxygen species directly. Additionally, pos5Delta, but not sod1 or sod2, cells displayed an anaerobic expression profile, indicating a defect in oxygen sensing that is specific to pos5, and is not a general stress-response. Finally, the pos5Delta expression profile is quite similar to the hap1Delta expression profile previously reported, which may indicate a shared mechanism.

In an amyotrophic lateral sclerosis (ALS) patient who also had an IgA gammopathy, autopsy studies identified the IgA in the surviving motor neurons. Further, the IgA bound the surface of isolated bovine motor neurons and inhibited neuronal proliferation in culture. To determine the pathologic basis of this IgA interaction with motor neurons, a neuroblastoma cDNA library was generated and screened with the IgA monoclonal antibody. Reactive clones were identified as flavin adenine dinucleotide (FAD) synthetase. To extend this finding to ALS in general, quantitative RT-PCRs were performed on blood samples from 26 ALS and 30 control blood samples to determine mRNA expression levels of FAD synthetase and other electron transport chain proteins, specifically riboflavin kinase (RFK), cytochrome C1 (CYC1), and succinate dehydrogenase complex subunit B (SDHB). All expression levels were measured against a control enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression levels for a non-respiratory chain protein (beta-actin) were also measured. We found that FAD synthetase expression levels were decreased in ALS samples compared to expression levels in controls (P = 0.0151). Expression levels for RFK, CYC1, and SDHB were also significantly decreased in the ALS group (P = 0.0025, P = 0.0002, and P < 0.0001, respectively). As control, expression levels for beta-actin did not show a significant difference between ALS and control groups (P = 0.2118). Our data show that a reduction in electron transport proteins, namely FAD synthetase, RFK, CYC1, and SDHB, is seen in patients with ALS. It is possible that this may have an effect on oxygen-dependent metabolic pathways. Human motor neurons may be particularly susceptible to injury if there is sub-optimal oxidative metabolism.

The recent generation of ceramide kinase (CerK)-deficient (Cerk (-/-)) mice as well as the identification of the potent CerK inhibitor NVP-231 have provided unprecedented opportunities to better understand CerK biology. Here we used skin dermal microendothelial cells (DMECs) and we show that CerK activity regulates their neovascularization in a matrigel environment in vitro. Capillary-like tube formation was significantly impaired in CerK-deficient cells or in wild-type (WT) cells treated with NVP-231 as compared with untreated WT cells. This was not the result of compromised proliferation or survival because Cerk (-/-) endothelial cells were able to migrate out of dermal fragments and grow in monolayer culture as well as their WT counterpart. Vascular endothelial growth factor, fibroblast growth factor or tumor necrosis factor could not rescue the angiogenesis defect observed in Cerk (-/-) DMEMs. Moreover, CerK ablation increased serum ceramide levels at the expense of dihydroceramide levels without affecting sphingosine, dihydrosphingosine, sphingosine-1-phosphate or dihydrosphingosine-1-phosphate levels. These observations collectively suggest that CerK-catalyzed formation of C1P may regulate angiogenesis by a novel mechanism that is independent of S1P formation and signaling.

Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) converts inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate upon Ag receptor activation and controls the fate and function of lymphocytes. To determine the role of Itpkb in B cell tolerance, Itpkb(-/-) mice were crossed to transgenic mice that express a BCR specific for hen egg lysozyme (IgHEL). B cells from Itpkb(-/-) IgHEL mice possess an anergic phenotype, hypoproliferate in response to cognate Ag, and yet they exhibit enhanced Ag-induced calcium signaling. In IgHEL transgenic mice that also express soluble HEL, lack of Itpkb converts anergy induction to deletion. These data establish Itpkb as a negative regulator of BCR signaling that controls the fate of developing B cells and tolerance induction.

Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Neurodegeneration associated with pantothenate kinase deficiency is an autosomal recessive condition caused by mutations in the pantothenate kinase 2 gene (PANK2). Clinical characteristics include progressive motor impairment and dementia. Medical treatment is limited and the dystonia tends to be refractory, making stereotactic surgery with placement of deep-brain electrodes an option that is being adopted with greater frequency in these patients. We report the case of a 32-year-old woman with severe dystonia associated with PANK2 protein deficiency. The patient was scheduled for stereotactic bilateral placement of electrodes in the medial globus pallidus, guided by computed tomography and under general anesthesia, to treat the debilitating dystonia and generalized stiffness associated with her condition. Anesthesia was maintained with propofol, rocuronium and remifentanil in perfusion during the intervention, which was uneventful. After the procedure, the patient was transferred to the intensive care unit and sedation was provided with remifentanil to allow slow, gradual emergence from anesthesia. The patient was discharged from hospital after placement of the implanted pulse generator, and subsequent follow-up showed improvement of the dystonia.

Sphingosine kinase 1 (SphK1) phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) and is a critical regulator of sphingolipid-mediated functions. Cell-based studies suggest a tumor-promoting function for the SphK1/S1P pathway. Also, our previous studies implicated the SphK1/S1P pathway in the induction of the arachidonic acid cascade, a major inflammatory pathway involved in colon carcinogenesis. Therefore, we investigated whether the SphK1/S1P pathway is necessary for mediating carcinogenesis in vivo. Here, we report that 89% (42/47) of human colon cancer samples stained positively for SphK1, whereas normal colon mucosa had negative or weak staining. Adenomas had higher expression of SphK1 vs. normal mucosa, and colon cancers with metastasis had higher expression of SphK1 than those without metastasis. In the azoxymethane (AOM) murine model of colon cancer, SphK1 and S1P were significantly elevated in colon cancer tissues compared to normal mucosa. Moreover, blood levels of S1P were higher in mice with colon cancers than in those without cancers. Notably, SphK1(-/-) mice subjected to AOM had significantly less aberrant crypt foci (ACF) formation and significantly reduced colon cancer development. These results are the first in vivo evidence that the SphK1/S1P pathway contributes to colon carcinogenesis and that inhibition of this pathway is a potential target for chemoprevention.

Introduction: Pantothenate kinase-associated neurodegenerative disease (PKAN) is a secondary generalized dystonia associated with an accumulation of iron in the basal ganglia and increased motor cortex excitability. A pilot study in three patients with secondary generalized dystonia had reported a reduced frequency of painful axial spasms following inhibitory 1-Hz repetitive transcranial magnetic stimulation (rTMS) applied over the premotor cortex., Patient and Methods: We compared the effects of real versus sham rTMS on the frequency of the complex movement pattern and the need for additional benzodiazepine medication in a 6-year-old male patient with PKAN. A 20-minute session of left premotor 1-Hz rTMS was performed daily on 5 consecutive days., Results: The occurrence of the complex movement pattern was gradually reduced from three to two attacks daily to one attack daily by real rTMS while sham rTMS had no effect. This reduction was obtained concomitantly with a similar reduction of additional benzodiazepines for both real and sham rTMS sessions., Conclusion: Inhibitory rTMS of the premotor cortex may be used to temporarily control motor symptoms in PKAN.

Ethanolamine kinase 2 (Eki2) was previously isolated from a differential expression screen designed to identify candidate genes involved in testis development and differentiation. In mouse, Eki2 is specifically up-regulated in Sertoli cells of the developing testis at the time of sex determination. Based on this expression profile, Eki2 was considered a good candidate testis-determining gene. To investigate a possible role of Eki2 in testis development, we have generated a mouse with targeted disruption of the Eki2 gene by using an EGFP replacement strategy. No abnormalities were detected in the Eki2-deficient mice with regard to embryonic and adult testis morphology, differentiation, function, or fertility. Furthermore, no significant differences were observed in litter sizes, pup mortality rates, or distribution of the sexes among the offspring. Ethanolamine kinases are involved in the biosynthesis of phosphatidylethanolamine, a major membrane phospholipid. Expression analysis indicates that the absence of an apparent phenotype in the Eki2-deficient mice may be due to compensation by Eki2-family members or the activation of an alternative pathway to generate phosphatidylethanolamine. Expression of EGFP in this mouse model enabled the isolation of gonad cell populations, providing a useful resource from which to obtain relatively pure early steroidogenic cells for further studies.

Mutated deoxyguanosine kinase (dGK), which catalyses the first step of the mitochondrial deoxypurine salvage pathway, accounts for a hepatocerebral variant of mitochondrial DNA (mtDNA) depletion syndromes. In order to elucidate the pathogenic mechanism of dGK deficiency, mitochondrial and cytoplasmic deoxyribonucleoside triphosphate (dNTP) pools in cycling and quiescent fibroblasts from a dGK deficient patient were measured. The mitochondrial dNTP pools were found to be imbalanced, mainly in quiescent cells due to decreased dGTP while mtDNA content and mitochondrial respiratory chain activities were concomitantly decreased. Supplementation of deoxyguanosine and deoxyadenosine normalized mitochondrial dNTP pools, mtDNA content and partially restored the MRC function. It is suggested that the cytoplasmic deoxycytine kinase supplemented with external substrates may compensate for the deficient dGK.

Abstract Sphingosine kinases (SKs) are key enzymes regulating the production of sphingosine-1-phosphate (S1P), which determines important cell responses including cell growth and death. Here we show that renal mesangial cells isolated from wild-type, SK-1(-/-), and SK-2(-/-) mice show a differential response to apoptotic stimuli. Wild-type mesangial cells responded to staurosporine with increased DNA fragmentation and caspase-3 processing, which was enhanced in SK-1(-/-) cells. In contrast, SK-2(-/-) cells were highly resistant to staurosporine-induced apoptosis. Furthermore, the basal phosphorylation and activity of the anti-apoptotic protein kinase B (PKB) and of its substrate Bad were decreased in SK-1(-/-) but not in SK-2(-/-) cells. Upon staurosporine treatment, phosphorylation of PKB and Bad decreased in wild-type and SK-1(-/-) cells, but remained high in SK-2(-/-) cells. In addition, the anti-apoptotic Bcl-X(L) was significantly upregulated in SK-2(-/-) cells, which may further contribute to the protective state of these cells. In summary, our data show that SK-1 and SK-2 have opposite effects on the capacity of mesangial cells to resist apoptotic stimuli. This is due to differential modulation of the PKB/Bad pathway and of Bcl-X(L) expression. Thus, subtype-selective targeting of SKs will be critical when considering these enzymes as therapeutic targets for the treatment of inflammation or cancer.