Lipophagy prevents activity‐dependent neurodegeneration due to dihydroceramide accumulation in vivo

Dihydroceramide desaturases are evolutionarily conserved enzymes that convert dihydroceramide (dhCer) to ceramide (Cer). While elevated Cer levels cause neurodegenerative diseases, the neuronal activity of its direct precursor, dhCer, remains unclear. We show that knockout of the fly dhCer desaturase gene, infertile crescent (ifc), results in larval lethality with increased dhCer and decreased Cer levels. Light stimulation leads to ROS increase and apoptotic cell death in ifc‐KO photoreceptors, resulting in activity‐dependent neurodegeneration. Lipid‐containing Atg8/LC3‐positive puncta accumulate in ifc‐KO photoreceptors, suggesting lipophagy activation. Further enhancing lipophagy reduces lipid droplet accumulation and rescues ifc‐KO defects, indicating that lipophagy plays a protective role. Reducing dhCer synthesis prevents photoreceptor degeneration and rescues ifc‐KO lethality, while supplementing downstream sphingolipids does not. These results pinpoint that dhCer accumulation is responsible for ifc‐KO defects. Human dhCer desaturase rescues ifc‐KO larval lethality, and rapamycin reverses defects caused by dhCer accumulation in human neuroblastoma cells, suggesting evolutionarily conserved functions. This study demonstrates a novel requirement for dhCer desaturase in neuronal maintenance in vivo and shows that lipophagy activation prevents activity‐dependent degeneration caused by dhCer accumulation.     

Ceramide metabolism is affected by obesity and diabetes in human adipose tissue

Ceramide is involved in development of insulin resistance. However, there are no data on ceramide metabolism in human adipose tissue. The aim of our study was to examine sphingolipid metabolism in fat tissue from obese nondiabetic (n = 11), obese diabetic (n = 11), and lean nondiabetic (n = 8) subjects. The content of ceramide (Cer), dihydroceramide (dhCer), sphingosine (SPH), sphinganine (SPA), sphingosine‐1‐phosphate (S1P; pmol/mg of protein), the expression (mRNA) and activity of key enzymes responsible for Cer metabolism: serine palmitoyltransferase (SPT), neutral and acidic sphingomyelinase (nSMase and aSMase, respectively), and neutral and acidic ceramidase (nCDase and aCDase, respectively) were examined in human adipose tissue. The contents of SPA and Cer were significantly lower whereas the content of dhCer was higher in both obese groups than the respective values in the lean subjects. The expression of examined enzymes was elevated in both obese groups. The SPT and CDases activity increased whereas aSMase activity deceased in both obese groups. We have found correlation between adipose tissue Cer content and plasma adiponectin concentration (r = 0.69, P < 0.001) and negative correlation between total Cer content and HOMA‐IR index (homeostasis model of insulin resistance) (r = ?0.67, P < 0.001). We have found that both obesity and diabetes affected pathways of sphingolipid metabolism in the adipose tissue. J. Cell. Physiol. 227: 550–557, 2012. © 2011 Wiley Periodicals, Inc.     

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.     

Dihydrosphingolipids are associated with steatosis and increased fibrosis damage in non-alcoholic fatty liver disease

Dihydrosphingolipids are lipids biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver, and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet induced NAFLD mouse model to study the association between this class of compounds and disease progression. Mice fed a high-fat diet were sacrificed at 22, 30 and 40 weeks to reproduce the full spectrum of histological damage found in human disease, steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients whose NAFLD severity was assessed histologically. To demonstrate the effect of dihydroceramides over NAFLD progression we treated mice with fenretinide an inhibitor of dihydroceramide desaturase-1 (DEGS1). Lipidomic analyses were performed using liquid chromatography-tandem mass spectrometry. Triglycerides, cholesteryl esters and dihydrosphingolipids were increased in the liver of model mice in association with the degree of steatosis and fibrosis. Dihydroceramides increased with the histological severity observed in liver samples of mice (0.024 ± 0.003 nmol/mg vs 0.049 ± 0.005 nmol/mg, non-NAFLD vs NASH-fibrosis, p < 0.0001) and patients (0.105 ± 0.011 nmol/mg vs 0.165 ± 0.021 nmol/mg, p = 0.0221). Inhibition of DEGS1 induce a four-fold increase in dihydroceramides improving steatosis but increasing the inflammatory activity and fibrosis. In conclusion, the degree of histological damage in NAFLD correlate with dihydroceramide and dihydrosphingolipid accumulation.Lay summaryAccumulation of triglyceride and cholesteryl ester lipids is the hallmark of non-alcoholic fatty liver disease. Using lipidomics, we examined the role of dihydrosphingolipids in NAFLD progression. Our results demonstrate that de novo dihydrosphingolipid synthesis is an early event in NAFLD and the concentrations of these lipids are correlated with histological severity in both mouse and human disease.     

Pulsatilla chinensis saponins cause liver injury through interfering ceramide/sphingomyelin balance that promotes lipid metabolism dysregulation and apoptosis

Background: P. chinensis saponins (PRS) are pentacyclic triterpenoid bioactive constituents from Pulsatilla chinensis (Bunge) Regel. In our previous study, PRS caused chronic liver injury (CLI) with the significant changes of lipid metabolites including sphingomyelin (SM) in serum after long-term administration. The SM in the hepatocytes membrane plays an indispensable role in maintaining cell membrane stability and regulating the extracellular and intracellular signal transduction. However, it is still unknown the pathway related to SM and the mechanism of CLI on hepatocyte.Purpose: The purpose of this study was to explore the hepatotoxicity mechanism of PRS in vivo and in vitro, to reveal the action of mechanism of SM and the pathway related to liver injury.Methods: SD rats were orally administered with PRS for 240 days and liver injury was evaluated by histological examinations. Metabolomics analysis was used to explore the liver metabolic pathway affected by PRS, and the expressions of related proteins were evaluated by western blots. To discover and elucidate the underlying mechanisms of metabolites changes induced by PRS at the cellular level, cellular morphology, MTT assays, western blots and cell membrane potential measurements were carried out using LO2 cells. Furthermore, the roles of SM and cholesterol (Chol) in hepatocyte injury were investigated individually in overload Chol and SM groups. Sphingolipid metabolic pathway related with ceramide/sphingomyelin (Cer/SM) balance was explored using cellular lipidomics and RT-PCR.Results: PRS gradually damaged the rat's liver in a time-dependent manner. The analysis of liver metabolism profiles showed that lipids metabolites were changed, including sphingolipid, bile acid, linoleic acid and fatty acid. We found that PRS induced apoptosis by interfering with bile acid-mediated sphingolipid metabolic pathway and Cer/SM balance in CLI. In in vitro experiments, PRS led to the increase of LDH leakage, depolarized cell membrane potential and caused cell membrane toxicity. Furthermore, PRS inducedG0/G1 phase cell cycle arrest in LO2 cells, simultaneously activated cellular extrinsic and intrinsic apoptosis pathways. PRS acted on SM and interfered with Cer/SM balance, which promote lipid metabolism dysregulation and apoptosis.Conclusion: PRS acted on SM to interfere Cer/SM balance on LO2 cell. Both in vivo and in vitro, PRS induced Cer/SM imbalance which promoted lipid metabolism disorder and apoptosis. Apoptosis and lipids changes gradually damaged the rats liver, and ultimately developed into CLI.     

C6-ceramide synergistically potentiates the anti-tumor effects of histone deacetylase inhibitors via AKT dephosphorylation and ��-tubulin hyperacetylation both in vitro and in vivo

Histone deacetylase inhibitors (HDACIs) have shown promising anti-tumor effects for a variety of malignancies, however, many tumors are reportedly resistant to them. In this study, we made a novel discovery that co-administration of HDACIs (Trichostatin A (TSA) and others) and exogenous cell-permeable short-chain ceramide (C6) results in striking increase in cancer cell death and apoptosis in multiple cancer cells. These events are associated with perturbations in diverse cell signaling pathways, including inactivation of Akt/mTOR and increase in α-tubulin acetylation (both in vivo and in vitro). TSA interacts in a highly synergistic manner with C6-ceramide to disrupt HDAC6/protein phosphatase 1 (PP1)/tubulin complex, to induce α-tubulin hyperacetylation, and to release and activate PP1, which then leads to AKT dephosphorylation and eventually causes cancer cell death. Interestingly, TSA itself results in short-term ceramide accumulation, which as a result of metabolic (glycosylation) removal, does not result in evident increase of cancer cell death. However, adding C6-ceramide led to a very pronounced increase in ceramide level and marked increase in cell death. Importantly, the effective synergistic anti-tumor activity of TSA plus C6-ceramide is also seen in in vivo mice xenograft pancreatic and ovarian cancer models, indicating that this regimen (HDACI plus C6-ceramide) may represent a more effective form of therapy against pancreatic and ovarian carcinoma.     

Dihydroceramide-based response to hypoxia

To understand the mechanisms of ceramide-based responses to hypoxia, we performed a mass spectrometry-based survey of ceramide species elicited by a wide range of hypoxic conditions (0.2-5% oxygen). We describe a rapid, time-dependent, marked up-regulation of dihydroceramides (DHCs) in mammalian cells and in the lungs of hypoxic rats. The increase affected all DHC species and was proportional with the depth and duration of hypoxia, ranging from 2- (1 h) to 10-fold (24 h), with complete return to normal after 1 h of reoxygenation at the expense of increased ceramides. We demonstrate that a DHC-based response to hypoxia occurs in a hypoxia-inducible factor-independent fashion and is catalyzed by the DHC desaturase (DEGS) in the de novo ceramide pathway. Both the impact of hypoxia on DHC molecular species and its inhibitory effect on cell proliferation were reproduced by knockdown of DEGS1 or DEGS2 by siRNA during normoxia. Conversely, overexpression of DEGS1 or DEGS2 attenuated the DHC accumulation and increased cell proliferation during hypoxia. Based on the amplitude and kinetics of DHC accumulation, the enzymatic desaturation of DHCs fulfills the criteria of an oxygen sensor across physiological hypoxic conditions, regulating the balance between biologically active components of ceramide metabolism.     

Molecular associations and surface-active properties of short- and long-N-acyl chain ceramides

The behaviour of N-hexadecanoylsphingosine (Cer16), N-hexanoylsphingosine (Cer6) and N-acetylsphingosine (Cer2) in aqueous media and in lipid-water systems, monolayers and bilayers has been comparatively examined using Langmuir balance and fluorescence techniques. Cer16 behaves as an insoluble non-swelling amphiphile, not partitioning into the air-water interface, thus not modifying the surface pressure of the aqueous solutions into which it is included. By contrast both Cer6 and Cer2 behave as soluble amphiphiles, up to approx. 100 μM. At low concentrations, they become oriented at the air-water interface, increasing surface pressure in a dose-dependent way up to ca. 5 μM bulk concentration. At higher concentrations, the excess ceramide forms micelles, critical micellar concentrations of both Cer6 and Cer2 being in the 5-6 μM range. When the air-water interface is occupied by a phospholipid, 6Cer2 and Cer6 become inserted in the phospholipid monolayer, causing a further increase in surface pressure. This increase is dose dependent, and reaches a plateau at ca. 2 μM ceramide bulk concentration. Both Cer2 and Cer6 become inserted in phospholipid monolayers with initial surface pressures of up to 43 and 46 mN m⁻¹, respectively, which ensures their capacity to become inserted into cell membranes whose monolayers are estimated to support a surface pressure of about 30 mN m⁻¹. Both Cer2 and Cer6, but not Cer16, had detergent-like properties, such as giving rise to phospholipid-ceramide mixed micelles, when added to phospholipid monolayers or bilayers. The short-chain ceramides form large aggregates and precipitate at concentrations above approx. 100 μM. These results are relevant in cell physiology studies in which short- and long-chain ceramides are sometimes used as equivalent molecules, in spite of their different biophysical behaviour.     

Influence of the estrous cycle on clock gene expression in reproductive tissues: Effects of fluctuating ovarian steroid hormone levels

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2::LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes. We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Bmal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression.     

Kinetics of spermatogenesis,cell-cycle analysis,and testis development in nymphs of the tick Dermacentor variabilis

Cytokinetic analyses were carried out on the testis of the American dog tick (Dermacentor variabilis) nymph during the developmental period which follows attachment to the host and feeding. The spermatogonial cell division cycle in vivo was determined: Tc, 22.8 hr; S, 6.7 hr; G₁, 8.2 hr; G₂, 4.0 hr; P, 3.5 hr; M, 0.4 hr. Histological techniques were used to determine the timing of spermatocyte differentiation and accumulation. This accumulation is markedly biphasic, occurring as two distinct waves during the developmental period. Models of germarial zone division-induction are presented, and the theoretical population curves generated by these models compared to the empirical data.Development of a previously-unreported secretory tissue in close association with the nymphal testis is described, and some correlations between germarial division activity and endocrine events known from related species presented.     

Methylmercury-induced neurotoxicity and apoptosis

Methylmercury is a widely distributed environmental toxicant with detrimental effects on the developing and adult nervous system. Due to its accumulation in the food chain, chronic exposure to methylmercury via consumption of fish and sea mammals is still a major concern for human health, especially developmental exposure that may lead to neurological alterations, including cognitive and motor dysfunctions. Mercury-induced neurotoxicity and the identification of the underlying mechanisms has been a main focus of research in the neurotoxicology field. Three major mechanisms have been identified as critical in methylmercury-induced cell damage including (i) disruption of calcium homeostasis, (ii) induction of oxidative stress via overproduction of reactive oxygen species or reduction of antioxidative defenses and (iii) interactions with sulfhydryl groups. In vivo and in vitro studies have provided solid evidence for the occurrence of neural cell death, as well as cytoarchitectural alterations in the nervous system after exposure to methylmercury. Signaling cascades leading to cell death induced by methylmercury involve the release of mitochondrial factors, such as cytochrome c and AIF with subsequent caspase-dependent or -independent apoptosis, respectively; induction of calcium-dependent proteases calpains; interaction with lysosomes leading to release of cathepsins. Interestingly, several pathways can be activated in parallel, depending on the cell type. In this paper, we provide an overview of recent findings on methylmercury-induced neurotoxicity and cell death pathways that have been described in neural and endocrine cell systems.     

End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

A phospholipase C/sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties; the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230-3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme.     

Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1–3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.     

C. elegans Germ Cells Show Temperature and Age-Dependent Expression of Cer1, a Gypsy/Ty3-Related Retrotransposon

Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology.     

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.     

Wolbachia Transfer from Rhagoletis cerasi to Drosophila simulans: Investigating the Outcomes of Host-Symbiont Coevolution

Wolbachia is an endosymbiont of diverse arthropod lineages that can induce various alterations of host reproduction for its own benefice. Cytoplasmic incompatibility (CI) is the most common phenomenon, which results in embryonic lethality when males that bear Wolbachia are mated with females that do not. In the cherry fruit fly, Rhagoletis cerasi, Wolbachia seems to be responsible for previously reported patterns of incompatibility between populations. Here we report on the artificial transfer of two Wolbachia variants (wCer1 and wCer2) from R. cerasi into Drosophila simulans, which was performed with two major goals in mind: first, to isolate wCer1 from wCer2 in order to individually test their respective abilities to induce CI in the new host; and, second, to test the theoretical prediction that recent Wolbachia-host associations should be characterized by high levels of CI, fitness costs to the new host, and inefficient transmission from mothers to offspring. wCer1 was unable to develop in the new host, resulting in its rapid loss after successful injection, while wCer2 was established in the new host. Transmission rates of wCer2 were low, and the infection showed negative fitness effects, consistent with our prediction, but CI levels were unexpectedly lower in the new host. Based on these parameter estimates, neither wCer1 nor wCer2 could be naturally maintained in D. simulans. The experiment thus suggests that natural Wolbachia transfer between species might be restricted by many factors, should the ecological barriers be bypassed.     

Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.     

Biochemistry of insect differentiation: A system for studying the mechanism of chitinase activity in vitro

Results obtained with an in vitro system for the study of chitinase are described. The system involves soluble enzyme protein(s) and an insoluble substrate preparation. With insect molting fluid chitinase, it shows properties that parallel those observed during in vivo breakdown of cuticle during the molt. For example, molting fluid chitinase activity not previously exposed to chitin is stronly and specifically adsorbed to the substrate, in contrast to other enzymatic activities including hexosaminidase (chitobiase) present in molting fluid. This leads to partial purification of molting fluid chitinase activity reflected in increased specific activity of chitinase associated with the insoluble chitin substrate; we have previously reported increase of specific chitinase activity of (deproteinized) cuticle resulting from its incubation with molting fluid (M. L. Bade and A. Stinson, 1978, Biochem. Biophys. Res. Commun.84, 381–388). Soluble end product is generated rapidly and linearly with time by the in vitro system; the end product is assumed to be N-acetylglucosamine since the specific radioactivity of this compound is unchanged during the 10 min required for assay. Molting fluid chitinase activity may involve a number of polypeptides ranging in molecular weight from 145,000 to less than 20,000 daltons. The system described gives results consistent with a processive mechanism for molting fluid chitinase, i.e., data are given demonstrating that molting fluid chitinase continues to act on the same chitin particle(s) with which it initially associates rather than diffusing freely from substrate particle to substrate particle, and the product of its action appears to be a monosaccharide rather than a mixture of oligosaccharides. Processive behavior for chitinase would be predicted from the known structure, and the in vivo measured rate of breakdown, of cuticle chitin during the molt; the preliminary nature of this conclusion, based on what is so far known about the structure of the substrate used in the in vitro system, is briefly discussed.     

Two Invaders Instead of One: The True Identity of Species under the Name Ceracis cucullatus (Coleoptera: Ciidae)

The Neotropical obligate fungivorous beetle Ceracis cucullatus (Mellié) has attracted attention of coleopterists due to the increasing number of records of populations in Africa. Although its disjunct populations have been interpreted as a cohesive taxonomic unity, previous comparisons between African and Neotropical specimens revealed differences in their external morphology, causing uncertainty about the true unity of the species. Here, we compare the external morphology of specimens named Cer. cucullatus from several localities of the Neotropical, Palearctic, Afrotropical, Afrotemperate and Oriental regions. As results, we reverse three previous junior synonymies of Cer. cucullatus, proposing Cer. lamellatus (Pic) and Cer. tabellifer (Mellié), both reinstated status and new combinations, as separate species. We also propose Enn. bilamellatum Pic as a new synonym of Cer. tabellifer. In face of these taxonomic changes, we identify Cer. tabellifer as the actual invasive species on African lands, instead of Cer. cucullatus as was previously accepted. Then, through historical records gathered from scientific collections and literature, and through examination of recently collected specimens from South Africa and Brazil, we provide data on host fungi and geographic distribution of Cer. tabellifer. Based on these data, we discuss possible explanations to the successful invasion of Cer. tabellifer in Africa and elsewhere and its potential threat to native faunas of ciids. This study helps to fulfil an old gap in the literature on biological invasions, with considerably more studies on predatory species, disease vectors or potential pests of agricultural crops, than on non-pest fungivorous organisms.