Thiamine Pyrophosphatase Research Articles

Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata.

Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C. glabrata, but the transcription factor CgPdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase (CgPMU3) that is critical for accessing external thiamine. Here, we demonstrate that CgPdc2 primarily regulates THI genes. In Saccharomyces cerevisiae, Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S. cerevisiae in standard growth conditions, but not in C. glabrata. We uncover cryptic cis elements in C. glabrata PDC promoters that still allow for regulation by ScPdc2, even when that regulation is not apparent in C. glabrata. C. glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S. cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C. glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3. CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20, CgPMU3, and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals.

Influence of cytomegalovirus infection on nucleoside diphosphatase activity in placenta

Introduction. Cytomegalovirus infection (CMVI) makes a significant contribution to the development of pregnancy pathologies, including dysfunction of the placenta. However, the mechanism of such an effect of CMV has not been precisely established. For the coordination of biochemical reactions in the placenta, an optimal concentration of substances, including nucleotides, is required. Nucleoside diphosphatase is an enzyme that catalyzes the hydrolysis of nucleotide diphosphates to nucleotide and phosphate.Aim. Determination of the activity of nucleoside diphosphatase in the placenta in physiological and complicated CMVI reactivation in the third trimester of pregnancy.Materials and methods. A study of 62 placentas obtained during childbirth at 38-40 weeks was carried out. The first group consisted of the placentas from women with CMVI reactivation in the third trimester of pregnancy and the second – the placenta of women without a history of infectious pathology. Diagnosis of CMVI was carried out by determining antibodies of class M and G by ELISA, as well as CMV DNA was detected by PCR. Histochemical analysis of nucleoside diphosphatase was performed according to the method of A.B.Novikoff and D.S.Goldfischer modified by Z.Lojda et al.Results. In the second group, the placenta nucleoside-diphosphatase had a pronounced activity in the syncytiotrophoblast and the connective tissue stroma of the villi. When CMVI was reactivated in the third trimester of pregnancy, a decrease in the intensity of the histochemical reaction to nucleoside-diphosphatase was found: the cytophotometric index in the first group significantly (p<0.001) decreased to 13.06±0.089 rel. units (in the second group it was 14.11±0.119 relative units).Conclusion. The decrease in the activity of nucleoside diphosphatase found during the reactivation of CMVI in the third trimester of pregnancy might indicate a decrease in nucleotide metabolism in the placenta. In our opinion, the study of enzymes involved in the exchange of nucleotides will help to reveal the mechanisms by which CMV causes disturbances in the functioning of the placenta, leading to the development of complications of pregnancy.

Organic secretion in ductal cells of the sublingual salivary gland of ferret: Histochemical observations

The presence of organic secretion in ductal cells of the sublingual salivary gland of ferret has been questioned, which prompted the present investigation. Paraffin or cryostat sections from aldehyde fixed or quenched sublingual glands of this species were tested for some amino acid residues, mucosubstances, oxidative and hydrolytic enzymes, and lectins. The glands showed inconspicuous ducts of simple appearances on routine histology. The histochemical procedures, however, revealed a granulated substance in the apical (periluminal) region of ductal cells, which contained tryptophan, disulphides, neutral mucosubstances, αFuc and GalNAc, and showed chloroacetate esterase activity. Occurrence of the substance varied between different ducts of the same gland and/or cells of the same duct. The ductal cells also showed diffuse peroxidase and acid phosphatase, and Golgi-like thiamine pyrophosphatase activities. Acetylcholinesterase-positive nerve fibres embraced the ducts. The results support a particular localisation of protein-bound amino acid residues and enzymatic catalytic activities indicative of organic secretion, possibly tissue kallikrein, in sublingual ductal cells of ferret.

The first comprehensive phylogenetic and biochemical analysis of NADH diphosphatases reveals that the enzyme from Tuber melanosporum is highly active towards NAD+

Nudix (for nucleoside diphosphatases linked to other moieties, X) hydrolases are a diverse family of proteins capable of cleaving an enormous variety of substrates, ranging from nucleotide sugars to NAD+-capped RNAs. Although all the members of this superfamily share a common conserved catalytic motif, the Nudix box, their substrate specificity lies in specific sequence traits, which give rise to different subfamilies. Among them, NADH pyrophosphatases or diphosphatases (NADDs) are poorly studied and nothing is known about their distribution. To address this, we designed a Prosite-compatible pattern to identify new NADDs sequences. In silico scanning of the UniProtKB database showed that 3% of Nudix proteins were NADDs and displayed 21 different domain architectures, the canonical architecture (NUDIX-like_zf-NADH-PPase_NUDIX) being the most abundant (53%). Interestingly, NADD fungal sequences were prominent among eukaryotes, and were distributed over several Classes, including Pezizomycetes. Unexpectedly, in this last fungal Class, NADDs were found to be present from the most common recent ancestor to Tuberaceae, following a molecular phylogeny distribution similar to that previously described using two thousand single concatenated genes. Finally, when truffle-forming ectomycorrhizal Tuber melanosporum NADD was biochemically characterized, it showed the highest NAD+/NADH catalytic efficiency ratio ever described.

Phosphohydrolases in Turbellaria Phagocata sibirica

Activity and properties of two phosphohydrolases, nucleoside diphosphatase (NDPase) and 5′-nucleotidase, were assayed in subcellular fractions of turbellaria Phagocata sibirica Sabussov (Turbellaria, Planariidae). The highest activity of N D Pase was observed in microsomes while that of 5′-nucleotidase in mitochondria and cytosolic fractions (12 000 and 105 000 g). The correlation between the rates of the relevant enzymatic reactions and concentrations of substrates and metal ions as well as the impact of various effectors and divalent cations (Mg2+, Mn2+, Ca2+, Zn2+, Cu2+) on phosphohydrolase activities were studied.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization.

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100mM Bis-Tris propane pH 6.0 and 50mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

A novel photoelectrochemical immunosensor by integration of nanobody and ZnO nanorods for sensitive detection of nucleoside diphosphatase kinase-A

Nucleoside diphosphatase kinase A (NDPK-A) is a metastasis-suppressor protein and a biomarker that act on a wide range cancer cells to inhibit the potential metastasis. Herein, we present a simple photoelectrochemical immunosensor based on ZnO nanorod arrays for the sensitive detection of NDPK-A. The ZnO nanorod arrays cosensitized with CdS nanoparticles and Mn2+ displayed a high and stable photocurrent response under irradiation. After anti-NPDK-A nanobodies were immobilized to the ZnO nanorod arrays, the proposed immunosensor can be utilized for detecting NPDK-A by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Accordingly, the well-designed immunosensor exhibited a low limit of detection (LOD) of 0.3 pg mL−1 and a wide linear range from 0.5 pg mL−1 to 10 μg mL−1. The R2 of the regression curve is 0.99782. Meanwhile, the good stability, reproducibility and specificity of the resulting photoelectrochemical biosensor are demonstrated. In addition, the presented work would offer a novel and simple approach for the detection of immunoreactions and provide new insights in popularizing the diagnosis of NPDK-A.

Integrative method for three-dimensional imaging of the entire Golgi apparatus by combining thiamine pyrophosphatase cytochemistry and array tomography using backscattered electron-mode scanning electron microscopy.

Thiamine pyrophosphatase (TPPase) cytochemistry is an established method for specific labeling of the trans-Golgi cisterns in tissue sections. Herein, we combined this enzyme cytochemical method with array tomography using scanning electron microscopy (SEM), a new imaging technique based on collection of backscattered electron (BSE) images of consecutive resin-embedded sections on glass slides, to detect the entire three-dimensional (3D) organization of the Golgi apparatus with sufficient spatial resolution. As the signal intensity of BSE depends on the atomic number of the materials, lead precipitates confined to the trans-Golgi cisterns after TPPase cytochemistry were clearly observed by BSE-mode SEM. The mild fixative used for TPPase cytochemistry also enabled accurate identification of target gonadotropes in the composite pituitary tissue by immunocytochemical staining. By 3D reconstruction of the entire trans-Golgi cisterns based on serial ultrathin section images of tissues after TPPase cytochemistry, we detected ultrastructural differences in the 3D configuration of the Golgi apparatus between cerebellar Purkinje cells and pituitary gonadotropes. The appropriate combination of enzyme cytochemistry and/or immunostaining with array tomography will further clarify the relationship between the organization and functional states of the Golgi apparatus.

Нуклеозиддифосфатазы цестод Bothriocephalus scorpii (Cestoda: Bothriocephalidae)

Objective of research: To study the activities and properties of nucleoside- diphosphatase (NDPase) in cestode Bothriocephalus scorpii.
 Materials and methods: Cestodes were homogenized with 10 vol. of extraction medium. NDPase was detected in mitochondria and microsomes with substrates (IDP, GDP, UDP). Inorganic phosphorus was determined by the method of Kochetov (1980). The effects of 10 anthelmintic drugs on the activity of NDPase were studied. 
 Results and discussion: It was found that the mitochondrial and microsomal fractions of cestodes B. scorpii have nucleoside diphosphatase activity. The activity of nucleoside diphosphatase depends on substrates and Mg2+ ions. The impact of various effectors and ions (Ca2+, Mn2+, Zn2+) on enzyme activity was determined. Effects of 10 anthelmintic drugs on activity of nucleoside diphosphatase were studied. The anthelmintics Bitionol and Trichlorophen have been proved effective.

Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings.

The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.

Novel Nucleoside Diphosphatase Contributes to Staphylococcus aureus Virulence

We identified SA1684 as a Staphylococcus aureus virulence gene using a silkworm infection model. The SA1684 gene product carried the DUF402 domain, which is found in RNA-binding proteins, and had amino acid sequence similarity with a nucleoside diphosphatase, Streptomyces coelicolor SC4828 protein. The SA1684-deletion mutant exhibited drastically decreased virulence, in which the LD50 against silkworms was more than 10 times that of the parent strain. The SA1684-deletion mutant also exhibited decreased exotoxin production and colony-spreading ability. Purified SA1684 protein had Mn(2+)- or Co(2+)-dependent hydrolyzing activity against nucleoside diphosphates. Alanine substitutions of Tyr-88, Asp-106, and Asp-123/Glu-124, which are conserved between SA1684 and SC4828, diminished the nucleoside diphosphatase activity. Introduction of the wild-type SA1684 gene restored the hemolysin production of the SA1684-deletion mutant, whereas none of the alanine-substituted SA1684 mutant genes restored the hemolysin production. RNA sequence analysis revealed that SA1684 is required for the expression of the virulence regulatory genes agr, sarZ, and sarX, as well as metabolic genes involved in glycolysis and fermentation pathways. These findings suggest that the novel nucleoside diphosphatase SA1684 links metabolic pathways and virulence gene expression and plays an important role in S. aureus virulence.

The microglial cell response to sterile nerve injury in the brain is regulated by IRF8

Abstract Microglia, the resident immune cells of the central nervous system (CNS), respond vigorously to insults such as nerve injury. We have shown previously in the normal CNS the properties of microglia are dramatically altered in the absence of IRF8. Here we examined whether IRF8 is involved in the microglial cell response to sterile nerve injury. Facial nerve axotomy (FNA) was performed in wild type (WT) and IRF8−/− (KO) mice and the brain removed at different days post-injury (dpi). In brains from IRF8 KO mice, nucleoside diphosphatase (NDPase) histochemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied but were present in comparable numbers with WT. In WT mice, a progressive increase in microglial cell activation was observed peaking at 7 dpi and was accompanied by dense staining for lectin, NDPase and CD11b. By contrast, in IRF8 KO mice, the microglial cell activation was grossly attenuated with the density of staining for lectin, NDPase and CD11b reduced significantly. The attenuated microglial cell activation in IRF8 KO mice was paralleled by a significant decrease in microglial cell proliferation at 7 and 14 dpi. The wrapping of individual motor neuron cell bodies in the axotomised facial nucleus by microglia involved in synaptic stripping and phagocytosis was incomplete in IRF8 KO mice and was accompanied by an increase in degenerated neurons. These findings show that in addition to maintenance of homeostatic properties, IRF8 also has an important role in regulating a number of key processes in the microglial cell response to neuronal injury.

The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.

The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three – GDP, IDP and UDP – were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca2+, Mg2+ and Mn2+ were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling.

Changes in GDPase/UDPase enzymatic activity in response to oxidative stress in four Candida species.

The terminal processing of proteins and lipids occurs in the Golgi apparatus and involves the transport of sugar nucleotides into the Golgi lumen by specific carriers and the accumulation of nucleoside diphosphates (NDPs) as a result of oligosaccharide-protein glycosyltransferase activity. NDPs are converted into the corresponding nucleoside monophosphates (NMPs) by nucleoside diphosphatases (NDPases), thus relieving inhibition of sugar transferases. In addition, NMPs are then exchanged for equimolecular amounts of cytosolic sugar nucleotides by antiport transport systems. NDPases, commonly GDPase and UDPase, thus play a critical role in glycoprotein maturation and may influence fungal pathogenesis, morphogenesis, and cell wall properties. Interest of this laboratory has recently focused on the effect of reactive oxygen species (ROS) on enzymes involved in detoxification of these oxidants and on the metabolism of biomolecules such as lipids, nucleic acids, and proteins in human pathogenic Candida species. We therefore consider it important to extend these studies to determine how GDPase and UDPase are affected after exposure of cells to oxidants such as menadione, a superoxide (O2 (•-))-generator, and H2O2. Results indicate that activity of both enzymes decrease in response to these agents suggesting that ROS may also affect other critical cell functions such as protein glycosylation.

202: Interferon regulator factor (IRF) 8 regulates the microglial response to neuronal injury

The transcription factor interferon regulatory factor (IRF) 8 has a key role in the cellular response to IFN-γ and is involved in myeloid cell differentiation. We have previously identified IRF8 to be a constitutive and IFN-γ-stimulated nuclear factor that regulates the homeostatic properties of microglia. Our study aimed to determine the role of IRF8 in the microglial response to sterile neuronal injury. Facial nerve axotomy (FNA) was performed in wildtype (WT) and IRF8 −/− (KO) mice and the brains removed at different times post-lesion. A subset of mice was injected with bromodeoxyuridine (BrdU) prior to retrieval of the brain. Changes in the facial nucleus (FN) were examined by immunohistochemistry and histochemistry. In brains from IRF8KO mice, nucleoside diphosphatase (NDPase) hisotchemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied. After FNA in WT mice, a progressive increase in microglial activation was observed in the lesioned FN peaking at day 7 and was accompanied by dense staining for Iba1, lectin, NDPase and CD11b. By contrast, in IRF8KO mice, the microglial response was markedly attenuated with little staining for Iba1, while the density of staining for lectin and NDPase was reduced significantly. The attenuated microglial response in IRF8KO mice was paralleled by a significant decrease at day 3 post-lesion in proliferation as monitored by BrdU, phosphohistone 3 and Ki67-positive cells. Furthermore, a decrease in PU.1-positive cells was observed in the FN of IRF8KO mice compared with WT. The wrapping of individual motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete in the absences of IRF8 in the axotomised FN. Quantitative analysis showed that in IRF8KO mice, the degeneration of axotomised motor neurons was significantly increased. These studies extend on our previous finding that IRF8 is a key homeostatic transcription regulator of microglial cell function in the healthy brain, and moreover, has a crucial role in the regulating the response to microglia to neuronal injury.

Effect of nicotine exposure on cellular and molecular parameters of vitamin B1 (thiamin) uptake by pancreatic acinar cells (896.3)

Pancreatic acinar cells take in thiamin by a specific carrier‐mediated process that involve both THTR‐1 and THTR‐2 (encoded by SLC19A2 and SLC19A3, respectively). In this study we examined the effect of chronic exposure of mouse pancreatic acinar in vivo (wild‐type and transgenic mice carrying the SLC19A2 and SLC19A3 promoters) and human pancreatic acinar cells in vitro to the cigarette smoke component nicotine on physiological and molecular parameters of the thiamin uptake process. Chronic exposure of mice to nicotine (0.77 mM nicotine in drinking water for 4 wks) leads to a significant decrease in thiamin uptake, expression of THTR‐1 and ‐2 protein, mRNA levels and activity of the SLC19A2 and SLC19A3 promoters expressed in transgenic mice. A decrease in the level of expression of protein and mRNA of the thiamin‐metabolizing enzymes TPKase and thiamin pyrophosphatase (TPPase) was observed. Similarly chronic exposure of human pancreatic acinar cells to nicotine (0.5 µM, 48 h) leads to a significant inhibition in thiamin uptake, as well as in the level of expression of THTR‐1 and ‐2 protein and mRNA. These results demonstrate, for the first time, that chronic exposure of pancreatic acinar cells to nicotine negatively impacts the physiological and molecular parameters of thiamin uptake and that this effect is exerted, at least in part, at the level of transcription of the SLC19A2 and SLC19A3 genes.Grant Funding Source: Supported by NIH grants AA 018071 and DK‐56061

Extracellular Nucleotide Catabolism by the Group B Streptococcus Ectonucleotidase NudP Increases Bacterial Survival in Blood

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

Human Liver Cell Trafficking Mutants: Characterization and Whole Exome Sequencing

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α’’. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Testicular effects of monensin, a golgi interfering agent in male rats

Objective: The present study was undertaken to explore the effects of monensin, a potent Golgi disturbing agent on male fertility. Methods: Male Wistar rats were administered monensin at the dose levels of 2.5, 5, and 10 mg/kg b wt. Animals were sacrificed after 67 days of the treatment. The activities of lactate dehydrogenase (LDH), ATPase, acid phosphatase and thiamine pyrophosphatase (TPPase) were measured in the testis. Cytochemical assay of Golgi body marker enzyme, thiamine pyrophosphatase was also performed. Ultrastructural changes in testis were studied by Transmission electron microscopy. Sperm number and motility were also examined. Results and discussion: The alterations in the activities of above mentioned enzymes indicate the pronounced effect of the drug on the functioning of spermatogenic cells. The findings from electron microscopy such as membrane disruption, swelling and disintegration of Golgi apparatus strongly suggest the interference of monensin with the functioning of Golgi apparatus in the spermatogenic cells. Data from the sperm number and motility as well as the fertility studies and the resulted litter size further points towards the antifertility effects of monensin in male rats. Conclusion: The findings from the present study strongly indicated the effects of monensin on the testis, involving alterations in key enzyme activities and changes at the ultrastructural level.

Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity

Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1∆ null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.