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Sample records for biochemical characterization mitochondrial

  1. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV

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    Han, Q.; Robinson, H.; Cai, T.; Tagle, D. A.; Li, J.

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  2. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV.

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    Han, Qian; Robinson, Howard; Cai, Tao; Tagle, Danilo A; Li, Jianyong

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  3. Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7.

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    Lantsman, Yelena; Tan, Kevin S W; Morada, Mary; Yarlett, Nigel

    2008-09-01

    A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.

  4. Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase: SUBSTRATE SPECIFICITY, DNA BRANCH MIGRATION, AND ABILITY TO OVERCOME BLOCKADES TO DNA UNWINDING.

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    Khan, Irfan; Crouch, Jack D; Bharti, Sanjay Kumar; Sommers, Joshua A; Carney, Sean M; Yakubovskaya, Elena; Garcia-Diaz, Miguel; Trakselis, Michael A; Brosh, Robert M

    2016-07-01

    Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.

  5. Unraveling Biochemical Pathways Affected by Mitochondrial Dysfunctions Using Metabolomic Approaches

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    Demine, Stéphane; Reddy, Nagabushana; Renard, Patricia; Raes, Martine; Arnould, Thierry

    2014-01-01

    Mitochondrial dysfunction(s) (MDs) can be defined as alterations in the mitochondria, including mitochondrial uncoupling, mitochondrial depolarization, inhibition of the mitochondrial respiratory chain, mitochondrial network fragmentation, mitochondrial or nuclear DNA mutations and the mitochondrial accumulation of protein aggregates. All these MDs are known to alter the capacity of ATP production and are observed in several pathological states/diseases, including cancer, obesity, muscle and neurological disorders. The induction of MDs can also alter the secretion of several metabolites, reactive oxygen species production and modify several cell-signalling pathways to resolve the mitochondrial dysfunction or ultimately trigger cell death. Many metabolites, such as fatty acids and derived compounds, could be secreted into the blood stream by cells suffering from mitochondrial alterations. In this review, we summarize how a mitochondrial uncoupling can modify metabolites, the signalling pathways and transcription factors involved in this process. We describe how to identify the causes or consequences of mitochondrial dysfunction using metabolomics (liquid and gas chromatography associated with mass spectrometry analysis, NMR spectroscopy) in the obesity and insulin resistance thematic. PMID:25257998

  6. Unraveling Biochemical Pathways Affected by Mitochondrial Dysfunctions Using Metabolomic Approaches

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    Stéphane Demine

    2014-09-01

    Full Text Available Mitochondrial dysfunction(s (MDs can be defined as alterations in the mitochondria, including mitochondrial uncoupling, mitochondrial depolarization, inhibition of the mitochondrial respiratory chain, mitochondrial network fragmentation, mitochondrial or nuclear DNA mutations and the mitochondrial accumulation of protein aggregates. All these MDs are known to alter the capacity of ATP production and are observed in several pathological states/diseases, including cancer, obesity, muscle and neurological disorders. The induction of MDs can also alter the secretion of several metabolites, reactive oxygen species production and modify several cell-signalling pathways to resolve the mitochondrial dysfunction or ultimately trigger cell death. Many metabolites, such as fatty acids and derived compounds, could be secreted into the blood stream by cells suffering from mitochondrial alterations. In this review, we summarize how a mitochondrial uncoupling can modify metabolites, the signalling pathways and transcription factors involved in this process. We describe how to identify the causes or consequences of mitochondrial dysfunction using metabolomics (liquid and gas chromatography associated with mass spectrometry analysis, NMR spectroscopy in the obesity and insulin resistance thematic.

  7. Characterizing multistationarity regimes in biochemical reaction networks.

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    Irene Otero-Muras

    Full Text Available Switch like responses appear as common strategies in the regulation of cellular systems. Here we present a method to characterize bistable regimes in biochemical reaction networks that can be of use to both direct and reverse engineering of biological switches. In the design of a synthetic biological switch, it is important to study the capability for bistability of the underlying biochemical network structure. Chemical Reaction Network Theory (CRNT may help at this level to decide whether a given network has the capacity for multiple positive equilibria, based on their structural properties. However, in order to build a working switch, we also need to ensure that the bistability property is robust, by studying the conditions leading to the existence of two different steady states. In the reverse engineering of biological switches, knowledge collected about the bistable regimes of the underlying potential model structures can contribute at the model identification stage to a drastic reduction of the feasible region in the parameter space of search. In this work, we make use and extend previous results of the CRNT, aiming not only to discriminate whether a biochemical reaction network can exhibit multiple steady states, but also to determine the regions within the whole space of parameters capable of producing multistationarity. To that purpose we present and justify a condition on the parameters of biochemical networks for the appearance of multistationarity, and propose an efficient and reliable computational method to check its satisfaction through the parameter space.

  8. A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

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    Vidone, Michele; Clima, Rosanna; Santorsola, Mariangela; Calabrese, Claudia; Girolimetti, Giulia; Kurelac, Ivana; Amato, Laura Benedetta; Iommarini, Luisa; Trevisan, Elisa; Leone, Marco; Soffietti, Riccardo; Morra, Isabella; Faccani, Giuliano; Attimonelli, Marcella; Porcelli, Anna Maria; Gasparre, Giuseppe

    2015-06-01

    Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  9. The clinical, biochemical and genetic features associated with RMND1-related mitochondrial disease

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    Ng, Yi Shiau; Alston, Charlotte L; Diodato, Daria;

    2016-01-01

    BACKGROUND: Mutations in the RMND1 (Required for Meiotic Nuclear Division protein 1) gene have recently been linked to infantile onset mitochondrial disease characterised by multiple mitochondrial respiratory chain defects. METHODS: We summarised the clinical, biochemical and molecular genetic......, developmental delay and lactic acidaemia are common clinical manifestations with disease onset under 2 years. Renal involvement is more prevalent than seizures (66% vs 44%). In addition, median survival time was longer in patients with renal involvement compared with those without renal disease (6 years vs 8...

  10. Biochemical indications of cerebral ischaemia and mitochondrial dysfunction in severe brain trauma analysed with regard to type of lesion

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    Nordström, Carl-Henrik; Nielsen, Troels Halfeld; Schalén, Wilhelm

    2016-01-01

    ), cerebral haemorrhagic contusion (CHC) and no mass lesion (NML). Altogether about 150,000 biochemical analyses were performed during the initial 96 h after trauma. Compromised aerobic metabolism occurred during 38 % of the study period. The biochemical pattern indicating mitochondrial dysfunction was more...

  11. Biochemical and histological characterization of tomato mutants

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    Carolina C. Monteiro

    2012-06-01

    Full Text Available Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT, we observed that the malondialdehyde (MDA content was enhanced in the diageotropica (dgt and lutescent (l mutants, whilst the highest levels of hydrogen peroxide (H2O2 were observed in high pigment 1 (hp1 and aurea (au mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT activity when compared to MT. Guaiacol peroxidase (GPOX was enhanced in both sitiens (sit and notabilis (not mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX. Based on PAGE analysis, the activities of glutathione reductase (GR isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD isoform III was reduced in leaves of sit, epi, Never ripe (Nr and green flesh (gf mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.Neste trabalho, analisamos as respostas bioquímicas inerentes ao sistema antioxidante, assim como propriedades morfológicas e anatômicas de mutantes fotomorfogenéticos e hormonais de tomateiro. Comparados ao não mutante Micro-Tom (MT, observamos que o conteúdo de malondialdeído (MDA aumentou nos mutantes diageotropica (dgt e lutescent (l, enquanto os maiores níveis de H2O2 foram encontrados nos mutantes high pigment 1 (hp1 e aurea (au. Análises de enzimas antioxidantes mostraram que todos os mutantes reduziram a atividade de catalase (CAT quando comparado a MT. A

  12. [Chronic progressive external ophthalmoplegia with mitochondrial anomalies. Clinical, histological, biochemical and genetic analysis (9 cases)].

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    Drouet, A

    1996-01-01

    We report the clinical signs and histological findings in nine patients with mitochondrial ocular myopathies. There were four males and five females. Of age ranging from 47 to 82 years. A more often asymetrical ptosis was in all cases of chronic progressive external ophtalmoplegia (CPEO), but muscle weakness in limbs was not usual. The prognosis in this group was good, but ubidecarenone (150 mg/d) used for two cases, did not improve ophtalmoplegia. The serum creatine kinase was normal in eight of nine cases and electromyography showed myopathic changes in three cases. Histoenzymatic analysis of the muscle biopsy and biochemical studies of mitochondria isolated from the muscle sample demonstrated mitochondrial myopathy associated with partial deficiency of complexes I and/or IV of the electron transfer chain. One of seven patients studied had single deletion by Southern blot analysis, in a heteroplasmic state and another an A-->G transition at position 3243 within the mitochondrial tRNA leu (UUR) gene. Chronic progressive external ophtalmoplegia, without large deletion, may have abnormality in other coding regions of mt DNA such as tRNA, rRNA or protein genes.

  13. Mitochondrial glutamate carriers from Drosophila melanogaster: biochemical, evolutionary and modeling studies.

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    Lunetti, Paola; Cappello, Anna Rita; Marsano, René Massimiliano; Pierri, Ciro Leonardo; Carrisi, Chiara; Martello, Emanuela; Caggese, Corrado; Dolce, Vincenza; Capobianco, Loredana

    2013-10-01

    The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.

  14. Biochemical and molecular characterization of hazelnut (Corylus avellana) seed lipoxygenases.

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    Santino, Angelo; De Paolis, Angelo; Gallo, Antonia; Quarta, Angela; Casey, Rod; Mita, Giovanni

    2003-11-01

    Plant lipoxygenases (LOXs) are a class of dioxygenases which display diverse functions in several physiological processes such as growth, development and response to biotic and abiotic stresses. Even though LOXs have been characterized from several plant species, the physiological role of seed LOXs is still unclear. With the aim to better clarify the occurrence of LOXs and their influence on hazelnut seed quality, we carried out the biochemical and molecular characterization of the main LOX isoforms expressed during seed development. A genomic clone containing a complete LOX gene was isolated and fully characterized. The 9887 bp sequence reported contains an open reading frame of 5334 bp encoding a putative polypeptide of 99 kDa. Semiquantitative RT-PCR carried out from RNAs extracted from seeds at different maturation stages showed that LOXs are mainly expressed at early developmental stages. These results were confirmed by LOX activity assays. Biochemical characterization of the reaction products of the hazelnut LOX indicated that it is a 9-LOX. Two cDNAs were isolated by RT-PCR carried out on total RNA from immature hazelnut seeds. Sequence analysis indicated that the two cDNAs are highly homologous (91.9% degree of identity) and one of these corresponded exactly to the genomic clone. The deduced amino acid sequences of the hazelnut LOXs showed that they are closely related to a previously reported almond LOX (79.5% identity) and, to a lesser extent, to some LOXs involved in plant responses to pathogens (cotton and tobacco LOXs, 75.5 and 74.6% identity, respectively). The physiological role of hazelnut LOXs and their role in influencing seed quality are also discussed.

  15. Neuroprotective Effect of Lycopene Against PTZ-induced Kindling Seizures in Mice: Possible Behavioural, Biochemical and Mitochondrial Dysfunction.

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    Bhardwaj, Manveen; Kumar, Anil

    2016-02-01

    Oxidative stress and mitochondrial dysfunction are the major contributing factors in the pathophysiology of various neurological disorders. Recently, antioxidant therapies aimed at reducing oxidative stress gained a considerable attention in epilepsy treatment. Lycopene, a carotenoid antioxidant, has received scientific interest in recent years. So, the present study has been designed to evaluate the neuroprotective effect of lycopene against the pentylenetetrazol (PTZ)-induced kindling epilepsy. Laca mice received lycopene (2.5, 5 and 10 mg/kg) and sodium valproate for a period of 29 days and PTZ (40 mg/kg i.p (Intraperitoneal)) injection on alternative days. Various behavioural (kindling score), biochemical parameters (lipid peroxidation, superoxide dismutase, reduced glutathione, catalase and nitrite) and mitochondrial enzyme complex activities (I, II and IV) were assessed in the brain. Results depicted that repeated administration of a sub-convulsive dose of PTZ (40 mg/kg) significantly increased kindling score, oxidative damage and impaired mitochondrial enzyme complex activities (I, II and IV) as compared with naive animals. Lycopene (5 and 10 mg/kg) and sodium valproate (100 mg/kg) treatment for a duration of 29 days significantly attenuated kindling score, reversed oxidative damage and restored mitochondrial enzyme complex activities (I, II and IV) as compared with control. Thus, present study demonstrates the neuroprotective potential of lycopene in PTZ-induced kindling in mice.

  16. Expression, purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase.

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    Wang, You; Xie, Juan-Juan; Han, Zhong; Liu, Jian-Hua; Liu, Xi-Peng

    2013-02-01

    We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg(2+) and Mn(2+). The optimal concentrations of ATP cofactor and Mg(2+) ion were 0.01-2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5' to the nick inhibited ligation more than those at the first position 3' to the nick. The mismatches at other positions 5' to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5' to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5' to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.

  17. Expression and biochemical characterization of recombinant human epididymis protein 4.

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    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

  18. Biochemical characterization of the DNA ligase I from Entamoeba histolytica.

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    Cardona-Felix, Cesar S; Pastor-Palacios, Guillermo; Cardenas, Helios; Azuara-Liceaga, Elisa; Brieba, Luis G

    2010-11-01

    DNA ligases play an essential role in DNA replication and repair. Herein, we report the cloning and biochemical characterization of DNA ligase I from the protozoan parasite Entamoeba histolytica (EhDNAligI). EhDNAligI is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I. This report shows that heterologous expressed EhDNAligI is able to perform the three conserved steps of a DNA ligation reaction: adenylation, binding to a 5'-phosphorylated nicked DNA substrate and sealing of the nick. EhDNAligI is strongly inhibited by NaCl and displays optimal activity at pH 7.5. EhDNAligI uses Mn2+ or Mg2+ as metal cofactors and ATP as nucleotide cofactor. EhDNAligI has a nicked DNA binding constant of 6.6microM and follows Michaelis-Menten steady-state kinetics with a K(m) ATP of 64nM and a k(cat) of 2.4min(-1). Accordingly to its properties as a family I DNA ligase, EhDNAligI is able to ligate a RNA strand upstream of a nucleic acid nick, but not in the downstream or the template position. We propose that EhDNAligI is involved in sealing DNA nicks during lagging strand synthesis and may have a role in base excision repair in E. histolytica.

  19. Biochemical and mechanical characterization of Nereis worm jaws

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    Broomell, Christopher C.

    The ultimate goal of biomimetics is to elucidate the design principles governing performance in biological materials and apply them to engineering systems. Successful transfer of these principles will require a thorough understanding of the complex interplay between molecular composition, organization and mechanical properties of the material. This dissertation describes the mechanical and biochemical characterization of jaws from the marine polychaete Nereis virens. Nereid jaws possess remarkable mechanical properties considering their predominantly organic composition. Hardness and stiffness are comparable to human dentin. However, in stark contrast to dentin, in Nereis these properties are achieved without mineralization. The role of metal ions in jaw sclerotization is addressed. In the pristine state, Zn ions are concentrated at the tip and toothed-edge of the jaw and are critical for hardness and modulus; both properties are reduced by ˜70% following Zn removal by treatment with EDTA. Furthermore, metal content in the jaw can be manipulated by soaking Zn-depleted samples in metal solutions; the comparative effects of treatment with alternative transition metals under both dry and hydrated conditions are described. The molecular composition of the jaw is also addressed. Protein comprises ˜90% of the jaw mass; amino acid analysis indicates that histidine is increased in the hardened, Zn-rich tip. The major protein component in Nereid jaw extracts is purified and characterized by partial peptide mapping and isolation of a partial clone from a jaw pulp cDNA library. Nvjp-1 is a 38 kDa glycine- histidine-rich protein and is believed to be the principle structural protein in the hardened jaw tip. The effects of selected environmental factors on Nvjp-1 structure and assembly are described. Transition from low to high pH is accompanied by changes in secondary structure and a significant molecular elongation. Furthermore, exposure to transition metals, notably Zn and

  20. A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders

    NARCIS (Netherlands)

    Rodenburg, R.J.T.; Schoonderwoerd, G.C.; Tiranti, V.; Taylor, R.W.; Rotig, A.; Valente, L.; Invernizzi, F.; Chretien, D.; He, L.; Backx, G.P.; Janssen, K.J.; Chinnery, P.F.; Smeets, H.J.M.; Coo, I.F. de; Heuvel, L.P. van den

    2013-01-01

    A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made

  1. Biochemical and structural characterization of the apicoplast dihydrolipoamide dehydrogenase of Plasmodium falciparum.

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    Laine, Larissa M; Biddau, Marco; Byron, Olwyn; Müller, Sylke

    2015-01-14

    PDC (pyruvate dehydrogenase complex) is a multi-enzyme complex comprising an E1 (pyruvate decarboxylase), an E2 (dihydrolipomide acetyltransferase) and an E3 (dihydrolipoamide dehydrogenase). PDC catalyses the decarboxylation of pyruvate and forms acetyl-CoA and NADH. In the human malaria parasite Plasmodium falciparum, the single PDC is located exclusively in the apicoplast. Plasmodium PDC is essential for parasite survival in the mosquito vector and for late liver stage development in the human host, suggesting its suitability as a target for intervention strategies against malaria. Here, PfaE3 (P. falciparum apicoplast E3) was recombinantly expressed and characterized. Biochemical parameters were comparable with those determined for E3 from other organisms. A homology model for PfaE3 reveals an extra anti-parallel β-strand at the position where human E3BP (E3-binding protein) interacts with E3; a parasite-specific feature that may be exploitable for drug discovery against PDC. To assess the biological role of Pfae3, it was deleted from P. falciparum and although the mutants are viable, they displayed a highly synchronous growth phenotype during intra-erythrocytic development. The mutants also showed changes in the expression of some mitochondrial and antioxidant proteins suggesting that deletion of Pfae3 impacts on the parasite's metabolic function with downstream effects on the parasite's redox homoeostasis and cell cycle.

  2. Biochemical characterization of legume seeds as ingredients in animal feed

    Energy Technology Data Exchange (ETDEWEB)

    Martín-Pedrosa, M.; Varela, A.; Guillamon, E.; Cabellos, B.; Burbano, C.; Gomez-Fernandez, J.; Mercado, E. de; Gomez-Izquierdo, E.; Cuadrado, C.; Muzquiz, M.

    2016-11-01

    The current European protein deficit is estimated as high as 70% of present needs. Because of the high protein content of their seeds, grain legumes are attractive candidates for lowering the deficiency in plant protein production. The objective of this work was to identify new sources of vegetable protein that would reduce our high dependence of soy, the main source of protein in the manufacture of feedstuffs. To achieve this goal, we determined the proximate composition, the bioactive components, as well as the antinutritional factors present in the studied seeds. In general, the protein, fat and carbohydrates content of legume seeds studied were within the limits found in the literature. The bioactive compounds detected in all the seeds were α-galactosides, myoinositol phosphates, protease inhibitors and phenols. IP6 (phytic acid) was the main inositol phosphate form in all the samples. The highest protease inhibitors content was detected in both Lathyrus cicera cultivars. Vicia ervilia and L. cicera cultivars showed low haemagglutinating activity (20.4 HU/g). The γ-glutamyl-S-ethenyl-cysteine content in Vicia narbonensis was around 16.0 mg/g. Both L. cicera varieties presented similar β-N-oxalyl-L-α, β-diaminopropionic acid content (0.80 mg/g). The two V. ervilia varieties showed high canavanine concentration (1.93-5.28 mg/g). Vicine was only detected in V. narbonensis cultivars (0.3 mg/g). The biochemical characterization carried out in this study allows us to know the limits of inclusion of these minor crop seeds in feed formulations in order to replace the soybean. (Author)

  3. Biochemical characterization of legume seeds as ingredients in animal feed

    Directory of Open Access Journals (Sweden)

    Mercedes Martín-Pedrosa

    2016-03-01

    Full Text Available The current European protein deficit is estimated as high as 70% of present needs. Because of the high protein content of their seeds, grain legumes are attractive candidates for lowering the deficiency in plant protein production. The objective of this work was to identify new sources of vegetable protein that would reduce our high dependence of soy, the main source of protein in the manufacture of feedstuffs. To achieve this goal, we determined the proximate composition, the bioactive components, as well as the antinutritional factors present in the studied seeds. In general, the protein, fat and carbohydrates content of legume seeds studied were within the limits found in the literature. The bioactive compounds detected in all the seeds were α-galactosides, myoinositol phosphates, protease inhibitors and phenols. IP6 (phytic acid was the main inositol phosphate form in all the samples. The highest protease inhibitors content was detected in both Lathyrus cicera cultivars. Vicia ervilia and L. cicera cultivars showed low haemagglutinating activity (20.4 HU/g. The γ-glutamyl-S-ethenyl-cysteine content in Vicia narbonensis was around 16.0 mg/g. Both L. cicera varieties presented similar β-N-oxalyl-L-α, β-diaminopropionic acid content (0.80 mg/g. The two V. ervilia varieties showed high canavanine concentration (1.93-5.28 mg/g. Vicine was only detected in V. narbonensis cultivars (0.3 mg/g. The biochemical characterization carried out in this study allows us to know the limits of inclusion of these minor crop seeds in feed formulations in order to replace the soybean.

  4. A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders

    OpenAIRE

    2013-01-01

    A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large varia...

  5. A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders.

    Science.gov (United States)

    Rodenburg, R J T; Schoonderwoerd, G C; Tiranti, V; Taylor, R W; Rötig, A; Valente, L; Invernizzi, F; Chretien, D; He, L; Backx, G P B M; Janssen, K J G M; Chinnery, P F; Smeets, H J; de Coo, I F; van den Heuvel, L P

    2013-01-01

    A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample.

  6. A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders

    Science.gov (United States)

    Rodenburg, R.J.T.; Schoonderwoerd, G.C.; Tiranti, V.; Taylor, R.W.; Rötig, A.; Valente, L.; Invernizzi, F.; Chretien, D.; He, L.; Backx, G.P.B.M.; Janssen, K.J.G.M.; Chinnery, P.F.; Smeets, H.J.; de Coo, I.F.; van den Heuvel, L.P.

    2013-01-01

    A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample. PMID:23164799

  7. Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins.

    NARCIS (Netherlands)

    Schwab, M.A.; Sauer, S.W.; Okun, J.G.; Nijtmans, L.G.J.; Rodenburg, R.J.T.; Heuvel, L.P.W.J. van den; Drose, S.; Brandt, U.; Hoffmann, G.F.; Laak, H.J. ter; Kolker, S.; Smeitink, J.A.M.

    2006-01-01

    Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternat

  8. Physicochemical and biochemical characterization of biosurfactants released by Lactobacillus strains

    NARCIS (Netherlands)

    Velraeds, MMC; vanderMei, HC; Reid, G; Busscher, HJ

    1996-01-01

    Biosurfactants from Lactobacillus casei subsp. rhamnosus 36 and ATCC 7469, Lactobacillus fermentum B54 and Lactobacillus acidophilus RC14 were isolated from bacteria in their mid-exponential (4-5 h) and stationary growth phases (18 h) and physicochemical and biochemical properties of the freeze-drie

  9. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Directory of Open Access Journals (Sweden)

    Alberto Cruz-Bermúdez

    Full Text Available The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids. Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  10. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  11. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J.; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A.

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations. PMID:26784702

  12. Ultrastructural and biochemical aspects of liver mitochondria during recovery from ethanol-induced alterations. Experimental evidence of mitochondrial division.

    Science.gov (United States)

    Koch, O. R.; Roatta de Conti, L. L.; Bolaños, L. P.; Stoppani, A. O.

    1978-01-01

    To study the morphologic and biochemical changes occuring in liver mitochondria during recovery from ethanol-induced injury, rats fed a 6-month high-alcohol regimen plus a nutritionally adequate diet which did not induce fatty liver were compared with isocalorically fed controls. After this period the alcohol-fed animals displayed striking ultrastructural changes of liver mitochondria and a decreased respiratory activity with succinate or malate-glutamate as substrate. On the contrary, the respiratory rate with I-glycerophosphate was 50% increased. Regression changes were studied after alcohol was withdrawn from the diet. Enlarged mitochondria rapidly disappeared (in 24 hours), although a few megamitochondria were still present after 8 days of abstinence. A similar recovery was observed for the functional alterations. At the end of the experimental period, only a slight decrease of the maximal respiratory rate using malate-glutamate as a substrate was noted. The ultrastructural findings and the morphometric data suggest that the way in which mitochondrial normalization takes place is based on partition of these organelles. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 1 Figure 2 Figure 13 PMID:623205

  13. Characterizing Autism Spectrum Disorders by Key Biochemical Pathways

    Directory of Open Access Journals (Sweden)

    Megha eSubramanian

    2015-09-01

    Full Text Available The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions.

  14. Characterizing autism spectrum disorders by key biochemical pathways.

    Science.gov (United States)

    Subramanian, Megha; Timmerman, Christina K; Schwartz, Joshua L; Pham, Daniel L; Meffert, Mollie K

    2015-01-01

    The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions.

  15. Biochemical characterization of exercise-trained porcine myocardium.

    Science.gov (United States)

    Laughlin, M H; Hale, C C; Novela, L; Gute, D; Hamilton, N; Ianuzzo, C D

    1991-07-01

    The purpose of this study was to determine whether cardiac biochemical adaptations are induced by chronic exercise training (ET) of miniature swine. Female Yucatan miniature swine were trained on a treadmill or were cage confined (C) for 16-22 wk. After training, the ET pigs had increased exercise tolerance, lower heart rates during exercise at submaximal intensities, moderate cardiac hypertrophy, increased coronary blood flow capacity, and increased oxidative capacity of skeletal muscle. Myosin from both the C and ET hearts was 100% of the V3 isozyme, and there were no differences between the myosin adenosine triphosphatase (ATPase) or myofibrillar ATPase activities of C and ET hearts. Also, the sarcoplasmic reticulum Ca(2+)-ATPase activity and Na(+)-Ca2+ exchange activity of sarcolemmal vesicles were the same in cardiac muscle of C and ET hearts. Finally, the glycolytic and oxidative capacity of ET cardiac muscle was not different from control, since phosphofructokinase, citrate synthase, and 3-hydroxyacyl-CoA dehydrogenase activities were the same in cardiac tissue from ET and C pigs. We conclude that endurance exercise training does not provide sufficient stress on the heart of a large mammal to induce changes in any of the three major cardiac biochemical systems of the porcine myocardium: the contractile system, the Ca2+ regulatory systems, or the metabolic system.

  16. Characterization of mitochondrial populations during stem cell differentiation.

    Science.gov (United States)

    Kerscher, Petra; Bussie, Blakely S; DeSimone, Katherine M; Dunn, David A; Lipke, Elizabeth A

    2015-01-01

    Mitochondrial dynamics play an important role in numerous physiological and pathophysiological phenomena in the developing and adult human heart. Alterations in structural aspects of cellular mitochondrial composition as a function of changes in physiology can easily be visualized using fluorescence microscopy. Commonly, mitochondrial location, number, and morphology are reported qualitatively due to the lack of automated and user-friendly computer-based analysis tools. Mitochondrial Quantification using MATLAB (MQM) is a computer-based tool to quantitatively assess these parameters by analyzing fluorescently labeled mitochondria within the cell; in particular, MQM provides numerical information on the number, area, and location of mitochondria within a cell in a time-efficient, automated, and unbiased way. This chapter describes the use of MQM's capabilities to quantify mitochondrial changes during human pluripotent stem cell (hPSC) differentiation into spontaneously contracting cardiomyocytes (SC-CMs), which follows physiological pathways of human heart development.

  17. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian;

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS...... classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...

  18. Molecular and biochemical characterization of Paragonimus westermani tyrosinase.

    Science.gov (United States)

    Bae, Y-A; Kim, S-H; Ahn, C-S; Kim, J-G; Kong, Y

    2015-05-01

    Trematode tyrosinases (TYRs) play a major role in the tanning process during eggshell formation. We investigated the molecular and biochemical features of Paragonimus westermani TYR (PwTYR). The PwTYR cDNA was composed of 1568-bp encompassing a 1422-bp-long open reading frame (474-amino acid polypeptide). A strong phylogenetic relationship with Platyhelminthes and Deuterostomian orthologues was evident. The recombinant PwTYR expressed in prokaryotic cells promptly oxidized diphenol substrates, with a preferential affinity toward ortho-positioned hydroxyl groups. It demonstrated fairly weak activity for monophenol compounds. Diphenol oxidase activity was augmented with an increase of pH from 5.0 to 8.0, while monophenol oxidase activity was highest at an acidic pH and gradually decreased as pH increased. Transcription profile of PwTYR was temporally upregulated along with worm development. PwTYR was specifically localized in vitellocytes and eggs. The results suggested that conversion of tyrosine to L-dihydroxyphenylalanine by PwTYR monophenol oxidase activity might be rate-limiting step during the sclerotization process of P. westermani eggs. The pH-dependent pattern of monophenol and diphenol oxidase activity further proposes that the initial hydroxylation might slowly but steadily progress in acidic secreted vesicles of vitellocytes and the second oxidation process might be rapidly accelerated by neural or weak alkaline pH environments within the ootype.

  19. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  20. Immunological and biochemical characterization of extracellular polysaccharides of mucoralean moulds.

    NARCIS (Netherlands)

    Ruiter, de G.A.

    1993-01-01

    In this thesis the characterization is described of the antigenic determinants (epitopes) of the extracellular polysaccharides (EPSs) from moulds belonging to the order of Mucorales. Detailed knowledge of the structure of these epitopes allows for further development of a new generation of methods f

  1. Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

    NARCIS (Netherlands)

    Natalia, Dessy; Vidilaseris, Keni; Satrimafitrah, Pasjan; Ismaya Purkan, Wangsa T.; Permentier, Hjalmar; Fibriansah, Guntur; Puspasari, Fernita; Nurachman, Zeily; Dijkstra, Bauke; Soemitro, Soetijoso

    2011-01-01

    Glucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6-6.4 and at 50A degrees C. T

  2. Bile salt hydrolase of Bifidobacterium longum - Biochemical and genetic characterization

    NARCIS (Netherlands)

    Tanaka, H; Hashiba, Honoo; Kok, Jan; Mierau, Igor

    2000-01-01

    A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized, Furthermore, we describe for the first time cloning and analysis of the gene encoding BSII (bsh) in a member of the genus Bifidobacterium. The enzyme has a native molecular weight of 125,000 to

  3. GENETIC CHARACTERIZATION OF ROMANIAN CATTLE BREEDS USING BIOCHEMICAL MARKERS

    Directory of Open Access Journals (Sweden)

    MARIANA REBEDEA

    2013-12-01

    Full Text Available The paper presents a genetic characterization of cattle breeds in Romania based onbiochemical markers in the blood and the milk. The surveyed breeds are: RomanianBlack Spotted Cattle (BNR, Romanian Spotted Cattle (BR, Romanian Brown (Band Romanian Steppe, and the markers identified are represented by some proteins,serum transferrin (Tf, serum albumins (Al, hemoglobin (Hb respectively-from theblood and beta-lactoglobulin (βLg-from the milk. In order to determine thegenotypes in the studied populations electrophoresis was used in three differentvariations, depending on the type of the protein, and the migration substrates usedwere starch and polyacrylamide. The identified genetic structures in the individualsfrom the surveyed breeds allowed their genetic characterization based on gene andgenotype frequencies, as well as using these data in establishing the identity andpaternity of the individuals in the surveyed breeds.

  4. Biochemical Characterization of Complexes with p21, a CDK Inhibitor

    Science.gov (United States)

    1998-08-01

    additional experiments to further characterize p28 and p40 , two potentially novel proteins that co-fractionated with p21 on glycerol gradients, sizing...well as with amino- and carboxy-terminal fragments of p21. Neither p28 nor p40 was captured in preliminary binding experiments, suggesting that these...an additional step of 1.0 HMGNB (25 mM Z 75 HEPES [pH 7.6], 1 M NaCI, 10% glycerol, 0.1% Nonidet P-40 [NP-40], 5 mM U P-mercaptoethanol, and 0.2 mM

  5. Biochemical characterization of GM1 micelles-Amphotericin B interaction.

    Science.gov (United States)

    Leonhard, Victoria; Alasino, Roxana V; Bianco, Ismael D; Garro, Ariel G; Heredia, Valeria; Beltramo, Dante M

    2015-01-01

    In this work a thorough characterization of the GM1 micelle-Amphotericin B (AmB) interaction was performed. The micelle formation as well as the drug loading occurs spontaneously, although influenced by the physicochemical conditions, pH and temperature. The chromatographic profile of GM1-AmB complexes at different molar ratios shows the existence of two populations. The differential absorbance of GM1, monomeric and aggregate AmB, allowed us to discriminate the presence of all of them in both fractions. Thus, we noted that at higher proportion of AmB in the complex, increases the larger population which is composed mainly of aggregated AmB. The physical behavior of these micelles shows that both GM1- AmB complexes were stable in solution for at least 30 days. However upon freeze-thawing or lyophilization-solubilization cycles, only the smallest population, enriched in monomeric AmB, showed a complete solubilization. In vitro, GM1-AmB micelles were significantly less toxic on cultured cells than other commercial micellar formulations as Fungizone, but had a similar behavior to liposomal formulations as Ambisome. Regarding the antifungal activity of the new formulation, it was very similar to that of other formulations. The characterization of these GM1-AmB complexes is discussed as a potential new formulation able to improve the antifungal therapeutic efficiency of AmB.

  6. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    Science.gov (United States)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data. PMID:27721652

  7. Characterization of mitochondrial function in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

    Science.gov (United States)

    Atlante, Anna; Favia, Maria; Bobba, Antonella; Guerra, Lorenzo; Casavola, Valeria; Reshkin, Stephan Joel

    2016-06-01

    Evidence supporting the occurrence of oxidative stress in Cystic Fibrosis (CF) is well established and the literature suggests that oxidative stress is inseparably linked to mitochondrial dysfunction. Here, we have characterized mitochondrial function, in particular as it regards the steps of oxidative phosphorylation and ROS production, in airway cells either homozygous for the F508del-CFTR allele or stably expressing wt-CFTR. We find that oxygen consumption, ΔΨ generation, adenine nucleotide translocator-dependent ADP/ATP exchange and both mitochondrial Complex I and IV activities are impaired in CF cells, while both mitochondrial ROS production and membrane lipid peroxidation increase. Importantly, treatment of CF cells with the small molecules VX-809 and 4,6,4'-trimethylangelicin, which act as "correctors" for F508del CFTR by rescuing the F508del CFTR-dependent chloride secretion, while having no effect per sè on mitochondrial function in wt-CFTR cells, significantly improved all the above mitochondrial parameters towards values found in the airway cells expressing wt-CFTR. This novel study on mitochondrial bioenergetics provides a springboard for future research to further understand the molecular mechanisms responsible for the involvement of mitochondria in CF and identify the proteins primarily responsible for the F508del-CFTR-dependent mitochondrial impairment and thus reveal potential novel targets for CF therapy.

  8. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    DEFF Research Database (Denmark)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with ...... was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s-1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s-1 μM-1 for TgTPS2. The kinetic parameters were in agreement with previously published data....

  9. Biochemical characterization of riboflavin carrier protein (RCP) in prostate cancer.

    Science.gov (United States)

    Johnson, Tanya; Ouhtit, Allal; Gaur, Rajiv; Fernando, Augusta; Schwarzenberger, Paul; Su, Joseph; Ismail, Mohamed F; El-Sayyad, Hassan I; Karande, Anjali; Elmageed, Zakaria Abd; Rao, Prakash; Raj, Madhwa

    2009-01-01

    Riboflavin carrier protein (RCP) is a growth- and development-specific protein. Here, we characterized the expression of this protein in prostate cancer by polyclonal and monoclonal antibodies against chicken RCP. RCP was localized to both androgen-dependent and independent prostate cancer cell lines. Compared to controls, RCP was over-expressed in all 45 prostate adenocarcinomas, irrespective of the Gleason's score or the stage of the disease. The identified RCP had a molecular weight of 38 kDa, similar to RCP purified from chicken. Presence of this protein was also confirmed by siRNA inhibition analysis. Antibodies to chicken RCP inhibited incorporation of tritiated thymidine into DNA and prevented riboflavin uptake in PC3 prostate cancer cells, suggesting a critical function of this protein in prostate cancer cell growth. These data suggest that RCP can be used as a tumor biomarker in prostate cancer.

  10. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    Directory of Open Access Journals (Sweden)

    Mariana Vieira Tomazett

    Full Text Available Abstract Carbonic anhydrases (CA belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4. In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1 and CA4 (rCA4, with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class and rCA4 (α-class were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration, arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

  11. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal alpha-amylase enzymes

    NARCIS (Netherlands)

    van der Kaaij, R. M.; Janecek, S.; van der Maarel, M. J. E. C.; Dijkhuizen, L.; Janeček, Š.

    2007-01-01

    Currently known fungal alpha-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal a-amylases. The phylogenetic analysis sho

  12. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

    NARCIS (Netherlands)

    Kaaij, R.M. van der; Janeček, Š.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2007-01-01

    Currently known fungal α-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal α-amylases. The phylogenetic analysis shows t

  13. Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR

    Directory of Open Access Journals (Sweden)

    Skjerve Eystein

    2009-12-01

    Full Text Available Abstract Background Bovine brucellosis caused by Brucella abortus is endemic in most large commercial and smallholder cattle farms of Zimbabwe, while brucellosis in other domestic animals is rare. The diagnosis of brucellosis is mainly accomplished using serological tests. However, some Brucella spp. have been isolated from clinical cases in the field and kept in culture collection but their biochemical profiles were not documented. We report biochemical profiling and AMOS-PCR characterization of some of these field isolates of Brucella originating from both commercial and smallholder cattle farming sectors of Zimbabwe. Findings Fourteen isolates of Brucella from culture collection were typed using biochemical profiles, agglutination by monospecific antisera, susceptibility to Brucella-specific bacteriophages and by AMOS-PCR that amplifies species- specific IS711. The results of the biochemical profiles for B. abortus biovar 1 (11 isolates and biovar 2 (2 isolates were consistent with those of reference strains. A single isolate from a goat originating from a smallholder mixed animal farm was identified as B. melitensis biovar 1. The AMOS-PCR produced DNA products of sizes 498 bp and 731 bp for B. abortus (biovar 1 and 2 and B. melitensis biovar 1, respectively. Conclusion We concluded that the biochemical profiles and AMOS-PCR characterization were consistent with their respective species and biovars. B. abortus biovar 1 is likely to be the predominant cause of brucellosis in both commercial and smallholder cattle farms in Zimbabwe.

  14. Biochemical characterization of RNase HⅡ from Aeropyrum pernix

    Institute of Scientific and Technical Information of China (English)

    Jingli Hou; Yufen Liu; Zheng Lu; Xipeng Liu; Jianhua Liu

    2012-01-01

    Aeropyrum pernix contains one homolog of ribonuclease H (RNase H),A.pernix RNase HⅡ (Ape-RNase HⅡ).Activity characterization showed that Ape-RNase HⅡ exhibited the highest activity in the presence of 5 mM Mn2+, 1 mM Co2+, or 10mM Mg2+, respectively;however,its cleavage efficiencies at different cleavage sites for Mn2+ and Mg2+ were different.Ape-RNase HⅡ cleaved 12-bp RNA/DNA substrates at multiple sites and the optimum pH value was 11.0.Moreover,16-bp DNAr4-DNA/DNA and 13-bp DNA-r1-DNA/DNA chimeric substrates were cleaved at DNA-RNA junction.ApeRNase HⅡ was thermostable and the stabilization was enhanced with increased salt concentration.This work is believed to be the first in vitro functional study of ApeRNase HⅡ and the results should contribute to the analysis of RNase H of other archaeal species.

  15. Biochemical characterization and growth patterns of new yeast isolates.

    Science.gov (United States)

    Djegui, Kadjogbé Y; Gachomo, Emma W; Hounhouigan, Djidjoho J; Kayodé, Adéchola P P; Kotchoni, Simeon O

    2014-08-01

    African sorghum opaque beers play a vital role in the diet of millions of consumers. In the current study we investigated the growth profiles of yeast strains isolated from kpete-kpete, a traditional starter used to produce tchoukoutou, an opaque sorghum beer in Benin. 10 yeast strains were isolated from sorghum beer starters and cultivated under both liquid and solid media for phenotypic growth characterization. All yeast isolates were able to grow both on solid and liquid media. Based on their growth profiles, the isolates were clustered into three groups: (i) the aggressive growth pattern (30%), (ii) the moderate growth pattern (50%), and (iii) the slow growth pattern (20%). Based on gene expression pattern, absorbance (A(600 nm)) and diameter of growth in both liquid and solid media respectively, yeast strains YK34, YK15 and YK48 were clustered in the first group, and referred to as the most aggressive growth strains, followed by group 2 (YK24, YK5, YK12, YK20, YK2) and group 3 (YK37, YK41). This growth pattern was confirmed by Invertase gene expression profiling of the yeasts showing group 1 with high level of Invertase gene expression followed by group 2 and group 3 respectively. Our results suggest that YK34, YK15 and YK48 and YK2 yeast strains constitute the best candidates in fermentation of sorghum beer production based on growth rate and assimilation of carbon and nitrogen sources.

  16. Efficient Characterization of Parametric Uncertainty of Complex (Biochemical Networks.

    Directory of Open Access Journals (Sweden)

    Claudia Schillings

    2015-08-01

    Full Text Available Parametric uncertainty is a particularly challenging and relevant aspect of systems analysis in domains such as systems biology where, both for inference and for assessing prediction uncertainties, it is essential to characterize the system behavior globally in the parameter space. However, current methods based on local approximations or on Monte-Carlo sampling cope only insufficiently with high-dimensional parameter spaces associated with complex network models. Here, we propose an alternative deterministic methodology that relies on sparse polynomial approximations. We propose a deterministic computational interpolation scheme which identifies most significant expansion coefficients adaptively. We present its performance in kinetic model equations from computational systems biology with several hundred parameters and state variables, leading to numerical approximations of the parametric solution on the entire parameter space. The scheme is based on adaptive Smolyak interpolation of the parametric solution at judiciously and adaptively chosen points in parameter space. As Monte-Carlo sampling, it is "non-intrusive" and well-suited for massively parallel implementation, but affords higher convergence rates. This opens up new avenues for large-scale dynamic network analysis by enabling scaling for many applications, including parameter estimation, uncertainty quantification, and systems design.

  17. Characterization of the biochemical properties of Campylobacter jejuni RNase III.

    Science.gov (United States)

    Haddad, Nabila; Saramago, Margarida; Matos, Rute G; Prévost, Hervé; Arraiano, Cecília M

    2013-11-25

    Campylobacter jejuni is a foodborne bacterial pathogen, which is now considered as a leading cause of human bacterial gastroenteritis. The information regarding ribonucleases in C. jejuni is very scarce but there are hints that they can be instrumental in virulence mechanisms. Namely, PNPase (polynucleotide phosphorylase) was shown to allow survival of C. jejuni in refrigerated conditions, to facilitate bacterial swimming, cell adhesion, colonization and invasion. In several microorganisms PNPase synthesis is auto-controlled in an RNase III (ribonuclease III)-dependent mechanism. Thereby, we have cloned, overexpressed, purified and characterized Cj-RNase III (C. jejuni RNase III). We have demonstrated that Cj-RNase III is able to complement an Escherichia coli rnc-deficient strain in 30S rRNA processing and PNPase regulation. Cj-RNase III was shown to be active in an unexpectedly large range of conditions, and Mn2+ seems to be its preferred co-factor, contrarily to what was described for other RNase III orthologues. The results lead us to speculate that Cj-RNase III may have an important role under a Mn2+-rich environment. Mutational analysis strengthened the function of some residues in the catalytic mechanism of action of RNase III, which was shown to be conserved.

  18. Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

    Full Text Available It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

  19. ZYMOGRAPHIC IDENTIFICATION AND BIOCHEMICAL CHARACTERIZATION OF CHITINASE AGAINST PHYTOFUNGAL PATHOGENS

    Directory of Open Access Journals (Sweden)

    Urja Pandya

    2014-08-01

    Full Text Available An endospore forming Gram positive bacterium (MBCU4 was isolated from a vermicompost amended soil, and confirmed as Bacillus subtilis through the 16S rRNA sequence analysis. An extracellular chitinase was detected from this strain of B. subtilis under specific environmental condition. An attempt was made to purify the enzyme by ammonium sulfate precipitation followed by DEAE sepharose CL-6B column chromatography. The purified enzyme was demonstrated as a single band, having the molecular weight 31kDa on SDS PAGE analysis and its activity in the gel was determined by clear zone on zymogram. Further characterization of the isolated enzymes has showed that this enzyme is most active at pH 6.0 and at the optimized temperature of 50 0C. The purified chitinase exhibited high degree of antifungal activity particularly by degrading their cell wall components of plant pathogens Macrophomina phaseolina (69.0% and Rhizoctonia solani (52.0%. It infers that the chitinase produced by B. subtilis could play an important role for biopesticidal activity.

  20. The mitochondrial genome of the fission yeast Schizosaccharomyces pombe : 5. Characterization of mitochondrial deletion mutants.

    Science.gov (United States)

    Ahne, F; Merlos-Lange, A M; Lang, B F; Wolf, K

    1984-09-01

    The three mutator strains ana (r)-8, ana (r)-14, and diu (r)-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe.

  1. Biochemical and structural characterization of Plasmodium falciparum glutamate dehydrogenase 2.

    Science.gov (United States)

    Zocher, Kathleen; Fritz-Wolf, Karin; Kehr, Sebastian; Fischer, Marina; Rahlfs, Stefan; Becker, Katja

    2012-05-01

    Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 Å resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs.

  2. Biochemical characterization of tektins from sperm flagellar doublet microtubules.

    Science.gov (United States)

    Linck, R W; Stephens, R E

    1987-04-01

    Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta-tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine-HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids.

  3. Biochemical and immunological characterization of recombinant allergen Lol p 1.

    Science.gov (United States)

    Tamborini, E; Faccini, S; Lidholm, J; Svensson, M; Brandazza, A; Longhi, R; Groenlund, H; Sidoli, A; Arosio, P

    1997-11-01

    Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des-(116-240)-Lol p 1, des-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17-18-residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.

  4. Mitochondrial functional changes characterization in young and senescent human adipose derived MSCs

    Directory of Open Access Journals (Sweden)

    Bernd Robert Stab II

    2016-12-01

    Full Text Available Mitochondria are highly dynamic organelles that in response to the cell’s bio-energetic state continuously undergo structural remodeling fission and fusion processes. This mitochondrial dynamic activity has been implicated in cell cycle, autophagy and age-related diseases. Adult tissue-derived mesenchymal stromal/stem cells present a therapeutic potential. However, to obtain an adequate mesenchymal stromal/stem cell number for clinical use, extensive in vitro expansion is required. Unfortunately, these cells undergo replicative senescence rapidly by mechanisms that are not well understood. Senescence has been associated with metabolic changes in the oxidative state of the cell, a process that has been also linked to mitochondrial fission and fusion events, suggesting an association between mitochondrial dynamic and senescence. In the present work, we studied the mitochondrial structural remodeling process of mesenchymal stromal/stem cells isolated from adipose tissue in vitro to determine if mitochondrial phenotypic changes are associated with mesenchymal stromal/stem cell senescence. For this purpose, mitochondrial dynamics and oxidative state of stromal/stem cell were compared between young and old cells. With increased cell passage, we observed a significant change in cell morphology that is associated with an increase in β-galactosidase activity. In addition, old cells (population doubling seven also showed increased mitochondrial mass, augmented superoxide production, and decreased mitochondrial membrane potential. These changes in morphology were related to slightly levels increases in mitochondrial fusion proteins, Mitofusion 1 (MFN1 and Dynamin-realted GTPase (OPA1. Collectively, our results showed that adipose tissue-derived MSCs at population doubling seven develop a senescent phenotype that is characterized by metabolic cell changes that can lead to mitochondrial fusion.

  5. Biochemical characterization of rhinovirus RNA-dependent RNA polymerase.

    Science.gov (United States)

    Hung, Magdeleine; Gibbs, Craig S; Tsiang, Manuel

    2002-11-01

    Human rhinoviruses (HRV) represent the single most important causative agent of the common cold. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome. We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus. The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E. coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity. The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined. The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism. A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme. In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity. However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template. Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation. The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical. The availability of recombinant rhinovirus RdRp in a

  6. Microbiological and biochemical characterization of fermented liquid feed samples from 40 Danish farms

    DEFF Research Database (Denmark)

    Canibe, Nuria; Pedersen, Anni Øyan; Jensen, Bent Borg;

    2010-01-01

    When feed and a liquid are mixed fermentation will spontaneously start. The microbial species dominating in the fermented mixture may vary depending on the environment and/or the ingredients being fermented. However, there is scarce knowledge on this subject. A study was carried out to investigate......' group. The biochemical characteristics and the microbiological composition to group level were determined. Furthermore, characterization of lactic acid bacteria and yeasts to species level was carried out. The biochemical characteristics and the composition of microbial groups of the two farm groups...

  7. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    Directory of Open Access Journals (Sweden)

    Maria Antonietta Ferrara

    2015-04-01

    Full Text Available A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH and Raman spectroscopy (RS. DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

  8. Structure and biochemical characterization of proliferating cellular nuclear antigen from a parasitic protozoon

    Energy Technology Data Exchange (ETDEWEB)

    Cardona-Felix, Cesar S.; Lara-Gonzalez, Samuel; Brieba, Luis G. (LNLS)

    2012-02-08

    Proliferating cellular nuclear antigen (PCNA) is a toroidal-shaped protein that is involved in cell-cycle control, DNA replication and DNA repair. Parasitic protozoa are early-diverged eukaryotes that are responsible for neglected diseases. In this work, a PCNA from a parasitic protozoon was identified, cloned and biochemically characterized and its crystal structure was determined. Structural and biochemical studies demonstrate that PCNA from Entamoeba histolytica assembles as a homotrimer that is able to interact with and stimulate the activity of a PCNA-interacting peptide-motif protein from E. histolytica, EhDNAligI. The data indicate a conservation of the biochemical mechanisms of PCNA-mediated interactions between metazoa, yeast and parasitic protozoa.

  9. Biochemical evidence for a mitochondrial genetic modifier in the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Liang, Min; Zhang, Chaofan; Zhao, Xiaoxu; He, Qiufen; Cui, Limei; Liu, Xiaoling; Sun, Yan-Hong; Fu, Qun; Ji, Yanchun; Bai, Yidong; Huang, Taosheng; Guan, Min-Xin

    2016-08-15

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease. Mitochondrial modifiers are proposed to modify the phenotypic expression of primary LHON-associated mitochondrial DNA (mtDNA) mutations. In this study, we demonstrated that the LHON susceptibility allele (m.14502T > C, p. 58I > V) in the ND6 gene modulated the phenotypic expression of primary LHON-associated m.11778G > A mutation. Twenty-two Han Chinese pedigrees carrying m.14502T > C and m.11778G > A mutations exhibited significantly higher penetrance of optic neuropathy than those carrying only m.11778G > A mutation. We performed functional assays using the cybrid cell models, generated by fusing mtDNA-less ρ(o) cells with enucleated cells from LHON patients carrying both m.11778G > A and m.14502T > C mutations, only m.14502T > C or m.11778G > A mutation and a control belonging to the same mtDNA haplogroup. These cybrids cell lines bearing m.14502T > C mutation exhibited mild effects on mitochondrial functions compared with those carrying only m.11778G > A mutation. However, more severe mitochondrial dysfunctions were observed in cell lines bearing both m.14502T > C and m.11778G > A mutations than those carrying only m.11778G > A or m.14502T > C mutation. In particular, the m.14502T > C mutation altered assemble of complex I, thereby aggravating the respiratory phenotypes associated with m.11778G > A mutation, resulted in a more defective complex I. Furthermore, more reductions in the levels of mitochondrial ATP and increasing production of reactive oxygen species were also observed in mutant cells bearing both m.14502T > C and m.11778G > A mutation than those carrying only 11778G > A mutation. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between primary and secondary mtDNA mutations.

  10. Characterization of mitochondrial haplogroups in a large population-based sample from the United States.

    Science.gov (United States)

    Mitchell, Sabrina L; Goodloe, Robert; Brown-Gentry, Kristin; Pendergrass, Sarah A; Murdock, Deborah G; Crawford, Dana C

    2014-07-01

    Mitochondrial DNA (mtDNA) haplogroups are valuable for investigations in forensic science, molecular anthropology, and human genetics. In this study, we developed a custom panel of 61 mtDNA markers for high-throughput classification of European, African, and Native American/Asian mitochondrial haplogroup lineages. Using these mtDNA markers, we constructed a mitochondrial haplogroup classification tree and classified 18,832 participants from the National Health and Nutrition Examination Surveys (NHANES). To our knowledge, this is the largest study to date characterizing mitochondrial haplogroups in a population-based sample from the United States, and the first study characterizing mitochondrial haplogroup distributions in self-identified Mexican Americans separately from Hispanic Americans of other descent. We observed clear differences in the distribution of maternal genetic ancestry consistent with proposed admixture models for these subpopulations, underscoring the genetic heterogeneity of the United States Hispanic population. The mitochondrial haplogroup distributions in the other self-identified racial/ethnic groups within NHANES were largely comparable to previous studies. Mitochondrial haplogroup classification was highly concordant with self-identified race/ethnicity (SIRE) in non-Hispanic whites (94.8 %), but was considerably lower in admixed populations including non-Hispanic blacks (88.3 %), Mexican Americans (81.8 %), and other Hispanics (61.6 %), suggesting SIRE does not accurately reflect maternal genetic ancestry, particularly in populations with greater proportions of admixture. Thus, it is important to consider inconsistencies between SIRE and genetic ancestry when performing genetic association studies. The mitochondrial haplogroup data that we have generated, coupled with the epidemiologic variables in NHANES, is a valuable resource for future studies investigating the contribution of mtDNA variation to human health and disease.

  11. Clinical, neuropathological, and biochemical characterization of the novel tau mutation P332S.

    Science.gov (United States)

    Deramecourt, Vincent; Lebert, Florence; Maurage, Claude-Alain; Fernandez-Gomez, Francisco-Jose; Dujardin, Simon; Colin, Morvane; Sergeant, Nicolas; Buée-Scherrer, Valérie; Clot, Fabienne; Ber, Isabelle Le; Brice, Alexis; Pasquier, Florence; Buée, Luc

    2012-01-01

    MAPT mutations cause autosomal dominant frontotemporal lobar degeneration. These diseases are characterized by considerable heterogeneity in their clinical, neuropathological, and biochemical presentations. We describe the full characterization of a family with autosomal dominant frontotemporal lobar degeneration caused by a novel MAPT mutation. Clinical, imaging, neuropathological, and biochemical data are presented. The proband was a woman who died at 85 years old, 25 years after the onset of a slowly progressive and isolated anarthria and opercular syndrome. The pathological examination of her brain showed marked atrophy of primary motor and premotor cortices, associated with predominant neuronal tau-positive lesions mimicking Pick bodies. At the biochemical level, the six tau isoforms aggregate to display a pathological triplet at 60, 64, and 69 kDa. Two of her sons presented at 48 and 50 years old with a right temporal variant of frontotemporal degeneration characterized by severe prosopagnosia, semantic impairment, and behavioral modifications. In these three patients, the molecular analysis of MAPT showed the c.1945C>T mutation on exon 11 resulting in the P332S substitution in tau sequence. This mutation changes the PGGG motif of the third repeat domain of the protein and therefore reduces the ability of tau to bind microtubule. From a clinical point of view, this mutation is associated with considerable intrafamilial phenotypic variation.

  12. Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA.

    Science.gov (United States)

    A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trny and rns and trnw and cox2 were identified by compa...

  13. Protective effect of montelukast against quinolinic acid/malonic acid induced neurotoxicity: possible behavioral, biochemical, mitochondrial and tumor necrosis factor-α level alterations in rats.

    Science.gov (United States)

    Kalonia, H; Kumar, P; Kumar, A; Nehru, B

    2010-11-24

    The present study has been designed to explore the protective effect of montelukast (leukotriene receptor antagonist) against intrastriatal quinolinic acid (QA; 300 nmol) and malonic acid (MA; 6 μmol) induced Huntington's like symptoms in rats. Quinolinic acid has been reported to induce excitotoxicity by stimulating the N-methyl-D-aspartate receptor, causing calcium overload which in turn leads to the neurodegeneration. On the other hand, MA, being a reversible inhibitor of mitochondrial enzyme complex-II, leads to energy crisis and free radical generation. Recent studies have reported the therapeutic potential of leukotriene receptor antagonists in different neurodegenerative disorders. However, their exact role is yet to be established. The present study accordingly, is an attempt to investigate the effect of montelukast against QA and MA induced behavioral, biochemical and molecular alterations in rat striatum. Oxidative stress, mitochondrial enzyme complex and tumor necrosis factor-alpha (TNF-α) were evaluated on day 21st and 14th post intrastriatal QA and MA treatment, respectively. Findings of the present study demonstrate significant alteration in the locomotor activity and motor coordination as well as oxidative burden (increased lipid peroxidation, nitrite concentration and decreased endogenous antioxidants), mitochondrial enzyme complex (I, II and IV) activities and TNF-α level, in both intrastriatal QA and MA treated animals. Further, montelukast (0.4, 0.8 mg/kg p.o.) treatment for 21 and 14 days respectively, attenuated the behavioral alterations, oxidative stress, mitochondrial dysfunction and TNF-α level in these models of Huntington's disease in a significant manner. In conclusion, the present study emphasizes the neuroprotective potential of montelukast in the therapeutic management of Huntington like symptoms.

  14. Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase.

    Science.gov (United States)

    Linassier, C; MacDougall, L K; Domin, J; Waterfield, M D

    1997-02-01

    Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. We have identified a representative member of each class of PI3Ks in Drosophila using a PCR-based approach. In the present paper we describe the molecular cloning of a PI3K from Drosophila, P13K_59F, that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologues, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes.

  15. Biochemical and serological characterization of mycoplasma strains isolated from the genital tracts of humans in Nigeria.

    Science.gov (United States)

    Agbakoba, N R; Adetosoye, A I; Adewole, I F

    2006-06-01

    Fifty-five (55) Mycoplasma strains isolated from the genital tracts of humans were biochemically characterized using various biochemical tests and also serologically identified by growth inhibition technique using 5 mycoplasma antisera namely M. hominis PG2 1: M. genitalium G37: M. penetrans GTU54 and 2 strains of M. fermentans PG18 (HRC 6-62-S-170 and MB713-501-069). Biochemically, 43 (78.2%) strains were identified as Mycoplasma hominis, 8 (14.5%) strains as M. fermentans and 4 (7.3%) as M. penetrans. The M. hominis strains hydrolyzed only arginine while the M. fermentans and M. penetrans strains in addition to arginine hydrolysis also broke down glucose fermentatively and oxidatively. The M. fermentans strains showed varying reactions to phosphatase activity and to the reduction of tetrazolium chloride. Serologically, 4 (7.3%) mycoplasma strains were confirmed as M. penetrans GTU54 and of the 8 M. fermentans strains, 4 (7.3%) were identified as M. fermentans PG18 serotype HRC 6-62-S-170 and the other 4 (7.3%) as M. fermentans PG18 serotype MB 713-501-069. Only 13 (30.2%) of the 43 M. hominis strains were identified as M. hominis serotype PG2 1. None was identified as M. genitalium. The heterogeneity of the mycoplasma strains especially M. hominis was observed in this study and the need for the use of multiple antisera in growth inhibition test is hereby supported.

  16. Characterization of nucleotide misincorporation patterns in the iceman's mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Cristina Olivieri

    Full Text Available BACKGROUND: The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation ("miscoding lesions" has been the object of extensive investigations. METHODOLOGY/PRINCIPAL FINDINGS: To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp mitochondrial (mt DNA sequences obtained from the 5,350-5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Otzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85% of the observed nucleotide misincorporations in ancient sequences. CONCLUSIONS/SIGNIFICANCE: This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine

  17. Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous▿ †

    OpenAIRE

    Linde López, Dolores; Macías Borrego, Isabel; Fernández Arrojo, Lucía; Plou Gasca, Francisco José; Antonio, Jiménez; Fernández Lobato, María

    2008-01-01

    An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T50 in the range 66 to 71°C) is higher than that exhibited by m...

  18. Biochemical and structural characterization of DNA ligases from bacteria and archaea

    Science.gov (United States)

    Pergolizzi, Giulia; Wagner, Gerd K.; Bowater, Richard P.

    2016-01-01

    DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterization. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5′-phosphate of the DNA end that will ultimately be joined to the 3′-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use β-nicotinamide adenine dinucleotide (β-NAD+) as their co-factor whereas those that are essential in other cells use adenosine-5′-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of β-NAD+ affords multiple opportunities for chemical modification. Several recent studies have synthesized novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes. PMID:27582505

  19. Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase.

    Science.gov (United States)

    Cheng, Tsung-Lin; Liao, Ching-Chun; Tsai, Wen-Hui; Lin, Chin-Chih; Yeh, Chin-Wei; Teng, Chiao-Fang; Chang, Wen-Tsan

    2009-08-01

    Citrate synthase (CS), the first and rate-limiting enzyme of the tricarboxylic acid (TCA) cycle, plays a decisive role in regulating energy generation of mitochondrial respiration. Most mitochondrial proteins are synthesized in the cytoplasm as preproteins with an amino (N)-terminal mitochondrial targeting sequence (MTS) that directs mitochondria-specific sorting of the preprotein. However, the MTS and targeting mechanism of the human CS protein are not fully characterized. The human CS gene is a single nuclear gene which transcribes into two mRNA variants, isoform a (CSa) and b (CSb), by alternative splicing of exon 2. CSa encodes 466 amino acids, including a putative N-terminal MTS, while CSb expresses 400 residues with a shorter N terminus, lacking the MTS. Our results indicated that CSa is localized in the mitochondria and the N-terminal 27 amino acids, including a well-conserved RXY downward arrow (S/A) motif (the RHAS sequence), can efficiently target the enhanced green fluorescent protein (EGFP) into the mitochondria. Furthermore, site-directed mutagenesis analysis of the conserved basic amino acids and serine/threonine residues revealed that the R9 residue is essential but all serine/threonine residues are dispensable in the mitochondrial targeting function. Moreover, RNA interference (RNAi)-mediated gene silencing of the preprotein import receptors, including TOM20, TOM22, and TOM70, showed that all three preprotein import receptors are required for transporting CSa into the mitochondria. In conclusion, we have experimentally identified the mitochondrial targeting sequence of human CSa and elucidated its targeting mechanism. These results provide an important basis for the study of mitochondrial dysfunction due to aberrant CSa trafficking.

  20. Biochemical characterization and antioxidant and antiproliferative activities of different Ganoderma collections.

    Science.gov (United States)

    Saltarelli, Roberta; Ceccaroli, Paola; Buffalini, Michele; Vallorani, Luciana; Casadei, Lucia; Zambonelli, Alessandra; Iotti, Mirco; Badalyan, Susanna; Stocchi, Vilberto

    2015-01-01

    The aim of this study was to conduct a molecular and biochemical characterization and to compare the antioxidant and antiproliferative activities of four Ganoderma isolates belonging to Ganoderma lucidum (Gl-4, Gl-5) and Ganoderma resinaceum (F-1, F-2) species. The molecular identification was performed by ITS and IGS sequence analyses and the biochemical characterization by enzymatic and proteomic approaches. The antioxidant activity of the ethanolic extracts was compared by three different methods and their flavonoid contents were also analyzed by high-performance liquid chromatography. The antiproliferative effect on U937 cells was determined by MTT assay. The studied mycelia differ both in the enzymatic activities and protein content. The highest content in total phenol and the highest antioxidant activity for DPPH free radical scavenging and chelating activity on Fe(2+) were observed with the Gl-4 isolate of G. lucidum. The presence of quercetin, rutin, myricetin, and morin as major flavonoids with effective antioxidant activity was detected. The ethanolic extracts from mycelia of G. lucidum isolates possess a substantial antiproliferative activity against U937 cells in contrast to G. resinaceum in which the antiproliferative effects were insignificant. This study provides a comparison between G. lucidum and G. resinaceum mycelial strains, and shows that G. resinaceum could be utilized to obtain several bioactive compounds.

  1. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    Science.gov (United States)

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  2. Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases.

    Directory of Open Access Journals (Sweden)

    Theresa Lindhout

    Full Text Available Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs, which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PSTMh. The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PSTMh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PSTMh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications.

  3. Structural and biochemical characterizations of an intramolecular tandem coiled coil protein.

    Science.gov (United States)

    Shin, Donghyuk; Kim, Gwanho; Kim, Gyuhee; Zheng, Xu; Kim, Yang-Gyun; Lee, Sangho

    2014-12-12

    Coiled coil has served as an excellent model system for studying protein folding and developing protein-based biomaterials. Most designed coiled coils function as oligomers, namely intermolecular coiled coils. However, less is known about structural and biochemical behavior of intramolecular coiled coils where coiled coil domains are covalently linked in one polypeptide. Here we prepare a protein which harbors three coiled coil domains with two short linkers, termed intramolecular tandem coiled coil (ITCC) and characterize its structural and biochemical behavior in solution. ITCC consists of three coiled coil domains whose sequences are derived from Coil-Ser and its domain swapped dimer. Modifications include positioning E (Glu) residue at "e" and K (Lys) at "g" positions throughout heptad repeats to enhance ionic interaction among its constituent coiled coil domains. Molecular modeling of ITCC suggests a compact triple helical bundle structure with the second and the third coiled coil domains forming a canonical coiled coil. ITCC exists as a mixture of monomeric and dimeric species in solution. Small-angle X-ray scattering reveals ellipsoidal molecular envelopes for both dimeric and monomeric ITCC in solution. The theoretically modeled structures of ITCC dock well into the envelopes of both species. Higher ionic strength shifts the equilibrium into monomer with apparently more compact structure while secondary structure remains unchanged. Taken together, our results suggest that our designed ITCC is predominantly monomeric structure through the enhanced ionic interactions, and its conformation is affected by the concentration of ionic species in the buffer.

  4. Compositional, microbiological, biochemical, volatile profile and sensory characterization of four Italian semi-hard goats' cheeses.

    Science.gov (United States)

    Di Cagno, Raffaella; Miracle, R Evan; De Angelis, Maria; Minervini, Fabio; Rizzello, Carlo G; Drake, Mary Anne; Fox, Patrick F; Gobbetti, Marco

    2007-11-01

    Four semi-hard Italian goats' milk cheeses, Flor di Capra (FC), Caprino di Cavalese (CC), Caprino di Valsassina (CV) and Capritilla (C), were compared for compositional, microbiological, biochemical, volatile profile and sensory characteristics. Mean values for the gross composition in part differed between cheeses. At the end of ripening, cheeses contained 7.98-8.51 log10 cfu/g of non-starter lactic acid bacteria. Lactobacillus paracasei, Lb. casei and Lb. plantarum were dominant in almost all cheeses. As shown by the Principal Component Analysis of RP-FPLC data for the pH 4.6-soluble fractions and by the determination of free amino acids, secondary proteolysis of CC and CV mainly differed from the other two cheeses. A total of 72 volatile components were identified by steam distillation-extraction followed by gas chromatography-mass spectrometry. Free fatty acids and esters qualitatively and quantitatively differentiated the profile of CV and CC, respectively. The lowest concentrations of volatile components characterized FC. Descriptive sensory analysis using 17 flavour attributes was carried out by a trained panel. Different flavour attributes distinguished the four goats' cheeses and relationships were found with volatile components, biochemical characteristics and technology.

  5. Structural, Biochemical, and Biophysical Characterization of Idelalisib Binding to Phosphoinositide 3-Kinase δ*

    Science.gov (United States)

    Somoza, John R.; Koditek, David; Villaseñor, Armando G.; Novikov, Nikolai; Wong, Melanie H.; Liclican, Albert; Xing, Weimei; Lagpacan, Leanna; Wang, Ruth; Schultz, Brian E.; Papalia, Giuseppe A.; Samuel, Dharmaraj; Lad, Latesh; McGrath, Mary E.

    2015-01-01

    Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib. PMID:25631052

  6. Development of a system for characterizing biomass quality of lignocellulosic feedstocks for biochemical conversion

    Science.gov (United States)

    Murphy, Patrick Thomas

    The purpose of this research was twofold: (i) to develop a system for screening lignocellulosic biomass feedstocks for biochemical conversion to biofuels and (ii) to evaluate brown midrib corn stover as feedstock for ethanol production. In the first study (Chapter 2), we investigated the potential of corn stover from bm1-4 hybrids for increased ethanol production and reduced pretreatment intensity compared to corn stover from the isogenic normal hybrid. Corn stover from hybrid W64A X A619 and respective isogenic bm hybrids was pretreated by aqueous ammonia steeping using ammonium hydroxide concentrations from 0 to 30%, by weight, and the resulting residues underwent simultaneous saccharification and cofermentation (SSCF) to ethanol. Dry matter (DM) digested by SSCF increased with increasing ammonium hydroxide concentration across all genotypes (P>0.0001) from 277 g kg-1 DM in the control to 439 g kg-1 DM in the 30% ammonium hydroxide pretreatment. The bm corn stover materials averaged 373 g kg-1 DM of DM digested by SSCF compared with 335 g kg-1 DM for the normal corn stover (Pcell-wall carbohydrate hydrolysis of corn stover, (ii) the lowest initial cell-wall carbohydrate concentration, (iii) the lowest dry matter remaining after pretreatment, and (iv) the highest amount of monosaccharides released during enzymatic hydrolysis. However, bm corn stover did not reduce the severity of aqueous ammonia steeping pretreatment needed to maximize DM hydrolysis during SSCF compared with normal corn stover. In the remaining studies (Chapters 3 thru 5), a system for analyzing the quality of lignocellulosic biomass feedstocks for biochemical conversion to biofuels (i.e., pretreatment, enzymatic hydrolysis, and fermentation) was developed. To accomplish this, a carbohydrate availability model was developed to characterize feedstock quality. The model partitions carbohydrates within a feedstock material into fractions based on their availability to be converted to fermentable

  7. Genetic and biochemical impairment of mitochondrial complex I activity in a family with Leber hereditary optic neuropathy and hereditary spastic dystonia

    Energy Technology Data Exchange (ETDEWEB)

    De Vries, D.D.; Oost, B.A. van [Univ. Hospital Nijmegen (Netherlands); Went, L.N.; Bruyn, G.W. [Univ. of Leiden (Netherlands)] [and others

    1996-04-01

    A rare form of Leber hereditary optic neuropathy (LHON) that is associated with hereditary spastic dystonia has been studied in a large Dutch family. Neuropathy and ophthalmological lesions were present together in some family members, whereas only one type of abnormality was found in others. mtDNA mutations previously reported in LHON were not present. Sequence analysis of the protein-coding mitochondrial genes revealed two previously unreported mtDNA mutations. A heteroplasmic A{yields}G transition at nucleotide position 11696 in the ND4 gene resulted in the substitution of an isoleucine for valine at amino acid position 312. A second mutation, a homoplasmic T{yields}A transition at nucleotide position 14596 in the ND6 gene, resulted in the substitution of a methionine for the isoleucine at amino acid residue 26. Biochemical analysis of a muscle biopsy revealed a severe complex I deficiency, providing a link between these unique mtDNA mutations and this rare, complex phenotype including Leber optic neuropathy. 80 refs., 2 figs., 3 tabs.

  8. Mitochondrial genome variations and functional characterization in Han Chinese families with schizophrenia.

    Science.gov (United States)

    Bi, Rui; Tang, Jinsong; Zhang, Wen; Li, Xiao; Chen, Shi-Yi; Yu, Dandan; Chen, Xiaogang; Yao, Yong-Gang

    2016-03-01

    The relationship between mitochondrial DNA (mtDNA) variants and schizophrenia has been strongly debated. To test whether mtDNA variants are involved in schizophrenia in Han Chinese patients, we sequenced the entire mitochondrial genomes of probands from 11 families with a family history and maternal inheritance pattern of schizophrenia. Besides the haplogroup-specific variants, we found 11 nonsynonymous private variants, one rRNA variant, and one tRNA variant in 5 of 11 probands. Among the nonsynonymous private variants, mutations m.15395 A>G and m.8536 A>G were predicted to be deleterious after web-based searches and in silico program affiliated analysis. Functional characterization further supported the potential pathogenicity of the two variants m.15395 A>G and m.8536 A>G to cause mitochondrial dysfunction at the cellular level. Our results showed that mtDNA variants were actively involved in schizophrenia in some families with maternal inheritance of this disease.

  9. Mitochondrial diseases caused by toxic compound accumulation: from etiopathology to therapeutic approaches

    OpenAIRE

    2015-01-01

    Mitochondrial disorders are a group of highly invalidating human conditions for which effective treatment is currently unavailable and characterized by faulty energy supply due to defective oxidative phosphorylation (OXPHOS). Given the complexity of mitochondrial genetics and biochemistry, mitochondrial inherited diseases may present with a vast range of symptoms, organ involvement, severity, age of onset, and outcome. Despite the wide spectrum of clinical signs and biochemical underpinnings ...

  10. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

    Directory of Open Access Journals (Sweden)

    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  11. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  12. Biochemical characterization of systemic bacteria in bananas, sensitivity to antibiotics and plant phytotoxicity during shoot proliferation

    Directory of Open Access Journals (Sweden)

    Janiffe Peres de Oliveira

    2016-04-01

    Full Text Available The objective of this work was to characterize the biochemically systemic bacterial isolated from banana plants, to evaluate the bacterial sensitivity to antibiotics, and to determine the phytotoxicity of banana shoots during in vitro proliferation. Systemic bacteria belonging to the Klebsiella and Aeromonas genera were isolated from the “Maravilha” (FHIA 01 AAAB, “Preciosa” (PV 4285 AAAB and “Thap Maeo” (AAB varieties and were then characterized. Tests of shoot sensitivity to antibiotics were performed, and the minimum inhibitory concentration (MIC and phytotoxic effects of selected antibiotics to plants were determined. Among the 20 antibiotics evaluated, the strains showed sensitivity to cefaclor, cefalexin, cefalotin, nalidixic acid, chloramphenicol, and vancomycin. However, during MIC determination, the best results were obtained with cefaclor, vancomycin or nalidixic acid alone in concentrations ranging from 512 to 1,024 mg L-1. In culture medium, cefaclor at 1,024 mg L-1 was the only antibiotic to affect the multiplication and the shoot survival in culture.

  13. Biochemical and genetic characterization of arazyme, an extracellular metalloprotease produced from Serratia proteamaculans HY-3.

    Science.gov (United States)

    Kwak, Jangyul; Lee, Kieun; Shin, Dong-Ha; Maeng, Jin-Soo; Park, Doo-Sang; Oh, Hyun Woo; Son, Kwang-Hee; Bae, Kyung-Sook; Park, Ho-Yong

    2007-05-01

    Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHl fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene (inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.

  14. Partial biochemical characterization of crude extract extracellular chitinase enzyme from Bacillus subtilis B 298

    Science.gov (United States)

    Lestari, P.; Prihatiningsih, N.; Djatmiko, H. A.

    2017-02-01

    Extraction and characterization of extracellular chitinase from Bacillus subtilis B 298 have been done. Growth curve determination of B. subtilis B 298, production curve determination of crude extract chitinase from B. subtilis B 298, and partial biochemical characterization of crude extract chitinase have been achieved in this study. Optimum growth of B. subtilis B 298 was achieved at logarithmic phase within 9 hours incubation time, so it was used as inoculum for enzyme production. According to production curve of the enzyme, it was known that incubation time which gave the highest chitinase activity of 15 hours with activity of 6.937 U/mL respectively. Effect of various temperatures on chitinase activity showed that optimum activity was achieved at 40°C with an activity of 5.764 U/mL respectively. Meanwhile, the optimum pH for chitinase activity was achieved at pH of 5.0 with an activity of 6.813 U/mL respectively. This enzyme was then classified as metalloenzyme due to the decline of the activity by EDTA addition. All divalent cations tested acted as inhibitors.

  15. Molecular cloning and biochemical characterization of iron superoxide dismutase from the rodent malaria parasite Plasmodium vinckei.

    Science.gov (United States)

    Prakash, Kirtika; Goyal, Manish; Soni, Awakash; Siddiqui, Arif Jamal; Bhardwaj, Jyoti; Puri, Sunil K

    2014-12-01

    Plasmodium parasite utilizes superoxide dismutase family proteins to limit the toxicity of reactive oxygen species, such as produced through hemoglobin degradation. These proteins play an important role in parasite survival during intra-erythrocytic phase. We have identified, and biochemically characterized a putative iron dependent superoxide dismutase from rodent malaria parasite Plasmodium vinckei (PvSOD1). The recombinant PvSOD1 protein was purified to homogeneity through a combination of affinity and gel filtration chromatography. Crosslinking, Native-PAGE and FPLC gel filtration analyses documented that PvSOD1 exists as a dimer in solution, a common feature shared by other Fe-SODs. PvSOD1 is cytosolic in localization and its expression is comparatively higher during trophozoite as compared to that of ring and schizont stages. Enzymatic activity of recombinant PvSOD1 was validated using conventional zymogram analyses and xanthine-xanthine oxidase system. Under optimal conditions, PvSOD1 was highly active and catalyzed the dismutation of superoxide radicals. Furthermore, PvSOD1 showed activity over a broad range of pH and temperature. Inhibition studies suggested that PvSOD1 was inactivated by hydrogen peroxide, and peroxynitrite, but not by cyanide and azide. Since, PvSOD1 plays a central role in oxidative defense mechanism, therefore, characterization of PvSOD1 will be exploited in the screening of new superoxide dismutase inhibitors for their antimalarial activity.

  16. Biochemical and Structural Characterization of the Human TL1A Ectodomain

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, C.; Yan, Q; Patskovsky, Y; Li, Z; Toro, R; Meyer, A; Cheng, H; Brenowitz, M; Nathenson, S; Almo, S

    2009-01-01

    TNF-like 1A (TL1A) is a newly described member of the TNF superfamily that is directly implicated in the pathogenesis of autoimmune diseases, including inflammatory bowel disease, atherosclerosis, and rheumatoid arthritis. We report the crystal structure of the human TL1A extracellular domain at a resolution of 2.5 {angstrom}, which reveals a jelly-roll fold typical of the TNF superfamily. This structural information, in combination with complementary mutagenesis and biochemical characterization, provides insights into the binding interface and the specificity of the interactions between TL1A and the DcR3 and DR3 receptors. These studies suggest that the mode of interaction between TL1A and DcR3 differs from other characterized TNF ligand/receptor complexes. In addition, we have generated functional TL1A mutants with altered disulfide bonding capability that exhibit enhanced solution properties, which will facilitate the production of materials for future cell-based and whole animal studies. In summary, these studies provide insights into the structure and function of TL1A and provide the basis for the rational manipulation of its interactions with cognate receptors.

  17. Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

    Institute of Scientific and Technical Information of China (English)

    Elaine de Paula Mendonca-Franqueiro; Eliane Candiani Arantes; Marcelo Dias-Baruffi; Suely Vilela Sampaio; Raquel de Melo Alves-Paiva; Marco Aurélio Sartim; Daniel Roberto Callejon; Helder Henrique Paiva; Gilmara Ausech Antonucci; José César Rosa; Adélia Cristina Oliveira Cintra; Jo(a)o José Franco

    2011-01-01

    Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycanbinding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability,which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

  18. Purification and partial biochemical characterization of polyphenol oxidase from mamey (Pouteria sapota).

    Science.gov (United States)

    Palma-Orozco, Gisela; Ortiz-Moreno, Alicia; Dorantes-Alvarez, Lidia; Sampedro, José G; Nájera, Hugo

    2011-01-01

    While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit (Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS-PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35°C. Kinetic constants for PPO 1 were K(m)=44 mM and K(m)=1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.

  19. Biochemical and molecular characterization of the gentisate transporter GenK in Corynebacterium glutamicum.

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    Ying Xu

    Full Text Available BACKGROUND: Gentisate (2,5-dihydroxybenzoate is a key ring-cleavage substrate involved in various aromatic compounds degradation. Corynebacterium glutamicum ATCC13032 is capable of growing on gentisate and genK was proposed to encode a transporter involved in this utilization by its disruption in the restriction-deficient mutant RES167. Its biochemical characterization by uptake assay using [(14C]-labeled gentisate has not been previously reported. METHODOLOGY/PRINCIPAL FINDINGS: In this study, biochemical characterization of GenK by uptake assays with [(14C]-labeled substrates demonstrated that it specifically transported gentisate into the cells with V(max and K(m of 3.06 ± 0.16 nmol/min/mg of dry weight and 10.71 ± 0.11 µM respectively, and no activity was detected for either benzoate or 3-hydoxybenzoate. When GenK was absent in strain RES167 ΔgenK, it retained 85% of its original transport activity at pH 6.5 compared to that of strain RES167. However, it lost 79% and 88% activity at pH 7.5 and 8.0, respectively. A number of competing substrates, including 3-hydroxybenzoate, benzoate, protocatechuate and catechol, significantly inhibited gentisate uptake by more than 40%. Through site-directed mutagenesis, eight amino acid residues of GenK, Asp-54, Asp-57 and Arg-386 in the hydrophobic transmembrane regions and Arg-103, Trp-309, Asp-312, Arg-313 and Ile-317 in the hydrophilic cytoplasmic loops were shown to be important for gentisate transport. When conserved residues Asp-54 and Asp-57 respectively were changed to glutamate, both mutants retained approximately 50% activity and were able to partially complement the ability of strain RES167 ΔgenK to grow on gentisate. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that GenK is an active gentisate transporter in Corynebacterium glutamicum ATCC13032. The GenK-mediated gentisate transport was also shown to be a limiting step for the gentisate utilization by this strain. This enhances our

  20. Biochemical Characterization of Mycobacterium tuberculosis DNA Repair Enzymes – Nfo, XthA and Nei2

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    Sailau Abeldenov

    2014-01-01

    Full Text Available Introduction: Tuberculosis (TB is a human disease caused by Mycobacterium tuberculosis (Mtb. Treatment of TB requires long-term courses of multi-drug therapies to eliminate subpopulations of bacteria, which sometimes persist against antibiotics. Therefore, understanding of the mechanism of Mtb antibiotic-resistance is extremely important. During infection, Mtb overcomes a variety of body defense mechanisms, including treatment with the reactive species of oxygen and nitrogen. The bases in DNA molecule are susceptible to the damages caused by reactive forms of intermediate compounds of oxygen and nitrogen. Most of this damage is repaired by the base excision repair (BER pathway. In this study, we aimed to biochemically characterize three Mtb DNA repair enzymes of BER pathway. Methods: XthA, nfo, and nei genes were identified in mycobacteria by homology search of genomic sequences available in the GenBank database. We used standard methods of genetic engineering  to clone and sequence Mtb genes, which coded Nfo, XthA and Nei2 repair enzymes. The protein products of Mtb genes were expressed and purified in Escherichia coli using affinity tags. The enzymatic activity of purified Nfo, XthA, and Nei2 proteins were measured using radioactively labeled DNA substrates containing various modified residues. Results: The genes end (Rv0670, xthA (Rv0427c, and nei (Rv3297 were PCR amplified using genomic DNA of Mtb H37Rv with primers that contain specific restriction sites. The amplified products were inserted into pET28c(+ expression vector in such a way that the recombinant proteins contain C-terminal histidine tags. The plasmid constructs were verified by sequencing and then transformed into the Escherichia coli BL21 (DE3 strain. Purification of recombinant proteins was performed using Ni2+ ions immobilized affinity column, coupled with the fast performance liquid chromatography machine AKTA. Identification of the isolated proteins was performed by

  1. Biochemical and Physiological Characterization: Development & Apply Optical Methods for Charaterizing Biochemical Protein-Protein Interactions in MR-1

    Energy Technology Data Exchange (ETDEWEB)

    Weiss, Shimon

    2006-08-30

    The objectives of this report are to: Develop novel site-specific protein labeling chemistries for assaying protein-protein interactions in MR-1; and development of a novel optical acquisition and data analysis method for characterizing protein-protein interactions in MR-1 model systems. Our work on analyzing protein-protein interactions in MR-1 is divided in four areas: (1) expression and labeling of MR-1 proteins; (2) general scheme for site-specific fluorescent labeling of expressed proteins; (3) methodology development for monitoring protein-protein interactions; and (4) study of protein-protein interactions in MR-1. In this final report, we give an account for our advances in all areas.

  2. Characterization of human mitochondrial ferritin promoter: identification of transcription factors and evidences of epigenetic control

    Science.gov (United States)

    Guaraldo, Michela; Santambrogio, Paolo; Rovelli, Elisabetta; di Savino, Augusta; Saglio, Giuseppe; Cittaro, Davide; Roetto, Antonella; Levi, Sonia

    2016-09-01

    Mitochondrial ferritin (FtMt) is an iron storage protein belonging to the ferritin family but, unlike the cytosolic ferritin, it has an iron-unrelated restricted tissue expression. FtMt appears to be preferentially expressed in cell types characterized by high metabolic activity and oxygen consumption, suggesting a role in protecting mitochondria from iron-dependent oxidative damage. The human gene (FTMT) is intronless and its promoter region has not been described yet. To analyze the regulatory mechanisms controlling FTMT expression, we characterized the 5‧ flanking region upstream the transcriptional starting site of FTMT by in silico enquiry of sequences conservation, DNA deletion analysis, and ChIP assay. The data revealed a minimal promoter region and identified the presence of SP1, CREB and YY1 as positive regulators, and GATA2, FoxA1 and C/EBPβ as inhibitors of the transcriptional regulation. Furthermore, the FTMT transcription is increased by acetylating and de-methylating agent treatments in K562 and HeLa cells. These treatments up-regulate FtMt expression even in fibroblasts derived from a Friedreich ataxia patient, where it might exert a beneficial effect against mitochondrial oxidative damage. The expression of FTMT appears regulated by a complex mechanism involving epigenetic events and interplay between transcription factors.

  3. Biochemical characterization of sirtuin 6 in the brain and its involvement in oxidative stress response.

    Science.gov (United States)

    Cardinale, Alessio; de Stefano, Maria Chiara; Mollinari, Cristiana; Racaniello, Mauro; Garaci, Enrico; Merlo, Daniela

    2015-01-01

    Sirtuin 6 (SIRT6) is a member of nicotinamide adenine dinucleotide-dependent deacetylase protein family and has been implicated in the control of glucose and lipid metabolism, cancer, genomic stability and DNA repair. Moreover, SIRT6 regulates the expression of a large number of genes involved in stress response and aging. The role of SIRT6 in brain function and neuronal survival is largely unknown. Here, we biochemically characterized SIRT6 in brain tissues and primary neuronal cultures and found that it is highly expressed in cortical and hippocampal regions and enriched in the synaptosomal membrane fraction. Immunoblotting analysis on cortical and hippocampal neurons showed that SIRT6 is downregulated during maturation in vitro, reaching the lowest expression at 11 days in vitro. In addition, SIRT6 overexpression in terminally differentiated cortical and hippocampal neurons, mediated by a neuron-specific recombinant adeno-associated virus, downregulated cell viability under oxidative stress condition. By contrast, under control condition, SIRT6 overexpression had no detrimental effect. Overall these results suggest that SIRT6 may play a role in synaptic function and neuronal maturation and it may be implicated in the regulation of neuronal survival.

  4. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    Science.gov (United States)

    Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

  5. Biochemical characterization, antimicrobial and hemolytic studies on skin mucus of fresh water spiny eel Mastacembelus armatus

    Institute of Scientific and Technical Information of China (English)

    Venkatachalam Uthayakumar; Venkatachalam Ramasubramanian; Dhanabalan Senthilkumar; Ramasamy Harikrishnan

    2012-01-01

    Objective: To characterize the biochemical, antimicrobial and hemolytic activities of Mastacembalus armatus skin mucus. Methods: Antimicrobial and antifungal activities of mucus extractions against human and fish pathogens were tested along with ampicillin as control. Hemolytic activity of the extraction was evaluated against sheep and cow blood cells. Amino acid and fatty acid profiles were analyzed by HPLC and gas chromatography in the mucus of fish. SDS-PAGE analysis of mucus and muscle tissue was done. Oneway-ANOVA was performed against all extraction and pathogens, amino acids and fatty acids. Result: All the mucus extracts exhibited higher inhibitory activity than antibiotic ampicillin against bacterial and fungal pathogens. The hemolytic activity was increased with higher mucus concentrations in both sheep and cow blood cells. The protein content soluble and insoluble fractions of mucus were 63.22 μg/g and 55.79μg/g, respectively. Out of 17 amino acids leucine was higher (8.54 mole %) in soluble gel, and glutamic acid was higher (6.92 mole %) in the insoluble gel, Histidine was very low (i.e. 0.20 mole%) both in soluble and (0.30 mole %) insoluble gel. In SDS-PAGE analysis, 6 bands of mucus and 9 bands of muscle were observed. Conclusions: The soluble and insoluble proteins are responsible for antimicrobial and hemolytic activity, these results indicate that mucus gel was prospective applications in fish and human therapeutics.

  6. State-dependent doubly weighted stochastic simulation algorithm for automatic characterization of stochastic biochemical rare events.

    Science.gov (United States)

    Roh, Min K; Daigle, Bernie J; Gillespie, Dan T; Petzold, Linda R

    2011-12-21

    In recent years there has been substantial growth in the development of algorithms for characterizing rare events in stochastic biochemical systems. Two such algorithms, the state-dependent weighted stochastic simulation algorithm (swSSA) and the doubly weighted SSA (dwSSA) are extensions of the weighted SSA (wSSA) by H. Kuwahara and I. Mura [J. Chem. Phys. 129, 165101 (2008)]. The swSSA substantially reduces estimator variance by implementing system state-dependent importance sampling (IS) parameters, but lacks an automatic parameter identification strategy. In contrast, the dwSSA provides for the automatic determination of state-independent IS parameters, thus it is inefficient for systems whose states vary widely in time. We present a novel modification of the dwSSA--the state-dependent doubly weighted SSA (sdwSSA)--that combines the strengths of the swSSA and the dwSSA without inheriting their weaknesses. The sdwSSA automatically computes state-dependent IS parameters via the multilevel cross-entropy method. We apply the method to three examples: a reversible isomerization process, a yeast polarization model, and a lac operon model. Our results demonstrate that the sdwSSA offers substantial improvements over previous methods in terms of both accuracy and efficiency.

  7. Extracellular α-Galactosidase from Trichoderma sp. (WF-3: Optimization of Enzyme Production and Biochemical Characterization

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    Aishwarya Singh Chauhan

    2015-01-01

    Full Text Available Trichoderma spp. have been reported earlier for their excellent capacity of secreting extracellular α-galactosidase. This communication focuses on the optimization of culture conditions for optimal production of enzyme and its characterization. The evaluation of the effects of different enzyme assay parameters such as stability, pH, temperature, substrate concentrations, and incubation time on enzyme activity has been made. The most suitable buffer for enzyme assay was found to be citrate phosphate buffer (50 mM, pH 6.0 for optimal enzyme activity. This enzyme was fairly stable at higher temperature as it exhibited 72% activity at 60°C. The enzyme when incubated at room temperature up to two hours did not show any significant loss in activity. It followed Michaelis-Menten curve and showed direct relationship with varying substrate concentrations. Higher substrate concentration was not inhibitory to enzyme activity. The apparent Michaelis-Menten constant (Km, maximum rate of reaction (Vmax, Kcat, and catalytic efficiency values for this enzyme were calculated from the Lineweaver-Burk double reciprocal plot and were found to be 0.5 mM, 10 mM/s, 1.30 U mg−1, and 2.33 U mg−1 mM−1, respectively. This information would be helpful in understanding the biophysical and biochemical characteristics of extracellular α-galactosidase from other microbial sources.

  8. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum.

    Science.gov (United States)

    Huang, Shi-Ping; Cheng, Hong-Mei; Wang, Peng; Zhu, Guo-Ping

    2016-02-26

    Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family.

  9. Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum

    Directory of Open Access Journals (Sweden)

    Shi-Ping Huang

    2016-02-01

    Full Text Available Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH from B. longum (BlIDH, a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP+-dependent IDH (NADP-IDH, with a 567- and 193-fold preference for NADP+ over NAD+ in the presence of Mg2+ and Mn2+, respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn2+ and 65 °C (with Mg2+, and pH 7.5 (with Mn2+ and pH 8.0 (with Mg2+. Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn2+ or Mg2+. Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP+ use by the IDH family.

  10. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI

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    Kristensen Torsten

    2009-05-01

    Full Text Available Abstract Background TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI, and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. Results The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. Conclusion The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

  11. Development and characterization of microbial biosensors for evaluating low biochemical oxygen demand in rivers.

    Science.gov (United States)

    Chee, Gab-Joo

    2013-12-15

    Five microorganisms were used to construct a biosensor for the evaluation of low biochemical oxygen demand (BOD) in rivers. Characterization and comparison of BOD biosensors were performed using two standard solutions: glucose and glutamic acid (GGA) and artificial wastewater (AWW). Pseudomonas putida SG10 demonstrated the best response when using AWW. Trichosporon cutaneum IFO10466, however, had an extremely poor response. When evaluating the biosensor response to each component of AWW, all of the microorganisms except T. cutaneum displayed the highest response to tannic acid. In a comparison of the two standard solutions for all the microorganisms, the biosensor responses of GGA were approximately three times higher than those of AWW were. In the BOD determination of environmental samples, the biosensor BOD values evaluated using AWW were slightly lower or equivalent to BOD5 values, whereas the biosensor BOD values evaluated using GGA were considerably lower. These results suggest that GGA is suitable for the detection of high BOD in industrial wastewaters and factory effluents, while AWW is suitable for the detection of low BOD in rivers.

  12. Immunohistochemical and Biochemical Characterization of Mucin in Pseudomyxoma Peritonei: A Case Study

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    Anwar S. Mall

    2011-01-01

    Full Text Available We previously reported the presence of MUC2, MUC5AC and, for the first time, MUC5B in a 58-year-old male with pseudomyxoma peritonei (PMP. This is a report on the biochemical and immunohistochemical characterization of mucin in a 50-year-old female with the same rare illness. A right oophorectomy and appendicectomy and a resection of the involved omentum were performed. Approximately a litre of crude material in the sol and gel phases was obtained from the patient during laparotomy. This was briefly homogenized in 6 M guanidinium hydrochloride and proteolytic inhibitors and purified by density gradient centrifugation in caesium chloride. At laparotomy it was noted that the patient had appendiceal and ovarian masses as well as extensive mucinous deposits in the omentum and peritoneum. A mucinous adenocarcinoma of the appendix and ovary was confirmed on histology. The cells expressed both sulphated and non-sulphated acidic mucins. The presence of MUC2, MUC5AC, MUC5B and α-1-acid glycoprotein was shown by Western blotting and MUC4 by immunohistochemical staining. MUC1 and MUC6 were not detectable in the tissue. The study confirms that MUC2, MUC5AC and MUC5B are produced in the mucus of patients with PMP. The expression of MUC4 in this disease has not been previously reported.

  13. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    Directory of Open Access Journals (Sweden)

    Gizeli S. Gimenez

    2014-01-01

    Full Text Available Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications.

  14. Biochemical and functional characterization of Parawixia bistriata spider venom with potential proteolytic and larvicidal activities.

    Science.gov (United States)

    Gimenez, Gizeli S; Coutinho-Neto, Antonio; Kayano, Anderson M; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Stábeli, Rodrigo G

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications.

  15. Metabolic Response of Human Osteoarthritic Cartilage to Biochemically Characterized Collagen Hydrolysates

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    Saskia Schadow

    2017-01-01

    Full Text Available The most frequent disease of the locomotor system is osteoarthritis (OA, which, as a chronic joint disease, might benefit more from nutrition than acute illnesses. Collagen hydrolysates (CHs are peptidic mixtures that are often used as nutraceuticals for OA. Three CHs were characterized biochemically and pharmacologically. Our biophysical (MALDI-TOF-MS, NMR, AFM and fluorescence assays revealed marked differences between CHs of fish (Peptan® F 5000, Peptan® F 2000 and porcine (Mobiforte® origin with respect to the total number of peptides and common peptides between them. Using a novel dual radiolabeling procedure, no CH modulated collagen biosynthesis in human knee cartilage explants. Peptan® F 2000 enhanced the activities of the aggrecanase ADMATS4 and ADMATS5 in vitro without loss of proteoglycan from cartilage explants; the opposite effect was observed with Mobiforte®. Interleukin (IL-6, matrix metalloproteinase (MMP-1, -3 and -13 levels were elevated in explants that were treated with Mobiforte® and Peptan® F 5000, but not with Peptan® F 2000. In conclusion, the heterogeneous peptide composition and disparate pharmacological effects between CHs suggest that the effect of a CH preparation cannot be extrapolated to other formulations. Thus, the declaration of a CH as a safe and effective nutraceutical requires a thorough examination of its pleiotropic effects.

  16. Metabolic Response of Human Osteoarthritic Cartilage to Biochemically Characterized Collagen Hydrolysates

    Science.gov (United States)

    Schadow, Saskia; Simons, Viktor S.; Lochnit, Guenter; Kordelle, Jens; Gazova, Zuzana; Siebert, Hans-Christian; Steinmeyer, Juergen

    2017-01-01

    The most frequent disease of the locomotor system is osteoarthritis (OA), which, as a chronic joint disease, might benefit more from nutrition than acute illnesses. Collagen hydrolysates (CHs) are peptidic mixtures that are often used as nutraceuticals for OA. Three CHs were characterized biochemically and pharmacologically. Our biophysical (MALDI-TOF-MS, NMR, AFM) and fluorescence assays revealed marked differences between CHs of fish (Peptan® F 5000, Peptan® F 2000) and porcine (Mobiforte®) origin with respect to the total number of peptides and common peptides between them. Using a novel dual radiolabeling procedure, no CH modulated collagen biosynthesis in human knee cartilage explants. Peptan® F 2000 enhanced the activities of the aggrecanase ADMATS4 and ADMATS5 in vitro without loss of proteoglycan from cartilage explants; the opposite effect was observed with Mobiforte®. Interleukin (IL)-6, matrix metalloproteinase (MMP)-1, -3 and -13 levels were elevated in explants that were treated with Mobiforte® and Peptan® F 5000, but not with Peptan® F 2000. In conclusion, the heterogeneous peptide composition and disparate pharmacological effects between CHs suggest that the effect of a CH preparation cannot be extrapolated to other formulations. Thus, the declaration of a CH as a safe and effective nutraceutical requires a thorough examination of its pleiotropic effects. PMID:28117674

  17. Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7.

    Science.gov (United States)

    Wu, Zhigang; Zhao, Guohui; Li, Tiehai; Qu, Jingyao; Guan, Wanyi; Wang, Jiajia; Ma, Cheng; Li, Xu; Zhao, Wei; Wang, Peng G; Li, Lei

    2016-05-01

    Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coliO55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galβ1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km= 9.2 min(-1)mM(-1)) as that toward GDP-colitose (kcat/Km= 12 min(-1)mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.

  18. Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

    Science.gov (United States)

    Benoit, Isabelle; Asther, Michèle; Bourne, Yves; Navarro, David; Canaan, Stéphane; Lesage-Meessen, Laurence; Herweijer, Marga; Coutinho, Pedro M; Asther, Marcel; Record, Eric

    2007-09-01

    The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.

  19. Biochemical and structural characterization of SplD protease from Staphylococcus aureus.

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    Michal Zdzalik

    Full Text Available Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1' with a consensus motif of R-(Y/W-(P/L-(T/L/I/V↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.

  20. Biochemical and structural characterization of neocartilage formed by mesenchymal stem cells in alginate hydrogels.

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    Magnus Ø Olderøy

    Full Text Available A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM. Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC immobilized in alginate hydrogels, and used immunohistochemistry (IHC and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG microscopy and focused ion beam/scanning electron microscopy (FIB/SEM to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide

  1. Toxicological and biochemical characterizations of malathion sensitivity in two field populations of Oxya chinensis (Orthoptera: Acridoidea)

    Institute of Scientific and Technical Information of China (English)

    MEI-LING YANG; HAI-HUA WU; YA-PING GUO; EN-BO MA

    2006-01-01

    We evaluate comparative toxicity of malathion in the two populations of the grasshopper Oxya chinensis, collected from Daixian and Fanshi of Shanxi province, China.General esterases and acetylcholinesterase (AChE) from the two populations were characterized and compared. LD50 of the Daixian population (7.58μg/g body weight) was 2.02-fold higher than that of the Fanshi population (3.75 μg/g body weight). General esterase-specific activities in the Daixian population were 1.91, 1.10 and 1.85-fold higher than those in the Fanshi population, when α-NA, α-NB and β-NA were used as a substrate, respectively.Kinetic studies of general esterase showed that Vmax values of general esterases hydrolyzing α-NA, α-NB and β-NA in the Daixian population were 2.15-, 1.12-, and 1.47-fold,respectively, higher than those in the Fanshi population. The AChE activity of the Fanshi population was 1.54-fold higher than that of the Daixian population. Kinetic analysis of AChE showed that significant differences were presented between the two populations in the Km values; and the Vmax value in the Fanshi population was higher than that in the Daixian population. Inhibition studies of AChE indicated that AChE from the Daixian population was 2.56-, 2.80-, and 2.29-fold less sensitive to inhibition by paraoxon, chlorpyrifos-oxon,and demeton-S-methyl, respectively, than that from the Fanshi population. These biochemical characterizations of general esterases and AChE were consistent with malathion bioassay in the two populations. It is inferred that the reduced sensitivity of altered AChE and increased general esterase activities play an important role in the differences of insusceptibility of Oxya chinensis to malathion between the two populations.

  2. 草鱼线粒体型超氧化物歧化酶的生化遗传特性%Genetic and biochemical characteristics of mitochondrial superoxide dismutase in grass carp Ctenopharyngodon idellus

    Institute of Scientific and Technical Information of China (English)

    颜勤; 罗琛

    2004-01-01

    , population genetics, and identification of different strains in the same species. However, the biochemical and genetic features of the mitochondrial superoxide dismutase have not been well characterized in fishes. In this study, we investigated the genetic phenotypes and biochemical features of grass carp Ctenopharyngodon idellus mitochondrial superoxide dismutase (fm-SOD) with polyacrylamide gradient gel electrophoresis. Our data revealed three isoforms of fm-SOD which were named fm-SOD 1, fm-SOD 2 and fm-SOD 3 according to their positions from the positive pole to the negative pole. The combination of three isoforms of fm-SOD constitute three distinct biochemical phenotypes. Phenotype 1 was only associated with the fastest migratory isoform fm-SOD 1 and phenotype 3 with the slowest migratory isoform fm-SOD 3, while phenotype 2 was associated with all three isoforms. In the wild group of grass carp, all three phenotypes were observed, whereas in the mito-gynogenetic group, only phenotypes 1and 3 were observed. The ratio of the three phenotypes in the wild group was consistent with the 1: 2: 1 ratio ofMendelian inheritance for 2 alleles of a single locus in the autosomal chromosomes. These results suggested that: (1) the mitochondrial superoxide dismutase gene in grass carp resided in chromosomes instead of mitochondrial DNA; (2) mitochondrial superoxide dismutase was encoded by a single locus; (3) there were at least two variant alleles in grass carp and (4) the fm-SOD is composed of two subunits. In addition, the fm-SOD was sensitive to the mixture of 15% ethanol and 25 % chloroform but resistant to H2O2, indicating that the fm-SOD in grass carp is Mn-SOD [ Acta Zoologica Sinica 50(3): 389-394, 2004].

  3. Generation and characterization of transgenic mice expressing mitochondrial targeted red fluorescent protein selectively in neurons: modeling mitochondriopathy in excitotoxicity and amyotrophic lateral sclerosis

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    Wang Yi

    2011-11-01

    Full Text Available Abstract Background Mitochondria have roles or appear to have roles in the pathogenesis of several chronic age-related and acute neurological disorders, including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Parkinson's disease, and cerebral ischemia, and could be critical targets for development of rational mechanism-based, disease-modifying therapeutics for treating these disorders effectively. A deeper understanding of neural tissue mitochondria pathobiologies as definitive mediators of neural injury, disease, and cell death merits further study, and the development of additional tools to study neural mitochondria will help achieve this unmet need. Results We created transgenic mice that express the coral (Discosoma sp. red fluorescent protein DsRed2 specifically in mitochondria of neurons using a construct engineered with a Thy1 promoter, specific for neuron expression, to drive expression of a fusion protein of DsRed2 with a mitochondrial targeting sequence. The biochemical and histological characterization of these mice shows the expression of mitochondrial-targeted DsRed2 to be specific for mitochondria and concentrated in distinct CNS regions, including cerebral cortex, hippocampus, thalamus, brainstem, and spinal cord. Red fluorescent mitochondria were visualized in cerebral cortical and hippocampal pyramidal neurons, ventrobasal thalamic neurons, subthalamic neurons, and spinal motor neurons. For the purpose of proof of principle application, these mice were used in excitotoxicity paradigms and double transgenic mice were generated by crossing Thy1-mitoDsRed2 mice with transgenic mice expressing enhanced-GFP (eGFP under the control of the Hlxb9 promoter that drives eGFP expression specifically in motor neurons and by crossing Thy1-mitoDsRed2 mice to amyotrophic lateral sclerosis (ALS mice expressing human mutant superoxide dismutase-1. Conclusions These novel transgenic mice will be a useful tool for better understanding

  4. Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

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    Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

    2010-11-01

    MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

  5. Characterization of the complete mitochondrial genome of flower-breeding Drosophila incompta (Diptera, Drosophilidae).

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    De Ré, F C; Wallau, G L; Robe, L J; Loreto, E L S

    2014-12-01

    Drosophila incompta belongs to the flavopilosa group of Drosophila, and has a restricted ecology, being adapted to flowers of Cestrum as feeding and oviposition sites. We sequenced, assembled, and characterized the complete mitochondrial genome (mtDNA) of D. incompta. In addition, we performed phylogenomic and polymorphism analyses to assess evolutionary diversification of this species. Our results suggest that this genome is syntenic with the other published mtDNA of Drosophila. This molecule contains 15,641 bp and encompasses two rRNA, 22 tRNA and 13 protein-coding genes. Regarding nucleotide composition, we found a high A-T bias (76.6 %). The recovered phylogenies indicate D. incompta in the virilis-repleta radiation, as sister to the virilis or repleta groups. The most interesting result is the high degree of polymorphism found throughout the D. incompta mitogenome, revealing pronounced intrapopulational variation. Furthermore, intraspecific nucleotide diversity levels varied between different regions of the genome, thus allowing the use of different mitochondrial molecular markers for analysis of population structure of this species.

  6. Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames

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    Hill Ryan E

    2009-08-01

    Full Text Available Abstract Background Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames alanine racemase (AlrBax, an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation. Results B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine of 101 U/mg. The crystal structure of unmodified AlrBax is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure. Conclusion The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.

  7. Characterization of changes in gene expression and biochemical pathways at low levels of benzene exposure.

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    Reuben Thomas

    Full Text Available Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML. Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC, we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from 10 ppm compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings.

  8. Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcus furiosus

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    Rahman M. Mizanur

    2008-01-01

    Full Text Available Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535 from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml–1 glycogen concentration was 52 U mg–1 and 31 U mg–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min–1 µg–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.

  9. Biochemical characterization of Anopheles gambiae SRPN6, a malaria parasite invasion marker in mosquitoes.

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    Chunju An

    Full Text Available Serine proteinase inhibitors of the serpin family are well known as negative regulators of hemostasis, thrombolysis and innate immune responses. Additionally, non-inhibitory serpins serve functions as chaperones, hormone transporters, or anti-angiogenic factors. In the African malaria mosquito, Anopheles gambiae s.s., at least three serpins (SRPNs are implicated in the innate immune response against malaria parasites. Based on reverse genetic and cell biological analyses, AgSRPN6 limits parasite numbers and transmission and has been postulated to control melanization and complement function in mosquitoes. This study aimed to characterize AgSRPN6 biophysically and determine its biochemical mode of action. The structure model of AgSRPN6, as predicted by I-Tasser showed the protein in the native serpin fold, with three central β-sheets, nine surrounding α-helices, and a protruding reactive center loop. This structure is in agreement with biophysical and functional data obtained from recombinant (r AgSRPN6, produced in Escherichia coli. The physical properties of purified rAgSRPN6 were investigated by means of analytical ultracentrifugation, circular dichroism, and differential scanning calorimetry tools. The recombinant protein exists predominantly as a monomer in solution, is composed of a mixture of α-helices and β-sheets, and has a mid-point unfolding temperature of 56°C. Recombinant AgSRPN6 strongly inhibited porcine pancreatic kallikrein and to a lesser extent bovine pancreatic trypsin in vitro. Furthermore, rAgSRPN6 formed inhibitory, SDS-stable, higher molecular weight complexes with prophenoloxidase-activating proteinase (PAP1, PAP3, and Hemolymph protein (HP6, which are required for melanization in the lepidopteran model organism, Manduca sexta. Taken together, our results strongly suggest that AgSRPN6 takes on a native serpin fold and is an inhibitor of trypsin-like serine proteinases.

  10. Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus.

    Science.gov (United States)

    García-Gómez, B I; Coronas, F I V; Restano-Cassulini, R; Rodríguez, R R; Possani, L D

    2011-07-01

    This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na+ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K+ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na+ currents verified here, this venom certainly warrant further investigation.

  11. Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

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    Concetta De Santi

    Full Text Available The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15 form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs.MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

  12. Genetic and biochemical characterization of human AP endonuclease 1 mutants deficient in nucleotide incision repair activity.

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    Aurore Gelin

    Full Text Available BACKGROUND: Human apurinic/apyrimidinic endonuclease 1 (APE1 is a key DNA repair enzyme involved in both base excision repair (BER and nucleotide incision repair (NIR pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells. METHODOLOGY/PRINCIPAL FINDING: We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3'-->5' exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase beta and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells. CONCLUSION/SIGNIFICANCE: Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.

  13. Molecular and biochemical characterization of the jasmonic acid methyltransferase gene from black cottonwood (Populus trichocarpa)

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    Zhao, Nan [ORNL; Yao, Jianzhuang [University of Tennessee, Knoxville (UTK); Chaiprasongsuk, Minta [University of Tennessee, Knoxville (UTK); Li, Guanglin [University of Tennessee, Knoxville (UTK); Guan, Ju [University of Tennessee, Knoxville (UTK); Tschaplinski, Timothy J [ORNL; Guo, Hong [University of Tennessee, Knoxville (UTK); Chen, Feng [University of Tennessee, Knoxville (UTK)

    2013-01-01

    Methyl jasmonate is a metabolite known to be produced by many plants and has roles in diverse biological processes. It is biosynthesized by the action of S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH family of methyltransferases. Herein is reported the isolation and biochemical characterization of a JMT gene from black cottonwood (Populus trichocarpa). The genome of P. trichocarpa contains 28 SABATH genes (PtSABATH1 to PtSABATH28). Recombinant PtSABATH3 expressed in Escherichia coli showed the highest level of activity with jasmonic acid (JA) among carboxylic acids tested. It was therefore renamed PtJMT1. PtJMT1 also displayed activity with benzoic acid (BA), with which the activity was about 22% of that with JA. PtSABATH2 and PtSABATH4 were most similar to PtJMT1 among all PtSABATHs. However, neither of them had activity with JA. The apparent Km values of PtJMT1 using JA and BA as substrate were 175 lM and 341 lM, respectively. Mutation of Ser-153 and Asn-361, two residues in the active site of PtJMT1, to Tyr and Ser respectively, led to higher specific activity with BA than with JA. Homology-based structural modeling indicated that substrate alignment, in which Asn-361 is involved, plays a role in determining the substrate specificity of PtJMT1. In the leaves of young seedlings of black cottonwood, the expression of PtJMT1 was induced by plant defense signal molecules methyl jasmonate and salicylic acid and a fungal elicitor alamethicin, suggesting that PtJMT1 may have a role in plant defense against biotic stresses. Phylogenetic analysis suggests that PtJMT1 shares a common ancestor with the Arabidopsis JMT, and functional divergence of these two apparent JMT orthologs has occurred since the split of poplar and Arabidopsis lineages.

  14. Biochemical and molecular characterization of high population density bacteria isolated from sunflower.

    Science.gov (United States)

    Guerra Pinheiro de Goes, Kelly Campos; de Castro Fisher, Maria Luisa; Cattelan, Alexandre José; Nogueira, Marco Antonio; Portela de Carvalho, Claudio Guilherme; Martinez de Oliveira, Andre Luiz

    2012-04-01

    Natural and beneficial associations between plants and bacteria have demonstrated potential commercial application for several agricultural crops. The sunflower has acquired increasing importance in Brazilian agribusiness owing to its agronomic characteristics such as the tolerance to edaphoclimatic variations, resistance to pests and diseases, and adaptation to the implements commonly used for maize and soybean, as well as the versatility of the products and by-products obtained from its cultivation. A study of the cultivable bacteria associated with two sunflower cultivars, using classical microbiological methods, successfully obtained isolates from different plant tissues (roots, stems, florets, and rhizosphere). Out of 57 plantgrowth- promoting isolates obtained, 45 were identified at the genus level and phylogenetically positioned based on 16S rRNA gene sequencing: 42 Bacillus (B. subtilis, B. cereus, B. thuringiensis, B. pumilus, B. megaterium, and Bacillus sp.) and 3 Methylobacterium komagatae. Random amplified polymorphic DNA (RAPD) analysis showed a broad diversity among the Bacillus isolates, which clustered into 2 groups with 75% similarity and 13 subgroups with 85% similarity, suggesting that the genetic distance correlated with the source of isolation. The isolates were also analyzed for certain growth-promoting activities. Auxin synthesis was widely distributed among the isolates, with values ranging from 93.34 to 1653.37 microM auxin per microng of protein. The phosphate solubilization index ranged from 1.25 to 3.89, and siderophore index varied from 1.15 to 5.25. From a total of 57 isolates, 3 showed an ability to biologically fix atmospheric nitrogen, and 7 showed antagonism against the pathogen Sclerotinia sclerotiorum. The results of biochemical characterization allowed identification of potential candidates for the development of biofertilizers targeted to the sunflower crop.

  15. Molecular and biochemical characterization of a beta-fructofuranosidase from Xanthophyllomyces dendrorhous.

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    Linde, Dolores; Macias, Isabel; Fernández-Arrojo, Lucía; Plou, Francisco J; Jiménez, Antonio; Fernández-Lobato, María

    2009-02-01

    An extracellular beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70 degrees C) and thermostability (with a T(50) in the range 66 to 71 degrees C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-beta-(2-->1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k(cat)/K(m) ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial beta-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter(-1). In addition, we isolated and sequenced the X. dendrorhous beta-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant beta-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.

  16. Mitochondrial disorders.

    Science.gov (United States)

    Zeviani, M; Tiranti, V; Piantadosi, C

    1998-01-01

    Mitochondrial respiration, the most efficient metabolic pathway devoted to energy production, is at the crosspoint of 2 quite different genetic systems, the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA). The latter encodes a few essential components of the mitochondrial respiratory chain and has unique molecular and genetic properties that account for some of the peculiar features of mitochondrial disorders. However, the perpetuation, propagation, and expression of mtDNA, the majority of the subunits of the respiratory complexes, as well as a number of genes involved in their assembly and turnover, are contained in the nuclear genome. Although mitochondrial disorders have been known for more than 30 years, a major breakthrough in their understanding has come much later, with the discovery of an impressive, ever-increasing number of mutations of mitochondrial DNA. Partial deletions or duplications of mtDNA, or maternally inherited point mutations, have been associated with well-defined clinical syndromes. However, phenotypes transmitted as mendelian traits have also been identified. These include clinical entities defined on the basis of specific biochemical defects, and also a few autosomal dominant or recessive syndromes associated with multiple deletions or tissue-specific depletion of mtDNA. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogenous. They range from lesions of single tissues or structures, such as the optic nerve in Leber hereditary optic neuropathy or the cochlea in maternally inherited nonsyndromic deafness, to more widespread lesions including myopathies, encephalomyopathies, cardiopathies, or complex multisystem syndromes. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights in this rapidly expanding area of human pathology.

  17. Characterization of changes in gene expression and biochemical pathways at low levels of benzene exposure

    NARCIS (Netherlands)

    Thomas, Reuben; Hubbard, Alan E.; McHale, Cliona M.; Zhang, Luoping; Rappaport, Stephen M.; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z.; Jinot, Jennifer; Sonawane, Babasaheb R.; Smith, Martyn T.

    2014-01-01

    Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across

  18. Biochemical characterization and evaluation of cytotoxicity of antistaphylococcal chimeric protein P128

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    George Shilpa E

    2012-06-01

    Full Text Available Abstract Background Antibiotic resistant S. aureus infection is a global threat. Newer approaches are required to control this organism in the current scenario. Cell wall degrading enzymes have been proposed as antibacterial agents for human therapy. P128 is a novel antistaphylococcal chimeric protein under development against S. aureus for human use which derives its bacterial cell wall degrading catalytic endopeptidase domain from ORF56, the Phage K tail-structure associated enzyme. Lead therapeutic entities have to be extensively characterized before they are assessed in animals for preclinical safety and toxicity. P128 is effective against antibiotic resistant strains as well as against a panel of isolates of global significance. Its efficacy against S. aureus in vivo has been established in our lab. Against this background, this study describes the characterization of this protein for its biochemical properties and other attributes. Results We evaluated the requirement or effect of divalent cations and the metal ion chelator, EDTA upon biological activity of P128. As the protein is intended for therapeutic use, we tested its activity in presence of body fluids and antibodies specific to P128. For the same reason, we used standard human cell lines to evaluate cytotoxic effects, if any. The divalent cations, calcium and magnesium at upto 25 mM and Zinc upto 2.5 mM neither inhibited nor enhanced P128 activity. Incubation of this protein with EDTA, human serum, plasma and blood also did not alter the antibacterial properties of the molecule. No inhibitory effect was observed in presence of hyper-immune sera raised against the protein. Finally, P128 did not show any cytotoxic effect on HEp2 and Vero cells at the highest concentration (5 mg/mL tested. Conclusions The results presented here throw light on several properties of protein P128. Taken together, these substantiate the potential of P128 for therapeutic use against S. aureus

  19. Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.

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    Sandra Pahr

    Full Text Available Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37 and in the eukaryotic system (BvTri a 37 as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN. Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of

  20. Mitochondrial DNA COI characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) from Paraguay and Uruguay.

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    Arnemann, J A; James, W J; Walsh, T K; Guedes, J V C; Smagghe, G; Castiglioni, E; Tay, W T

    2016-04-07

    Since its detection in Brazil in 2013, the Old World cotton bollworm Helicoverpa armigera has been reported in Argentina, Paraguay, and Bolivia. Here we present evidence extending the South American range of H. armigera to Uruguay, using polymerase chain reaction and sequencing of the partial mitochondrial DNA (mtDNA) cytochrome oxidase I region. Molecular characterization of this gene region from individuals from Paraguay also supports previous morphological identification of H. armigera in Paraguay. Shared mtDNA haplotypes in H. armigera from Brazil, Uruguay, and Paraguay were identified. Additional surveying of populations in this region will be imperative to better monitor and understand factors that are underpinning its presence and successful adaptation in these South American regions. We discuss our findings with respect to the development of resistance pest management strategies of this invasive insect pest in a predominantly monoculture soybean crop landscape in the Southern Cone region.

  1. Cloning, characterization, and expression of Cytochrome b (Cytb)-a key mitochondrial gene from Prorocentrum donghaiense

    Institute of Scientific and Technical Information of China (English)

    ZHAO Liyuan; MI Tiezhu; ZHEN Yu; YU Zhigang

    2012-01-01

    Mitochondrial cytochrome b (Cytb),one of the few proteins encoded by the mitochondrial DNA,plays an important role in transferring electrons.As a mitochondrial gene,it has been widely used for phylogenetic analysis.Previously,a 949-bp fragment of the coding gene and mRNA editing were characterized from Prorocentrum donghaiense,which might prove useful for resolving P.donghaiense from closely related species.However,the full-length coding region has not been characterized.In this study,we used rapid amplification of cDNA ends (RACE) to obtain full-length,1124 bp cDNA.Cytb transcript contained a standard initiation codon ATG,but did not have a recognizable stop codon.Homology comparison showed that the P.donghaiense Cytb had a high sequence identity to Cytb sequences from other dinoflagellate species.Phylogenetic analysis placed Cytb from P.donghaiense in the clade of dinofiagellates and it clustered together strongly with that from P.minimum.Based on the full-length sequence,we inferred 32 editing events at different positions,accounting for 2.93% of the Cytb gent.34.4% (11) of the changes were A to G,25% (8) were T to C,and 25% (8) were C to U,with smaller proportions of G to C and G to A edits (9.4% (3) and 6.2% (2),respectively).The expression level of the Cytb transcript was quantified by real-time PCR with a TaqMan probe at different times during the whole growth phase.The average Cytb transcript was present at 39.27±7.46 copies of cDNA per cell during the whole growth cycle,and the expression of Cytb was relatively stable over the different phases.These results deepen our understanding of the structure and characteristics of Cytb in P.donghaiense,and confirmed that Cytb in P.donghaiense is a candidate reference gene for studying the expression of other genes.

  2. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

    Science.gov (United States)

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg

    2014-01-01

    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  3. Biochemical characterization of enzyme fidelity of influenza A virus RNA polymerase complex.

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    Shilpa Aggarwal

    Full Text Available BACKGROUND: It is widely accepted that the highly error prone replication process of influenza A virus (IAV, together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol, is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized. PRINCIPAL FINDINGS: Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT of human immunodeficiency virus (HIV-1 and murine leukemia virus (MuLV, which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg(++ with Mn(++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn(++. Finally, when the IAV nucleoprotein (NP was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity. SIGNIFICANCE: Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be

  4. Structural And Biochemical Characterization of the Therapeutic A. Variabilis Phenylalanine Ammonia Lyase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, L.; Gamez, A.; Archer, H.; Abola, E.E.; Sarkissian, C.N.; Fitzpatrick, P.; Wendt, D.; Zhang, Y.; Vellard, M.; Bliesath, J.; Bell, S.; Lemont, J.; Scriver, C.R.; Stevens, R.C.

    2009-05-26

    We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides

  5. Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing

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    Vasudeo Zambare, Archana Zambare, Kasiviswanath Muthukumarappan, Lew P. Christopher

    2011-01-01

    Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.

  6. BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF RECOMBINANT YEAST PROTEASOME MATURATION FACTOR UMP1

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    Bebiana Sá-Moura

    2013-04-01

    We show that recombinant Ump1 is purified as a mixture of different oligomeric species and thatoligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein.Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump1 shows characteristics of anintrinsically disordered protein, which might become structured only upon interaction with the proteasomesubunits.

  7. NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

    Science.gov (United States)

    Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A; Li, Wei; Leoni, Valerio; Schon, Eric A; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

    2014-06-03

    Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans.

  8. Biochemical abnormalities in Pearson syndrome.

    Science.gov (United States)

    Crippa, Beatrice Letizia; Leon, Eyby; Calhoun, Amy; Lowichik, Amy; Pasquali, Marzia; Longo, Nicola

    2015-03-01

    Pearson marrow-pancreas syndrome is a multisystem mitochondrial disorder characterized by bone marrow failure and pancreatic insufficiency. Children who survive the severe bone marrow dysfunction in childhood develop Kearns-Sayre syndrome later in life. Here we report on four new cases with this condition and define their biochemical abnormalities. Three out of four patients presented with failure to thrive, with most of them having normal development and head size. All patients had evidence of bone marrow involvement that spontaneously improved in three out of four patients. Unique findings in our patients were acute pancreatitis (one out of four), renal Fanconi syndrome (present in all patients, but symptomatic only in one), and an unusual organic aciduria with 3-hydroxyisobutyric aciduria in one patient. Biochemical analysis indicated low levels of plasma citrulline and arginine, despite low-normal ammonia levels. Regression analysis indicated a significant correlation between each intermediate of the urea cycle and the next, except between ornithine and citrulline. This suggested that the reaction catalyzed by ornithine transcarbamylase (that converts ornithine to citrulline) might not be very efficient in patients with Pearson syndrome. In view of low-normal ammonia levels, we hypothesize that ammonia and carbamylphosphate could be diverted from the urea cycle to the synthesis of nucleotides in patients with Pearson syndrome and possibly other mitochondrial disorders.

  9. Biochemical and nutritional characterization of three prickly pear species with different ripening behavior.

    Science.gov (United States)

    Hernández-Pérez, Talia; Carrillo-López, Armando; Guevara-Lara, Fidel; Cruz-Hernández, Andrés; Paredes-López, Octavio

    2005-12-01

    Biochemical and nutritional changes were studied during the ripening process of three Opuntia morphospecies with different ripening behavior: Naranjona (O. ficus-indica), Blanca Cristalina (Opuntia sp.), and Esmeralda (Opuntia sp.) of early, early-intermediate, and intermediate-late ripening, respectively. In loss of fresh weight, Naranjona showed the highest values, while in Blanca Cristalina and Esmeralda, a discrete weight loss was found. No significant differences were found among morphospecies in soluble solids, total titratable acidity and pH during the postharvest days. Blanca Cristalina and Esmeralda showed an increase in the content of carotenoids, while these diminished in Naranjona. The cell wall enzymes evaluated showed particular behaviors during the ripening of each morphospecies suggesting a fine biochemical control and not a clear relationship between fruit softening and enzyme activity. This study provides basic information on prickly pear ripening, in order to understand this process for its control and for improving shelf life.

  10. Characterization of bean (Phaseolus vulgaris L.) ecotype "Fagiolo occhio nero di Oliveto Citra" using agronomic, biochemical and molecular approaches.

    Science.gov (United States)

    Zaccardelli, Massimo; Pentangelo, Alfonso; Tripodi, Pasquale

    2013-09-15

    Common bean (Phaseolus vulgaris) is the most important grain legume and plays a significant role in human nutrition being a major source of dietary protein and representing a rich source of minerals and certain vitamins. Several large germplasm collections have been established, which contain large amounts of genetic diversity, including wild and domesticated species. In this study agronomic, biochemical and molecular characterization of landrace bean named "Fagiolo occhio nero di Oliveto Citra" (Phaseolus vulgaris L.), is described. Seeds were obtained by local farmers and field trials were carried out during years 2009-2010, in the typical cultivation site (Oliveto Citra, Salerno Province), using two different densities of investment. During 2011, in order to evaluate the performance in different environments, field trials were conducted in three localities (Battipaglia, Oliveto Citra and Controne). Data analysis shows good adaptability across locations and similar grain yield using two spacing's of seeds. Morphological characterization and molecular analysis, using AFLP and Minisatellite molecular markers, were performed on ten "biotypes" collected from local farmers. Seeds characterization showed variability on the violet area surrounding the hilum (named as eye) while markers have provided useful information on relationships between biotypes. Biochemical analysis, which includes the contents of protein, minerals and antioxidants, shows how the composition is consistent with respect to other landraces and commercial cultivars. The landrace under study revealed genetic stability and good adaptation to cultivated environment with best performance in the native area. In addition, the bio-agronomic characteristics are in accord with studies reported in literature.

  11. BIOCHEMICAL AND PHYSIOLOGICAL CHARACTERIZATION OF OIL PALM INTERSPECIFIC HYBRIDS (Elaeis oleifera x Elaeis guineensis GROWN IN HYDROPONICS

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    Yurany Dayana Rivera

    2013-09-01

    Full Text Available The interspecific hybrid, Elaeis oleifera x Elaeis guineensis (OxG is an alternative for improving the competitiveness and sustainability of the Latin American oil palm agro-industry, because of its partial resistance to some lethal diseases and also because of the high quality of its oil. A comparative characterization was conducted of the physiological and biochemical performance of seedlings of six OxG hybrids grown in hydroponics. Gas exchange, vegetative growth, protein, sugar and photosynthetic pigment content, and antioxidant system activity were determined. With the exception of gas exchange, the other variables showed significant differences between materials. The ‘U1273’ and ‘U1737’ materials showed greater vegetative growth with no expression of biochemical traits, while the ‘U1914’and ‘U1990’ materials showed high levels of reducing and total sugars, photosynthetic pigments, and antioxidant system activities, characteristics that could confer them adaptation to stress conditions. With the standardized hydroponics technique, the optimal conditions for the growth of seedlings were ensured, the differences between materials and hybrid crosses were established, so those with promising features from the physiological and biochemical standpoint were identified. Finally, it could be used to study in a simple, fast, clean and inexpensive way, the effect of levels and sources of mineral nutrients on the growth and development of oil palm.

  12. Isolation and biochemical characterization of the microsomal fraction from the digestive gland of mussel Mytilus galloprovincialis Lam.

    Science.gov (United States)

    Viarengo, A; Pertica, M; Mancinelli, G; Palmero, S; Orunesu, M

    1986-01-01

    A procedure to prepare microsomes from the mussel digestive gland is proposed. The data concerning the biochemical characterization of this subcellular fraction shows a typical RNA:protein ratio, but the presence of hydrolytic enzymes was also found; therefore a mixture of hydrolase inhibitors to study the different biochemical characteristics was used. The biochemical data demonstrate that glucose-6-phosphatase activity (G6Pase), a typical microsomal marker in mammalian cells, is not present in mussel digestive gland microsomes but a high non-specific phosphatase activity was detected. Benzo[a]pyrene hydroxylase activity was found to be present although in a minimal amount. The evaluation of the molecular weight of the rRNA demonstrates that the larger ribosomal subunit contains RNA of Mr 1.40 X 10(-6) (approximately 26S) and the smaller subunit is composed of RNA of Mr 0.65 X 10(-6) (18S). The data from mussel digestive gland microsomes was compared with that experimentally obtained from rat liver microsomes and discussed from a functional or an evolutionary point of view.

  13. Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

    Science.gov (United States)

    Su, C S; Meyer, S A

    1991-01-01

    A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.

  14. Characterization of the Complete Mitochondrial Genome Sequence of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae from China

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    Guo-Hua Liu, Chun Li, Jia-Yuan Li, Dong-Hui Zhou, Rong-Chuan Xiong, Rui-Qing Lin, Feng-Cai Zou, Xing-Quan Zhu

    2012-01-01

    Full Text Available Sparganosis, caused by the plerocercoid larvae of members of the genus Spirometra, can cause significant public health problem and considerable economic losses. In the present study, the complete mitochondrial DNA (mtDNA sequence of Spirometra erinaceieuropaei from China was determined, characterized and compared with that of S. erinaceieuropaei from Japan. The gene arrangement in the mt genome sequences of S. erinaceieuropaei from China and Japan is identical. The identity of the mt genomes was 99.1% between S. erinaceieuropaei from China and Japan, and the complete mtDNA sequence of S. erinaceieuropaei from China is slightly shorter (2 bp than that from Japan. Phylogenetic analysis of S. erinaceieuropaei with other representative cestodes using two different computational algorithms [Bayesian inference (BI and maximum likelihood (ML] based on concatenated amino acid sequences of 12 protein-coding genes, revealed that S. erinaceieuropaei is closely related to Diphyllobothrium spp., supporting classification based on morphological features. The present study determined the complete mtDNA sequences of S. erinaceieuropaei from China that provides novel genetic markers for studying the population genetics and molecular epidemiology of S. erinaceieuropaei in humans and animals.

  15. Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis

    Directory of Open Access Journals (Sweden)

    Jolly Emmitt R

    2005-11-01

    Full Text Available Abstract Background A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. Results Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. Conclusion We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

  16. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR,HSR) were isolated from rabbit leg muscle using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).

  17. Characterization of 67 mitochondrial tRNA gene rearrangements in the Hymenoptera suggests that mitochondrial tRNA gene position is selectively neutral.

    Science.gov (United States)

    Dowton, Mark; Cameron, Stephen L; Dowavic, Jessica I; Austin, Andy D; Whiting, Michael F

    2009-07-01

    We present entire sequences of two hymenopteran mitochondrial genomes and the major portion of three others. We combined these data with nine previously sequenced hymenopteran mitochondrial genomes. This allowed us to infer and analyze the evolution of the 67 mitochondrial gene rearrangements so far found in this order. All of these involve tRNA genes, whereas four also involve larger (protein-coding or ribosomal RNA) genes. We find that the vast majority of mitochondrial gene rearrangements are independently derived. A maximum of four of these rearrangements represent shared, derived organizations, whereas three are convergently derived. The remaining mitochondrial gene rearrangements represent new mitochondrial genome organizations. These data are consistent with the proposal that there are an enormous number of alternative mitochondrial genome organizations possible and that mitochondrial genome organization is, for the most part, selectively neutral. Nevertheless, some mitochondrial genes appear less mobile than others. Genes close to the noncoding region are generally more mobile but only marginally so. Some mitochondrial genes rearrange in a pattern consistent with the duplication/random loss model, but more mitochondrial genes move in a pattern inconsistent with this model. An increased rate of mitochondrial gene rearrangement is not tightly associated with the evolution of parasitism. Although parasitic lineages tend to have more mitochondrial gene rearrangements than nonparasitic lineages, there are exceptions (e.g., Orussus and Schlettererius). It is likely that only a small proportion of the total number of mitochondrial gene rearrangements that have occurred during the evolution of the Hymenoptera have been sampled in the present study.

  18. Biochemical and immunohistochemical characterization of feline spongiform encephalopathy in a German captive cheetah.

    Science.gov (United States)

    Eiden, Martin; Hoffmann, Christine; Balkema-Buschmann, Anne; Müller, Matthias; Baumgartner, Katrin; Groschup, Martin H

    2010-11-01

    Feline spongiform encephalopathy (FSE) is a transmissible spongiform encephalopathy that affects domestic cats (Felis catus) and captive wild members of the family Felidae. In this report we describe a case of FSE in a captive cheetah from the zoological garden of Nuremberg. The biochemical examination revealed a BSE-like pattern. Disease-associated scrapie prion protein (PrP(Sc)) was widely distributed in the central and peripheral nervous system, as well as in the lymphoreticular system and in other tissues of the affected animal, as demonstrated by immunohistochemistry and/or immunoblotting. Moreover, we report for the first time the use of the protein misfolding cyclic amplification technique for highly sensitive detection of PrP(Sc) in the family Felidae. The widespread PrP(Sc) deposition suggests a simultaneous lymphatic and neural spread of the FSE agent. The detection of PrP(Sc) in the spleen indicates a potential for prion infectivity of cheetah blood.

  19. Biochemical characterization of consortium compost of toxic weeds Parthenium hysterophorus and Eichhornia crassipe.

    Science.gov (United States)

    Khaket, Tejinder Pal; Singh, Mangal; Dhanda, Suman; Singh, Talwinder; Singh, Jasbir

    2012-11-01

    Parthenium hysterophorus and Eichhornia crassipes are two uncontrolled weeds with high concentration of N, P, K, Zn and Fe that makes them suitable for composting. Three types of compost viz. Parthenium and Eichhornia each alone as well as combined were prepared. Biochemical and enzymatic analysis of the compost in addition to seed germination was performed. Phenols, organic carbon, C/N and C/P ratios were found to decrease significantly while N, P, K, polyphenol oxidase increased significantly in combined compost. Furthermore, seed germination test of Vigna radiata and Triticum seeds, revealed a significant increase in root, shoot length and germination index in 60days old combined compost. It can be concluded that combined composting of Parthenium with Eichhornia not only reduces the allelopathic effect but also increases its nutrient quality and thus could be promising for organic farming and bioremediation.

  20. Cytogenetic and biochemical characterization of scots pine trees differing in resin productivity

    Energy Technology Data Exchange (ETDEWEB)

    Butorina, A.K.; Muraya, L.S.; Ryazantseva, L.A.; Vysotskii, A.A.

    1988-11-01

    The groups of Scots pine trees differing in resin productivity were compared on the basis of meiotic abnormalities. Analysis of variance by Fisher's criterion showed significant differences for these parameters between the group of high resin producing trees and the combined group of medium and low resin producing forms. These differences are related with the predominance of gene and genomic mutations in the former group, in which the higher frequency of meiotic abnormalities is genetically determined. Significant differences have been also observed between the group for the ratio for sucrose and reducing sugars in the year-old needle in the mid-vegetative period. Maximum effect of this biochemical parameter on resin productivity in pine compared to other parameters studied has been established. It is suggested that the high resin productivity of mutant is the response of the organism to metabolic disbalance caused by the mutant genes.

  1. Characterizing genetic diversity of contemporary pacific chickens using mitochondrial DNA analyses.

    Directory of Open Access Journals (Sweden)

    Kelsey Needham Dancause

    Full Text Available BACKGROUND: Mitochondrial DNA (mtDNA hypervariable region (HVR sequences of prehistoric Polynesian chicken samples reflect dispersal of two haplogroups--D and E--by the settlers of the Pacific. The distribution of these chicken haplogroups has been used as an indicator of human movement. Recent analyses suggested similarities between prehistoric Pacific and South American chicken samples, perhaps reflecting prehistoric Polynesian introduction of the chicken into South America. These analyses have been heavily debated. The current distribution of the D and E lineages among contemporary chicken populations in the Western Pacific is unclear, but might ultimately help to inform debates about the movements of humans that carried them. OBJECTIVES: We sought to characterize contemporary mtDNA diversity among chickens in two of the earliest settled archipelagos of Remote Oceania, the Marianas and Vanuatu. METHODS: We generated HVR sequences for 43 chickens from four islands in Vanuatu, and for 5 chickens from Guam in the Marianas. RESULTS: Forty samples from Vanuatu and three from Guam were assigned to haplogroup D, supporting this as a Pacific chicken haplogroup that persists in the Western Pacific. Two haplogroup E lineages were observed in Guam and two in Vanuatu. Of the E lineages in Vanuatu, one was identical to prehistoric Vanuatu and Polynesian samples and the other differed by one polymorphism. Contrary to our expectations, we observed few globally distributed domesticate lineages not associated with Pacific chicken dispersal. This might suggest less European introgression of chickens into Vanuatu than expected. If so, the E lineages might represent lineages maintained from ancient Pacific chicken introductions. The Vanuatu sample might thus provide an opportunity to distinguish between maintained ancestral Pacific chicken lineages and replacement by global domesticates through genomic analyses, which could resolve questions of contemporary

  2. Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco

    Institute of Scientific and Technical Information of China (English)

    Yun WANG; Mei ZHANG; Ke KE; Ying Tang LU

    2005-01-01

    By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 (accession number AY971376), which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526 nmol/min/mg and 210 μg/ml respectively with calf thymus histone (fraction Ⅲ, abbreviated to histone Ⅲs) as substrate. For substrate syntide-2, NtCPK5 showed a higher. Vmax of 2008 nmol/min/mg and a lower Km of 30 μM. The K0.5 of calcium activation was 0.04 μM or 0.06 μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.

  3. Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1.

    Science.gov (United States)

    Davis, Mindy I; Gross, Stefan; Shen, Min; Straley, Kimberly S; Pragani, Rajan; Lea, Wendy A; Popovici-Muller, Janeta; DeLaBarre, Byron; Artin, Erin; Thorne, Natasha; Auld, Douglas S; Li, Zhuyin; Dang, Lenny; Boxer, Matthew B; Simeonov, Anton

    2014-05-16

    Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.

  4. Characteristics of mitochondrial calpains.

    Science.gov (United States)

    Ozaki, Taku; Tomita, Hiroshi; Tamai, Makoto; Ishiguro, Sei-Ichi

    2007-09-01

    Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.

  5. The impact of mitochondrial aldehyde dehydrogenase (ALDH2) activation by Alda-1 on the behavioral and biochemical disturbances in animal model of depression.

    Science.gov (United States)

    Stachowicz, Aneta; Głombik, Katarzyna; Olszanecki, Rafał; Basta-Kaim, Agnieszka; Suski, Maciej; Lasoń, Władysław; Korbut, Ryszard

    2016-01-01

    The etiology of depression remains still unclear. Recently, it has been proposed, that mitochondrial dysfunction may be associated with development of mood disorders, such as depression, bipolar disorder and anxiety disorders. Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, a small-molecule activator of ALDH2, on depressive- and anxiety-like behaviors in an animal model of depression - the prenatally stressed rats - using behavioral, molecular and proteomic methods. Prolonged Alda-1 administration significantly increased the climbing time, tended to reduce the immobility time and increased the swimming time of the prenatally stressed rats in the forced swim test. Moreover, treatment of prenatally stressed rats with Alda-1 significantly increased number of entries into the open arms of the maze and the time spent therein, as assessed by elevated plus-maze test. Such actions were associated with reduction of plasma 4-HNE-protein content, decrease of TNF-α mRNA and increase of PGC-1α (regulator of mitochondrial biogenesis) mRNA level in the frontal cortex and hippocampus of the prenatally stressed rats as well as with normalization of peripheral immune parameters and significant changes in expression of 6 and 4 proteins related to mitochondrial functions in the frontal cortex and hippocampus, respectively. Collectively, ALDH2 activation by Alda-1 led to a significant attenuation of depressive- and anxiety-like behaviors in the prenatally stressed rats. The pattern of changes suggested mitoprotective effect of Alda-1, however the exact functional consequences of the revealed alterations require further investigation.

  6. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

    Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

  7. Biochemical and Biophysical Characterization of Recombinant Yeast Proteasome Maturation Factor UMP1

    Directory of Open Access Journals (Sweden)

    Bebiana Sá-Moura

    2013-04-01

    Full Text Available Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump1 is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump1 is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump1 in E. coli for biophysical and structural analysis.We show that recombinant Ump1 is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatics tools, biochemical and structural analysis revealed that Ump1 shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.

  8. Fluorescence lifetime imaging for the characterization of the biochemical composition of atherosclerotic plaques

    Science.gov (United States)

    Phipps, Jennifer; Sun, Yinghua; Saroufeem, Ramez; Hatami, Nisa; Fishbein, Michael C.; Marcu, Laura

    2011-09-01

    This study investigates the ability of a flexible fiberoptic-based fluorescence lifetime imaging microscopy (FLIM) technique to resolve biochemical features in plaque fibrotic cap associated with plaque instability and based solely on fluorescence decay characteristics. Autofluorescence of atherosclerotic human aorta (11 autopsy samples) was measured at 48 locations through two filters, F377: 377/50 and F460: 460/60 nm (center wavelength/bandwidth). The fluorescence decay dynamic was described by average lifetime (τ) and four Laguerre coefficients (LECs) retrieved through a Laguerre deconvolution technique. FLIM-derived parameters discriminated between four groups [elastin-rich (ER), elastin and macrophage-rich (E+M), collagen-rich (CR), and lipid-rich (LR)]. For example, τF377 discriminated ER from CR (R = 0.84); τF460 discriminated E+M from CR and ER (R = 0.60 and 0.54, respectively); LEC-1F377 discriminated CR from LR and E+M (R = 0.69 and 0.77, respectively); P 87% (all cases) and sensitivity as high as 86%. Current results demonstrate for the first time that clinically relevant features (e.g., ratios of lipid versus collagen versus elastin) can be evaluated with a flexible-fiber based FLIM technique without the need for fluorescence intensity information or contrast agents.

  9. Purification and partial biochemical characterization of polyphenol oxidase from mango (Mangifera indica cv. Manila).

    Science.gov (United States)

    Palma-Orozco, Gisela; Marrufo-Hernández, Norma A; Sampedro, José G; Nájera, Hugo

    2014-10-08

    Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20-70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM.

  10. Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria.

    Science.gov (United States)

    Di Salvo, Luciana P; Silva, Esdras; Teixeira, Kátia R S; Cote, Rosalba Esquivel; Pereyra, M Alejandra; García de Salamone, Inés E

    2014-12-01

    Azospirillum is a plant growth-promoting rhizobacteria (PGPR) genus vastly studied and utilized as agriculture inoculants. Isolation of new strains under different environmental conditions allows the access to the genetic diversity and improves the success of inoculation procedures. Historically, the isolation of this genus has been performed by the use of some traditional culture media. In this work we characterized the physiology and biochemistry of five different A. brasilense strains, commonly used as cereal inoculants. The aim of this work is to contribute to pose into revision some concepts concerning the most used protocols to isolate and characterize this bacterium. We characterized their growth in different traditional and non-traditional culture media, evaluated some PGPR mechanisms and characterized their profiles of fatty acid methyl esters and carbon-source utilization. This work shows, for the first time, differences in both profiles, and ACC deaminase activity of A. brasilense strains. Also, we show unexpected results obtained in some of the evaluated culture media. Results obtained here and an exhaustive knowledge revision revealed that it is not appropriate to conclude about bacterial species without analyzing several strains. Also, it is necessary to continue developing studies and laboratory techniques to improve the isolation and characterization protocols.

  11. Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro

    DEFF Research Database (Denmark)

    Jaeger, K E; Kharazmi, A; Høiby, N

    1991-01-01

    on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused...... concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect...... chromatography revealed spherical particles with diameters ranging from 5 to 20 nm. Biochemical characterization and SDS polyacrylamide gel electrophoresis suggested that these particles consisted of protein and carbohydrate including lipopolysaccharide with the major enzyme activity being lipase. Various...

  12. Biochemical characterization of Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus, a Gram-negative developmental bacterium.

    Science.gov (United States)

    Udo, H; Inouye, M; Inouye, S

    1997-01-03

    Pkn2, a protein Ser/Thr kinase, from the developmental bacterium Myxococcus xanthus was expressed under a T7 promoter in Escherichia coli and purified. Purified Pkn2 retained the autophosphorylation activity with the Km value of 177 microM for ATP and 73 nmol/min/mg for Vmax. The optimum pH and temperature were determined to be 7.5 and 35 degrees C, respectively. The autophosphorylation activity was inhibited by staurosporine with the IC50 value of 400 nM while H-7 and genistein had little effect on this kinase. Pkn2 appears to be unique for its higher manganese dependence. This is the first biochemical characterization of the prokaryotic protein Ser/Thr kinase.

  13. Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway

    Energy Technology Data Exchange (ETDEWEB)

    Hicks, Katherine A.; Ealick, Steven E.

    2016-05-25

    HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

  14. Characterization of the complete mitochondrial genome of the king pigeon (Columba livia breed king).

    Science.gov (United States)

    Zhang, Rui-Hua; He, Wen-Xiao; Xu, Tong

    2015-06-01

    The king pigeon is a breed of pigeon developed over many years of selective breeding primarily as a utility breed. In the present work, we report the complete mitochondrial genome sequence of king pigeon for the first time. The total length of the mitogenome was 17,221 bp with the base composition of 30.14% for A, 24.05% for T, 31.82% for C, and 13.99% for G and an A-T (54.22 %)-rich feature was detected. It harbored 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one non-coding control region (D-loop region). The arrangement of all genes was identical to the typical mitochondrial genomes of pigeon. The complete mitochondrial genome sequence of king pigeon would serve as an important data set of the germplasm resources for further study.

  15. Complete mitochondrial DNA sequence of the endangered fish (Bahaba taipingensis): Mitogenome characterization and phylogenetic implications.

    Science.gov (United States)

    Zhao, Linlin; Gao, Tianxiang; Lu, Weihua

    2015-01-01

    To understand the systematic status of Bahaba taipingensis within Sciaenidae, the complete mitochondrial genome (mitogenome) sequence of Chinese bahaba has recently been determined by long PCR and primer walking methods. The complete mitochondrial genome is 16500 bp in length and contains 37 mitochondrial genes (13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes) as well as a control region (CR) as other bony fishes. Within the control region, we identified the extended termination associated sequence domain (ETAS), the central conserved sequence block domain (CSB-D, SCB-E and CSB-F) and the conserved sequence block domain (CSB-1, CSB-2 and CSB-3). Phylogenetic analyses revealed that Bahaba taipingensis is more closely related to Pseudosciaeniae than Argyrosominae and Sciaeninae. Additionally, Bahaba taipingensis is the sister taxon of Miichthys miiuy, and those two are sister to Collichthys plus Larimichthys.

  16. Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization.

    Science.gov (United States)

    Mazzo, Francesca; Zwart, Ruud; Serratto, Giulia Maia; Gardinier, Kevin M; Porter, Warren; Reel, Jon; Maraula, Giovanna; Sher, Emanuele

    2016-08-01

    Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that

  17. Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

    Science.gov (United States)

    Perfetto, Rosa; Del Prete, Sonia; Vullo, Daniela; Sansone, Giovanni; Barone, Carmela; Rossi, Mosè; Supuran, Claudiu T; Capasso, Clemente

    2017-12-01

    A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 10(5) s(-1) and kcat/Km of 3.6 × 10(7) M(-1) × s(-1). The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.

  18. Microsatellite loci and the complete mitochondrial DNA sequence characterized through next generation sequencing and de novo genome assembly for the critically endangered orange-bellied parrot, Neophema chrysogaster.

    Science.gov (United States)

    Miller, Adam D; Good, Robert T; Coleman, Rhys A; Lancaster, Melanie L; Weeks, Andrew R

    2013-01-01

    A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.

  19. Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization.

    Science.gov (United States)

    Challier, Emilse; Lisa, María-Natalia; Nerli, Bibiana B; Calcaterra, Nora B; Armas, Pablo

    2014-01-01

    Cellular nucleic acid binding protein (CNBP) is a highly conserved multi-zinc knuckle protein that enhances c-MYC expression, is related to certain human muscular diseases and is required for proper rostral head development. CNBP binds to single-stranded DNA (ssDNA) and RNA and acts as nucleic acid chaperone. Despite the advances made concerning CNBP biological roles, a full knowledge about the structure-function relationship has not yet been achieved, likely due to difficulty in obtaining pure and tag-free CNBP. Here, we report a fast, simple, reproducible, and high-performance expression and purification protocol that provides recombinant tag-free CNBP from Escherichia coli cultures. We determined that tag-free CNBP binds its molecular targets with higher affinity than tagged-CNBP. Furthermore, fluorescence spectroscopy revealed the presence of a unique and conserved tryptophan, which is exposed to the solvent and involved, directly or indirectly, in nucleic acid binding. Size-exclusion HPLC revealed that CNBP forms homodimers independently of nucleic acid binding and coexist with monomers as non-interconvertible forms or in slow equilibrium. Circular dichroism spectroscopy showed that CNBP has a secondary structure dominated by random-coil and β-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBP structural stability increased in the presence of single-stranded nucleic acid targets similar to other unstructured nucleic acid chaperones. Altogether, data suggest that CNBP is a flexible protein with interspersed structured zinc knuckles, and acquires a more rigid structure upon nucleic acid binding.

  20. Molecular and biochemical characterization of xrs mutants defective in Ku80.

    Science.gov (United States)

    Singleton, B K; Priestley, A; Steingrimsdottir, H; Gell, D; Blunt, T; Jackson, S P; Lehmann, A R; Jeggo, P A

    1997-01-01

    The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities. PMID:9032253

  1. Biochemical, serological, and virulence characterization of clinical and oyster Vibrio parahaemolyticus isolates.

    Science.gov (United States)

    Jones, Jessica L; Lüdeke, Catharina H M; Bowers, John C; Garrett, Nancy; Fischer, Markus; Parsons, Michele B; Bopp, Cheryl A; DePaola, Angelo

    2012-07-01

    In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2α or T3SS2β genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2α genes, and all isolates negative for tdh and positive for trh possessed all T3SS2β genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2α, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2β genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.

  2. Biochemical characterization of embryogenic calli of Vanilla planifolia in response to two years of thidiazuron treatment.

    Science.gov (United States)

    Kodja, Hippolyte; Noirot, Michel; Khoyratty, Shahnoo S; Limbada, Hafsah; Verpoorte, Robert; Palama, Tony Lionel

    2015-11-01

    Vanilla planifolia embryogenic calli were cultured for two years on a medium containing thidiazuron (TDZ). Due to the presence of TDZ, these calli were under permanent chemical treatment and the differentiation of adventitious shoots from protocorm-like-bodies (PLBs) was blocked. When embryogenic calli were transferred onto a medium without TDZ, shoot organogenesis and plantlet regeneration occurred. To gain better knowledge about the biochemical and molecular processes involved in the morphoregulatory role of TDZ, hormonal and metabolomic analyses were performed. Our results indicate that in the presence of TDZ, embryogenic calli contained a high amount of abscisic acid (ABA) essentially metabolized into abscisic acid glucosyl ester (ABAGE) and phaseic acid (PA), which was the most abundant. When transferred onto a medium without TDZ, shoot regeneration and development take place in four stages that include: embryogenic calli growth, differentiation of PLBs from meristmatic cells zones (MCZ), shoot organogenesis from PLBs and the elongation of well-formed shoots. From a hormonal perspective, the significant reduction in ABA metabolism and its readjustment in the ABAGE pathway triggered PLBs formation. However, this first morphogenesis was stimulated by a strong reduction in IAA metabolism. The organogenesis of PLBs into shoots is associated with an increase in ABA catabolism and a gradual shift in cellular metabolism towards shoot differentiation. Thus, the initiation of the elongation process in shoots is correlated with an alteration in metabolite composition, including an increase in energy reserves (sucrose/starch) and a rapid decrease in alanine content. Our data highlighted the relationship between endogenous hormone signalling, carbohydrate metabolism and shoot organogenesis in Orchid plants.

  3. Further biochemical characterization of an Na+ pump inhibitor purified from human urine.

    Science.gov (United States)

    Crabos, M; Grichois, M L; Guicheney, P; Wainer, I W; Cloix, J F

    1987-01-02

    An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.

  4. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

    Directory of Open Access Journals (Sweden)

    Guillaume Manat

    Full Text Available Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2 catalyze the dephosphorylation of C55-PP molecules on the same (outer side of the plasma membrane.

  5. Expressional and Biochemical Characterization of Rice Disease Resistance Gene Xa3/Xa26 Family

    Institute of Scientific and Technical Information of China (English)

    Songjie Xu; Yinglong Cao; Xianghua Li; Shiping Wang

    2007-01-01

    The rice (Oryza sativa L.) Xa3/Xa26 gene, conferring race-specific resistance to bacterial blight disease and encoding a leucine-rich repeat (LRR) receptor kinase-like protein, belongs to a multigene family consisting of tandem clustered homologous genes, colocalizing with several uncharacterized genes for resistance to bacterial blight or fungal blast. To provide more information on the expressional and biochemical characteristics of the Xa3/Xa26 family, we analyzed the family members. Four Xa3/Xa26 family members in the indica rice variety Teqing, which carries a bacterial blight resistance gene with a chromosomal location tightly linked to Xa3/Xa26, and five Xa3/Xa26 family members in the japonica rice variety Nipponbare, which carries at least one uncharacterized blast resistance gene, were constitutively expressed in leaf tissue. The result suggests that some of the family members may be candidates of these uncharacterized resistance genes. At least five putative N-glycosylation sites in the LRR domain of XA3/XA26 protein are not glycosylated. The XA3/XA26 and its family members MRKa and MRKc all possess the consensus sequences of paired cysteines, which putatively function in dimerization of the receptor proteins for signal transduction, immediately before the first LRR and immediately after the last LRR. However, no homo-dimer between the XA3/XA26 molecules or hetero-dimer between XA3/XA26 and MRKa or MRKc were formed, indicating that XA3/XA26 protein might function either as a monomer or a hetero-dimer formed with other protein outside of the XA3/XA26 family. These results provide valuable information for further extensive investigation into this multiple protein family.

  6. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae

    Institute of Scientific and Technical Information of China (English)

    Xin Zhang; Kun Yan Zhu

    2013-01-01

    Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control.However,our understanding of biochemical properties of insect CHSs has been very limited.We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito,Anopheles gambiae.Our study,which represents the first time to use a nonradioactive method to assay CHS activity in an insect species,determined the optimal conditions for measuring the enzyme activity,including pH,temperature,and concentrations of the substrate uridine diphosphate N-acetyl-D-glucosamine (UDPGlcNAc) and Mg++.The optimal pH was about 6.5-7.0,and the highest activity was detected at temperatures between 37℃ and 44℃.Dithithreitol is required to prevent melanization of the enzyme extract.CHS activity was enhanced at low concentration of GlcNAc,but inhibited at high concentrations.Proteolytic activation of the activity is significant both in the 500×g supernatant and the 40 000×g pellet.Our study revealed only slight in vitro inhibition ofA.gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5μmol/L) examined.There was no in vitro inhibition by polyoxin D at any concentration examined.Furthermore,we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined.Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A.gambiae.

  7. Biochemical Characterization of SFC-1, a class A carbapenem-hydrolyzing beta-lactamase.

    Science.gov (United States)

    Fonseca, Fátima; Sarmento, Ana Cristina; Henriques, Isabel; Samyn, Bart; van Beeumen, Jozef; Domingues, Pedro; Domingues, Maria Rosário; Saavedra, Maria José; Correia, António

    2007-12-01

    The carbapenem-hydrolyzing beta-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

  8. Biochemical Characterization of SFC-1, a Class A Carbapenem-Hydrolyzing β-Lactamase▿

    Science.gov (United States)

    Fonseca, Fátima; Sarmento, Ana Cristina; Henriques, Isabel; Samyn, Bart; van Beeumen, Jozef; Domingues, Pedro; Domingues, Maria Rosário; Saavedra, Maria José; Correia, António

    2007-01-01

    The carbapenem-hydrolyzing β-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam. PMID:17875998

  9. Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs)

    NARCIS (Netherlands)

    Bastiaan-Net, S.; Chanput, W.; Hertz, A.; Zwittink, R.D.; Mes, J.J.; Wichers, H.J.

    2013-01-01

    In this study two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganode

  10. Single arginine mutation in two yeast isocitrate dehydrogenases: biochemical characterization and functional implication.

    Science.gov (United States)

    Song, Ping; Wei, Huanhuan; Cao, Zhengyu; Wang, Peng; Zhu, Guoping

    2014-01-01

    Isocitrate dehydrogenase (IDH), a housekeeping gene, has drawn the attention of cancer experts. Mutation of the catalytic Arg132 residue of human IDH1 (HcIDH) eliminates the enzyme's wild-type isocitrate oxidation activity, but confer the mutant an ability of reducing α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). To examine whether an analogous mutation in IDHs of other eukaryotes could cause similar effects, two yeast mitochondrial IDHs, Saccharomyces cerevisiae NADP+-IDH1 (ScIDH1) and Yarrowia lipolytica NADP+-IDH (YlIDH), were studied. The analogous Arg residues (Arg148 of ScIDH1 and Arg141 of YlIDH) were mutated to His. The Km values of ScIDH1 R148H and YlIDH R141H for isocitrate were determined to be 2.4-fold and 2.2-fold higher, respectively, than those of the corresponding wild-type enzymes. The catalytic efficiencies (kcat/Km) of ScIDH1 R148H and YlIDH R141H for isocitrate oxidation were drastically reduced by 227-fold and 460-fold, respectively, of those of the wild-type enzymes. As expected, both ScIDH1 R148H and YlIDH R141H acquired the neomorphic activity of catalyzing α-KG to 2-HG, and the generation of 2-HG was confirmed using gas chromatography/time of flight-mass spectrometry (GC/TOF-MS). Kinetic analysis showed that ScIDH1 R148H and YlIDH R141H displayed 5.2-fold and 3.3-fold higher affinities, respectively, for α-KG than the HcIDH R132H mutant. The catalytic efficiencies of ScIDH1 R148H and YlIDH R141H for α-KG were 5.5-fold and 4.5-fold, respectively, of that of the HcIDH R132H mutant. Since the HcIDH Arg132 mutation is associated with the tumorigenesis, this study provides fundamental information for further research on the physiological role of this IDH mutation in vivo using yeast.

  11. Single arginine mutation in two yeast isocitrate dehydrogenases: biochemical characterization and functional implication.

    Directory of Open Access Journals (Sweden)

    Ping Song

    Full Text Available Isocitrate dehydrogenase (IDH, a housekeeping gene, has drawn the attention of cancer experts. Mutation of the catalytic Arg132 residue of human IDH1 (HcIDH eliminates the enzyme's wild-type isocitrate oxidation activity, but confer the mutant an ability of reducing α-ketoglutarate (α-KG to 2-hydroxyglutarate (2-HG. To examine whether an analogous mutation in IDHs of other eukaryotes could cause similar effects, two yeast mitochondrial IDHs, Saccharomyces cerevisiae NADP+-IDH1 (ScIDH1 and Yarrowia lipolytica NADP+-IDH (YlIDH, were studied. The analogous Arg residues (Arg148 of ScIDH1 and Arg141 of YlIDH were mutated to His. The Km values of ScIDH1 R148H and YlIDH R141H for isocitrate were determined to be 2.4-fold and 2.2-fold higher, respectively, than those of the corresponding wild-type enzymes. The catalytic efficiencies (kcat/Km of ScIDH1 R148H and YlIDH R141H for isocitrate oxidation were drastically reduced by 227-fold and 460-fold, respectively, of those of the wild-type enzymes. As expected, both ScIDH1 R148H and YlIDH R141H acquired the neomorphic activity of catalyzing α-KG to 2-HG, and the generation of 2-HG was confirmed using gas chromatography/time of flight-mass spectrometry (GC/TOF-MS. Kinetic analysis showed that ScIDH1 R148H and YlIDH R141H displayed 5.2-fold and 3.3-fold higher affinities, respectively, for α-KG than the HcIDH R132H mutant. The catalytic efficiencies of ScIDH1 R148H and YlIDH R141H for α-KG were 5.5-fold and 4.5-fold, respectively, of that of the HcIDH R132H mutant. Since the HcIDH Arg132 mutation is associated with the tumorigenesis, this study provides fundamental information for further research on the physiological role of this IDH mutation in vivo using yeast.

  12. Isolation and Biochemical Characterization of a Glucose Dehydrogenase from a Hay Infusion Metagenome

    OpenAIRE

    Alexander Basner; Garabed Antranikian

    2014-01-01

    Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate ex...

  13. Label-free biochemical characterization of bovine sperm cells using Raman microscopy

    Science.gov (United States)

    De Luca, A. C.; Managò, S.; Ferrara, M. A.; Sirleto, L.; Puglisi, R.; Balduzzi, D.; Galli, A.; Rendina, I.; Ferraro, P.; Coppola, G.

    2014-02-01

    The current study relates to a Raman spectroscopy-based method for addressing the problem of sex assessment in mammals. A direct method for sex predetermination in animals is based on the X- and Y-bearing sperm cells sorting before insemination. Our Raman spectroscope allows distinguishing and characterizing the difference between X- and Y-bearing sperm cells by detecting and analyzing their Raman spectra in a non-invasive and non-destructive way.

  14. General Biochemical Characterization of Thermostable Extracellular β-Amylase from Clostridium thermosulfurogenes

    OpenAIRE

    Hyun, H H; Zeikus, J G

    1985-01-01

    Clostridium thermosulfurogenes, an anaerobic bacterium which ferments starch into ethanol at 62°C, produced an active extracellular amylase and contained intracellular glucoamylase but not pullulanase activity. The extracellular amylase was purified 2.4-fold, and its general physicochemical and catalytic properties were examined. The extracellular amylase was characterized as a β-amylase (1,4-α-d-glucan maltohydrolase) based on demonstration of exocleavage activity and the production of malto...

  15. Biochemical Characterization of a First Fungal Esterase from Rhizomucor miehei Showing High Efficiency of Ester Synthesis

    OpenAIRE

    Yu Liu; Haibo Xu; Qiaojuan Yan; Shaoqing Yang; Xiaojie Duan; Zhengqiang Jiang

    2013-01-01

    BACKGROUND: Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis. METHODOLOGY/PRINCIPAL FINDINGS: A novel esterase-encoding gene from Rhizomucor miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide be...

  16. Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3

    OpenAIRE

    He Meng; Xiaojie Zhang; Genggeng Yu; Soo Jung Lee; Y Eugene Chen; Igor Prudovsky; Wang, Michael M.

    2012-01-01

    Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth mu...

  17. Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome.

    Science.gov (United States)

    Basner, Alexander; Antranikian, Garabed

    2014-01-01

    Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

  18. Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome.

    Directory of Open Access Journals (Sweden)

    Alexander Basner

    Full Text Available Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P, the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

  19. Combining measurements to estimate properties and characterization extent of complex biochemical mixtures; applications to Heparan Sulfate

    Science.gov (United States)

    Pradines, Joël R.; Beccati, Daniela; Lech, Miroslaw; Ozug, Jennifer; Farutin, Victor; Huang, Yongqing; Gunay, Nur Sibel; Capila, Ishan

    2016-04-01

    Complex mixtures of molecular species, such as glycoproteins and glycosaminoglycans, have important biological and therapeutic functions. Characterization of these mixtures with analytical chemistry measurements is an important step when developing generic drugs such as biosimilars. Recent developments have focused on analytical methods and statistical approaches to test similarity between mixtures. The question of how much uncertainty on mixture composition is reduced by combining several measurements still remains mostly unexplored. Mathematical frameworks to combine measurements, estimate mixture properties, and quantify remaining uncertainty, i.e. a characterization extent, are introduced here. Constrained optimization and mathematical modeling are applied to a set of twenty-three experimental measurements on heparan sulfate, a mixture of linear chains of disaccharides having different levels of sulfation. While this mixture has potentially over two million molecular species, mathematical modeling and the small set of measurements establish the existence of nonhomogeneity of sulfate level along chains and the presence of abundant sulfate repeats. Constrained optimization yields not only estimations of sulfate repeats and sulfate level at each position in the chains but also bounds on these levels, thereby estimating the extent of characterization of the sulfation pattern which is achieved by the set of measurements.

  20. Biochemical and Molecular Characterization of a Barley Seed ß-Glucosidase

    DEFF Research Database (Denmark)

    Leah, R.; Kigel, J.; Svendsen, I.

    1995-01-01

    A 60-kDa ß-glucosidase (BGQ60) was purified and characterized from seeds of barley (Hordeum vulgare L.). BGQ60 catalytic activity was restricted to the cleavage of short-chain oligosaccharides composed of(1, 2) -,(1, 2, 3) -, and/or(1, 2, 3, 4) -ß-linked glucose or mannose units. These oligosacch......A 60-kDa ß-glucosidase (BGQ60) was purified and characterized from seeds of barley (Hordeum vulgare L.). BGQ60 catalytic activity was restricted to the cleavage of short-chain oligosaccharides composed of(1, 2) -,(1, 2, 3) -, and/or(1, 2, 3, 4) -ß-linked glucose or mannose units....... These oligosaccharides are the primary products of endosperm cell wall polysaccharide hydrolysis by other enzymes. In keeping with this, complete hydrolysis of the major polysaccharide of barley starchy endosperm cell wall, (1-3,1-4)-ß-glucan, to free glucose was shown to require the concerted action of endo-(1-3,1-4)-ß...... interrupted by 9 introns. The complete nucleotide sequence of the bgq60 structural gene represents the first characterized plant gene encoding a ß-glucosidase. The barley BGQ60 is a novel plant ß-glucosidase with a hitherto undescribed specific enzymatic activity. The possible biological functions of BGQ60...

  1. Genetic and biochemical characterization of carotenoid biosynthesis mutants of Rhodobacter capsulatus.

    Science.gov (United States)

    Armstrong, G A; Schmidt, A; Sandmann, G; Hearst, J E

    1990-05-15

    We have used genetic and biochemical techniques to study carotenoid biosynthesis (crt) mutants of Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium. All nine identified crt genes are located within the 46-kilobase pair photosynthesis gene cluster, and eight of the crt genes form a subcluster. We have studied the operon structure of the crt gene cluster using transposon Tn5.7 mutants. The Tn5.7 insertion sites in 10 mutants have been mapped to high resolution (25-267 base pairs) by Southern hybridization. Two insertions each map within the coding regions of the crtA, crtC, crtE, and crtF genes, and one insertion lies within the crtI gene. The insertion in crtI is not polar on the downstream crtB gene, suggesting that crtI and crtB may form two separate operons. Another insertion located in the 5' noncoding region between the divergent crtA and crtI genes has no effect on wild-type pigmentation and apparently lies between the promoters for these operons. A Tn5.7 mutation in the 3' region of crtA yields a bacteriochlorophyll-minus phenotype, while a 5' insertion affects only carotenoid biosynthesis. Regulatory signals for transcription of a downstream operon required for bacteriochlorophyll biosynthesis may thus overlap the coding region of crtA. We also present the first evidence for the functions of the crtB, crtE, and crtJ gene products using a new in vitro assay for the incorporation of [14C]isopentenyl pyrophosphate into carotenoid precursors and phytoene in cell-free extracts. Extracts from a crtE mutant accumulate [14C]prephytoene pyrophosphate, while those from crtB and crtJ mutants accumulate [14C]geranylgeranyl pyrophosphate. We therefore propose that CrtE is the phytoene synthetase and that CrtB, and possibly CrtJ, are components of the prephytoene pyrophosphate synthetase.

  2. Biochemical compositional and technological characterizations of black and white myrtle ( Myrtus communis L.) fruits.

    Science.gov (United States)

    Hacıseferoğulları, Haydar; Ozcan, Mehmet Musa; Arslan, Derya; Unver, Ahmet

    2012-02-01

    Biochemical and technological properties were determined in developing Myrtus communis L. fruits (myrtle) from Mersin to investigate potential uses. Completely ripe black and white fruits contained ash, crude protein, crude oil, water- and alcohol soluble extracts, tartaric, malic and citric acids, and minerals including Ca, K, P, Mg and Na. Proximate compounds (%) for black and white fruits were: 7.47 and 6.36 protein, 3.487 and 3.453 oil, 3.02 and 2.30 ash, 24.28 and 26.09 dry matter, respectively. Fruits were found to be rich in some minerals such as Ca (6719.88 mg/kg and 4676.14 mg/kg), K (22647.78 mg/kg and 18339.84 mg/kg), Mg (2145.19 mg/kg and 1408.88 mg/kg), Na (3336.16 mg/kg and 2976.59 mg/kg) and P (4336.07 mg/kg and 3927.4 mg/kg). Also, physical properties such as length (14.94 mm and 13.64 mm), mass (0.94 g and 0.94 g), geometric mean diameter (12.73 mm and 12.31 mm), sphericity (0.85 and 0.90), diameter (11.76 and 11.70), projected area (1.48 cm(2) and 1.65 cm(2)), kernel density (757.47 kg/m(3)and 752.09 kg/m(3)), porosity (41.41% and 39.05%), bulk density (426.50 kg/m(3) and 431.05 kg/m(3)), terminal velocity (8.42 m/s and 8.49 m/s), volume (1.32 mm(3) and 1.35 mm(3)), repture strenth (1.77 N and 2.06 N), static (0.26-0.33 and 0.20-0.28) and dynamic coefficient (0.22-0.29 and 0.17-0.24) of friction of black myrtle and white myrtle fruits species were measured at 75.72% and 73.91% moisture content levels, respectively. These results show that both myrtle fruits may be useful for the evaluation of dietary information in important food crops.

  3. Ixodes ricinus tick lipocalins: identification, cloning, phylogenetic analysis and biochemical characterization.

    Directory of Open Access Journals (Sweden)

    Jérôme Beaufays

    Full Text Available BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Screening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for "Lipocalin from I. ricinus" and numbered from 1 to 14 (LIR1 to LIR14. According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50-70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4. CONCLUSIONS/SIGNIFICANCE: This work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C

  4. GM1-gangliosidosis in American black bears: clinical, pathological, biochemical and molecular genetic characterization.

    Science.gov (United States)

    Muthupalani, Sureshkumar; Torres, Paola A; Wang, Betty C; Zeng, Bai Jin; Eaton, Samuel; Erdelyi, Ildiko; Ducore, Rebecca; Maganti, Rajanikarath; Keating, John; Perry, Bain J; Tseng, Florina S; Waliszewski, Nicole; Pokras, Mark; Causey, Robert; Seger, Rita; March, Philip; Tidwell, Amy; Pfannl, Rolf; Seyfried, Thomas; Kolodny, Edwin H; Alroy, Joseph

    2014-04-01

    G(M1)-gangliosidosis is a rare progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of lysosomal β-galactosidase. We have identified seven American black bears (Ursus americanus) found in the Northeast United States suffering from G(M1)-gangliosidosis. This report describes the clinical features, brain MRI, and morphologic, biochemical and molecular genetic findings in the affected bears. Brain lipids were compared with those in the brain of a G(M1)-mouse. The bears presented at ages 10-14 months in poor clinical condition, lethargic, tremulous and ataxic. They continued to decline and were humanely euthanized. The T(2)-weighted MR images of the brain of one bear disclosed white matter hyperintensity. Morphological studies of the brain from five of the bears revealed enlarged neurons with foamy cytoplasm containing granules. Axonal spheroids were present in white matter. Electron microscopic examination revealed lamellated membrane structures within neurons. Cytoplasmic vacuoles were found in the liver, kidneys and chondrocytes and foamy macrophages within the lungs. Acid β-galactosidase activity in cultured skin fibroblasts was only 1-2% of control values. In the brain, ganglioside-bound sialic acid was increased more than 2-fold with G(M1)-ganglioside predominating. G(A1) content was also increased whereas cerebrosides and sulfatides were markedly decreased. The distribution of gangliosides was similar to that in the G(M1)-mouse brain, but the loss of myelin lipids was greater in the brain of the affected bear than in the brain of the G(M1) mouse. Isolated full-length cDNA of the black bear GLB1 gene revealed 86% homology to its human counterpart in nucleotide sequence and 82% in amino acid sequence. GLB1 cDNA from liver tissue of an affected bear contained a homozygous recessive T(1042) to C transition inducing a Tyr348 to His mutation (Y348H) within a highly conserved region of the GLB1 gene. The coincidence of several

  5. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays.

    Science.gov (United States)

    Barrientos, Antoni; Fontanesi, Flavia; Díaz, Francisca

    2009-10-01

    The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, the assays describe methods that form a biochemical characterization of the OXPHOS system in cells and mitochondria isolated from cultured cells or tissues.

  6. Identification and characterization of a mitochondrial unfolded protein response transcription factor ATFS-1 in Litopenaeus vannamei.

    Science.gov (United States)

    Chen, Yong-Gui; Yue, Hai-Tao; Zhang, Ze-Zhi; Yuan, Feng-Hua; Bi, Hai-Tao; Yuan, Kai; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-07-01

    A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence.

  7. Detailed structural and biochemical characterization of the nexin-dynein regulatory complex

    OpenAIRE

    Oda, Toshiyuki; Yanagisawa, Haruaki; Kikkawa, Masahide

    2015-01-01

    The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo–electron tomograph...

  8. Proteolytic cleavage of stingray phospholipase A2: Isolation and biochemical characterization of an active N-terminal form

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    Mejdoub Hafedh

    2011-07-01

    Full Text Available Abstract Background Mammalian GIB-PLA2 are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. The aim of this study was to check some biochemical and structural properties of a marine stingray phospholipase A2 (SPLA2. Results The effect of some proteolytic enzymes on SPLA2 was checked. Chymotrypsin and trypsin were able to hydrolyze SPLA2 in different ways. In both cases, only N-terminal fragments were accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic and chymotryptic attack generated 13 kDa and 12 kDa forms of SPLA2, respectively. Interestingly, the SPLA2 13 kDa form was inactive, whereas the SPLA2 12 kDa form conserved almost its full phospholipase activity. In the absence of bile slats both native and 12kDa SPLA2 failed to catalyse the hydrolysis of PC emulsion. When bile salts were pre-incubated with the substrate, the native kinetic protein remained linear for more than 25 min, whereas the 12 kDa form activity was found to decrease rapidly. Furthermore, The SPLA2 activity was dependent on Ca2+; other cations (Mg2+, Mn2+, Cd2+ and Zn2+ reduced the enzymatic activity notably, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. Conclusions Although marine and mammal pancreatic PLA2 share a high amino acid sequence homology, polyclonal antibodies directed against SPLA2 failed to recognize mammal PLA2 like the dromedary pancreatic one. Further investigations are needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of phospholipases.

  9. Characterization of blood biochemical markers during aging in the Grey Mouse Lemur (Microcebus murinus: impact of gender and season

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    Marchal Julia

    2012-11-01

    Full Text Available Abstract Background Hematologic and biochemical data are needed to characterize the health status of animal populations over time to determine the habitat quality and captivity conditions. Blood components and the chemical entities that they transport change predominantly with sex and age. The aim of this study was to utilize blood chemistry monitoring to establish the reference levels in a small prosimian primate, the Grey Mouse Lemur (Microcebus murinus. Method In the captive colony, mouse lemurs may live 10–12 years, and three age groups for both males and females were studied: young (1–3 years, middle-aged (4–5 years and old (6–10 years. Blood biochemical markers were measured using the VetScan Comprehensive Diagnostic Profile. Because many life history traits of this primate are highly dependent on the photoperiod (body mass and reproduction, the effect of season was also assessed. Results The main effect of age was observed in blood markers of renal functions such as creatinine, which was higher among females. Additionally, blood urea nitrogen significantly increased with age and is potentially linked to chronic renal insufficiency, which has been described in captive mouse lemurs. The results demonstrated significant effects related to season, especially in blood protein levels and glucose rates; these effects were observed regardless of gender or age and were likely due to seasonal variations in food intake, which is very marked in this species. Conclusion These results were highly similar with those obtained in other primate species and can serve as references for future research of the Grey Mouse Lemur.

  10. Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation.

    Science.gov (United States)

    Troncoso-Ponce, M A; Rivoal, J; Dorion, S; Moisan, M-C; Garcés, R; Martínez-Force, E

    2011-03-01

    A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation.

  11. Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

    Science.gov (United States)

    Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-03-01

    The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.

  12. Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

    Science.gov (United States)

    Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

    2012-07-01

    Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.

  13. Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.

    Science.gov (United States)

    Soriano-Maldonado, Pablo; Andújar-Sánchez, Montserrat; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier; Martínez-Rodríguez, Sergio

    2015-05-01

    N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as "Acylase Process". In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55-65 °C, with Co(2+) as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.

  14. Overexpression, purification and biochemical characterization of the wound-induced leucine aminopeptidase of tomato.

    Science.gov (United States)

    Gu, Y Q; Holzer, F M; Walling, L L

    1999-08-01

    Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A). While the expression of LapA genes are well characterized, the specificity of the LAP-A enzyme has not been studied. The LAP-A preprotein and mature polypeptide were overexpressed in Escherichia coli. PreLAP-A was not processed and was inactive accumulating in inclusion bodies. In contrast, 55-kDa mature LAP-A subunits assembled into an active, 357-kDa enzyme in E. coli. LAP-A from E. coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E. coli PepA) homologues. Similar to the porcine and E. coli enzymes, the tomato LAP-A had high temperature and pH optima. Mn2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow-binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs. The substrate specificities of porcine, E. coli and tomato LAPs were determined using amino-acid-p-nitroanilide and -beta-naphthylamide substrates. The tomato LAP-A preferentially hydrolyzed substrates with N-terminal Leu, Met and Arg residues. LAP-A had substantially lower levels of activity on other chromogenic substrates. Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted.

  15. Detailed structural and biochemical characterization of the nexin-dynein regulatory complex.

    Science.gov (United States)

    Oda, Toshiyuki; Yanagisawa, Haruaki; Kikkawa, Masahide

    2015-01-15

    The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo-electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.

  16. A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

    Directory of Open Access Journals (Sweden)

    Masakazu Kohda

    2016-01-01

    Full Text Available Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4 as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3 and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21 as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

  17. Cloning, characterization, and expression of Cytochrome b ( Cytb)—a key mitochondrial gene from Prorocentrum donghaiense

    Science.gov (United States)

    Zhao, Liyuan; Mi, Tiezhu; Zhen, Yu; Yu, Zhigang

    2012-05-01

    Mitochondrial cytochrome b (Cytb), one of the few proteins encoded by the mitochondrial DNA, plays an important role in transferring electrons. As a mitochondrial gene, it has been widely used for phylogenetic analysis. Previously, a 949-bp fragment of the coding gene and mRNA editing were characterized from Prorocentrum donghaiense, which might prove useful for resolving P. donghaiense from closely related species. However, the full-length coding region has not been characterized. In this study, we used rapid amplification of cDNA ends (RACE) to obtain full-length, 1 124 bp cDNA. Cytb transcript contained a standard initiation codon ATG, but did not have a recognizable stop codon. Homology comparison showed that the P. donghaiense Cytb had a high sequence identity to Cytb sequences from other dinoflagellate species. Phylogenetic analysis placed Cytb from P. donghaiense in the clade of dinoflagellates and it clustered together strongly with that from P. minimum. Based on the full-length sequence, we inferred 32 editing events at different positions, accounting for 2.93% of the Cytb gene. 34.4% (11) of the changes were A to G, 25% (8) were T to C, and 25% (8) were C to U, with smaller proportions of G to C and G to A edits (9.4% (3) and 6.2% (2), respectively). The expression level of the Cytb transcript was quantified by real-time PCR with a TaqMan probe at different times during the whole growth phase. The average Cytb transcript was present at 39.27±7.46 copies of cDNA per cell during the whole growth cycle, and the expression of Cytb was relatively stable over the different phases. These results deepen our understanding of the structure and characteristics of Cytb in P. donghaiense, and confirmed that Cytb in P. donghaiense is a candidate reference gene for studying the expression of other genes.

  18. Biochemical and genetical characterization of nitrate reductase deficient mutants of Petunia.

    Science.gov (United States)

    Steffen, A; Schieder, O

    1984-08-01

    Four NR(-) lines were selected by their resistance to 100 mM chlorate from X-ray irradiated protoplasts of haploid Petunia hybrida var. Mitchell. The four cell lines were characterized by the presence of xanthine dehydrogenase activity and by complementation tests via protoplast fusion. One mutant (line 1) was classified as defective in the NR apoprotein (tentatively, nia-type) and the other three (lines 2, 3, 4) in the molybdenum cofactor (tentatively, cnx-type). Some NR activity (15 %) could be restored by adding unphysiologically high concentrations of molybdate to the culture medium in two of the cnx-lines (lines 3 and 4). The third cnx-line (line 2) had no NR activity. A complementation analysis via protoplast fusion confirmed that the mutants comprised 3 non-allelic groups. From these results it can be concluded that these NR(-) mutants are recessive and that two of the cnx-mutants (lines 3, 4) are allelic.

  19. Biochemical characterization of a trypanosomatid isolated from the plant Amaranthus retroflexus

    Directory of Open Access Journals (Sweden)

    Clotilde Marín

    2000-10-01

    Full Text Available A protozoan flagelate has recently been isolated from Amaranthus retroflexus. This plant grows near economically important crops in southeastern Spain, which are known to be parasitized by Phytomonas spp. The present study focuses on the characterization of the energy metabolism of this new isolate. These flagellates utilize glucose efficiently as their primary energy source, although they are unable to completely degrade it. They excrete ethanol, acetate, glycine, and succinate in lower amount, as well as ammonium. The presence of glycosomes was indicated by the early enzymes of the glycolytic pathway, one enzyme of the glycerol pathway (glycerol kinase, and malate dehydrogenase. No evidence of a fully functional citric-acid cycle was found. In the absence of catalase activity, these flagellates showed significant superoxide dismutase activity located in the glycosomal and cytosolic fractions. These trypanosomes, despite being morphologically and metabolically similar to other Phytomonas isolated from the same area, showed significant differences, suggesting that they are phylogenetically different species.

  20. Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.

    Science.gov (United States)

    Inoue, Hiroyuki; Tamura, Takashi; Ehara, Nagisa; Nishito, Akira; Nakayama, Yumi; Maekawa, Makiko; Imada, Katsumi; Tanaka, Hidehiko; Inagaki, Kenji

    2002-08-27

    An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.

  1. Biochemical characterization and helix stabilizing properties of HSNP-C' from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    2000-01-19

    Helix stabilizing nucleoid protein HSNP-C' from the thermophilic archaeon Sulfolobus acidocaldarius has been characterized with respect to its interactions with nucleic acids by gel retardation assay, affinities to immobilized matrices, electron microscopy, and fluorescence titration. The amino acids implicated in the DNA binding site of the protein have been shown by selectively modifying specific amino acyl functional groups and looking at their effects on the DNA binding properties of the protein. Lysine, arginine, tryptophan, and tyrosine residues of the protein HSNP-C' were modified with pyridoxal-5-phosphate; 2,3-butanedione; BNPS-skatole; and tetranitromethane, respectively. The modification of residues was assessed according to standard procedures. The effect of the chemical modification on the function of the protein HSNP-C' with respect to DNA protein interactions was studied and the results indicate the definite involvement of tyrosines and also the significant involvement of the flanking tryptophan residues in the DNA binding domain on the protein.

  2. Biochemical characterization of the surface-associated lipase of Staphylococcus saprophyticus.

    Science.gov (United States)

    Sakinç, Türkân; Kleine, Britta; Gatermann, Sören G

    2007-09-01

    Staphylococcus saprophyticus, an important cause of urinary tract infections, produces a surface-associated lipase, Ssp. In contrast to other lipases, Ssp is a protein that is present in high amounts on the surface of the bacteria and it was shown that it is a true lipase. Characterization of S. saprophyticus lipase (Ssp) showed that it is more similar to Staphylococcus aureus lipase and Staphylococcus epidermidis lipase than to Staphylococcus hyicus lipase and Staphylococcus simulans lipase. Ssp showed an optimum of lipolytic activity at pH 6 and lost its activity at pH>8 or pH<5. The present results show that Ssp activity is dependent on Ca(2+). Consequently, activity increased c. 10-fold in the presence of 2 mM Ca(2+). Optimal activity was reached at 30 degrees C. It was also observed that the enzymatic activity of Ssp depends strongly on the acyl chain length of the substrate molecule.

  3. Biochemical characterization of trans-sialidase TS1 variants from Trypanosoma congolense

    Directory of Open Access Journals (Sweden)

    Dietz Frank

    2011-07-01

    Full Text Available Abstract Background Animal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense. Results Based on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the N-glycans on fetuin catalyzed by the trans-sialidases investigated. Conclusions The identification and characterization of a trans-sialidase gene family of the African parasite T. congolense has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the

  4. Biochemical characterization of GDP-L-fucose de novo synthesis pathway in fungus Mortierella alpina

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Yan [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Perepelov, Andrei V. [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation); Wang, Haiyan [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Zhang, Hao [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China); Knirel, Yuriy A. [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation); Wang, Lei [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Chen, Wei, E-mail: weichen@jiangnan.edu.cn [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China)

    2010-01-22

    Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. L-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-L-fucose (the nucleotide-activated form of L-fucose) in M. alpina. Genes encoding GDP-D-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-L-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k{sub m} values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 {sup o}C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-L-fucose biosynthetic pathway in fungi.

  5. Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog

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    Zaccolo Manuela

    2008-06-01

    Full Text Available Abstract Background A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Iα, PKA-IIα, PKA-IIβ in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging. Results The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor. Conclusion The novel analog combines good membrane permeability- comparable to 8-Br-cAMP – with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.

  6. Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix.

    Science.gov (United States)

    Yoshimura, Yukihiro; Araki, Aya; Maruta, Hitomi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-12-21

    Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.

  7. Characterization of a Dairy Gyr herd with respect to its mitochondrial DNA (mt DNA origin

    Directory of Open Access Journals (Sweden)

    Anibal Eugênio Vercesi Filho

    2010-01-01

    Full Text Available The Zebu breeds were introduced in Brazil mainly in the last century by imports from the Indian subcontinent. When the Zebu cattle arrived, the national herd suffered a significative change by backcrossing the national cows of taurine origin with Zebu sires. These processes created a polymorphism in the mitochondrial DNA (mtDNA in the Zebu animals with are in a major part derived from backcrossing and sharing mtDNA of taurine origin. To verify the maternal origin of cows belonging to the Dairy Gyr herd of APTA, Mococa 60 females were analyzed and 33 presented mtDNA from Bos taurus origin and 27 presented mtDNA from Bos indicus origin. None of these animals presented patterns of both mtDNA origins, indicating absence of heteroplasmy for these mitochondrial genotypes.

  8. Genetic characterization of Meigu goat (Capra hircus) based on the mitochondrial DNA.

    Science.gov (United States)

    Duan, Xiaoyue; Zhang, Hao; Li, Haijun; Niu, Lili; Wang, Linjie; Li, Li; Zhang, Hongping; Zhong, Tao

    2016-01-01

    Meigu goat (Capra hircus) is one of the indigenous goat breeds in China. Our research findings revealed that the entire mitochondrial genome of Meigu goat was 16,643 bp in length. The contents of A, C, T and G in the mitochondrial genome were 33.59%, 26.05%, 27.31% and 13.05%, respectively. The mitogenome of meigu goat contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region. Components of the Meigu goat's mitogenome were similar to those of other Capra hircus in gene arrangement and composition. These results could provide essential information for molecular phylogenetic and evolutionary analyses of domestic goats.

  9. Characterization of the complete mitochondrial genomes from Polycladida (Platyhelminthes) using next-generation sequencing.

    Science.gov (United States)

    Aguado, M Teresa; Grande, Cristina; Gerth, Michael; Bleidorn, Christoph; Noreña, Carolina

    2016-01-10

    The complete mitochondrial genomes of three polycladids, the acotylean Hoploplana elisabelloi and the cotyleans Enchiridium sp. and Prosthiostomum siphunculus have been assembled with high coverage from Illumina sequencing data. The mt genomes contain 36 genes including 12 of the 13 protein-coding genes characteristic for metazoan mitochondrial genomes, two ribosomal RNA genes, and 22 transfer RNA genes. Gene annotation, gene order, genetic code, start and stop codons and codon bias have been identified. In comparison with the well investigated parasitic Neodermata, our analysis reveals a great diversity of gene orders within Polycladida and Platyhelminthes in general. By analyzing representative genomes of the main groups of Platyhelminthes we explored the phylogenetic relationships of this group. The phylogenetic analyses strongly supported the monophyly of Polycladida, and based on a small taxon sampling suggest the monophyly of Acotylea and Cotylea.

  10. Complete sequence of the mitochondrial genome of Odontamblyopus rubicundus (Perciformes: Gobiidae): genome characterization and phylogenetic analysis.

    Science.gov (United States)

    Liu, Tianxing; Jin, Xiaoxiao; Wang, Rixin; Xu, Tianjun

    2013-12-01

    Odontamblyopus rubicundus is a species of gobiid fishes, inhabits muddy-bottomed coastal waters. In this paper, the first complete mitochondrial genome sequence of O. rubicundus is reported. The complete mitochondrial genome sequence is 17119 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a control region and an L-strand origin as in other teleosts. Most mitochondrial genes are encoded on H-strand except for ND6 and seven tRNA genes. Some overlaps occur in protein-coding genes and tRNAs ranging from 1 to 7 bp. The possibly nonfunctional L-strand origin folded into a typical stem-loop secondary structure and a conserved motif (5'-GCCGG-3') was found at the base of the stem within the tRNACys gene. The TAS, CSB-2 and CSB-3 could be detected in the control region. However, in contrast to most of other fishes, the central conserved sequence block domain and the CSB-1 could not be recognized in O. rubicundus, which is consistent with Acanthogobius hasta (Gobiidae). In addition, phylogenetic analyses based on different sequences of species of Gobiidae and different methods showed that the classification of O. rubicundus into Odontamblyopus due to morphology is debatable.

  11. Characterization of the complete mitochondrial genome of the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Wu, Qiu-Ling; Cui, Wen-Xia; Wei, Shu-Jun

    2015-02-01

    The complete mitochondrial genome of the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae) was determined (GenBank accession No. KF163965). The length of this mitochondrial genome is 15,377 bp with an A + T content of 82.5%. There are 37 typical animal mitochondrial genes, that is, 13 protein-coding, 2 rRNA and 22 tRNA gene and an A + T-rich region. The tRNA gene trnM was rearranged to the upstream of the trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Ten protein-coding genes stop with termination codon TAA, whereas three protein-coding gene use incomplete stop codon T. The A + T-region is located between rrnS and trnM with a length of 332 bp and A + T content of 94.88%.

  12. Complete sequence of the mitochondrial genome of Odontamblyopus rubicundus (Perciformes: Gobiidae): genome characterization and phylogenetic analysis

    Indian Academy of Sciences (India)

    Tianxing Liu; Xiaoxiao Jin; Rixin Wang; Tianjun Xu

    2013-12-01

    Odontamblyopus rubicundus is a species of gobiid fishes, inhabits muddy-bottomed coastal waters. In this paper, the first complete mitochondrial genome sequence of O. rubicundus is reported. The complete mitochondrial genome sequence is 17119 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, a control region and an L-strand origin as in other teleosts. Most mitochondrial genes are encoded on H-strand except for ND6 and seven tRNA genes. Some overlaps occur in protein-coding genes and tRNAs ranging from 1 to 7 bp. The possibly nonfunctional L-strand origin folded into a typical stem-loop secondary structure and a conserved motif (5′-GCCGG-3′) was found at the base of the stem within the $tRNA^{Cys}$ gene. The TAS, CSB-2 and CSB-3 could be detected in the control region. However, in contrast to most of other fishes, the central conserved sequence block domain and the CSB-1 could not be recognized in O. rubicundus, which is consistent with Acanthogobius hasta (Gobiidae). In addition, phylogenetic analyses based on different sequences of species of Gobiidae and different methods showed that the classification of O. rubicundus into Odontamblyopus due to morphology is debatable.

  13. Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus.

    Science.gov (United States)

    Pastorino, Boris A M; Peyrefitte, Christophe N; Almeras, Lionel; Grandadam, Marc; Rolland, Dominique; Tolou, Hugues J; Bessaud, Maël

    2008-02-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemic fever, rash and polyarthralgia in Africa and Asia. Although it is known since the 1950s, new epidemiological and clinical features reported during the recent outbreak in the Indian Ocean can be regarded as the emergence of a new disease. Numerous severe forms of the infection have been described that put emphasis on the lack of efficient antiviral therapy. Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 proteinase is an essential enzyme whose proteolytic activity is critical for virus replication. In this work, a recombinant CHIKV nsP2pro and a C-terminally truncated variant were expressed in Escherichia coli and purified by metal-chelate chromatography. The enzymatic properties of the proteinase were then determined using specific synthetic fluorogenic substrates. This study constitutes the first characterization of a recombinant CHIKV nsP2 cysteine protease, which may be useful for future drug screening.

  14. Biochemical Characterization of Tunisian Cichorium Intybus L. Roots and Optimization of Ultrasonic Inulin Extraction

    Directory of Open Access Journals (Sweden)

    Youkabed Ouederni Zarroug

    2016-11-01

    Full Text Available In this study Cichorium intybus L. roots were tested for its chemical composition, antioxidant activity, and phenolic profile. Optimization of ultrasonic inulin extraction using response surface methodology (RSM was further investigated. Chicory roots were found to have high value of total carbohydrates (70.43%, soluble fiber (66.93, Neutral detergent fiber (NDF (33.07%, potassium (380 mg/100g, calcium (540 mg/100g and sodium (140 mg/100g. Chicory roots exhibit a high content of flavonoids, polyphenols, and tannins. Antioxidant activity measurement reveals the capacity of Chicory roots to scavenge diphenylpicrylhydrazyl (DPPH radicals. Phenolic acids profile shows the abundance of vanillic acid (19.64% followed by protocatechuic acid (15.67%. The effect of three independent variables namely extraction time, the ratio of water to raw material and temperature on inulin extraction was studied. Optimum deciding responses were Inulin content, Total Soluble Solids (TSS content and Water produced inulin yield. The optimal ultrasonic extraction conditions were: extraction time 87 min, liquid to solid ratio 38 (ml/g and ultrasonic temperature 61 ̊ C. Under these conditions, the inulin content, TSS content and produced inulin yield were 35.92%, 24.72%, and 32.53%, respectively. The produced inulin was characterized by the Fourier infrared transformation (FTIR and observed by means of scanning electron microscopy (SEM.  

  15. Genetic and Biochemical Characterization of Monokaryotic Progeny Strains of Button Mushroom (Agaricus bisporus).

    Science.gov (United States)

    Kwon, Hyuk Woo; Choi, Min Ah; Yun, Yeo Hong; Oh, Youn-Lee; Kong, Won-Sik; Kim, Seong Hwan

    2015-03-01

    To promote the selection of promising monokaryotic strains of button mushroom (Agaricus bisporus) during breeding, 61 progeny strains derived from basidiospores of two different lines of dikaryotic parental strains, ASI1038 and ASI1346, were analyzed by nucleotide sequencing of the intergenic spacer I (IGS I) region in their rDNA and by extracellular enzyme assays. Nineteen different sizes of IGS I, which ranged from 1,301 to 1,348 bp, were present among twenty ASI1346-derived progeny strains, while 15 different sizes of IGS I, which ranged from 700 to 1,347 bp, were present among twenty ASI1038-derived progeny strains. Phylogenetic analysis of the IGS sequences revealed that different clades were present in both the ASI10388- and ASI1346-derived progeny strains. Plating assays of seven kinds of extracellular enzymes (β-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease) also revealed apparent variation in the ability to produce extracellular enzymes among the 40 tested progeny strains from both parental A. bisporus strains. Overall, this study demonstrates that characterization of IGS I regions and extracellular enzymes is useful for the assessment of the substrate-degrading ability and heterogenicity of A. bisporus monokaryotic strains.

  16. General Biochemical Characterization of Thermostable Extracellular beta-Amylase from Clostridium thermosulfurogenes.

    Science.gov (United States)

    Hyun, H H; Zeikus, J G

    1985-05-01

    Clostridium thermosulfurogenes, an anaerobic bacterium which ferments starch into ethanol at 62 degrees C, produced an active extracellular amylase and contained intracellular glucoamylase but not pullulanase activity. The extracellular amylase was purified 2.4-fold, and its general physicochemical and catalytic properties were examined. The extracellular amylase was characterized as a beta-amylase (1,4-alpha-d-glucan maltohydrolase) based on demonstration of exocleavage activity and the production of maltose with a beta-anomeric configuration from starch. The beta-amylase activity was stable and optimally active at 80 and 75 degrees C, respectively. The pH optimum for activity and the pH stability range was 5.5 to 6 and 3.5 to 6.5, respectively. The apparent [S](0.5V) and V(max) for beta-amylase activity on starch was 1 mg/ml and 60 U/mg of protein. Similar to described beta-amylase, the enzyme was inhibited by p-chloromercuribenzoate, Cu, and Hg; however, alpha- and beta-cyclodextrins were not competitive inhibitors. The beta-amylase was active and stable in the presence of air or 10% (vol/vol) ethanol. The beta-amylase and glucoamylase activities enabled the organism to actively ferment raw starch in the absence of significant pullulanase or alpha-amylase activity.

  17. Crab digestive lipase acting at high temperature: purification and biochemical characterization.

    Science.gov (United States)

    Cherif, Slim; Fendri, Ahmed; Miled, Nabil; Trabelsi, Heykel; Mejdoub, Hafedh; Gargouri, Youssef

    2007-08-01

    In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates have taken place and this had led to the emergence of some interesting new applications of these enzymes. However, much less is known about lipases from crustaceans. A lipolytic activity was located in the crab digestive glands (hepatopancreas), from which a crab digestive lipase (CDL) was purified. Pure CDL has a molecular mass of 65kDa as determined by SDS/PAGE analysis. Unlike known digestive lipases, CDL displayed its maximal activity on long and short-chain triacylglycerols at a temperature of 60 degrees C. A specific activity of 500U/mg or 130U/mg was obtained with TC(4) or olive oil as substrate, respectively. Only 10% of the maximal activity was detected at 37 degrees C. The enzyme retained 80% of its maximal activity when incubated during 10 min at 60 degrees C, and was completely inactivated at a temperature higher than 65 degrees C. Interestingly, neither colipase, nor bile salts were detected in the crab hepatopancreas. Which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produce bile salts. No similarity between the 13 N-terminal amino acid residues of CDL was found with those of known other digestive lipases.

  18. Biochemical characterization of a first fungal esterase from Rhizomucor miehei showing high efficiency of ester synthesis.

    Directory of Open Access Journals (Sweden)

    Yu Liu

    Full Text Available BACKGROUND: Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis. METHODOLOGY/PRINCIPAL FINDINGS: A novel esterase-encoding gene from Rhizomucor miehei (RmEstA was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL family IV and showing highest similarity (44% to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45°C, respectively. The enzyme was stable to 50°C, under a broad pH range (5.0-10.6. RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg(-1 and 228 U mg(-1 for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92% conversion yield when immobilized on AOT-based organogel. CONCLUSION: RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor miehei.

  19. HHF35, a muscle-actin-specific monoclonal antibody. I. Immunocytochemical and biochemical characterization.

    Science.gov (United States)

    Tsukada, T; Tippens, D; Gordon, D; Ross, R; Gown, A M

    1987-01-01

    A monoclonal antibody to muscle cell actin isotypes was produced and characterized. Immunocytochemical analysis of methanol-Carnoy's-fixed, paraffin-embedded human tissue revealed that this antibody, termed HHF35, reacts with skeletal muscle cells, cardiac muscle cells, smooth muscle cells, pericytes, and myoepithelial cells, but is nonreactive with endothelial, epithelial, neural, or connective tissue cells. When assayed by indirect immunofluorescence, HHF35 reacts with microfilament bundles from various cultured mammalian smooth muscle cells, but does not react with cultured human dermal fibroblasts or various epithelial tumor cell lines. In one-dimensional gel electrophoresis immunoblot experiments this antibody detects a 42-kd polypeptide from tissue extracts of uterus, ileum, aorta, diaphragm, and heart and extract from smooth muscle cells. The antibody also reacts with a comigrating 42-kd band of highly purified rabbit skeletal muscle actin. HHF35 is nonreactive on immunoblots of extracts from all tested nonmuscle cell extracts. Immunoelectrophoresis followed by immunoblotting performed in the presence of urea and reducing agents reveals recognition of the alpha isoelectrophoretic variant of actin from skeletal, cardiac, and smooth muscle sources and of the gamma variant from smooth muscle sources. Because HHF35 reacts with virtually all muscle cells, it will be useful as a marker for muscle and muscle-derived cells.

  20. Biochemical characterization and distribution of glutathione S-transferases in leaping mullet (Liza saliens).

    Science.gov (United States)

    Sen, A; Kirikbakan, A

    2004-09-01

    In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.

  1. Efficient Characterization of Parametric Uncertainty of Complex (Bio)chemical Networks.

    Science.gov (United States)

    Schillings, Claudia; Sunnåker, Mikael; Stelling, Jörg; Schwab, Christoph

    2015-08-01

    Parametric uncertainty is a particularly challenging and relevant aspect of systems analysis in domains such as systems biology where, both for inference and for assessing prediction uncertainties, it is essential to characterize the system behavior globally in the parameter space. However, current methods based on local approximations or on Monte-Carlo sampling cope only insufficiently with high-dimensional parameter spaces associated with complex network models. Here, we propose an alternative deterministic methodology that relies on sparse polynomial approximations. We propose a deterministic computational interpolation scheme which identifies most significant expansion coefficients adaptively. We present its performance in kinetic model equations from computational systems biology with several hundred parameters and state variables, leading to numerical approximations of the parametric solution on the entire parameter space. The scheme is based on adaptive Smolyak interpolation of the parametric solution at judiciously and adaptively chosen points in parameter space. As Monte-Carlo sampling, it is "non-intrusive" and well-suited for massively parallel implementation, but affords higher convergence rates. This opens up new avenues for large-scale dynamic network analysis by enabling scaling for many applications, including parameter estimation, uncertainty quantification, and systems design.

  2. Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori.

    Science.gov (United States)

    Mitsudome, Takumi; Mon, Hiroaki; Xu, Jian; Li, Zhiqing; Lee, Jae Man; Patil, Anandrao Ashok; Masuda, Atsushi; Iiyama, Kazuhiro; Morokuma, Daisuke; Kusakabe, Takahiro

    2015-03-01

    DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn(2+) and Mn(2+). Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.

  3. Biochemical and molecular characterization of Avena indolines and their role in kernel texture.

    Science.gov (United States)

    Gazza, Laura; Taddei, Federica; Conti, Salvatore; Gazzelloni, Gloria; Muccilli, Vera; Janni, Michela; D'Ovidio, Renato; Alfieri, Michela; Redaelli, Rita; Pogna, Norberto E

    2015-02-01

    Among cereals, Avena sativa is characterized by an extremely soft endosperm texture, which leads to some negative agronomic and technological traits. On the basis of the well-known softening effect of puroindolines in wheat kernel texture, in this study, indolines and their encoding genes are investigated in Avena species at different ploidy levels. Three novel 14 kDa proteins, showing a central hydrophobic domain with four tryptophan residues and here named vromindoline (VIN)-1,2 and 3, were identified. Each VIN protein in diploid oat species was found to be synthesized by a single Vin gene whereas, in hexaploid A. sativa, three Vin-1, three Vin-2 and two Vin-3 genes coding for VIN-1, VIN-2 and VIN-3, respectively, were described and assigned to the A, C or D genomes based on similarity to their counterparts in diploid species. Expression of oat vromindoline transgenes in the extra-hard durum wheat led to accumulation of vromindolines in the endosperm and caused an approximate 50 % reduction of grain hardness, suggesting a central role for vromindolines in causing the extra-soft texture of oat grain. Further, hexaploid oats showed three orthologous genes coding for avenoindolines A and B, with five or three tryptophan residues, respectively, but very low amounts of avenoindolines were found in mature kernels. The present results identify a novel protein family affecting cereal kernel texture and would further elucidate the phylogenetic evolution of Avena genus.

  4. Atypical BSE in Germany--proof of transmissibility and biochemical characterization.

    Science.gov (United States)

    Buschmann, A; Gretzschel, A; Biacabe, A-G; Schiebel, K; Corona, C; Hoffmann, C; Eiden, M; Baron, T; Casalone, C; Groschup, Martin H

    2006-10-31

    Intensive active surveillance has uncovered two atypical German BSE cases in older cattle which resemble the two different atypical BSE phenotypes that have recently been described in France (designated H-type) and Italy (designated L-type or BASE). The H-type is characterized by a significantly higher molecular size, but a conventional glycopattern of the proteinase K treated abnormal prion protein (PrP(Sc)), while the L-type PrP(Sc) has only a slightly lower molecular size and a distinctly different glycopattern. In this paper we describe the successful transmission of both German atypical BSE cases to transgenic mice overexpressing bovine PrP(C). Upon challenge with the L-type, these mice developed BSE after a substantially shorter incubation period than any classical BSE transmission using these mice to date. In contrast, the incubation period was distinctly prolonged when these mice were challenged with the H-type. PrP(Sc) accumulated in the brains of these mice were of the same atypical BSE type that had been used for the transmission. These atypical cases suggest the possible existence of sporadic BSE cases in bovines. It is thus feasible that the BSE epidemic in the UK could have also been initiated by an intraspecies transmission from a sporadic BSE case.

  5. Identification and biochemical characterization of Leishmania strains isolated in Peru, Mexico, and Spain.

    Science.gov (United States)

    Rodríguez-González, Isabel; Marín, Clotilde; Vargas, Franklin; Córdova, Ofelia; Barrera, Mario; Gutiérrez-Sánchez, Ramón; Alunda, Jose María; Sánchez-Moreno, Manuel

    2006-01-01

    Eight Leishmania promastigotes were isolated from different geographical areas: three (LP1, LP2, and LP3) from the provincial department La Libertad and the fourth (LP4) from the department of Cajamarca (northern Peru); another three (LM1, LM2, and LM3) in the province of Campeche (Mexico); and the last (LS1) from a clinical case of a dog in Madrid (Spain). The isolates were characterized by carbohydrate cell-surface residues using agglutinations with four purified lectins, by isoenzyme analysis using different isoenzymes, by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases and by the final metabolite patterns after in vitro culture. These isolates were compared with four reference strains and typified as: Leishmania (Leishmania) donovani, two strains of L. (L.) infantum, and one species of L. (Viania) peruviana. According to our results and the statistical study, the Peruvian isolates represent three different strains: one would be L. (V.) peruviana, another the strain isolated in Cajamarca (LP4) and the third would include the three strains from the department of La Libertad (LP1, LP2, and LP3), these latter three isolates being phylogenetically closer to the reference strain L. (L.) donovani. Meanwhile, the three isolates from Mexico form a group with close phylogenetic relationships to each other. The isolate from Spain belongs to the species L. (L.) infantum. Thus, a close correlation was drawn between the identity of each strain and its geographical origin.

  6. Evaluation and biochemical characterization of a distinctive pyoverdin from a pseudomonas isolated from chickpea rhizosphere

    Directory of Open Access Journals (Sweden)

    Neelam Tank

    2012-06-01

    Full Text Available Microbial siderophores confiscate the available ferric ions around the roots and trigger a reaction resulting in plant growth promotion. In our study, a high level of siderophore production was observed from a newly isolated Pseudomonas sp. from the rhizosphere of Chickpea plants. Under an iron depleted condition in Standard Succinic acid medium a 1000 µgmL-1 of siderophore production was achieved. Increasing the concentration of iron showed an inverse relationship between growth and siderophore production. Fourier Transform Infrared Spectroscopy (FTIR analysis of the purified crystals, its UV spectral analysis and High Pressure Liquid Chromatography (HPLC revealed the identity of the siderophore as similar to that of pyoverdin with distinctive characters. Electron spray ionization mass spectroscopy (ESIMS shows presence of abundance of A1 ions (419 m/z and branching of amino acids from B1-B5. This pyoverdin contains a cyclic tetra peptide but Serine and Arginine are missing. Based on our analysis and deviations from the reported structure of pyoverdin it is suggested that this pseudomonas produces distinctly characterized pyoverdin siderophore.

  7. Ribbing disease: radiographic and biochemical characterization, lack of response to pamidronate

    Energy Technology Data Exchange (ETDEWEB)

    Ziran, Navid [Department of Orthopedic Surgery, University of Rochester, Rochester, New York (United States); Hill, Suvimol [Department of Radiology, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland (United States); Wright, Mary E.; Kovacs, Joseph [Department of Critical Care Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland (United States); Robey, Pamela Gehron [Craniofacial and Skeletal Diseases Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland (United States); Wientroub, Shlomo [Department of Pediatric Orthopedic Surgery, Dana Children' s Hospital, Tel-Aviv Medical Center, Tel-Aviv (Israel); Collins, Michael T. [Craniofacial and Skeletal Diseases Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland (United States); CSDB/NIDCR/NIH, Building 30 Room 228, MSC 4320, Bethesda, MD 20892-4320 (United States)

    2002-12-01

    Ribbing disease is a rare form of sclerosing dysplasia characterized by benign endosteal and periosteal bone growth confined to the diaphyses of the long bones, usually the tibiae and femora. The onset is usually after puberty and the most common presentation is pain that is usually self-limited, but may progress. The etiology and optimal treatment for the disease are unknown. We present the case of a 39-year-old Hispanic man with clinical and radiological manifestations of Ribbing disease. Radiographs and CT imaging demonstrated typical cortical thickening in the mid-diaphyses of the tibiae bilaterally that correlated with intense tracer uptake on {sup 99m}Tc-MDP bone scans. MRI demonstrated cortical thickening and abnormal marrow signal consistent with marrow edema. Bone marrow edema may explain the pain frequently associated with the disease. Multiple serum and urine markers of bone metabolism were within normal limits. In an effort to ameliorate pain, the patient was treated with the bisphosphonate, pamidronate. In spite of treatment, pain increased, requiring additional and larger doses of analgesics. Serial radiographs, CT, bone scans, and MRI all demonstrated disease progression with pamidronate treatment. In this report we present for the first time the finding of bone marrow edema with MRI as well as disease progression during intravenous pamidronate treatment. (orig.)

  8. Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae).

    Science.gov (United States)

    Pallela, Ramjee; Bojja, Sreedhar; Janapala, Venkateswara Rao

    2011-07-01

    Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and under-used resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries.

  9. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

    Science.gov (United States)

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.

  10. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano (Toronto); (Colorado)

    2011-12-14

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  11. Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca

    Directory of Open Access Journals (Sweden)

    Gargouri Youssef

    2011-02-01

    Full Text Available Abstract Background Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. Results A marine stingray phospholipase A2 (SPLA2 was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. Conclusions SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.

  12. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

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    Gaëtan Vanier

    Full Text Available Monoclonal antibodies (mAbs represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.

  13. Biochemical and molecular characterization of the tegument protein RT10 from Raillietina tetragona.

    Science.gov (United States)

    Chen, Li; Li, Haiyun

    2014-03-01

    Tegument antigens of tapeworm play an important role in modulation of host response and parasite survival. Characterizing appropriate antigens for parasite infection diagnosis and vaccination is rational and could have both economic and epidemiological significance in poultry industry. In the present study, a major protoscolex homologue (named RT10) of Echinococcus and Taenia spp. was amplified from Raillietina tetragona cestode. The RT10 cDNA was 1,877 bp long containing an open reading frame of 1,683 bp nucleotides, which encoded a deduced protein of 560 amino acids with an isoelectric point of 6.33. Secondary structure analysis demonstrated that RT10 was both hydrophilic and antigenic, and possessed N-terminal FERM domain and C-terminal ERM domain, respectively. With the same structural properties of previously reported antigens from Echinococcus and Taenia spp., RT10 tegument antigen had a more than 82% similarity in nucleotide level with initially reported antigens from Echinococcus and Taenia spp., and a more than 83% similarity in protein level, with the highest similarity of 85.2% to Taenia antigen H17g. In addition, phylogenetic analysis illustrated a high consistency between different genus antigens and evolutionary branching. Although the detailed function of RT10 is still unknown, the high sequence conservation and structural similarity to formerly identified tegument antigens from Echinococcus and Taenia spp. suggested that RT10 may play a similar role as the previous reported antigens between cestode and host. It is significant to clarify the antigenic and serodiagnostic characteristics in the subsequent work.

  14. Sperm evaluation and biochemical characterization of cat seminal plasma collected by electroejaculation and urethral catheterization.

    Science.gov (United States)

    Zambelli, Daniele; Raccagni, Ramona; Cunto, Marco; Andreani, Giulia; Isani, Gloria

    2010-11-01

    This paper aimed to evaluate cat seminal plasma protein profile (with SDS-page) and determine differences in seminal plasma composition from ejaculates obtained using urethral catheterization after pharmacological induction (UrCaPI) and electroejaculation (EE). In addition, this study evaluates whether the recovery method affected seminal plasma protein and zinc concentrations. A single ejaculation was collected from 17 mixed-breed cats by EE (5/21) or UrCaPI (12/21), while 4/21 cats underwent four sperm collections once every four days using EE and UrCaPI techniques alternately. The semen parameters evaluated were: volume, percentage of motility and progressive motility, morphology, and sperm concentration. After centrifugation, the seminal plasma obtained was stored at -80 °C and later used to measure protein and zinc concentrations, and to determine protein profile by SDS-polyacrylamide gel electrophoresis (PAGE). The results obtained indicate that cat seminal plasma protein profile is characterized by many protein bands (>30) with a molecular weight ranging from 3.5 to 200 kDa, and that the recovery method influences the seminal plasma protein profile: EE is related to the absence of two proteins (P55 and P14), and alters three protein bands (P200, P80, P28). The collection technique also affected zinc concentration (mg/dL) and protein concentration (g/dL) which were significantly higher (P < 0.01) in samples collected by UrCaPI; on the contrary the total Zn and protein amount/ejaculate were not significantly different in samples collected by both technique (P < 0.05).

  15. β-D-xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme.

    Science.gov (United States)

    Ratnadewi, Anak Agung Istri; Fanani, Muchzainal; Kurniasih, Sari Dewi; Sakka, Makiko; Wasito, Eddy Bagus; Sakka, Kazuo; Nurachman, Zeily; Puspaningsih, Ni Nyoman Tri

    2013-08-01

    The gene encoding a thermostable β-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-β-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

  16. Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein.

    Science.gov (United States)

    Lecours, Katia; Tremblay, Marie-Hélène; Gagné, Marie-Eve Laliberté; Gagné, Stéphane M; Leclerc, Denis

    2006-05-01

    Papaya mosaic virus (PapMV) is a flexuous rod shape virus made of 1400 subunits that assemble around a plus sense genomic RNA. The structure determination of PapMV and of flexuous viruses in general is a major challenge for both NMR and X-ray crystallography. In this report, we present the characterization of a truncated version of the PapMV coat protein (CP) that is suitable for NMR study. The deletion of the N-terminal 26 amino acids of the PapMV CP (CP27-215) generates a monomer that can be expressed to high level and easily purified for production of an adequate NMR sample. The RNA gel shift assay showed that CP27-215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6x His tag at the C-terminus improved the solubility of the monomer and allowed its concentration to 0.2 mM. The CD spectra of the truncated and the wild-type proteins were similar, suggesting that both proteins are well ordered and have a similar secondary structure. CP27-215 was 15N labeled for NMR studies and a 2D 1H-15N-HSQC spectrum confirmed the presence of a well-ordered structure and the monomeric form of the protein. These results show that CP27-215 is amenable to a complete and exhaustive NMR study that should lead to the first three-dimensional structure determination of a flexuous rod shape virus.

  17. Extraction of lipase from Burkholderia cepacia by PEG/Phosphate ATPS and its biochemical characterization

    Directory of Open Access Journals (Sweden)

    Giovana da Silva Padilha

    2012-02-01

    Full Text Available This work aimed to study the partitioning of a lipase produced by Burkholderia cepacia in PEG/Phosphate aqueous two phase system (ATPS and its characterization. Lipase was produced by B. cepacia strains in a fermenter. Enzyme partitioning occurred at pH 6.0 and 8.0, using PEG 1500 and 6000 on two tie lines. Metal ions, pH and temperature effects on enzyme activity were evaluated. Five milliliter of 7.5% olive oil emulsion with 2.5% gumarabic in 0.1M sodium phosphate buffer at pH 8.0 and 37ºC were used for the activity determinations. Results showed that crude stratum from B. cepacia was partitioned by PEG1500/phosphate ATPS at pH 6.0 or 8.0 for, which the partitioning coefficients were 108-and 209-folds. Lipase presented optimal activity conditions at 37ºC and pH 8.0; it showed pH-stability for 4 h of incubation at different pH values at 37ºC. Metal ions such as Mn2+ , Co2+, I-and Ca2+ sustained enzymatic activities; however, it was inhibited by the presence of Fe2+, Hg2+ and Al3+ . Km and Vmax values were 0.258 U/mg and 43.90 g/L, respectively. A molecular weight of 33 kDa and an isoelectric point at pH 5.0 were determined by SDS-PAGE and IFS electrophoresis, respectively.

  18. Biochemical and molecular characterization of the calcineurin in Echinococcus granulosus larval stages.

    Science.gov (United States)

    Nicolao, María Celeste; Cumino, Andrea C

    2015-06-01

    Calcineurin (CaN) is a Ca(2+)-calmodulin activated serine-threonine protein phosphatase that couples the local or global calcium signals, thus controlling important cellular functions in physiological and developmental processes. The aim of this study was to characterize CaN in Echinococcus granulosus (Eg-CaN), a human cestode parasite of clinical importance, both functionally and molecularly. We found that the catalytic subunit isoforms have predicted sequences of 613 and 557 amino acids and are substantially similar to those of the human counterpart, except for the C-terminal end. We also found that the regulatory subunit consists of 169 amino acids which are 87% identical to the human ortholog. We cloned a cDNA encoding for one of the two catalytic subunit isoforms of CaN (Eg-can-A1) as well as the only copy of the Eg-can-B gene, both constitutively transcribed in all Echinococcus larval stages and responsible for generating a functionally active heterodimer. Eg-CaN native enzyme has phosphatase activity, which is enhanced by Ca(2+)/Ni(2+) and reduced by cyclosporine A and Ca(2+) chelators. Participation of Eg-CaN in exocytosis was demonstrated using the FM4-64 probe and Eg-CaN-A was immunolocalized in the cytoplasm of tegumental cells, suckers and excretory bladder of protoscoleces. We also showed that the Eg-can-B transcripts were down-regulated in response to low Ca(2+) intracellular level, in agreement with decreased enzyme activity. Confocal microscopy revealed a striking pattern of Eg-CaN-A in discrete fluorescent spots in the protoscolex posterior bladder and vesicularized protoscoleces beginning the vesicular differentiation. In contrast, Eg-CaN-A was undetectable during the pre-microcyst closing stage while a high DDX-like RNA helicase expression was evidenced. Finally, we identified and analyzed the expression of CaN-related endogenous regulators.

  19. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

    Science.gov (United States)

    Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

    2014-09-01

    Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

  20. Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

    DEFF Research Database (Denmark)

    Lombard, David B; Alt, Frederick W; Cheng, Hwei-Ling;

    2007-01-01

    Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble...... mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial...... deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis...

  1. Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

    Directory of Open Access Journals (Sweden)

    Ryu Yeon-Woo

    2010-06-01

    Full Text Available Abstract Background Erythrose reductase (ER catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(PH as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110. Results The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could

  2. Biochemical and biological characterization of two serine proteinases from Colombian Crotalus durissus cumanensis snake venom.

    Science.gov (United States)

    Patiño, Arley Camilo; Pereañez, Jaime Andrés; Gutiérrez, José María; Rucavado, Alexandra

    2013-03-01

    Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 μg and 0.571 μg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.

  3. Biochemical and Structural Characterization of Mycobacterium tuberculosis beta-Lactamase with the Carbapenems Ertapenem and Doripenem

    Energy Technology Data Exchange (ETDEWEB)

    L Tremblay; F Fan; J Blanchard

    2011-12-31

    Despite the enormous success of {beta}-lactams as broad-spectrum antibacterials, they have never been widely used for the treatment of tuberculosis (TB) due to intrinsic resistance that is caused by the presence of a chromosomally encoded gene (blaC) in Mycobacterium tuberculosis. Our previous studies of TB BlaC revealed that this enzyme is an extremely broad-spectrum {beta}-lactamase hydrolyzing all {beta}-lactam classes. Carbapenems are slow substrates that acylate the enzyme but are only slowly deacylated and can therefore act also as potent inhibitors of BlaC. We conducted the in vitro characterization of doripenem and ertapenem with BlaC. A steady-state kinetic burst was observed with both compounds with magnitudes proportional to the concentration of BlaC used. The results provide apparent K{sub m} and k{sub cat} values of 0.18 {micro}M and 0.016 min{sup -1} for doripenem and 0.18 {micro}M and 0.017 min{sup -1} for ertapenem, respectively. FTICR mass spectrometry demonstrated that the doripenem and ertapenem acyl-enzyme complexes remain stable over a time period of 90 min. The BlaC-doripenem covalent complex obtained after a 90 min soak was determined to 2.2 {angstrom}, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 {angstrom}. The 1.3 {angstrom} diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original {Delta}2-pyrroline ring was tautomerized to generate the {Delta}1-pyrroline ring. The isomerization leads to the flipping of the carbapenem hydroxyethyl group to hydrogen bond to carboxyl O2 of Glu166. The hydroxyethyl flip results in both the decreased basicity of Glu166 and a significant increase in the distance between carboxyl O2 of Glu166 and the catalytic water molecule, slowing hydrolysis.

  4. Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

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    Ákos Tóth

    Full Text Available Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T. Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5, their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs (PTDP-Tc, TEEP-Tf, DPGT-Th. All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial

  5. Mitochondrial and Cell Death Mechanisms in Neurodegenerative Diseases

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    Lee J. Martin

    2010-03-01

    Full Text Available Alzheimer’s disease (AD, Parkinson’s disease (PD and amyotrophic lateral sclerosis (ALS are the most common human adult-onset neurodegenerative diseases. They are characterized by prominent age-related neurodegeneration in selectively vulnerable neural systems. Some forms of AD, PD, and ALS are inherited, and genes causing these diseases have been identified. Nevertheless, the mechanisms of the neuronal cell death are unresolved. Morphological, biochemical, genetic, as well as cell and animal model studies reveal that mitochondria could have roles in this neurodegeneration. The functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations, triggering neurodegeneration according to a cell death matrix theory. In AD, alterations in enzymes involved in oxidative phosphorylation, oxidative damage, and mitochondrial binding of Aβ and amyloid precursor protein have been reported. In PD, mutations in putative mitochondrial proteins have been identified and mitochondrial DNA mutations have been found in neurons in the substantia nigra. In ALS, changes occur in mitochondrial respiratory chain enzymes and mitochondrial cell death proteins. Transgenic mouse models of human neurodegenerative disease are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria and the mitochondrial permeability transition pore. This review summarizes how mitochondrial pathobiology might contribute to neuronal death in AD, PD, and ALS and could serve as a target for drug therapy.

  6. Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei.

    Science.gov (United States)

    Kafková, Lucie; Ammerman, Michelle L; Faktorová, Drahomíra; Fisk, John C; Zimmer, Sara L; Sobotka, Roman; Read, Laurie K; Lukes, Julius; Hashimi, Hassan

    2012-10-01

    A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

  7. Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

    Science.gov (United States)

    Ker, De-Sheng; Pang, Sze Lei; Othman, Noor Farhan; Kumaran, Sekar; Tan, Ee Fun; Krishnan, Thiba; Chan, Kok Gan; Othman, Roohaida

    2017-01-01

    Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation

  8. Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77.

    Science.gov (United States)

    Tsuji, Kohsei; Yoon, Ki-Seok; Ogo, Seiji

    2016-03-01

    Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHE(S77)). Interestingly, the ADHE(S77) was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH(4))(2)SO(4) without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration.

  9. Isolation and biochemical characterization of a galactoside binding lectin from Bauhinia variegata candida (BvcL) seeds.

    Science.gov (United States)

    Silva, José A; Damico, Daniela C S; Baldasso, Paulo A; Mattioli, Marcelo A; Winck, Flávia V; Fraceto, Leonardo F; Novello, José C; Marangoni, Sérgio

    2007-04-01

    A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized D: -lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.

  10. Production and Biochemical Characterization of a High Maltotetraose (G4 Producing Amylase from Pseudomonas stutzeri AS22

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    Hana Maalej

    2014-01-01

    Full Text Available Amylase production and biochemical characterization of the crude enzyme preparation from Pseudomonas stutzeri AS22 were evaluated. The highest α-amylase production was achieved after 24 hours of incubation in a culture medium containing 10 g/L potato starch and 5 g/L yeast extract, with initial pH 8.0 at 30°C under continuous agitation at 200 rpm. The optimum temperature and pH for the crude α-amylase activity were 60°C and 8.0, respectively. The effect of different salts was evaluated and it was found that both α-amylase production and activity were Ca2+-dependent. The amylolytic preparation was found to catalyze exceptionally the formation of very high levels of maltotetraose from starch (98%, w/w in the complete absence of glucose since the initial stages of starch hydrolysis (15 min and hence would have a potential application in the manufacturing of maltotetraose syrups.

  11. Purification and Biochemical Characterization of Three Myotoxins from Bothrops mattogrossensis Snake Venom with Toxicity against Leishmania and Tumor Cells

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    Andréa A. de Moura

    2014-01-01

    Full Text Available Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s, one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like, BmatTX-II (Lys49 PLA2-like, and BmatTX-III (Asp49 PLA2. The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T and SK-BR-3 (breast adenocarcinoma cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

  12. Biochemical, genetic, and epidemiologic characterization of Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) strains associated with Brazilian purpuric fever.

    Science.gov (United States)

    Brenner, D J; Mayer, L W; Carlone, G M; Harrison, L H; Bibb, W F; Brandileone, M C; Sottnek, F O; Irino, K; Reeves, M W; Swenson, J M

    1988-08-01

    Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and BPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates from 42 persons in towns where BPF cases occurred, and control strains from 32 persons in towns without BPF cases were characterized biochemically, genetically, and epidemiologically. Results indicated that a single clone was responsible for all BPF cases identified in six Brazilian towns from 1984 through 1986. All of 15 (100%) case strains were the same clone as was 1 of 32 (3%) control strains (P = less than 10(-8). Isolates of the clone were preferentially intrarelated by DNA hybridization (99% relatedness, hydroxyapatite method at 60 and 75 degrees C) and were separable from other H. influenzae biogroup aegyptius strains (approximately 90% relatedness at 60 degrees C and 82% relatedness at 75 degrees C). All isolates of the BPF clone and no other strains contained a 24-megadalton plasmid of restriction endonuclease type 3031, were of a single multilocus enzyme mobility type, were of a single sodium dodecyl sulfate-polyacrylamide gel electrophoresis type, and were in one of two ribosomal DNA restriction patterns. All BPF clone isolates reacted with monoclonal antibodies produced from a case strain; only 3 of 62 (5%) other strains reacted with this monoclonal antibody. Ninety percent of BPF clone strains and 27% of other strains were relatively resistant to sulfamethoxazole-trimethoprim.

  13. Biochemical characterization of Toxoplasma gondii 1-Cys peroxiredoxin 2 with mechanistic similarities to typical 2-Cys Prx.

    Science.gov (United States)

    Deponte, Marcel; Becker, Katja

    2005-03-01

    TgPrx2 represents a recently discovered cytosolic 1-Cys peroxiredoxin (Prx) from the intracellular parasite Toxoplasma gondii. Over-expression of the respective gene confers protection against H(2)O(2), suggesting that the protein possesses peroxidase activity. According to the current nomenclature eukaryotic typical and atypical 2-Cys Prx contain a second conserved resolving cysteine residue whereas 1-Cys Prx work on the basis of a monothiol mechanism. Only a few 1-Cys peroxiredoxins have been biochemically characterized to date. Here we describe the mechanistic characterization of TgPrx2 in vitro, including site directed mutagenesis studies, gel filtration chromatography, and molecular modeling. TgPrx2 has general antioxidant properties as indicated by its ability to protect glutamine synthetase against a dithiothreitol Fe(3+)-catalyzed oxidation system. However, TgPrx2 does not reduce H(2)O(2) nor tert-butyl hydroperoxide at the expense of glutaredoxin, thioredoxin or glutathione. Cys(47) was identified as the active site cysteine residue. Most interestingly, Cys(47) was found to form an intermolecular disulfide with Cys(209) from the C-terminal domain of a second subunit which acts as the resolving cysteine. This is a mechanism analogous to typical peroxiredoxins. In contrast to the latter, however, dimeric TgPrx2 does not oligomerize to decamers but is able to form tetramers and hexamers which are non-covalently associated. To our knowledge, TgPrx2 is the first eukaryotic 'so called' 1-Cys peroxiredoxin shown to act on the basis of a 2-Cys mechanism. Our data indicate that mechanistic studies are essential for classifying peroxiredoxins.

  14. Mitochondrial helicases and mitochondrial genome maintenance

    DEFF Research Database (Denmark)

    Aamann, Maria Diget; de Souza-Pinto, Nadja C; Kulikowicz, Tomasz;

    2010-01-01

    Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases...... have been studied in detail; however, the roles of specific helicases in mitochondrial biology remain poorly characterized. This review presents important recent advances in identifying and characterizing mitochondrial helicases, some of which also operate in the nucleus....

  15. Genetic characterization of clinical acanthamoeba isolates from Japan using nuclear and mitochondrial small subunit ribosomal RNA.

    Science.gov (United States)

    Rahman, Md Moshiur; Yagita, Kenji; Kobayashi, Akira; Oikawa, Yosaburo; Hussein, Amjad I A; Matsumura, Takahiro; Tokoro, Masaharu

    2013-08-01

    Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

  16. Characterization of complete mitochondrial genome of Dezhou donkey (Equus asinus) and evolutionary analysis.

    Science.gov (United States)

    Sun, Yan; Jiang, Qiang; Yang, Chunhong; Wang, Xiuge; Tian, Fang; Wang, Yinchao; Ma, Yong; Ju, Zhihua; Huang, Jinming; Zhou, Xiangshan; Zhong, Jifeng; Wang, Changfa

    2016-05-01

    Mitochondrial DNA (mtDNA) has been widely used in species identification and genetic diversification. Comparisons among mtDNAs of closely related species are valuable for phylogenetic studies. However, only the partial mtDNA Cytb gene and the D-loop sequences were used to analysis the phylogenetic relationship between donkey breeds due to lack of complete mitochondrial genome. Dezhou donkey, as a bigger somatotype ass, is one of Chinese domestic donkey breeds, and used by many places as breeding stock. To further investigate the phylogenetic relationship of Dezhou donkey with other breeds, the complete mtDNA was firstly sequenced and de novo assembled using next generation sequence data from total genomic DNA. The genome was 16,813 bp in length (NCBI submission number: KT182635) and contained 13 protein coding genes, 2 ribosomal RNA genes, 25 transfer RNA genes, and 1 control region. Based on the novel complete mtDNA sequence, the sequences of 13 protein coding genes and 2 ribosomal RNA genes were amplifying in other 2 Dezhou donkeys and 3 Yunnan donkeys, respectively. The pattern of genetic variation in horse, wild ass and domestic donkeys among these 15 genes indicated the sequence polymorphisms. The more accurate phylogenetic relationships of donkey species (Dezhou donkey, Yunnan donkey and previously published donkeys) were first obtained using the combined sequences of 12S rRNA+16S rRNA+13 protein-coding genes. Molecular-based phylogeny supported the hypothesis that Chinese domestic donkey breeds may have originated from Somali wild ass, not from Asian wild ass by analyzing mitochondrial genomes.

  17. Sequence and characterization of six mitochondrial subgenomes from Globodera rostochiensis: multipartite structure is conserved among close nematode relatives.

    Science.gov (United States)

    Gibson, Tracey; Blok, Vivian C; Dowton, Mark

    2007-09-01

    Recently, a multipartite mitochondrial genome was characterized in the potato cyst nematode, Globodera pallida. Six subgenomic circles were detectable by PCR, while full-length genomes were not. We investigate here whether this subgenomic organization occurs in a close relative of G. pallida. We amplified and sequenced one entire mitochondrial subgenome from the cyst-forming nematode, Globodera rostochiensis. Comparison of the noncoding region of this subgenome with those reported previously for G. pallida facilitated the design of amplification primers for a range of subgenomes from G. rostochiensis. We then randomly sequenced five subgenomic fragments, each representative of a unique subgenome. This study indicates that the multipartite structure reported for G. pallida is conserved in G. rostochiensis. A comparison of subgenomic organization between these two Globodera species indicates a considerable degree of overlap between them. Indeed, we identify two subgenomes with an organization identical with that reported for G. pallida. However, other subgenomes are unique to G. rostochiensis, although some of these have blocks of genes comparable to those in G. pallida. Dot-plot comparisons of pairs of subgenomes from G. rostochiensis indicate that the different subgenomes share fragments with high sequence identity. We interpret this as evidence that recombination is operating in the mitochondria of G. rostochiensis.

  18. Cloning, Expression and Characterization of Mitochondrial Manganese Superoxide Dismutase from the Whitefly, Bemisia tabaci

    Directory of Open Access Journals (Sweden)

    Xian-Long Gao

    2013-01-01

    Full Text Available A mitochondrial manganese superoxide dismutase from an invasive species of the whitefly Bemisia tabaci complex (Bt-mMnSOD was cloned and analyzed. The full length cDNA of Bt-mMnSOD is 1210 bp with a 675 bp open reading frame, corresponding to 224 amino acids, which include 25 residues of the mitochondrial targeting sequence. Compared with various vertebrate and invertebrate animals, the MnSOD signature (DVWEHAYY and four conserved amino acids for manganese binding (H54, H102, D186 and H190 were observed in Bt-mMnSOD. Recombinant Bt-mMnSOD was overexpressed in Escherichia coli, and the enzymatic activity of purified mMnSOD was assayed under various temperatures. Quantitative real-time PCR analysis with whiteflies of different development stages showed that the mRNA levels of Bt-mMnSOD were significantly higher in the 4th instar than in other stages. In addition, the in vivo activities of MnSOD in the whitefly were measured under various conditions, including exposure to low (4 °C and high (40 °C temperatures, transfer from a favorable to an unfavorable host plant (from cotton to tobacco and treatment with pesticides. Our results indicate that the whitefly MnSOD plays an important role in cellular stress responses and anti-oxidative processes and that it might contribute to the successful worldwide distribution of the invasive whitefly.

  19. Cloning, expression and characterization of mitochondrial manganese superoxide dismutase from the Whitefly, Bemisia tabaci.

    Science.gov (United States)

    Gao, Xian-Long; Li, Jun-Min; Wang, Yong-Liang; Jiu, Min; Yan, Gen-Hong; Liu, Shu-Sheng; Wang, Xiao-Wei

    2013-01-07

    A mitochondrial manganese superoxide dismutase from an invasive species of the whitefly Bemisia tabaci complex (Bt-mMnSOD) was cloned and analyzed. The full length cDNA of Bt-mMnSOD is 1210 bp with a 675 bp open reading frame, corresponding to 224 amino acids, which include 25 residues of the mitochondrial targeting sequence. Compared with various vertebrate and invertebrate animals, the MnSOD signature (DVWEHAYY) and four conserved amino acids for manganese binding (H54, H102, D186 and H190) were observed in Bt-mMnSOD. Recombinant Bt-mMnSOD was overexpressed in Escherichia coli, and the enzymatic activity of purified mMnSOD was assayed under various temperatures. Quantitative real-time PCR analysis with whiteflies of different development stages showed that the mRNA levels of Bt-mMnSOD were significantly higher in the 4th instar than in other stages. In addition, the in vivo activities of MnSOD in the whitefly were measured under various conditions, including exposure to low (4 °C) and high (40 °C) temperatures, transfer from a favorable to an unfavorable host plant (from cotton to tobacco) and treatment with pesticides. Our results indicate that the whitefly MnSOD plays an important role in cellular stress responses and anti-oxidative processes and that it might contribute to the successful worldwide distribution of the invasive whitefly.

  20. Integrated analysis of genetic, behavioral, and biochemical data implicates neural stem cell-induced changes in immunity, neurotransmission and mitochondrial function in Dementia with Lewy Body mice.

    Science.gov (United States)

    Lakatos, Anita; Goldberg, Natalie R S; Blurton-Jones, Mathew

    2017-03-10

    We previously demonstrated that transplantation of murine neural stem cells (NSCs) can improve motor and cognitive function in a transgenic model of Dementia with Lewy Bodies (DLB). These benefits occurred without changes in human α-synuclein pathology and were mediated in part by stem cell-induced elevation of brain-derived neurotrophic factor (BDNF). However, instrastriatal NSC transplantation likely alters the brain microenvironment via multiple mechanisms that may synergize to promote cognitive and motor recovery. The underlying neurobiology that mediates such restoration no doubt involves numerous genes acting in concert to modulate signaling within and between host brain cells and transplanted NSCs. In order to identify functionally connected gene networks and additional mechanisms that may contribute to stem cell-induced benefits, we performed weighted gene co-expression network analysis (WGCNA) on striatal tissue isolated from NSC- and vehicle-injected wild-type and DLB mice. Combining continuous behavioral and biochemical data with genome wide expression via network analysis proved to be a powerful approach; revealing significant alterations in immune response, neurotransmission, and mitochondria function. Taken together, these data shed further light on the gene network and biological processes that underlie the therapeutic effects of NSC transplantation on α-synuclein induced cognitive and motor impairments, thereby highlighting additional therapeutic targets for synucleinopathies.

  1. Molecular characterization of Taenia multiceps isolates from Gansu Province, China by sequencing of mitochondrial cytochrome C oxidase subunit 1.

    Science.gov (United States)

    Li, Wen Hui; Jia, Wan Zhong; Qu, Zi Gang; Xie, Zhi Zhou; Luo, Jian Xun; Yin, Hong; Sun, Xiao Lin; Blaga, Radu; Fu, Bao Quan

    2013-04-01

    A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.

  2. Isolation and characterization of a Ca/sup 2 +/ carrier candidate from calf heart inner mitochondrial membrane

    Energy Technology Data Exchange (ETDEWEB)

    Jeng, A.Y.

    1979-01-01

    A protein was isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance assay based on the relative binding properties of Ca/sup 2 +/, Mn/sup 2 +/, and Mg/sup 2 +/ to the protein. Partial delipidation of the protein was performed by using either the organic solvent extraction procedure or the silicic acid column chromatography. Control experiments indicated that the Ca/sup 2 +/ transport properties of the isolated protein were not due to the contaminating phospholipids. A complete delipidation procedure was developd by using Sephadex LH-20 column chromatography. Further characterization of the physical and chemical properties of the delipidated protein showed that delipidated protein becomes more hydrophobic in the presence of Ca/sup 2 +/ and alkaline pH in the organic solvent extraction experiments. Two possible models of calciphorin-mediated Ca/sup 2 +/ transport in mitochondria are proposed. (PCS)

  3. Characterization of the mitochondrial genome of the threatened alpine butterfly, Parnassius nomion (Lepidoptera: Papilionidae).

    Science.gov (United States)

    Dong, Wan-Wei; Jiang, Guo-Fang

    2016-01-01

    The sequenced mitochondrial genome of the threatened alpine butterfly Parnassius nomion includes 13 protein-coding genes (ND1-6, COI-III, ATP6, ATP8, ND4, ND4L, CTYB), 2 ribosomal RNAs (12 S and 16 S), 22 transfer RNAs, which is 14,547 bp in length. Its gene order and orientation are identical to the common type found in most of other completely sequenced lepidopteran mitogenomes. All protein-coding genes are initiated by ATN codons, except for COI, which uses CGA as its start codon. Eleven PCGs use the standard TAA as their termination codon, and COI, COII use the incomplete termination codon T.

  4. Characterization of the mitochondrial genome of the cabbage webworm, Hellula undalis (Lepidoptera: Pyralidae).

    Science.gov (United States)

    Dong, Wan-Wei; Feng, Xiao-Jing; Huang, Guo-Hua; Jiang, Guo-Fang

    2016-01-01

    The sequenced mitochondrial genome of the cabbage webworm Hellula undalis includes 13 protein-coding genes (PCGs) (nad1-6, cox1-3, atp6, atp8, nad4L and cob), two ribosomal RNAs (12S and 16S) and 19 transfer RNAs, which is 14,678 bp in length. Its gene order and orientation are identical to the common types found in most of the other completely sequenced lepidopteran mitogenomes. Thirteen PCGs start with a typical ATN codon, while cox1 uses CGA as its start codon. Some PCGs use the standard TAA as their termination codon, while others use the incomplete termination codon T (cox1, cox2 and nad4).

  5. [Purification of the mitochondrial calcium uniporter from the beef heart and characterization of its properties].

    Science.gov (United States)

    Gritsenko, E N; Kutyshenko, V P; Saris, N E-L; Wahlsten, M; Jokela, J; Mironova, G D

    2010-01-01

    A low-molecular-weight component (LMC) inducing selective transport of calcium across the bilayer lipid membrane has been isolated from mitochondria of the bovine heart by the method developed in our laboratory, which excludes the use of detergents and proteolytic enzymes. It was shown that, in the presence of 10 mM CaCl2, LMC forms conduction channels in the membrane multiples of 5 pS. The specific inhibitor of mitochondrial calcium uniporter, ruthenium red, closes Ca2(+)-induced channels formed in the membrane by LMC. In the absence of calcium or in the presence of potassium ions only, the component is incapable of forming channels of conduction. It was shown using nuclear magnetic resonance that LMC is a complex consisting of lipids, amino acids, and sugars with a molecular weight of 1-2 kDa.

  6. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    Science.gov (United States)

    Fernández-García, Yaiza; Reguera, Juan; Busch, Carola; Witte, Gregor; Sánchez-Ramos, Oliberto; Betzel, Christian; Cusack, Stephen; Günther, Stephan; Reindl, Sophia

    2016-06-01

    Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

  7. Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization.

    Science.gov (United States)

    Jackson, Alana J; Hershey, David M; Chesnut, Taylor; Xu, Meimei; Peters, Reuben J

    2014-07-01

    It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral ent-kaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, a synthetic biology approach was applied to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a "splicing" error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, it is possible to assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway.

  8. Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.

    Directory of Open Access Journals (Sweden)

    Adam E Brummett

    Full Text Available The osmolyte dimethylsulfoniopropionate (DMSP is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS, a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121. Measurements of metal binding affinity and catalytic activity indicate that Fe(II is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II per monomer. Electronic absorption and electron paramagnetic resonance (EPR studies show an interaction between NO and Fe(II-DddW, with NO binding to the EPR silent Fe(II site giving rise to an EPR active species (g = 4.29, 3.95, 2.00. The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

  9. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    Directory of Open Access Journals (Sweden)

    Yaiza Fernández-García

    2016-06-01

    Full Text Available Andes virus (ANDV is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

  10. Morphological, molecular, and biochemical characterization of astaxanthin-producing green microalga Haematococcus sp. KORDI03 (Haematococcaceae, Chlorophyta) isolated from Korea.

    Science.gov (United States)

    Kim, Ji Hyung; Affan, Abu; Jang, Jiyi; Kang, Mee-Hye; Ko, Ah-Ra; Jeon, Seon-Mi; Oh, Chulhong; Heo, Soo-Jin; Lee, Youn-Ho; Ju, Se-Jong; Kang, Do-Hyung

    2015-02-01

    A unicellular red microalga was isolated from environmental freshwater in Korea, and its morphological, molecular, and biochemical properties were characterized. Morphological analysis revealed that the isolate was a unicellular biflagellated green microalga that formed a non-motile, thick-walled palmelloid or red aplanospore. To determine the taxonomical position of the isolate, its 18S rRNA and rbcL genes were sequenced and phylogenetic analysis was performed. We found that the isolate was clustered together with other related Haematococcus strains showing differences in the rbcL gene. Therefore, the isolated microalga was classified into the genus Haematococcus, and finally designated Haematococcus sp. KORDI03. The microalga could be cultivated in various culture media under a broad range of pH and temperature conditions. Compositions of the microalgal cellular components were analyzed, and its protein, carbohydrate, and lipid compositions were estimated to be 21.1 ± 0.2%, 48.8 ± 1.8%, and 22.2 ± 0.9%, respectively. In addition, D-glucose and D-mannose were the dominant monosaccharides in the isolate, and its amino acids were composed mainly of aspartic acid, glutamic acid, alanine, and leucine. Moreover, several polyunsaturated fatty acids accounted for about 80% of the total fatty acids in Haematococcus sp. KORDI03, and the astaxanthin content in the red aplanospores was estimated to be 1.8% of the dry cell weight. To the best of our knowledge, this is the first report of an Haematococcus sp. isolated from Korea, which may be used for bioresource production in the microalgal industry.

  11. Biochemical Characterization and Crystal Structures of a Fungal Family 3 β-Glucosidase, Cel3A from Hypocrea jecorina*

    Science.gov (United States)

    Karkehabadi, Saeid; Helmich, Kate E.; Kaper, Thijs; Hansson, Henrik; Mikkelsen, Nils-Egil; Gudmundsson, Mikael; Piens, Kathleen; Fujdala, Meredith; Banerjee, Goutami; Scott-Craig, John S.; Walton, Jonathan D.; Phillips, George N.; Sandgren, Mats

    2014-01-01

    Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)-β-d-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A). Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 β-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn208 and Asn310. In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms. PMID:25164811

  12. Biochemical characterization and crystal structures of a fungal family 3 β-glucosidase, Cel3A from Hypocrea jecorina.

    Science.gov (United States)

    Karkehabadi, Saeid; Helmich, Kate E; Kaper, Thijs; Hansson, Henrik; Mikkelsen, Nils-Egil; Gudmundsson, Mikael; Piens, Kathleen; Fujdala, Meredith; Banerjee, Goutami; Scott-Craig, John S; Walton, Jonathan D; Phillips, George N; Sandgren, Mats

    2014-11-07

    Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)-β-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A). Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 β-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn(208) and Asn(310). In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms.

  13. UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors.

    Science.gov (United States)

    Zinsser, Veronika L; Lindert, Steffen; Banford, Samantha; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J

    2015-03-01

    Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

  14. Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization

    Science.gov (United States)

    Jackson, Alana J.; Hershey, David M.; Chesnut, Taylor; Xu, Meimei; Peters, Reuben J.

    2014-01-01

    It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral ent-kaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, we applied a synthetic biology approach to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a “splicing” error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, we can assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally novel enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway. PMID:24810014

  15. Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization.

    Science.gov (United States)

    Mohammadian, Mahdi; Fathi-Roudsari, Mehrnoosh; Mollania, Nasrin; Badoei-Dalfard, Arastoo; Khajeh, Khosro

    2010-08-01

    Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K(M) and k(cat) were calculated 535 microM and 127 s(-1) for ABTS, 53 microM and 3 s(-1) for 2, 6-DMP and 5 microM and 20 s(-1) for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80 degrees C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70 degrees C and 2.4-fold after 10 min preincubation at 80 degrees C. Preincubation of the enzyme in 70 degrees C for 30 min raised the activity four-fold with ABTS as the substrate. Also, L-dopa was used as a substrate. The enzyme was able to oxidize L-dopa with the K(M) and k(cat) of 1,493 microM and 194 s(-1), respectively.

  16. Biochemical characterization and spatio-temporal expression of myo-inositol oxygenase (MIOX from wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Anshu Alok

    2015-12-01

    Full Text Available Myo-inositol oxygenase (MIOX catalyzes the conversion of myo-inositol into d-glucuronic acid. The present study demonstrates isolation of MIOX cDNA (TaMIOX from wheat (Triticum aestivum L. with open reading frame of 912 bp encoding 303 amino acid polypeptides with a molecular mass of 35.2 kDa. Phylogenetic analysis of TaMIOX across kingdoms confirmed the close relationship with Triticum urartu and Aegilops tauschii. Secondary structure of TaMIOX consists of α-helixes (42.9%, β-turns (7.26% joined by extended strands (14.85%, and 37 random coils (34.94%. Three-dimensional structure of TaMIOX suggested its close functional and structural resemblance with known MIOX. Catalytic activity of the purified TaMIOX is 3.47 μkatal at pH 8.0 and 35 °C with Michaelis constant 5.6 mM. Differential expression pattern of TaMIOX was observed in leaves, root, stem, seed and seed developmental stages. Leaves showed a significantly higher transcript accumulation followed by root, stem and seed. Expression of TaMIOX during seed development stages showed higher expression at later stage and suggested that expression of TaMIOX was significantly higher in endosperm as compared with aleurone. The exogenous application of myo-inositol enhanced the expression of TaMIOX in leaves and roots suggested myo-inositol acts as an inducer for the TaMIOX expression. The present study reports molecular, structural and biochemical characterizations of MIOX in wheat which might play an important role in myo-inositol oxidation pathway.

  17. Characterization of mitochondrial genome of Chinese wild mulberry silkworm, Bomyx mandarina(Lepidoptera:Bombycidae)

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The complete mitochondrial genome of Chinese Bombyx mandarina(ChBm) was determined.The cir-cular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement iden-tical to that of Bombyx mori(B.mori) and Japanese Bombyx mandarina(JaBm) except for two addi-tional tRNA-like structures:tRNASer(TGA)-like and tRNAIle(TAT)-like.All protein-coding sequences are initi-ated with a typical ATN codon except for the COI gene, which has a 4-bp TTAG putative initiator codon.Eleven of 13 protein-coding genes(PCGs) have a complete termination codon(all TAA), but the re-maining two genes terminate with incomplete codons.All tRNAs have the typical clover-leaf structures of mitochondrial tRNAs, with the exception of tRNASer(TGA)-like, with a four stem-and-loop structure.The length of the A+T-rich region of ChBm is 484 bp, shorter than those of JaBm(747 bp) and B.mori(494―499 bp).Phylogenetic analysis among B.mori, ChBm, JaBm, and Antheraea pernyi(Anpe) showed that B.mori is more closely related to ChBm than JaBm.The earliest divergence time estimate for B.mori-ChBm and B.mori-JaBm is about 1.08±0.18―1.41±0.24 and 1.53±0.20―2.01±0.26 Mya, respec-tively.ChBm and JaBm diverged around 1.11±0.16―1.45±0.21 Mya.

  18. Biochemical Characterization of CPS-1, a Subclass B3 Metallo-β-Lactamase from a Chryseobacterium piscium Soil Isolate

    DEFF Research Database (Denmark)

    Gudeta, Dereje Dadi; Pollini, Simona; Docquier, Jean-Denis;

    2016-01-01

    CPS-1 is a subclass B3 metallo-β-lactamase from a Chryseobacterium piscium isolated from soil, showing 68 % amino acid identity to GOB-1 enzyme. CPS-1 was overproduced in Escherichia coli Rosetta (DE3), purified by chromatography and biochemically characterized. This enzyme exhibits a broad spect...... spectrum substrate profile including penicillins, cephalosporins and carbapenems, which overall resembles those of L1, GOB-1 and acquired subclass B3 enzymes AIM-1 and SMB-1.......CPS-1 is a subclass B3 metallo-β-lactamase from a Chryseobacterium piscium isolated from soil, showing 68 % amino acid identity to GOB-1 enzyme. CPS-1 was overproduced in Escherichia coli Rosetta (DE3), purified by chromatography and biochemically characterized. This enzyme exhibits a broad...

  19. Isolation and Biochemical Characterizations of Mid Gut Microbiota of Culex (Culex quinquefasciatus Mosquitoes in Some Urban Sub Urban & Rural Areas of West Bengal.

    Directory of Open Access Journals (Sweden)

    Rahul Kumar

    2013-06-01

    Full Text Available Mosquitoes, in general are medically important vectors of many diseases like Malaria, Dengue and Filariasis, which are a great challenge for public health in many countries. All animals and plants establish symbiotic relationship with microbes. Mosquitoes can be considered as an holobiont units in which the host (mosquito and its microbiota are involved in complex reciprocal multipartite interaction such as host reproduction and survival, protection against natural enemies. This naturally acquired microbial flora can modulate the mosquitos’ vectorial capacity by inhibiting the development of pathogen. But enough care has not been under taken regarding the biochemical characterization of Culex mosquitoes (Culex quinquefasciatus in West Bengal. Therefore a preliminary investigation have been undertaken for the determination of biochemical characterization such as gram staining, pattern of growth, detection of economically important enzyme as well as antibiotic susceptibility assay of midgut bacterial isolates of Culex (Culex quinquefasciatus in some urban, sub-urban and rural areas of West Bengal.

  20. Biochemical characterization of nuclear receptors for vitamin D{sub 3} and glucocorticoids in prostate stroma cell microenvironment

    Energy Technology Data Exchange (ETDEWEB)

    Hidalgo, Alejandro A. [Laboratory of Molecular Endocrinology, Department of Physiopathology, University of Concepcion, Concepcion (Chile); Department of Molecular Pharmacology and Therapeutics, NY (United States); Montecinos, Viviana P.; Paredes, Roberto; Godoy, Alejandro S.; McNerney, Eileen M.; Tovar, Heribelt; Pantoja, Diego [Laboratory of Molecular Endocrinology, Department of Physiopathology, University of Concepcion, Concepcion (Chile); Johnson, Candace [Department of Molecular Pharmacology and Therapeutics, NY (United States); Trump, Donald [Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY (United States); Onate, Sergio A., E-mail: sergio.onate@udec.cl [Laboratory of Molecular Endocrinology, Department of Physiopathology, University of Concepcion, Concepcion (Chile); Department of Urology, State University of New York at Buffalo, NY (United States)

    2011-08-19

    Highlights: {yields} Fibroblasts from benign and carcinoma-associated stroma were biochemically characterized for VDR and GR function as transcription factors in prostate stroma cell microenvironment. {yields} Decreased SRC-1/CBP coactivators recruitment to VDR and GR may result in hormone resistance to 1,25D{sub 3} in stromal cell microenvironment prostate cancer. {yields} 1a,25-Dyhidroxyvitamin D{sub 3} (1,25D{sub 3}) and glucocorticoids, either alone or in combination, may not be an alternative for 'some' advanced prostate cancers that fails androgen therapies. -- Abstract: The disruption of stromal cell signals in prostate tissue microenvironment influences the development of prostate cancer to androgen independence. 1{alpha},25-Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) and glucocorticoids, either alone or in combination, have been investigated as alternatives for the treatment of advanced prostate cancers that fails androgen therapies. The effects of glucocorticoids are mediated by the intracellular glucocorticoid receptor (GR). Similarly, the effect of 1,25D{sub 3} is mediated by the 1,25D{sub 3} nuclear receptor (VDR). In this study, fibroblasts from benign- (BAS) and carcinoma-associated stroma (CAS) were isolated from human prostates to characterize VDR and GR function as transcription factors in prostate stroma. The VDR-mediated transcriptional activity assessed using the CYP24-luciferase reporter was limited to 3-fold induction by 1,25D{sub 3} in 9 out of 13 CAS (70%), as compared to >10-fold induction in the BAS clinical sample pair. Expression of His-tagged VDR (Ad-his-VDR) failed to recover the low transcriptional activity of the luciferase reporter in 7 out of 9 CAS. Interestingly, expression of Ad-his-VDR successfully recovered receptor-mediated induction in 2 out of the 9 CAS analyzed, suggesting that changes in the receptor protein itself was responsible for decreased response and resistance to 1,25D{sub 3} action. Conversely, VDR

  1. Characterization of the complete mitochondrial genomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae).

    Science.gov (United States)

    Chai, Huan-Na; Du, Yu-Zhou; Zhai, Bao-Ping

    2012-01-01

    The complete mitochondrial genomes (mitogenomes) of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae) were determined and analyzed. The circular genomes were 15,388 bp long for C. medinalis and 15,395 bp long for C. suppressalis. Both mitogenomes contained 37 genes, with gene order similar to that of other lepidopterans. Notably, 12 protein-coding genes (PCGs) utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; the cox1, cox2, and nad4 genes in the two mitogenomes had the truncated termination codons T, T, and TA, respectively, but the nad5 gene was found to use T as the termination codon only in the C. medinalis mitogenome. Additionally, the codon distribution and Relative Synonymous Codon Usage of the 13 PCGs in the C. medinalis mitogenome were very different from those in other pyralid moth mitogenomes. Most of the tRNA genes had typical cloverleaf secondary structures. However, the dihydrouridine (DHU) arm of the trnS1(AGN) gene did not form a stable stem-loop structure. Forty-nine helices in six domains, and 33 helices in three domains were present in the secondary structures of the rrnL and rrnS genes of the two mitogenomes, respectively. There were four major intergenic spacers, except for the A+T-rich region, spanning at least 12 bp in the two mitogenomes. The A+T-rich region contained an 'ATAGT(A)'-like motif followed by a poly-T stretch in the two mitogenomes. In addition, there were a potential stem-loop structure, a duplicated 25-bp repeat element, and a microsatellite '(TA)(13)' observed in the A+T-rich region of the C. medinalis mitogenome. A poly-T motif, a duplicated 31-bp repeat element, and a 19-bp triplication were found in the C. suppressalis mitogenome. However, there are many differences in the A+T-rich regions between the C. suppressalis mitogenome sequence in the present study and previous reports. Finally, the phylogenetic relationships of these insects were reconstructed based on amino acid

  2. Characterization of the Complete Mitochondrial Genomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae

    Directory of Open Access Journals (Sweden)

    Huan-Na Chai, Yu-Zhou Du, Bao-Ping Zhai

    2012-01-01

    Full Text Available The complete mitochondrial genomes (mitogenomes of Cnaphalocrocis medinalis and Chilo suppressalis (Lepidoptera: Pyralidae were determined and analyzed. The circular genomes were 15,388 bp long for C. medinalis and 15,395 bp long for C. suppressalis. Both mitogenomes contained 37 genes, with gene order similar to that of other lepidopterans. Notably, 12 protein-coding genes (PCGs utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; the cox1, cox2, and nad4 genes in the two mitogenomes had the truncated termination codons T, T, and TA, respectively, but the nad5 gene was found to use T as the termination codon only in the C. medinalis mitogenome. Additionally, the codon distribution and Relative Synonymous Codon Usage of the 13 PCGs in the C. medinalis mitogenome were very different from those in other pyralid moth mitogenomes. Most of the tRNA genes had typical cloverleaf secondary structures. However, the dihydrouridine (DHU arm of the trnS1(AGN gene did not form a stable stem-loop structure. Forty-nine helices in six domains, and 33 helices in three domains were present in the secondary structures of the rrnL and rrnS genes of the two mitogenomes, respectively. There were four major intergenic spacers, except for the A+T-rich region, spanning at least 12 bp in the two mitogenomes. The A+T-rich region contained an 'ATAGT(A'-like motif followed by a poly-T stretch in the two mitogenomes. In addition, there were a potential stem-loop structure, a duplicated 25-bp repeat element, and a microsatellite '(TA13' observed in the A+T-rich region of the C. medinalis mitogenome. A poly-T motif, a duplicated 31-bp repeat element, and a 19-bp triplication were found in the C. suppressalis mitogenome. However, there are many differences in the A+T-rich regions between the C. suppressalis mitogenome sequence in the present study and previous reports. Finally, the phylogenetic relationships of these insects were reconstructed based on

  3. Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor.

    Science.gov (United States)

    Drakulic, Srdja; Wang, Liping; Cuéllar, Jorge; Guo, Qing; Velázquez, Gilberto; Martín-Benito, Jaime; Sousa, Rui; Valpuesta, José M

    2014-01-01

    Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s). To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand.

  4. "Stiff neonate" with mitochondrial DNA depletion and secondary neurotransmitter defects.

    LENUS (Irish Health Repository)

    Moran, Margaret M

    2011-12-01

    Mitochondrial disorders comprise a heterogenous group. A neonate who presented with episodes of severe truncal hypertonia and apnea progressed to a hypokinetic rigid syndrome characterized by hypokinesia, tremulousness, profound head lag, absent suck and gag reflexes, brisk deep tendon reflexes, ankle and jaw clonus, and evidence of autonomic dysfunction. Analysis of cerebrospinal fluid neurotransmitters from age 7 weeks demonstrated low levels of amine metabolites (homovanillic acid and 5-hydroxyindoleacetic acid), tetrahydrobiopterin, and pyridoxal phosphate. Mitochondrial DNA quantitative studies on muscle homogenate demonstrated a mitochondrial DNA depletion disorder. Respiratory chain enzymology demonstrated decreased complex IV activity. Screening for mitochondrial DNA rearrangement disorders and sequencing relevant mitochondrial genes produced negative results. No clinical or biochemical response to treatment with pyridoxal phosphate, tetrahydrobiopterin, or l-dopa occurred. The clinical course was progressive, and the patient died at age 19 months. Mitochondrial disorders causing secondary neurotransmitter diseases are usually severe, but are rarely reported. This diagnosis should be considered in neonates or infants who present with hypertonia, hypokinesia rigidity, and progressive neurodegeneration.

  5. Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica for PCR-RFLP Based Species Identification

    Directory of Open Access Journals (Sweden)

    Chandra Mohan Siddappa

    2013-01-01

    Full Text Available Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species.

  6. Characterization and comparison of mitochondrial DNAs and rRNAs from Penicillium urticae and P. chrysogenum.

    Science.gov (United States)

    Sekiguchi, J; Ohsaki, T; Yamamoto, H; Koichi, K; Shida, T

    1990-03-01

    Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P. urticae and 3.5 kb for P. chrysogenum. These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.

  7. Characterization of the complete mitochondrial genomes of two whipworms Trichuris ovis and Trichuris discolor (Nematoda: Trichuridae).

    Science.gov (United States)

    Liu, Guo-Hua; Wang, Yan; Xu, Min-Jun; Zhou, Dong-Hui; Ye, Yong-Gang; Li, Jia-Yuan; Song, Hui-Qun; Lin, Rui-Qing; Zhu, Xing-Quan

    2012-12-01

    For many years, whipworms (Trichuris spp.) have been described with a relatively narrow range of both morphological and biometrical features. Moreover, there has been insufficient discrimination between congeners (or closely related species). In the present study, we determined the complete mitochondrial (mt) genomes of two whipworms Trichuris ovis and Trichuris discolor, compared them and then tested the hypothesis that T. ovis and T. discolor are distinct species by phylogenetic analyses using Bayesian inference, maximum likelihood and maximum parsimony) based on the deduced amino acid sequences of the mt protein-coding genes. The complete mt genomes of T. ovis and T. discolor were 13,946 bp and 13,904 bp in size, respectively. Both mt genomes are circular, and consist of 37 genes, including 13 genes coding for proteins, 2 genes for rRNA, and 22 genes for tRNA. The gene content and arrangement are identical to that of human and pig whipworms Trichuris trichiura and Trichuris suis. Taken together, these analyses showed genetic distinctiveness and strongly supported the recent proposal that T. ovis and T. discolor are distinct species using nuclear ribosomal DNA and a portion of the mtDNA sequence dataset. The availability of the complete mtDNA sequences of T. ovis and T. discolor provides novel genetic markers for studying the population genetics, diagnostics and molecular epidemiology of T. ovis and T. discolor.

  8. Molecular characterization of Saudi local chicken strains using mitochondrial DNA markers.

    Science.gov (United States)

    Yacoub, H A; Ramadan, H A I; Baeshen, Nabih A; Sadek, Mahmoud Abdel; Abou Alsoud, M E

    2015-08-01

    The current study was carried out to investigate and estimate the genetic diversity of native breeds based on cytochrome b (cyt-b) gene of mitochondrial DNA information. The obtained sequences of cyt-b gene segment have TAA as a stop codon at 488 position with no insertions or deletion in all individuals of both native chicken strains. The blast results showed that no variation was found among individuals within both native chicken strains, but when a comparison was established among them and other species of genus Gallus the variation is exploring, additionally many mutant sites were detected as single nucleotide polymorphisms (SNPs) in different sites. The phylogenetic trees exhibited three different groups. The results revealed that the native chicken strains were closely related to the cluster of Gallus gallus and subspecies of Gallus, suggesting that they may be separated from the same origin. According to this result and previously studies, the native chicken strains are genetically closer to Gallus gallus and it could be successfully distinguished from the other wild types of Gallus chicken based on cyt-b gene information. We recommended that the governmental concerns for native chicken strain should be enhanced to screen its genetic structure for large scale in the Kingdom of Saudi Arabia.

  9. Genetic characterization of Golden mahseer (Tor putitora) populations using mitochondrial DNA markers.

    Science.gov (United States)

    Sati, Jyoti; Kumar, Rohit; Sahoo, Prabhati Kumari; Patiyal, Rabindar S; Ali, Shahnawaz; Barat, Ashoktaru

    2015-02-01

    Golden Mahseer (Tor putitora) is an economically important fish of India and Southeast Asia. The present study examined the genetic variations between seven geographically isolated populations of T. putitora using Cyt b (Cytochrome b) and ATPase6/8 gene sequences of mitochondrial DNA. Analysis of 133 sequences of Cyt b (1141 bp) and 130 sequences of ATPase6/8 gene (842 bp) revealed 47 and 44 haplotypes, respectively. The estimated haplotype and nucleotide diversity was high in River Jia Bhoreli (Bhalukpong) population (h = 1.00000, π = 0.007121 for Cyt b and h = 0.90441 π = 0.004867 for ATPase6/8). Results of AMOVA indicated that majority of the genetic variations in both genes were due to variation among populations (60.79% for Cyt b and 51.41% for ATPase6/8 gene). The pairwise F(ST) comparison and neighbor-joining tree revealed high genetic divergence of River Jia Bhoreli population from other populations. The understanding of genetic variations of T. putitora populations will play a key role in conservation and management of this endangered fish species.

  10. Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes.

    Science.gov (United States)

    Chitimia, Lidia; Lin, Rui-Qing; Cosoroaba, Iustin; Wu, Xiang-Yun; Song, Hui-Qun; Yuan, Zi-Guo; Zhu, Xing-Quan

    2010-11-01

    In the present study, samples representing three hard tick species and one soft tick species, namely Dermacentor marginatus, Haemaphysalis punctata, Ixodes ricinus and Argas persicus from southwestern Romania, and one hard tick, Haemaphysalis longicornis, from China were characterized genetically by a portion of mitochondrial cytochrome c oxidase subunit 1 gene (pcox1) and a portion of nicotinamide adenine dinucleotide dehydrogenase subunit 5 gene (pnad5). The pcox1 and pnad5 were amplified separately from individual ticks by PCR, sequenced and analyzed. The length of pcox1 and pnad5 sequences of all samples was 732 and 519 bp, respectively. The intra-specific sequence variation in De. marginatus was 0.1-1.0% for pcox1 and 0.2-1.2% for pnad5, whereas in Ha. punctata it was 0.4-1.9% for pcox1 and 0.4-1.0% for pnad5. For the tick species examined in the present study, sequence comparison revealed that the inter-specific sequence differences were higher: 15.9-27.6% for pcox1 and 20.3-42.4% for pnad5. This suggests that the cox1 and nad5 sequences could provide useful genetic markers for the specific identification and genetic characterization of ticks in Romania and elsewhere.

  11. Mitochondrial Dynamics in Mitochondrial Diseases

    Directory of Open Access Journals (Sweden)

    Juan M. Suárez-Rivero

    2016-12-01

    Full Text Available Mitochondria are very versatile organelles in continuous fusion and fission processes in response to various cellular signals. Mitochondrial dynamics, including mitochondrial fission/fusion, movements and turnover, are essential for the mitochondrial network quality control. Alterations in mitochondrial dynamics can cause neuropathies such as Charcot-Marie-Tooth disease in which mitochondrial fusion and transport are impaired, or dominant optic atrophy which is caused by a reduced mitochondrial fusion. On the other hand, mitochondrial dysfunction in primary mitochondrial diseases promotes reactive oxygen species production that impairs its own function and dynamics, causing a continuous vicious cycle that aggravates the pathological phenotype. Mitochondrial dynamics provides a new way to understand the pathophysiology of mitochondrial disorders and other diseases related to mitochondria dysfunction such as diabetes, heart failure, or Hungtinton’s disease. The knowledge about mitochondrial dynamics also offers new therapeutics targets in mitochondrial diseases.

  12. Pigmentation in sand pear (Pyrus pyrifolia) fruit: biochemical characterization, gene discovery and expression analysis with exocarp pigmentation mutant.

    Science.gov (United States)

    Wang, Yue-zhi; Zhang, Shujun; Dai, Mei-song; Shi, Ze-bin

    2014-05-01

    Exocarp color of sand pear is an important trait for the fruit production and has caused our concern for a long time. Our previous study explored the different expression genes between the two genotypes contrasting for exocarp color, which indicated the different suberin, cutin, wax and lignin biosynthesis between the russet- and green-exocarp. In this study, we carried out microscopic observation and Fourier transform infrared spectroscopy analysis to detect the differences of tissue structure and biochemical composition between the russet- and green-exocarp of sand pear. The green exocarp was covered with epidermis and cuticle which was replaced by a cork layer on the surface of russet exocarp, and the chemicals of the russet exocarp were characterized by lignin, cellulose and hemicellulose. We explored differential gene expression between the russet exocarp of 'Niitaka' and its green exocarp mutant cv. 'Suisho' using Illumina RNA-sequencing. A total of 559 unigenes showed different expression between the two types of exocarp, and 123 of them were common to the previous study. The quantitative real time-PCR analysis supports the RNA-seq-derived gene with different expression between the two types of exocarp and revealed the preferential expression of these genes in exocarp than in mesocarp and fruit core. Gene ontology enrichment analysis revealed divorced expression of lipid metabolic process genes, transport genes, stress responsive genes and other biological process genes in the two types of exocarp. Expression changes in lignin metabolism-related genes were consistent with the different pigmentation of russet and green exocarp. Increased transcripts of putative genes involved the suberin, cutin and wax biosynthesis in 'Suisho' exocarp could facilitate deposition of the chemicals and take a role in the mutant trait responsible for the green exocarp. In addition, the divorced expression of ATP-binding cassette transporters involved in the trans

  13. Field and Laboratory Studies on Pathological and Biochemical Characterization of Microcystin-Induced Liver and Kidney Damage in the Phytoplanktivorous Bighead Carp

    Directory of Open Access Journals (Sweden)

    Li Li

    2008-01-01

    Full Text Available Field and experimental studies were conducted to investigate pathological characterizations and biochemical responses in the liver and kidney of the phytoplanktivorous bighead carp after intraperitoneal (i.p. administration of microcystins (MCs and exposure to natural cyanobacterial blooms in Meiliang Bay, Lake Taihu. Bighead carp in field and laboratory studies showed a progressive recovery of structure and function in terms of histological, cellular, and biochemical features. In laboratory study, when fish were i.p. injected with extracted MCs at the doses of 200 and 500 μg MC-LReq/kg body weight, respectively, liver pathology in bighead carp was observed in a time dose-dependent manner within 24 h postinjection and characterized by disruption of liver structure, condensed cytoplasm, and the appearance of massive hepatocytes with karyopyknosis, karyorrhexis, and karyolysis. In comparison with previous studies on other fish, bighead carp in field study endured higher MC doses and longer-term exposure, but displayed less damage in the liver and kidney. Ultrastructural examination in the liver revealed the presence of lysosome proliferation, suggesting that bighead carp might eliminate or lessen cell damage caused by MCs through lysosome activation. Biochemically, sensitive responses in the antioxidant enzymes and higher basal glutathione concentrations might be responsible for their powerful resistance to MCs, suggesting that bighead carp can be used as biomanipulation fish to counteract cyanotoxin contamination.

  14. Detection and Biochemical Characterization of Microorganisms in Milk and Cocoa powder samples by FTIR and subsequent production of Bacteriocin from Lactobacillus

    Directory of Open Access Journals (Sweden)

    Ramalingam C

    2013-03-01

    Full Text Available Cocoa and milk powder samples were taken from a confectionery and tested for presence of microbes (harmful and pathogenic.Biochemical characterization of isolated microbes was carried out for confirmation. Lactobacillus was isolated from milk powder. When a culture of Lactobacillus sp. was inoculated into milk and incubated at room temperature, it multiplies and converts lactose to lactic acid. Fourier transform infrared spectroscopy was used to study the variation of functional group peaks in milk by the action of Lactobacillus sp. The spectral changes were also observed. Our main aim of this project is the production of bacteriocin from isolated lactobacillus species; it showed broad range of antibacterial activity against some food borne pathogens like staphyloccus, Ecoli, streptococcus, Enterococcus etc. The bacteriocin is purified by ammonium sulfate precipitate and dialysis. Biochemically it was pure protein moiety. Maximum bacteriocin concentration was found after dialysis. Project revealed the possibility of using bacteriocin as food preservative.

  15. Advanced glycation end product ligands for the receptor for advanced glycation end products: Biochemical characterization and formation kinetics

    NARCIS (Netherlands)

    Valencia, J.V.; Weldon, S.C.; Quinn, D.; Kiers, G.H.; Groot, J. de; TeKoppele, J.M.; Hughes, T.E.

    2004-01-01

    Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown.

  16. Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina

    NARCIS (Netherlands)

    Hiseni, A.; Arends, I.W.C.E.; Otten, L.G.

    2011-01-01

    Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their subs

  17. Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

    NARCIS (Netherlands)

    van der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaarl, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2007-01-01

    In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain SI on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by

  18. Characterization of a Leber's hereditary optic neuropathy (LHON) family harboring two primary LHON mutations m.11778G>A and m.14484T>C of the mitochondrial DNA.

    Science.gov (United States)

    Catarino, Claudia B; Ahting, Uwe; Gusic, Mirjana; Iuso, Arcangela; Repp, Birgit; Peters, Katrin; Biskup, Saskia; von Livonius, Bettina; Prokisch, Holger; Klopstock, Thomas

    2016-10-06

    Leber's hereditary optic neuropathy (LHON) is an inherited mitochondrial disease that usually leads to acute or subacute bilateral central vision loss. In 95% of cases, LHON is caused by one of three primary mutations of the mitochondrial DNA (mtDNA), m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, or m.3460G>A in the MT-ND1 gene. Here we characterize clinically, genetically, and biochemically a LHON family with multiple patients harboring two of these primary LHON mutations, m.11778G>A homoplasmic and m.14484T>C heteroplasmic. The unusually low male-to-female ratio of affected family members is also seen among the other patients previously reported with two primary LHON mutations m.11778G>A and m.14484T>C. While the index patient had very late onset of symptoms at 75years and severe visual loss, her two daughters had both onset in childhood (6 and 9years), with moderate to mild visual loss. A higher degree of heteroplasmy of the m.14484T>C mutation was found to correlate with an earlier age at onset in this family. Ours is the first LHON family harboring two primary LHON mutations where functional studies were performed in several affected family members. A more pronounced bioenergetic defect was found to correlate with an earlier age at onset. The patient with the earliest age at onset had a more significant complex I dysfunction than all controls, including the LHON patient with only the m.11778G>A mutation, suggesting a synergistic effect of the two primary LHON mutations in this patient.

  19. Characterization of Fasciola spp. in Myanmar on the basis of spermatogenesis status and nuclear and mitochondrial DNA markers.

    Science.gov (United States)

    Ichikawa, Madoka; Bawn, Saw; Maw, Ni Ni; Htun, Lat Lat; Thein, Myint; Gyi, Aung; Sunn, Kyaw; Katakura, Ken; Itagaki, Tadashi

    2011-12-01

    Fasciola spp. in Myanmar were characterized on the basis of spermatogenesis status and DNA markers of nuclear internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1). We collected 88 adult flukes from Yangon, Lashio, and Myitkyina. Spermatogenesis status was analyzed by the presence of sperm in the seminal vesicles, and 8 aspermic and 80 spermic flukes were detected. The flukes were identified on the basis of spermatogenesis status and ITS1 types which were analyzed by a PCR-RFLP method, and 80 spermic flukes were identified as F. gigantica. A very low detection rate of aspermic Fasciola sp. indicated that they are not established in Myanmar. In phylogenetic analyses, the 7 aspermic Fasciola sp. from Myitkyina displayed a haplotype in nad1 sequence, which was identical to that of aspermic Fasciola sp. from other Asian countries including China. Therefore, they were probably introduced from China through an infected domestic ruminant. On the other hand, 17 nad1 haplotypes detected in F. gigantica belonged to 2 clades unique to Myanmar, each with a distinct founder haplotype in a network analysis. This indicated a unique history of F. gigantica introduction into Myanmar involving ancient artificial movements of domestic ruminants.

  20. Purification, immobilization, and biochemical characterization of l-arginine deiminase from thermophilic Aspergillus fumigatus KJ434941: anticancer activity in vitro.

    Science.gov (United States)

    El-Sayed, Ashraf S A; Hassan, Mohamed N; Nada, Hend M S

    2015-01-01

    l-Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G1 phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1-fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG-ADI, and Dex-ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG-ADI displays twofold resistance to thermal denaturation (t1/2 13.9 h), than free ADI (t1/2 6.9 h), while at 70°C, the thermal stability of PEG-ADI was increased by 1.7-fold, with similar stability to Dex-ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG-ADI and Dex-ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG-ADI, and Dex-AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP-G2, and MCF7, in vitro. The free and PEG-ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex-ADI. The free ADI had IC50 value 22.0, 16.6, and 13.9 U/mL, while Dex-ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG-2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG-2 was increased by five-, three-, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti-ADI immunoglobulins in vivo. The in vivo half-life time of free ADI, PEG

  1. Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides

    Directory of Open Access Journals (Sweden)

    Pragya Tiwari

    2014-01-01

    Full Text Available Gymnema sylvestre R.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator, is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed in Escherichia coli and biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene from G. sylvestre R.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants.

  2. Molecular cloning and biochemical characterization of a recombinant sterol 3-O-glucosyltransferase from Gymnema sylvestre R.Br. catalyzing biosynthesis of steryl glucosides.

    Science.gov (United States)

    Tiwari, Pragya; Sangwan, Rajender Singh; Asha; Mishra, B N; Sabir, Farzana; Sangwan, Neelam S

    2014-01-01

    Gymnema sylvestre R.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator), is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp) contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed in Escherichia coli and biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene from G. sylvestre R.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants.

  3. Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Cvetić Antić, Tijana; Vučinić, Željko

    2015-01-01

    Comparative biochemical characterization of class III peroxidase activity tightly bound to the cell walls of maize roots was performed. Ionically bound proteins were solubilized from isolated walls by salt washing, and the remaining covalently bound peroxidases were released, either by enzymatic digestion or by a novel alkaline extraction procedure that released covalently bound alkali-resistant peroxidase enzyme. Solubilized fractions, as well as the salt-washed cell wall fragments containing covalently bound proteins, were analyzed for peroxidase activity. Peroxidative and oxidative activities indicated that peroxidase enzymes were predominately associated with walls by ionic interactions, and this fraction differs from the covalently bound one according to molecular weight, isozyme patterns, and biochemical parameters. The effect of covalent binding was evaluated by comparison of the catalytic properties of the enzyme bound to the salt-washed cell wall fragments with the corresponding solubilized and released enzyme. Higher thermal stability, improved resistance to KCN, increased susceptibility to H2O2, stimulated capacity of wall-bound enzyme to oxidize indole-3-acetic acid (IAA) as well as the difference in kinetic parameters between free and bound enzymes point to conformational changes due to covalent binding. Differences in biochemical properties of ionically and covalently bound peroxidases, as well as the modulation of the enzyme properties as a result of covalent binding to the walls, indicate that these two fractions of apoplastic peroxidases play different roles.

  4. Identification of novel mutations in five patients with mitochondrial encephalomyopathy.

    Science.gov (United States)

    Valente, Lucia; Piga, Daniela; Lamantea, Eleonora; Carrara, Franco; Uziel, Graziella; Cudia, Paola; Zani, Anna; Farina, Laura; Morandi, Lucia; Mora, Marina; Spinazzola, Antonella; Zeviani, Massimo; Tiranti, Valeria

    2009-05-01

    MELAS, MERRF, LHON and NARP, are well-established mitochondrial syndromes associated with specific point mutations of mitochondrial DNA (mtDNA). However, these recurrent mtDNA mutations account for only a minority of mitochondrial disease cases. To evaluate the impact of novel mtDNA mutations, we performed mtDNA sequence analysis in muscle and other tissues of 240 patients with different mitochondrial neuromuscular syndromes. We identified a total of 33 subjects with novel, private or uncommon mutations. Among these, five novel mutations were found in both paediatric and adult cases. We here report on the clinical description of these patients, as well as the biochemical and molecular genetic characterization of the corresponding mutations. Patients 1 and 2 showed changes in ND genes, patient 3 carried a heteroplasmic deletion in the COI gene, patients 4 and 5 carried heteroplasmic mutations in tRNA(Trp) and tRNA(Phe), respectively. Altogether, these data indicate that mtDNA analysis must become part of the routine screening for mitochondrial disorders.

  5. Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle

    OpenAIRE

    2008-01-01

    A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 ± 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 ± 13.5 days). Further, C-BSE was ...

  6. A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual mitochondrial genetic etiology

    Energy Technology Data Exchange (ETDEWEB)

    Mackey, D. (Royal Children' s Hospital, Melbourne (Australia)); Howell, N. (Univ. of Texas, Galveston (United States))

    1992-12-01

    The Tas2 and Vic2 Australian families are affected with a variant of Leber hereditary optic neuropathy (LHON). The risk of developing the optic neuropathy shows strict maternal inheritance, and the opthalmological changes in affected family members are characteristic of LHON. However, in contrast to the common form of the disease, members of these two families show a high frequency of vision recovery. To ascertain the mitochondrial genetic etiology of the LHON in these families, both (a) the nucleotide sequences of the seven mitochondrial genes encoding subunits of respiratory-chain complex I and (b) the mitochondrial cytochrome b gene were determined for representatives of both families. Neither family carries any of the previously identified primary mitochondrial LHON mutations: ND4/11778, ND1/3460, or ND1/4160. Instead, both LHON families carry multiple nucleotide changes in the mitochondrial complex I genes, which produce conservative amino acid changes. From the available sequence data, it is inferred that the Vic2 and Tas2 LHON families are phylogenetically related to each other and to a cluster of LHON families in which mutations in the mitochondrial cytochrome b gene have been hypothesized to play a primary etiological role. However, sequencing analysis establishes that the Vic2 and Tas2 LHON families do not carry these cytochrome b mutations. There are two hypotheses to account for the unusual mitochondrial genetic etiology of the LHON in the Tas2 and Vic2 LHON families. One possibility is that there is a primary LHON mutation within the mitochondrial genome but that it is at a site that was not included in the sequencing analyses. Alternatively, the disease in these families may result from the cumulative effects of multiple secondary LHON mutations that have less severe phenotypic consequences. 29 refs., 3 figs., 3 tabs.

  7. The Role of Mitochondrial Dysfunction in Psychiatric Disease

    Science.gov (United States)

    Scaglia, Fernando

    2010-01-01

    Mitochondrial respiratory chain disorders are a group of genetically and clinically heterogeneous disorders caused by the biochemical complexity of mitochondrial respiration and the fact that two genomes, one mitochondrial and one nuclear, encode the components of the respiratory chain. These disorders can manifest at birth or present later in…

  8. Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids.

    Science.gov (United States)

    Bakonyi, Daniel; Hummel, Werner

    2017-04-01

    A gene encoding a novel 7α-specific NADP(+)-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP(+) using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type.

  9. Mitochondrial Diseases

    Science.gov (United States)

    ... disorder, something goes wrong with this process. Mitochondrial diseases are a group of metabolic disorders. Mitochondria are ... cells and cause damage. The symptoms of mitochondrial disease can vary. It depends on how many mitochondria ...

  10. Mitochondrial Myopathies

    Science.gov (United States)

    ... which stimulates normal beating of the heart. Cardiac muscle damage also may occur. People with mitochondrial disorders may need to have regular examina- tions by a cardiologist. Other potential health issues Some people with mitochondrial disease experience ...

  11. Mitochondrial haplogroups

    DEFF Research Database (Denmark)

    Benn, Marianne; Schwartz, Marianne; Nordestgaard, Børge G;

    2008-01-01

    Rare mutations in the mitochondrial genome may cause disease. Mitochondrial haplogroups defined by common polymorphisms have been associated with risk of disease and longevity. We tested the hypothesis that common haplogroups predict risk of ischemic cardiovascular disease, morbidity from other...

  12. Mitochondrial genetics

    OpenAIRE

    Chinnery, Patrick Francis; Hudson, Gavin

    2013-01-01

    Introduction In the last 10 years the field of mitochondrial genetics has widened, shifting the focus from rare sporadic, metabolic disease to the effects of mitochondrial DNA (mtDNA) variation in a growing spectrum of human disease. The aim of this review is to guide the reader through some key concepts regarding mitochondria before introducing both classic and emerging mitochondrial disorders. Sources of data In this article, a review of the current mitochondrial genetics literature was con...

  13. Mitochondrial diseases caused by toxic compound accumulation: from etiopathology to therapeutic approaches.

    Science.gov (United States)

    Di Meo, Ivano; Lamperti, Costanza; Tiranti, Valeria

    2015-07-20

    Mitochondrial disorders are a group of highly invalidating human conditions for which effective treatment is currently unavailable and characterized by faulty energy supply due to defective oxidative phosphorylation (OXPHOS). Given the complexity of mitochondrial genetics and biochemistry, mitochondrial inherited diseases may present with a vast range of symptoms, organ involvement, severity, age of onset, and outcome. Despite the wide spectrum of clinical signs and biochemical underpinnings of this group of dis-orders, some common traits can be identified, based on both pathogenic mechanisms and potential therapeutic approaches. Here, we will review two peculiar mitochondrial disorders, ethylmalonic encephalopathy (EE) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), caused by mutations in the ETHE1 and TYMP nuclear genes, respectively. ETHE1 encodes for a mitochondrial enzyme involved in sulfide detoxification and TYMP for a cytosolic enzyme involved in the thymidine/deoxyuridine catabolic pathway. We will discuss these two clinical entities as a paradigm of mitochondrial diseases caused by the accumulation of compounds normally present in traces, which exerts a toxic and inhibitory effect on the OXPHOS system.

  14. Biochemical and molecular characterization of the Cyclophyllidean cestode, Cotugnia cuneata (Meggit, 1924), an endoparasite of domestic pigeons, Columba livia domestica.

    Science.gov (United States)

    Biswal, Debraj; Nandi, Anadi Prasad; Chatterjee, Soumendranath

    2014-03-01

    Analysis of the major biochemical constituents of Cotugnia cuneata revealed that the total protein, carbohydrate, glycogen and lipid contents (as percentage of dry weight) were 25.22 ± 0.93, 32.90 ± 0.30, 21.33 ± 0.99 and 9.94 ± 0.42 respectively. The results showed that the carbohydrate content was the highest followed by protein and lipid contents respectively. Glycogen content was relatively high which showed that carbohydrate was mainly present in the form of glycogen in these cestodes. The A + T and G + C contents were obtained as 49.82 and 50.18 % respectively. The phylogenetic tree showed that C. cuneata branched with its closest cluster comprising of Raillietina tunetensis, Raillietina australis, Fuhrmannetta malakartis and Raillietina sonini with 99 % bootstrap support.

  15. Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease

    Energy Technology Data Exchange (ETDEWEB)

    Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander (Debrecen); (NCI); (Florida); (Suan Sunandha)

    2012-10-23

    Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K{sub i}, depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.

  16. Cultivation and biochemical characterization of heterotrophic bacteria associated with phytoplankton bloom in the Amundsen sea polynya, Antarctica

    Science.gov (United States)

    Choi, Seon-Bin; Kim, Jong-Geol; Jung, Man-Young; Kim, So-Jeong; Min, Ui-Gi; Si, Ok-Ja; Park, Soo-Je; Yeon Hwang, Chung; Park, Jisoo; Lee, SangHoon; Rhee, Sung-Keun

    2016-01-01

    Polynyas are a key ecosystem for carbon cycling in the Antarctic Ocean due to the intensive primary production. Most of the knowledge regarding the bacterioplankton community in the Antarctic Ocean that is responsible for re-mineralization of fixed carbon comes from metagenomic analyses. Here, the extinction-dilution method was used to obtain representative heterotrophs from a polynya in the Amundsen Sea, Antarctica, and their biochemical potential for carbon re-mineralization were assessed. All 23 strains have close relatives belonging to type strains within the following genera (number of strains; % 16S rRNA gene sequence similarity): Bizionia (4; >97.8%), Leeuwenhoekiella (1; 96.2%), Pseudoalteromonas (14; >98.5%), Pseudomonas (1; 99.4%) and Sulfitobacter (3; 100%), which were also observed in 454 pyrosequencing-based analysis of 16S rRNA gene sequences of the polynya. Although sequence reads related to Polaribacter were the most common, Polaribacter strains could only be obtained from colonies cultured on agar plates. The strain of Leeuwenhoekiella showed a prominent ability in hydrolyzing diverse esters, amides, and glycosides while the strains of Pseudoalteromonas, Polaribacter, and Bizionia showed extracellular enzyme activities only on a narrow range of amides. The strains of Leeuwenhoekiella, Pseudoalteromonas, and Sulfitobacter utilized various labile carbon sources: carbohydrates, organic acids, amino acids, and peptides. The most frequent isolates, strains of Pseudoaltermonas, showed marked differences in terms of their potential to utilize different types of labile carbon sources, which may reflect high genomic diversity. The strains of Bizionia and Pseudomonas did not utilize carbohydrates. Unique biochemical properties associated with extracellular hydrolase activities and labile carbon utilization were revealed for dominant culturable heterotrophs which gives insights into their roles in active re-mineralization of fixed carbons in polynya.

  17. Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle.

    Science.gov (United States)

    Masujin, Kentaro; Shu, Yujing; Yamakawa, Yoshio; Hagiwara, Ken'ichi; Sata, Tetsutaro; Matsuura, Yuichi; Iwamaru, Yoshifumi; Imamura, Morikazu; Okada, Hiroyuki; Mohri, Shirou; Yokoyama, Takashi

    2008-01-01

    A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 +/- 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 +/- 13.5 days). Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion inoculated mice showed no clinical signs up to 649 days. Severe vacuolation and a widespread and uniform distribution of PrP(Sc) were pathologically observed in the brain of BSE/JP24 prion affected TgBoPrP mice. The molecular weight and glycoform ratio of PrP(Sc) in BSE/JP24 were different from those in C-BSE, and PrP(Sc) in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE. These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties reported for that of C-BSE. Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 +/- 3.1 days). This result implies that BSE/JP24 prion has newly emerged and showed the possibility that L-type BSE prion might be classified into multiple strains.

  18. Biochemical research elucidating metabolic pathways in Pneumocystis*

    Directory of Open Access Journals (Sweden)

    Kaneshiro E.S.

    2010-12-01

    Full Text Available Advances in sequencing the Pneumocystis carinii genome have helped identify potential metabolic pathways operative in the organism. Also, data from characterizing the biochemical and physiological nature of these organisms now allow elucidation of metabolic pathways as well as pose new challenges and questions that require additional experiments. These experiments are being performed despite the difficulty in doing experiments directly on this pathogen that has yet to be subcultured indefinitely and produce mass numbers of cells in vitro. This article reviews biochemical approaches that have provided insights into several Pneumocystis metabolic pathways. It focuses on 1 S-adenosyl-L-methionine (AdoMet; SAM, which is a ubiquitous participant in numerous cellular reactions; 2 sterols: focusing on oxidosqualene cyclase that forms lanosterol in P. carinii; SAM:sterol C-24 methyltransferase that adds methyl groups at the C-24 position of the sterol side chain; and sterol 14α-demethylase that removes a methyl group at the C-14 position of the sterol nucleus; and 3 synthesis of ubiquinone homologs, which play a pivotal role in mitochondrial inner membrane and other cellular membrane electron transport.

  19. Genetically Determined Insulin Resistance is Characterized by Down-Regulation of Mitochondrial Oxidative Metabolism in Human Skeletal Muscle

    DEFF Research Database (Denmark)

    Kristensen, Jonas M; Skov, Vibe; Wojtaszewski, Jørgen

    2010-01-01

    Transcriptional profiling of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated a co-ordinated down-regulation of oxidative phosphorylation (OxPhos) genes, suggesting a link between insulin resistance and mitochondrial dysfunction. However, whether...... mitochondrial dysfunction is a cause or consequence of insulin resistance remains to be clarified. In the present study, we tested the hypothesis that mitochondrial oxidative metabolism was down-regulated in skeletal muscle of patients with genetically determined insulin resistance. Skeletal muscle biopsies.......02), and complex V (ATP5B; p=0.005). Our data demonstrate that genetically determined insulin resistance is associated with a co-ordinated down-regulation of OxPhos components both at the transcriptional and translational level. These findings suggest that an impaired biological response to insulin in skeletal...

  20. DECREASED SYNTHESIS AND INEFFICIENT MITOCHONDRIAL IMPORT OF HSP60 IN A PATIENT WITH A MITOCHONDRIAL ENCEPHALOMYOPATHY

    NARCIS (Netherlands)

    HUCKRIEDE, A; AGSTERIBBE, E

    1994-01-01

    In a recent paper (Agsteribbe et al. (1993) Biochem. Biophys. Res. Commun. 193, 146-154) we suggested deficiency of heat shock protein 60 (hsp60) as the possible cause of a systemic mitochondrial encephalomyopathy with multiple deficiency of mitochondrial enzymes. In this paper we present new data w

  1. Molecular Cloning, Biochemical Characterization, and Antitumor Properties of a Novel L-Asparaginase from Synechococcus elongatus PCC6803.

    Science.gov (United States)

    Kebeish, R; El-Sayed, A; Fahmy, H; Abdel-Ghany, A

    2016-10-01

    L-asparaginase (EC 3.5.1.1), which catalyzes the deamidation of L-asparagine to L-aspartic acid and ammonia, has been widely used as a key therapeutic tool in the treatment of tumors. The current commercially available L-asparaginases, produced from bacteria, have signs of toxicity and hypersensitivity reactions during the course of tumor therapy. Therefore, searching for L-asparaginases with unique biochemical properties and fewer adverse effects was the objective of this work. In this study, cyanobacterial strain Synechococcus elongatus PCC6803 was found as a novel source of L-asparaginase. The L-asparaginase gene coding sequence (gi:939195038) was cloned and expressed in E. coli BL21(DE3), and the recombinant protein (Se.ASPII) was purified by affinity chromatography. The enzyme has high affinity towards L-asparagine and shows very weak affinity towards L-glutamine. The enzymatic properties of the recombinant enzyme were investigated, and the kinetic parameters (Km, Vmax) were measured. The pH and temperature dependence profiles of the novel enzyme were analyzed. The work was extended to measure the antitumor properties of the novel enzyme against different human tumor cell lines.

  2. Differential Expression of Laccase Genes in Pleurotus ostreatus and Biochemical Characterization of Laccase Isozymes Produced in Pichia pastoris.

    Science.gov (United States)

    Park, Minsa; Kim, Minseek; Kim, Sinil; Ha, Byeongsuk; Ro, Hyeon-Su

    2015-09-01

    In this study, transcriptome analysis of twelve laccase genes in Pleurotus ostreatus revealed that their expression was differentially regulated at different developmental stages. Lacc5 and Lacc12 were specifically expressed in fruiting bodies and primordia, respectively, whereas Lacc6 was expressed at all developmental stages. Lacc1 and Lacc3 were specific to the mycelial stage in solid medium. In order to investigate their biochemical characteristics, these laccases were heterologously expressed in Pichia pastoris using the pPICHOLI-2 expression vector. Expression of the laccases was facilitated by intermittent addition of methanol as an inducer and sole carbon source, in order to reduce the toxic effects associated with high methanol concentration. The highest expression was observed when the recombinant yeast cells were grown for 5 days at 15℃ with intermittent addition of 1% methanol at a 12-hr interval. Investigation of enzyme kinetics using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate revealed that the primordium-specific laccase Lacc12 was 5.4-fold less active than Lacc6 at low substrate concentration with respect to ABTS oxidation activity. The optimal pH and temperature of Lacc12 were 0.5 pH units and 5℃ higher than those of Lacc6. Lacc12 showed maximal activity at pH 3.5 and 50℃, which may reflect the physiological conditions at the primordiation stage.

  3. Construct design, biophysical, and biochemical characterization of the fusion core from mouse hepatitis virus (a coronavirus) spike protein.

    Science.gov (United States)

    Xu, Yanhui; Cole, David K; Lou, Zhiyong; Liu, Yiwei; Qin, Lan; Li, Xu; Bai, Zhihong; Yuan, Fang; Rao, Zihe; Gao, George F

    2004-11-01

    Membrane fusion between virus and host cells is the key step for enveloped virus entry and is mediated by the viral envelope fusion protein. In murine coronavirus, mouse hepatitis virus (MHV), the spike (S) protein mediates this process. Recently, the formation of anti-parallel 6-helix bundle of the MHV S protein heptad repeat (HR) regions (HR1 and HR2) has been confirmed, implying coronavirus has a class I fusion protein. This bundle is also called fusion core. To facilitate the solution of the crystal structure of this fusion core, we deployed an Escherichia coli in vitro expression system to express the HR1 and HR2 regions linked together by a flexible linker as a single chain (named 2-helix). This 2-helix polypeptide subsequently assembled into a typical 6-helix bundle. This bundle has been analyzed by a series of biophysical and biochemical techniques and confirmed that the design technique can be used for coronavirus as we successfully used for members of paramyxoviruses.

  4. Biochemical characterization of blood plasma of coronary artery disease patients by in vitro high-resolution proton NMR spectroscopy

    Indian Academy of Sciences (India)

    Anu Malik; Uma Sharma; R Lakshmy; Rajiv Narang; Naranamanglam R Jagannathan

    2015-03-01

    This study aimed to investigate the biochemical profile of blood plasma of patients with coronary artery disease (CAD) and angiographically normal subjects (controls) to determine biomarkers for their differentiation. In this double blind study, 5 mL venous blood was drawn before angiography from CAD patients (n=60) and controls (n=13) comprising angiography normal individuals. In vitro high-resolution NMR spectroscopy of these blood plasma samples was carried out at 400 MHz, and intensity data were analysed with partial least square discriminant analysis. Categorization of subjects as controls or CAD patients and the patients further as single vessel disease (SVD), double vessel disease (DVD) and triple vessel disease (TVD) was done at the end of the study based on their angiography reports. Raised levels of lipids, alanine (Ala) and isoleucine/leucine/valine (Ile/Leu/Val) were observed in CAD patients compared with controls. Partial least square discriminant analysis showed separation between controls vs CAD patients. TVD patients showed increased levels of Ile/Leu/Val and Ala compared with controls and SVD. Alanine, Ile/Leu/Val, and LDL/VLDL appear as possible biomarkers for distinguishing between controls and patients with SVD and TVD. A metabolic adaptation of myocardium may play a role in raising the Ala level.

  5. Biochemical Characterization and Molecular Modeling of Pancreatic Lipase from a Cartilaginous Fish, the Common Stingray (Dasyatis pastinaca).

    Science.gov (United States)

    Bouchaâla, Emna; BouAli, Madiha; Ben Ali, Yassine; Miled, Nabil; Gargouri, Youssef; Fendri, Ahmed

    2015-05-01

    In order to identify fish enzymes displaying novel biochemical properties, we have chosen the common stingray (Dasyatis pastinaca), one of the most primitive living jawed aquatic vertebrates as a starting biological material to purify a lipase. A stingray pancreatic lipase (SPL) was purified from delipidated pancreatic powder. The SPL molecular weight was around 55 kDa which is slightly higher than that of known classical pancreatic lipases (50 kDa). This increase in the molecular weight was due to glycosylation. Like classic pancreatic lipases, SPL was found to be much more active on short-chain triacylglycerols than on long-chain ones. Natural detergents act as inhibitors of the SPL activity. This inhibition can be reversed by the addition of stingray colipase. Starting from total pancreatic messenger RNAs (mRNAs), partial stingray pancreatic lipase complementary DNA (cDNA) was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) and cloned into the PGEM-T vector. Partial amino acid sequence of the SPL was homologous to that of Japanese eel, porcine, and human pancreatic lipases. A 3D structure model of the sequenced part of SPL was built using the 3D structure of porcine pancreatic lipase as template, since both lipases shared an amino acid sequence identity of 60%.

  6. Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina.

    Science.gov (United States)

    Hiseni, Aida; Arends, Isabel W C E; Otten, Linda G

    2011-08-01

    Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1'-(HO)(2)-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K (m) and V (max) values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h(-1) mg(-1) for RgCrtC and 9.5 μM and 0.15 nmol h(-1) mg(-1) for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene.

  7. Anaerobic digestion of grape pomace: Biochemical characterization of the fractions and methane production in batch and continuous digesters.

    Science.gov (United States)

    El Achkar, Jean H; Lendormi, Thomas; Hobaika, Zeina; Salameh, Dominique; Louka, Nicolas; Maroun, Richard G; Lanoisellé, Jean-Louis

    2016-04-01

    In this study, we have estimated the biogas and methane production from grape pomace (variety Cabernet Franc). The physical and chemical characteristics of the raw material were determined, and the structural polysaccharides were identified and analyzed by the Van Soest method. Batch anaerobic digestions were carried out to assess the methane production of the grape pomace, pulp and seeds. The obtained cumulative methane productions are 0.125, 0.165 and 0.052 Nm(3) kg COD(-1) for grape pomace, pulps and seeds, respectively. The effect of grinding on the methane potential of the substrates, as a mechanical pretreatment, was evaluated. We found that it increased the anaerobic biodegradability for grape pomace, pulp and seeds by 13.1%, 4.8% and 22.2%, respectively. On the other hand, the methane potential of the grape pomace was determined in a laboratory pilot plant (12L) continuously mixed with an organic loading rate of 2.5 kg COD m(3) d(-1) and a hydraulic retention time of 30 days. The corresponding biogas production was 6.43 × 10(-3) Nm(3) d(-1), with a methane content of 62.3%. Thus, the pilot plant's efficiency compared to that achieved in the batch process was 81.2%. Finally, a significant correlation was found between the biochemical content and methane production.

  8. Characterization of postmortem biochemical changes in rabbit plasma using ATR-FTIR combined with chemometrics: A preliminary study

    Science.gov (United States)

    Zhang, Ji; Li, Bing; Wang, Qi; Li, Chengzhi; Zhang, Yinming; Lin, Hancheng; Wang, Zhenyuan

    2017-02-01

    Postmortem interval (PMI) determination is one of the most challenging tasks in forensic medicine due to a lack of accurate and reliable methods. It is especially difficult for late PMI determination. Although many attempts with various types of body fluids based on chemical methods have been made to solve this problem, few investigations are focused on blood samples. In this study, we employed an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) technique coupled with principle component analysis (PCA) to monitor biochemical changes in rabbit plasma with increasing PMI. Partial least square (PLS) model was used based on the spectral data for PMI prediction in an independent sample set. Our results revealed that postmortem chemical changes in compositions of the plasma were time-dependent, and various components including proteins, lipids and nucleic acids contributed to the discrimination of the samples at different time points. A satisfactory prediction within 48 h postmortem was performed by the combined PLS model with a good fitting between actual and predicted PMI of 0.984 and with an error of ± 1.92 h. In consideration of the simplicity and portability of ATR-FTIR, our preliminary study provides an experimental and theoretical basis for application of this technique in forensic practice.

  9. Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

    Science.gov (United States)

    Galdino, Alexsandro Sobreira; Silva, Roberto Nascimento; Lottermann, Muriele Taborda; Álvares, Alice Cunha Morales; de Moraes, Lídia Maria Pepe; Torres, Fernando Araripe Gonçalves; de Freitas, Sonia Maria; Ulhoa, Cirano José

    2011-01-01

    An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH4+ and inhibited by Cu+2 and Hg+2. Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to Km values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1w, points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels. PMID:21490699

  10. Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Alexsandro Sobreira Galdino

    2011-01-01

    Full Text Available An extracellular alpha-amylase (Amy1 whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH4+ and inhibited by Cu+2 and Hg+2. Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to Km values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1w, points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.

  11. 柚皮苷改善束缚性应激引起的小鼠生化指标及行为变化的一氧化氮调节机制%Amelioration of immobilization stress-induced biochemical and behavioral alterations and mitochondrial dysfunction by naringin in mice: possible mechanism of nitric oxide modulation

    Institute of Scientific and Technical Information of China (English)

    Gollapalle L.Viswanatha; Hanumanthappa Shylaja; K.Sadashiva Sandeep Rao; Yathiraj Ashwini; V. Ramaiah Santhosh Kumar; C. Gangadharaiah Mohan; Venkate Gowda Sunil; M. Venkateshappa Sarvesh Kumar; Subbanna Rajesh

    2011-01-01

    Objective:The present study was undertaken to evaluate the effects of naringin on immobilization stress-induced biochemical-behavioral changes and mitochondrial dysfunction in mice.Methods:Mice were randomized and grouped based on body weights.Respective drug treatments were given for 14 d,and on the 15th day all the animals were subjected to a 6-hour immobilization stress; then all the animals were subjected to various behavioral paradigms and were sacrificed.Various biochemical parameters and mitochondrial functions were analyzed using brain homogenate.Results:The 6-hour acute immobilization stress significantly altered the behavioral (anxiety and memory) and biochemical parameters coupled with mitochondrial dysfunction in mice.Fourteen days pretreatment with naringin (50 and 100 mg/kg,per oral) significantly inhibited the behavioral and biochemical alterations and mitochondrial dysfunction caused by acute immobilization stress (P<0.05).Further,pretreatment with L-arginine (50 mg/kg,intraperitoneally),a nitric oxide precursor,reversed the protective effect of naringin (P<0.05).In addition,pretreatment with NG-nitro-L-arginine methyl ester (5 mg/kg,intraperitoneally) caused potentiation in the protective effect of naringin.Conclusion:These results suggest the possible involvement of nitrergic pathway in the protective effect of naringin against immobilization stress-induced behavioral,biochemical and mitochondrial dysfunctions in mice.%目的:研究柚皮苷对束缚性应激引起的小鼠生化指标改变、行为变化及线粒体功能紊乱的影响.方法:将小鼠按照体质量随机分组,分别给予不同药物治疗14 d.在第15天让所有小鼠接受束缚性应激刺激6h,然后在接受各种不同的行为测试后处死.取小鼠的大脑匀浆进行各种生化指标测量和线粒体功能分析.结果:6h的急性束缚性应激刺激能显著改变小鼠的行为(焦虑和记忆),引起生化指标变化和线粒体功

  12. [Molecular genetic studies of mitochondrial ornithine transporter deficiency (HHH syndrome)].

    Science.gov (United States)

    Tsujino, S; Miyamoto, T; Kanazawa, N

    2001-11-01

    Mitochondrial ornithine transporter deficiency has been called HHH syndrome, because this disorder is characterized by three biochemical abnormalities; hyperornithinemia, hyperammonemia, and homocitrullinuria, and presents with various neurological symptoms; mental retardation, spastic paraparesis with pyramidal signs, cerebellar ataxia and episodic disturbance of consciousness or coma due to hyperammonemia. We identified four mutations in the mitochondrial ornithine transporter gene (ORNT1) of Japanese patients with HHH syndrome. These include a nonsense mutation (R179X), associated with exon skipping, missense mutations (G27E, P126R), and an insertion of AAC between codons 228 and 229, leading to an insertion of amino acid Asn. Especially, R179X was detected 4 of 7 Japanese patients (8 of 14 alleles), implying that this is a common mutation in Japanese population.

  13. Mitochondrial vasculopathy

    Institute of Scientific and Technical Information of China (English)

    Josef Finsterer; Sinda Zarrouk-Mahjoub

    2016-01-01

    Mitochondrial disorders(MIDs)are usually multisystem disorders(mitochondrial multiorgan disorder syndrome)either on from onset or starting at a point during the disease course.Most frequently affected tissues are those with a high oxygen demand such as the central nervous system,the muscle,endocrine glands,or the myocardium.Recently,it has been shown that rarely alsothe arteries may be affected(mitochondrial arteriopathy).This review focuses on the type,diagnosis,and treat-ment of mitochondrial vasculopathy in MID patients.A literature search using appropriate search terms was carried out.Mitochondrial vasculopathy manifests as either microangiopathy or macroangiopathy.Clinical manifestations of mitochondrial microangiopathy include leukoencephalopathy,migraine-like headache,stroke-like episodes,or peripheral retinopathy.Mitochondrial macroangiopathy manifests as atherosclerosis,ectasia of arteries,aneurysm formation,dissection,or spontan-eous rupture of arteries.The diagnosis relies on the documentation and confirmation of the mitochondrial metabolic defect or the genetic cause after exclusion of non-MID causes.Treatment is not at variance compared to treatment of vasculopathy due to non-MID causes.Mitochondrial vasculopathy exists and manifests as micro-or macroangiopathy.Diagnosing mitochondrial vasculopathy is crucial since appropriate treatment may prevent from severe complications.

  14. Morphological, biochemical and molecular characterization of Herpetomonas samuelpessoai camargoi n. subsp., a trypanosomatid isolated from the flower of the squash Cucurbita moschata.

    Science.gov (United States)

    Fiorini, J E; Takata, C S; Teofilo, V M; Nascimento, L C; Faria-e-Silva, P M; Soares, M J; Teixeira, M M; De Souza, W

    2001-01-01

    We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas-specific molecular markers based on spliced-leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed.

  15. Biochemical characterization of human gluconokinase and the proposed metabolic impact of gluconic Acid as determined by constraint based metabolic network analysis

    DEFF Research Database (Denmark)

    Rohatgi, Neha; Nielsen, Tine Kragh; Bjørn, Sara Petersen

    2014-01-01

    and strict specificity towards gluconate out of 122 substrates tested. In order to evaluate the metabolic impact of gluconate in humans we modeled gluconate metabolism using steady state metabolic network analysis. The results indicate that significant metabolic flux changes in anabolic pathways linked......The metabolism of gluconate is well characterized in prokaryotes where it is known to be degraded following phosphorylation by gluconokinase. Less is known of gluconate metabolism in humans. Human gluconokinase activity was recently identified proposing questions about the metabolic role...... to the hexose monophosphate shunt (HMS) are induced through a small increase in gluconate concentration. We argue that the enzyme takes part in a context specific carbon flux route into the HMS that, in humans, remains incompletely explored. Apart from the biochemical description of human gluconokinase...

  16. Transcriptional and biochemical markers in transplanted Perca flavescens to characterize cadmium- and copper-induced oxidative stress in the field

    Energy Technology Data Exchange (ETDEWEB)

    Defo, Michel A. [Institut National De La Recherche Scientifique (INRS), Centre Eau Terre Environnement, 490 De La Couronne, Québec, QC G1K 9A9 (Canada); Bernatchez, Louis [Institut De Biologie Intégrative Et Des Systèmes (IBIS), Université Laval, Québec, QC G1V 0A6 (Canada); Campbell, Peter G.C.; Couture, Patrice [Institut National De La Recherche Scientifique (INRS), Centre Eau Terre Environnement, 490 De La Couronne, Québec, QC G1K 9A9 (Canada)

    2015-05-15

    Highlights: • Four-weeks exposure is sufficient to increase kidney metal levels in wild perch. • Cd and Cu affected indicators of retinoid metabolism and oxidative stress in fish. • Multi-level biological approaches are needed when assessing fish metal toxicology. • Changes at molecular level do not always mean changes at the functional level. • Wild juvenile perch may partly adjust to metal contamination by plastic responses. - Abstract: Despite recent progress achieved in elucidating the mechanisms underlying local adaptation to pollution, little is known about the evolutionary change that may be occurring at the molecular level. The goal of this study was to examine patterns of gene transcription and biochemical responses induced by metal accumulation in clean yellow perch (Perca flavescens) and metal depuration in contaminated fish in a mining and smelting region of Canada. Fish were collected from a reference lake (lake Opasatica) and a Cd, Cu and Zn contaminated lake (lake Dufault) located in the Rouyn-Noranda region (Qc, Canada) and caged for one or four weeks in their own lake or transplanted in the other lake. Free-ranging fish from the same lakes were also collected. Kidney Cd and Cu concentrations in clean fish caged in the contaminated lake increased with the time of exposure, but metal depuration did not occur in contaminated fish caged in the clean lake. After 4 weeks, the major retinoid metabolites analysed, the percentage of free dehydroretinol (dROH) and the retinol dehydrogenase-2 (rdh-2) transcription level in liver decreased in clean fish transplanted into the metal-contaminated lake, suggesting that metal exposure negatively impacted retinoid metabolism. However, we observed an increase in almost all of the retinoid parameters analysed in fish from the metal-impacted lake caged in the same lake, which we interpret as an adaptation response to higher ambient metal concentration. In support of this hypothesis, liver transcription levels

  17. Structural and Biochemical Characterization of the Francisella tularensis Pathogenicity Regulator, Macrophage Locus Protein A (MglA.

    Directory of Open Access Journals (Sweden)

    Bonnie J Cuthbert

    Full Text Available Francisella tularensis is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase (Kb pathogenicity island (FPI that is regulated by the macrophage locus protein A (MglA and the stringent starvation protein A (SspA. These proteins interact with both RNA polymerase (RNAP and the pathogenicity island gene regulator (PigR to activate FPI transcription. However, the molecular mechanisms involved are not well understood. Indeed, while most bacterial SspA proteins function as homodimers to activate transcription, F. tularensis SspA forms a heterodimer with the MglA protein, which is unique to F. tularensis. To gain insight into MglA function, we performed structural and biochemical studies. The MglA structure revealed that it contains a fold similar to the SspA protein family. Unexpectedly, MglA also formed a homodimer in the crystal. Chemical crosslinking and size exclusion chromatography (SEC studies showed that MglA is able to self-associate in solution to form a dimer but that it preferentially heterodimerizes with SspA. Finally, the MglA structure revealed malate, which was used in crystallization, bound in an open pocket formed by the dimer, suggesting the possibility that this cleft could function in small molecule ligand binding. The location of this binding region relative to recently mapped PigR and RNAP interacting sites suggest possible roles for small molecule binding in MglA and SspA•MglA function.

  18. Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae.

    Science.gov (United States)

    Narayanan, Shalini; Beckham, Simone A; Davies, John K; Roujeinikova, Anna

    2014-12-01

    DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ(32))-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ(32), RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a V max of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min(-1) under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ(32) and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ(32) is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli.

  19. Genetic and biochemical characterization of the chromosomal class A beta-lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica.

    Science.gov (United States)

    Walckenaer, Estelle; Poirel, Laurent; Leflon-Guibout, Véronique; Nordmann, Patrice; Nicolas-Chanoine, Marie-Hélène

    2004-01-01

    Enterobacterial strains of Raoultella spp. display a penicillinase-related beta-lactam resistance pattern suggesting the presence of a chromosomal bla gene. From whole-cell DNA of Raoultella planticola strain ATCC 33531(T) and Raoultella ornithinolytica strain ATCC 31898(T), bla genes were cloned and expressed into Escherichia coli. Each gene encoded an Ambler class A beta-lactamase, named PLA-1 and ORN-1 for R. planticola and R. ornithinolytica, respectively. These beta-lactamases (291 amino acids), with the same pI value of 7.8, had a shared amino acid identity of 94%, 37 to 47% identity with the majority of the chromosome-encoded class A beta-lactamases previously described for Enterobacteriaceae, and 66 to 69% identity with the two beta-lactamases LEN-1 and SHV-1 from Klebsiella pneumoniae. However, the highest identity percentage (69 to 71%) was found with the plasmid-mediated beta-lactamase TEM-1. PLA-1, which displayed very strong hydrolytic activity against penicillins, also displayed significant hydrolytic activity against cefepime and, to a lesser extent, against cefotaxime and aztreonam, but there was no hydrolytic activity against ceftazidime. Such a substrate profile suggests that the Raoultella beta-lactamases PLA-1 and ORN-1 should be classified into the group 2be of the beta-lactamase classification of K. Bush, G. A. Jacoby, and A. A. Medeiros (Antimicrob. Agents Chemother. 39:1211-1233, 1995). The highly homologous regions upstream of the bla(PLA-1A) and bla(ORN-1A) genes comprised a nucleotide sequence identical to the -35 region and another one very close to the -10 region of the bla(LEN-1) gene. From now on, as the bla gene sequences of the most frequent Raoultella and Klebsiella species are available, the bla gene amplification method can be used to differentiate these species from each other, which the biochemical tests currently carried out in the clinical laboratory are unable to do.

  20. Metagenomic and biochemical characterizations of sulfur oxidation metabolism in uncultured large sausage-shaped bacterium in hot spring microbial mats.

    Directory of Open Access Journals (Sweden)

    Satoshi Tamazawa

    Full Text Available So-called "sulfur-turf" microbial mats in sulfide containing hot springs (55-70°C, pH 7.3-8.3 in Japan were dominated by a large sausage-shaped bacterium (LSSB that is closely related to the genus Sulfurihydrogenibium. Several previous reports proposed that the LSSB would be involved in sulfide oxidation in hot spring. However, the LSSB has not been isolated yet, thus there has been no clear evidence showing whether it possesses any genes and enzymes responsible for sulfide oxidation. To verify this, we investigated sulfide oxidation potential in the LSSB using a metagenomic approach and subsequent biochemical analysis. Genome fragments of the LSSB (a total of 3.7 Mb sequence including overlapping fragments were obtained from the metagenomic fosmid library constructed from genomic DNA of the sulfur-turf mats. The sequence annotation clearly revealed that the LSSB possesses sulfur oxidation-related genes coding sulfide dehydrogenase (SD, sulfide-quinone reductase and sulfite dehydrogenase. The gene encoding SD, the key enzyme for sulfide oxidation, was successfully cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme clearly showed SD activity with optimum temperature and pH of 60°C and 8.0, respectively, which were consistent with the environmental conditions in the hot spring where the sulfur-turf thrives. Furthermore, the affinity of SD to sulfide was relatively high, which also reflected the environment where the sulfide could be continuously supplied. This is the first report showing that the LSSB harbors sulfide oxidizing metabolism adapted to the hot spring environment and can be involved in sulfide oxidation in the sulfur-turf microbial mats.

  1. Biochemical and molecular characterization of a flavonoid 3-O-glycosyltransferase responsible for anthocyanins and flavonols biosynthesis in Freesia hybrida

    Directory of Open Access Journals (Sweden)

    Wei eSun

    2016-03-01

    Full Text Available The glycosylation of flavonoids increases their solubility and stability in plants. Flowers accumulate anthocyanidin and flavonol glycosides which are synthesized by UDP-sugar flavonoid glycosyltransferases (UFGTs. In our previous study, a cDNA clone (Fh3GT1 encoding UFGT was isolated from Freesia hybrida, which was preliminarily proved to be invovled in cyanidin 3-O-glucoside biosynthesis. Here, a variety of anthocyanin and flavonol glycosides were detected in flowers and other tissues of F. hybrida, implying the versatile roles of Fh3GT1 in flavonoids biosynthesis. To further unravel its multi-functional roles, integrative analysis between gene expression and metabolites was investigated. The results showed expression of Fh3GT1 was positively related to the accumulation of anthocyanins and flavonol glycosides, suggesting its potential roles in the biosynthesis of both flavonoid glycosides. Subsequently, biochemical analysis results revealed that a broad range of flavonoid substrates including flavonoid not naturally occurred in F. hybrida could be recognized by the recombinant Fh3GT1. Both UDP-glucose and UDP-galactose could be used as sugar donors by recombinant Fh3GT1, although UDP-galactose was transferred with relatively low activity. Furthermore, regiospecificity analysis demonstrated that Fh3GT1 was able to glycosylate delphinidin at the 3-, 4'- and 7- positions in a sugar-dependent manner. And the introduction of Fh3GT1 into Arabidopsis UGT78D2 mutant successfully restored the anthocyanins and flavonols phenotypes caused by lost-of-function of the 3GT, indicating that Fh3GT1 functions as a flavonoid 3-O-glucosyltransferase in vivo. In summary, these results demonstrate that Fh3GT1 is a flavonoid 3-O-glycosyltransferase using UDP-glucose as the preferred sugar donor and may involve in flavonoid glycosylation in F. hybrida.

  2. Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme.

    Science.gov (United States)

    Chang, Shu-Wei; Lee, Guan-Chiun; Shaw, Jei-Fu

    2006-08-09

    Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZalphaC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.

  3. Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion

    Science.gov (United States)

    Bovine papillomatous digital dermatitis (DD) is the leading cause of lameness in dairy cattle and represents a serious welfare and economic burden. Found primarily in high production dairy cattle worldwide, DD is characterized by the development of an often painful red, raw ulcerative or papillomato...

  4. Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

    Science.gov (United States)

    Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

    2011-01-01

    Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

  5. Bedside diagnosis of mitochondrial dysfunction in aneurysmal subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Jacobsen, A.; Nielsen, T. H.; Nilsson, O.;

    2014-01-01

    to diagnose and separate cerebral ischemia and mitochondrial dysfunction bedside. The study explores whether cerebral biochemical variables in SAH patients most frequently exhibit a pattern indicating ischemia or mitochondrial dysfunction. Methods - In 55 patients with severe SAH, intracerebral microdialysis...... was performed during neurocritical care with bedside analysis and display of glucose, pyruvate, lactate, glutamate, and glycerol. The biochemical patterns observed were compared to those previously described in animal studies of induced mitochondrial dysfunction as well as the pattern obtained in patients...... with recirculated cerebral infarcts. Results - In 29 patients, the biochemical pattern indicated mitochondrial dysfunction while 10 patients showed a pattern of cerebral ischemia, six of which also exhibited periods of mitochondrial dysfunction. Mitochondrial dysfunction was observed during 5162 h. An ischemic...

  6. Synthesis, stereochemical determination and biochemical characterization of the enantiomeric phosphate esters of the novel immunosuppressive agent FTY720.

    Science.gov (United States)

    Hale, Jeffrey J; Yan, Lin; Neway, William E; Hajdu, Richard; Bergstrom, James D; Milligan, James A; Shei, Gan-Ju; Chrebet, Gary L; Thornton, Rosemary A; Card, Deborah; Rosenbach, Mark; HughRosen; Mandala, Suzanne

    2004-09-15

    The novel immunosuppressive agent FTY720 (1) is phosphorylated in vivo in a variety of species yielding an active metabolite that is an agonist of four of the five known G-protein-coupled sphingosine-1-phosphate (S1P) receptors. A synthesis amenable to producing gram quantities of the stereoisomeric phosphate esters, a determination of their absolute stereochemistry via an enantioselective synthesis and their characterization as S1P receptor agonists and antagonists is reported.

  7. Biochemical, genetic, and epidemiologic characterization of Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) strains associated with Brazilian purpuric fever.

    OpenAIRE

    Brenner, D J; Mayer, L W; Carlone, G M; Harrison, L. H.; Bibb, W F; Brandileone, M. C.; Sottnek, F O; K. Irino; Reeves, M W; Swenson, J M

    1988-01-01

    Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and BPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates f...

  8. Physicochemical, biochemical and sensory properties for the characterization of Petrovská klobása (traditional fermented sausage

    Directory of Open Access Journals (Sweden)

    Ikonić Predrag M.

    2010-01-01

    Full Text Available A study was carried out on a typical homemade Petrovská klobasá in order to characterize this traditional dry-fermented sausage, to provide a basis for establishing the quality standard and protecting designation of origin. This paper reviews the chemical composition, some physicochemical, proteolytic and sensory parameters of Petrovská klobasá made by five manufacturers chosen as representatives. Beside the differences between sausages made by different manufacturers the main properties of this traditional product were though recognized. Compared to other dry-fermented sausages Petrovská klobasá is characterized by a high content of protein (23.36-30.45% and low contents of NaCl (2.99-3.28%. With some minor exceptions, the values of other chemical parameters are within the range of those observed for various dry-cured sausages. Weight loss during the processing is high (up to 45.71% and pH value (~ 5.4 corresponds to the values for this parameter in other European traditional fermented sausages. Contents of different nitrogen fractions show that Petrovská klobasá undergoes significant proteolytic changes. At the end of ripening, Petrovská klobasá is characterized by aromatic and spicy-hot flavor, dark-red color and hard consistency.

  9. Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus

    Directory of Open Access Journals (Sweden)

    Spielman Andrew I

    2004-07-01

    Da which may be interpreted as a component of a multimeric receptor/channel complex. Conclusions The data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata.

  10. Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.

    Science.gov (United States)

    Wang, Maoliang; Basu, Amartya; Palm, Thomas; Hua, Jack; Youngster, Stephen; Hwang, Lisa; Liu, Hsien-Ching; Li, Xiguang; Peng, Ping; Zhang, Yue; Zhao, Hong; Zhang, Zhihua; Longley, Clifford; Mehlig, Mary; Borowski, Virna; Sai, Prakash; Viswanathan, Manickam; Jang, Eun; Petti, Gerald; Liu, Sam; Yang, Karen; Filpula, David

    2006-01-01

    Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics

  11. Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome.

    Directory of Open Access Journals (Sweden)

    Julianne H Grose

    Full Text Available Small Heat Shock Proteins (sHSPs are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2, which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait and a human cardiac library (prey coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 "cardiac interactome" to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID. A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is

  12. Mitochondrial optic neuropathy: In vivo model of neurodegeneration and neuroprotective strategies

    Directory of Open Access Journals (Sweden)

    Julio C Rojas

    2010-03-01

    Full Text Available Julio C Rojas, Francisco Gonzalez-LimaDepartments of Psychology, Pharmacology and Toxicology, University of Texas at Austin, Austin, TX, USAAbstract: This review summarizes the characteristics of a rodent toxicologic model of optic neuropathy induced by the mitochondrial complex I inhibitor rotenone. This model has been developed to fulfill the demand for a drug-screening tool providing a sound mechanistic context to address the role of mitochondrial dysfunction in the pathogenesis of neurodegenerative disorders. It features biochemical, structural, and functional retinal deficits that resemble those of patients with Leber’s hereditary optic neuropathy, a mitochondrial disease characterized by selective degeneration of retinal ganglion cells, and for which an environmental component is believed to play a major triggering role. The available data support the efficiency, sensitivity, and versatility of the model for providing insights into the mechanisms of neurodegeneration, including mitochondrial dysfunction, oxidative stress and excitotoxicity. Screening work with this model has provided proof-of-principle that interventions targeting the electron transport chain, such as USP methylene blue and near-infrared light therapy, are effective at preventing neurodegeneration induced by mitochondrial dysfunction in vivo. Prospective developments of this model include the use of neuronal reporter genes for in vivo non-invasive assessment of retinal degeneration at different time points, and its combination with genetic approaches to elucidate the synergism of environmental and genetic factors in neurodegeneration.Keywords: animal model, neuroprotection, mitochondrial dysfunction, visual function, oxidative stress, cytochrome oxidase

  13. Characterization of PHB1 and its role in mitochondrial maturation and yolk platelet degradation during development of Artemia embryos.

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    Xiang Ye

    Full Text Available BACKGROUND: To cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown. PRINCIPAL FINDING: Our study investigated the role of prohibitin 1 (PHB1 in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein. CONCLUSIONS/SIGNIFICANCE: PHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.

  14. Fluence- and time-dependant lysosomal and mitochondrial damage induced by LS11 PDT characterized with light scattering

    Science.gov (United States)

    Wilson, Jeremy D.; Foster, Thomas H.

    2007-02-01

    Light scattering from cells originates from sub-cellular organelles. Our measurements of angularly resolved light scattering have demonstrated that at 633 nm, the dominant scattering centers within EMT6 cells are mitochondria and lysosomes. To assess their specific contributions, we have used photodynamic therapy (PDT) to induce organelle-specific perturbations within intact cells. We have developed a coated sphere scattering model for mitochondrial swelling in response to ALA- and Pc 4-PDT, and in the case of Pc 4-PDT we have used this model to map the scattering responses into clonogenic cell survival. More recently, we demonstrated the ability to measure the size, scattering contribution, and refractive index of lysosomes within cells by exploiting the localization and high extinction of the photosensitizer LS11 and an absorbing sphere scattering model. Here we report on time- and fluence-dependant scattering measurements from cells treated with LS11-PDT. LS11-PDT causes rapid lysosomal disruption, as quantified by uptake of acridine orange, and can induce downstream effects including release of mitochondrial cytochrome c preceding the loss of mitochondrial membrane potential (Reiners et al., Cell Death Differ. 9:934, 2002). Using scattering and these various methods of analysis, we observed that the induction of lysosomal morphology changes requires a fluence significantly higher than that reported for cell killing. At lower fluences, we observe that at 1 h after irradiation there is significant mitochondrial swelling, consistent with the onset of cytochrome c-induced cell death, while the morphology of lysosomes remains unchanged. We also expand on the ideas of lysosomal staining to demonstrate the sensitivity of scattering measurements at different wavelengths to different organelle populations.

  15. Gestational diabetes is characterized by reduced mitochondrial protein expression and altered calcium signaling proteins in skeletal muscle.

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    Kristen E Boyle

    Full Text Available The rising prevalence of gestational diabetes mellitus (GDM affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM and obese pregnant women with normal glucose tolerance (ONGT. Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I subunits (NDUFS3, NDUFV2 and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4 in OGDM (n = 6 vs. ONGT (n = 6. Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (-60-75% in the OGDM (n = 8 compared with ONGT (n = 10 subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.

  16. Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the kappa-opioid receptor.

    Science.gov (United States)

    Yan, Feng; Bikbulatov, Ruslan V; Mocanu, Viorel; Dicheva, Nedyalka; Parker, Carol E; Wetsel, William C; Mosier, Philip D; Westkaemper, Richard B; Allen, John A; Zjawiony, Jordan K; Roth, Bryan L

    2009-07-28

    Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934-11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315(7.38) was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315(7.38). 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315(7.38) and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.

  17. Biochemical characterization of a novel L-asparaginase from Paenibacillus barengoltzii being suitable for acrylamide reduction in potato chips and mooncakes.

    Science.gov (United States)

    Shi, Ran; Liu, Yu; Mu, Qing; Jiang, Zhengqiang; Yang, Shaoqing

    2017-03-01

    A novel L-asparaginase gene (PbAsnase) from Paenibaeillus barengoltzii CAU904 was cloned and expressed in Escherichia coli. The L-asparaginase gene was 1011bp encoding 336 amino acids. Multiple sequence alignment of PbAsnase with other known L-asparaginases revealed that the enzyme showed high similarities with some Rhizobial-type L-asparaginases, sharing the highest identity of 32% with a characterized L-asparaginase from Rhizobium etli CFN 42, suggesting that it should be a novel L-asparaginase. The recombinant L-asparaginase (PbAsnase) was purified to homogeneity and biochemically characterized. The purified enzyme was optimally active at pH 8.5 and 45°C, respectively. It was stable within pH 5.5-10.0 and at temperatures below 55°C. PbAsnase exhibited strict substrate specificity towards L-asparagine (35.2U/mg), with Km and Vmax values of 3.6mM and 162.2μmol/min/mg, respectively, but displayed trace activity towards L-glutamine. Moreover, the application potential of PbAsnase on acrylamide migration in potato chips and mooncakes was evaluated. The pretreatment by PbAsnase significantly decreased the acrylamide contents in potato chips and mooncakes by 86% and 52%, respectively. The unique properties of PbAsnase may make it a good candidate in industries, especially in food safety.

  18. Isolation of Sporothrix schenckii MNT1 and the biochemical and functional characterization of the encoded α1,2-mannosyltransferase activity.

    Science.gov (United States)

    Hernández-Cervantes, Arturo; Mora-Montes, Héctor M; Álvarez-Vargas, Aurelio; Jiménez, Diana F Díaz; Robledo-Ortiz, Claudia I; Flores-Carreón, Arturo

    2012-09-01

    Sporothrix (Sp.) schenckii is a pathogenic fungus that infects humans and animals, and is responsible for the disease named sporotrichosis. The cell wall of this fungus has glycoproteins with a high content of mannose and rhamnose units, which are synthesized by endoplasmic reticulum- and Golgi-localized glycosyltransferases. Little is known about the enzymic machinery involved in the synthesis of these oligosaccharides in Sp. schenckii, or the genes encoding these activities. This is in part because of the lack of an available genome sequence for this organism. Using a partial genomic DNA library we identified SsMNT1, whose predicted product has significant similarity to proteins encoded by members of the Saccharomyces (Sa.) cerevisiae KRE2/MNT1 gene family. In order to biochemically characterize the putative enzyme, SsMNT1 was heterologously expressed in the methylotrophic yeast Pichia pastoris. Recombinant SsMnt1 showed Mn(2+)-dependent mannosyltransferase activity and the ability to recognize as acceptors α-methyl mannoside, mannose, Man(5)GlcNAc(2) oligosaccharide and a variety of mannobiosides. The characterization of the enzymic products generated by SsMnt1 revealed that the enzyme is an α1,2-mannosyltransferase that adds up to two mannose residues to the acceptor molecule. Functional complementation studies were performed in Sa. cerevisiae and Candida albicans mutants lacking members of the KRE2/MNT1 gene family, demonstrating that SsMnt1 is involved in both the N- and O-linked glycosylation pathways, but not in phosphomannan elaboration.

  19. Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

    Science.gov (United States)

    Eram, Mohammad S; Wong, Alton; Oduaran, Erica; Ma, Kesen

    2015-12-01

    Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

  20. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

    Science.gov (United States)

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site. PMID:27213370

  1. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

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    Saúl Gómez-Manzo

    2016-05-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I, Vanua-Lava (Class II and Viangchan (Class II. For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT. Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.

  2. Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Banaś, Antoni

    2016-08-01

    Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols.

  3. Mitochondrial myopathy and myoclonic epilepsy

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    Walter O. Arruda

    1990-03-01

    Full Text Available The authors describe a family (mother, son and two daughters with mitochondrial myopathy. The mother was asymptomatic. Two daughters had lactic acidosis and myoclonic epilepsy, mild dementia, ataxia, weakness and sensory neuropathy. The son suffered one acute hemiplegic episode due to an ischemic infarct in the right temporal region. All the patients studied had hypertension. EEG disclosed photomyoclonic response in the proband patient. Muscle biopsy disclosed ragged-red fibers and abnormal mitochondria by electron microscopy. Biochemical analysis showed a defect of cytochrome C oxidase in mitochondria isolated from skeletal muscle. Several clinical and genetic aspects of the mitochondrial encephalomyopathies are discussed.

  4. Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307

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    Azmi Kifaya

    2012-06-01

    Full Text Available Abstract Background Many cases of cutaneous leishmaniasis (CL have been recorded in the Jenin District based on their clinical appearance. Here, their parasites have been characterized in depth. Methods Leishmanial parasites isolated from 12 human cases of CL from the Jenin District were cultured as promastigotes, whose DNA was extracted. The ITS1 sequence and the 7SL RNA gene were analysed as was the kinetoplast minicircle DNA (kDNA sequence. Excreted factor (EF serotyping and multilocus enzyme electrophoresis (MLEE were also applied. Results This extensive characterization identified the strains as Leishmania tropica of two very distinct sub-types that parallel the two sub-groups discerned by multilocus microsatellite typing (MLMT done previously. A high degree of congruity was displayed among the results generated by the different analytical methods that had examined various cellular components and exposed intra-specific heterogeneity among the 12 strains. Three of the ten strains subjected to MLEE constituted a new zymodeme, zymodeme MON-307, and seven belonged to the known zymodeme MON-137. Ten of the 15 enzymes in the profile of zymodeme MON-307 displayed different electrophoretic mobilities compared with the enzyme profile of the zymodeme MON-137. The closest profile to that of zymodeme MON-307 was that of the zymodeme MON-76 known from Syria. Strains of the zymodeme MON-307 were EF sub-serotype A2 and those of the zymodeme MON-137 were either A9 or A9B4. The sub-serotype B4 component appears, so far, to be unique to some strains of L. tropica of zymodeme MON-137. Strains of the zymodeme MON-137 displayed a distinctive fragment of 417 bp that was absent in those of zymodeme MON-307 when their kDNA was digested with the endonuclease RsaI. kDNA-RFLP after digestion with the endonuclease MboI facilitated a further level of differentiation that partially coincided with the geographical distribution of the human cases from which the strains

  5. Cloning, expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13.

    Science.gov (United States)

    Eminoğlu, Ayşenur; Vullo, Daniela; Aşık, Aycan; Çolak, Dilşat Nigar; Supuran, Claudiu T; Çanakçı, Sabriye; Osman Beldüz, Ali

    2016-12-01

    A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co(2+) affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 °C and pH of 8.3: kcat of 4.8 × 10(5) s(-1) and kcat/Km of 5.6 × 10(7) M(-1) × s(-1). This activity was potently inhibited by acetazolamide which showed a KI of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO3 biomineralization processes.

  6. Cloning, expression and biochemical characterization of recombinant superoxide dismutase from Antarctic psychrophilic bacterium Pseudoalteromonas sp. ANT506.

    Science.gov (United States)

    Wang, Quan-Fu; Wang, Yi-Fan; Hou, Yan-Hua; Shi, Yong-Lei; Han, Han; Miao, Miao; Wu, Ying-Ying; Liu, Yuan-Ping; Yue, Xiao-Na; Li, Yu-Jin

    2016-07-01

    In this study, a superoxide dismutase gene (PsSOD) from Pseudoalteromonas sp. ANT506 was cloned and over expressed in Escherichia coli. The PsSOD has an open reading frame of 582 bp with a putative product of 193 amino acid residue and an estimated molecular size of 21.4 kDa. His-tagged PsSOD was subsequently purified 12.6-fold by Ni-affinity chromatography and the yield of 22.9%. The characterization of the purified rPsSOD exhibited maximum activity at 30 °C and pH 8.0. The enzyme exhibited 13.9% activity at 0 °C and had high-thermo lability at higher than 50 °C. rPsSOD exhibited well capability to 2.5 M NaCl (62.4%). These results indicated that rPsSOD exhibited special catalytic properties.

  7. Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

    2010-03-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

  8. Identification and biochemical characterization of a thermostable malate dehydrogenase from the mesophile Streptomyces coelicolor A3(2).

    Science.gov (United States)

    Ge, Ya-Dong; Cao, Zheng-Yu; Wang, Zong-Da; Chen, Lu-Lu; Zhu, You-Ming; Zhu, Guo-Ping

    2010-01-01

    We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD(+)-specific and displayed about 400-fold (k(cat)) and 1,050-fold (k(cat)/K(m)) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (K(i)=5.8 mM) and excess L-malate (K(i)=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe(2+) and Zn(2+). Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.

  9. Biochemical characterization of the Caenorhabditis elegans FBF.CPB-1 translational regulation complex identifies conserved protein interaction hotspots.

    Science.gov (United States)

    Menichelli, Elena; Wu, Joann; Campbell, Zachary T; Wickens, Marvin; Williamson, James R

    2013-02-22

    Caenorhabditis elegans CPB-1 (cytoplasmic polyadenylation element binding protein homolog-1) and FBF (fem-3 mRNA binding factor) are evolutionary conserved regulators of mRNA translation that belong to the CPEB (cytoplasmic polyadenylation element binding) and PUF (Pumilio and FBF) protein families, respectively. In hermaphrodite worms, CPB-1 and FBF control key steps during germline development, including stem cell maintenance and sex determination. While CPB-1 and FBF are known to interact, the molecular basis and function of the CPB-1⋅FBF complex are not known. The surface of CPB-1 that interacts with FBF was localized using in vivo and in vitro methods to a 10-residue region at the N-terminus of the protein and these residues are present in the FBF-binding protein GLD-3 (germline development defective-3). PUF proteins are characterized by the presence of eight α-helical repeats (PUF repeats) arranged side by side in an elongated structure. Critical residues for CPB-1 binding are found in the extended loop that connects PUF repeats 7 and 8. The same FBF residues also mediate binding to GLD-3, indicating a conserved binding mode between different protein partners. CPB-1 binding was competitive with GLD-3, suggestive of mutual exclusivity in vivo. RNA binding measurements demonstrated that CPB-1 alters the affinity of FBF for specific RNA sequences, implying a functional model where the coregulatory protein CPB-1 modulates FBF target selection.

  10. Purification and Biochemical Characterization of D-hydantoinase and D-N-carbamoylase from Burkholderia cepecia, njut01

    Institute of Scientific and Technical Information of China (English)

    李家璜; 李苏平; 严明; 姚忠; 欧阳平凯

    2005-01-01

    Hydantoinase and N-carbamoylase play important roles in the production of optically pure amino acids from racemic 5-monosubstituted hydantoins. In this report, hydantoinase and the N-carbamoylase from Burkholderia cepecia, njut01 were purified to homogeneity by chromatography (Pharmacia Explorer 100 system). The substrate specificity, enantioselectivity, pH dependence of activity and temperature stability of the activity were characterized. The results show that the hydantoinase and N-carbamoylase induced from Burkholderia cepecia, njut01 are both strict D-stereo selective enzymes. They both hydrolyze substrates with side chains containing aliphatic and aromatic residues with higher activity and affinity toward aromatic than aliphatic substituted substrates. The hydantoinase is a homotetramer with subtmit molecular weight near 52,000 and is active between pH 6.5 and 10 with an optimum near pH 9.0. The enzyme is active at temperatures up to 60~C, however, it appears instable at higher temperatures. The subunit molecular weight of N-carbamoylase is about 35KD. The N-carbamoylase is active in the pH range from 6.0 to 9.5. The optira-pH is 7.2 and the optinfizing bioconversion temperature of the N-carbamyolase is 52℃.

  11. Inhibition and Biochemical Characterization of Methicillin-Resistant Staphylococcus aureus Shikimate Dehydrogenase: An in Silico and Kinetic Study

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    Claudia Avitia-Domínguez

    2014-04-01

    Full Text Available Methicillin-resistant Staphylococcus auerus (MRSA strains are having a major impact worldwide, and due to their resistance to all β-lactams, an urgent need for new drugs is emerging. In this regard, the shikimate pathway is considered to be one of the metabolic features of bacteria and is absent in humans. Therefore enzymes involved in this route, such as shikimate dehydrogenase (SDH, are considered excellent targets for discovery of novel antibacterial drugs. In this study, the SDH from MRSA (SaSDH was characterized. The results showed that the enzyme is a monomer with a molecular weight of 29 kDa, an optimum temperature of 65 °C, and a maximal pH range of 9–11 for its activity. Kinetic studies revealed that SDH showed Michaelis-Menten kinetics toward both substrates (shikimate and NADP+. Initial velocity analysis suggested that SaSDH catalysis followed a sequential random mechanism. Additionally, a tridimensional model of SaSDH was obtained by homology modeling and validated. Through virtual screening three inhibitors of SaSDH were found (compounds 238, 766 and 894 and their inhibition constants and mechanism were obtained. Flexible docking studies revealed that these molecules make interactions with catalytic residues. The data of this study could serve as starting point in the search of new chemotherapeutic agents against MRSA.

  12. Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

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    Xi'en Chen

    2013-12-01

    Full Text Available Protein phosphatase 5 (PP5 is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5 was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5 was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.

  13. Heterologous expression and biochemical characterization of an endo-β-1,4-glucanase from Thermobifida fusca.

    Science.gov (United States)

    Yan, Peng'an; Su, Lingqia; Chen, Jian; Wu, Jing

    2013-01-01

    The endoglucanase Cel5A from Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The carboxymethyl cellulase (CMCase) activity in shake flasks and 3-L fermentation scale reached 46.8 and 656.6 IU/mL, respectively. The CMCase activity in 3-L fermentation scale represented the highest yield of T. fusca Cel5A reported so far. Recombinant Cel5A was purified and characterized in detail. The optimum temperature of recombinant enzyme was 80 °C, and the half-life of the enzyme was 132 H at 50 °C and 65 H at 60 °C. The activity of recombinant Cel5A was retained more than 90% over the range of pH 5.0-10.0 with maximal activity at pH 5.5. Using carboxymethyl cellulose as the substrate, the Km and Vmax values were 5.1 mg/mL and 48.7 IU/mg, respectively. The enzyme showed superstability in surfactants and was retained above 90% activity after treatment with sodium dodecyl sulfate, linear alkyl benzene sulfonate, fatty alcohol polyoxyethylene (9) ether, and polyoxyethylene (10) nonyl phenyl ether at 25 °C for 1 H, indicating that the enzyme could be a valuable component in detergents. The potential mechanism of this stability was investigated by analysis of the electrostatic potential of the surface of the enzyme.

  14. Mitochondrial biogenesis: pharmacological approaches.

    Science.gov (United States)

    Valero, Teresa

    2014-01-01

    diseases do not have exclusively a mitochondrial origin but they might have an important mitochondrial component both on their onset and on their development. This is the case of type 2 diabetes or neurodegenerative diseases. Type 2 diabetes is characterized by a peripheral insulin resistance accompanied by an increased secretion of insulin as a compensatory system. Among the explanations about the origin of insulin resistance Mónica Zamora and Josep A. Villena (Department of Experimental and Health Sciences, Universitat Pompeu Fabra / Laboratory of Metabolism and Obesity, Universitat Autònoma de Barcelona, Spain) [5] consider the hypothesis that mitochondrial dysfunction, e.g. impaired (mitochondrial) oxidative capacity of the cell or tissue, is one of the main underlying causes of insulin resistance and type 2 diabetes. Although this hypothesis is not free of controversy due to the uncertainty on the sequence of events during type 2 diabetes onset, e.g. whether mitochondrial dysfunction is the cause or the consequence of insulin resistance, it has been widely observed that improving mitochondrial function also improves insulin sensitivity and prevents type 2 diabetes. Thus restoring oxidative capacity by increasing mitochondrial mass appears as a suitable strategy to treat insulin resistance. The effort made by researchers trying to understand the signaling pathways mediating mitochondrial biogenesis has uncovered new potential pharmacological targets and opens the perspectives for the design of suitable treatments for insulin resistance. In addition some of the current used strategies could be used to treat insulin resistance such as lifestyle interventions (caloric restriction and endurance exercise) and pharmacological interventions (thiazolidinediones and other PPAR agonists, resveratrol and other calorie restriction mimetics, AMPK activators, ERR activators). Mitochondrial biogenesis is of special importance in modern neurochemistry because of the broad spectrum

  15. Understanding the role of PknJ in Mycobacterium tuberculosis: biochemical characterization and identification of novel substrate pyruvate kinase A.

    Directory of Open Access Journals (Sweden)

    Gunjan Arora

    Full Text Available Reversible protein phosphorylation is a prevalent signaling mechanism which modulates cellular metabolism in response to changing environmental conditions. In this study, we focus on previously uncharacterized Mycobacterium tuberculosis Ser/Thr protein kinase (STPK PknJ, a putative transmembrane protein. PknJ is shown to possess autophosphorylation activity and is also found to be capable of carrying out phosphorylation on the artificial substrate myelin basic protein (MyBP. Previous studies have shown that the autophosphorylation activity of M. tuberculosis STPKs is dependent on the conserved residues in the activation loop. However, our results show that apart from the conventional conserved residues, additional residues in the activation loop may also play a crucial role in kinase activation. Further characterization of PknJ reveals that the kinase utilizes unusual ions (Ni(2+, Co(2+ as cofactors, thus hinting at a novel mechanism for PknJ activation. Additionally, as shown for other STPKs, we observe that PknJ possesses the capability to dimerize. In order to elucidate the signal transduction cascade emanating from PknJ, the M. tuberculosis membrane-associated protein fraction is treated with the active kinase and glycolytic enzyme Pyruvate kinase A (mtPykA is identified as one of the potential substrates of PknJ. The phospholabel is found to be localized on serine and threonine residue(s, with Ser(37 identified as one of the sites of phosphorylation. Since Pyk is known to catalyze the last step of glycolysis, our study shows that the fundamental pathways such as glycolysis can also be governed by STPK-mediated signaling.

  16. Molecular and biochemical approaches for characterization of antifungal trait of a potent biocontrol agent Bacillus subtilis RP24.

    Science.gov (United States)

    Grover, Minakshi; Nain, Lata; Singh, Shashi Bala; Saxena, Anil Kumar

    2010-02-01

    Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma-Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4'-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.

  17. Molecular characterization of Aedes aegypti (L. (Diptera: Culicidae of Easter Island based on analysis of the mitochondrial ND4 gene

    Directory of Open Access Journals (Sweden)

    Claudia Andrea Núñez

    2016-06-01

    Full Text Available ABSTRACT Aedes aegypti mosquitoes are the main vector of viruses Dengue, Zika and Chikungunya. Shortly after the first report of the dengue vector Ae. aegypti in Easter Island (Rapa Nui in late 2000, the first disease outbreak dengue occurred. Viral serotyping during the 2002 outbreak revealed a close relationship with Pacific DENV-1 genotype IV viruses, supporting the idea that the virus most likely originated in Tahiti. Mitochondrial NADH dehydrogenase subunit 4 (ND4 DNA sequences generated from 68 specimens of Ae. aegypti from Easter Island reporting a unique finding of a single maternal lineage of Ae. aegypti on Easter Island.

  18. Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A.

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    Jui-Hsin Huang

    Full Text Available Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB specifically bind to unknown glycoprotein(s on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1-411, middle (residues 296-701, and C-terminal portions (residues 524-911 of the RBD (F1, F2 and F3, respectively were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a are capable of binding to the cell surface of both Vero and Caco-2 cells; (b possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii the fragments exhibit various levels

  19. The complete mitochondrial genome of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae): genome characterization and phylogenetic considerations.

    Science.gov (United States)

    Xu, Tian-Jun; Cheng, Yuan-Zhi; Liu, Xue-Zhu; Shi, Ge; Wang, Ri-Xin

    2011-01-01

    The complete mitochondrial genome sequence of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae) was determined and phylogenetic analysis was conducted to elucidate the evolutionary relationship of the marbled rockfish with other Sebastinae species. This mitochondrial genome, consisting of 17301 bp, is highly similar to that of most other vertebrates, containing the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one putative control region. Most of the genes are encoded on the H-strand, while the ND6 and seven tRNA genes (for Gln, Ala, Asn, Tyr, Ser (UCA), Glu, and Pro) are encoded on the L-strand. The reading frame of two pairs of genes overlapped on the same strand (the ATPase 8 and 6 genes overlapped by ten nucleotides; ND4L and ND4 genes overlapped by seven nucleotides). The possibly nonfunctional light-strand replication origin folded into a typical stem-loop secondary structure and a conserved motif (5'-GCCGG-3') was found at the base of the stem within the tRNA(Cys) gene. An extent termination-associated sequence (ETAS) and conserved sequence blocks (CSB) were identified in the control region, except for CSB-1; unusual long tandem repeats were found at the 3' end of the control region. Phylogenetic analyses supported the view that Sebastinae comprises four genera (Sebates, Hozukius, Helicolenus, and Sebasticus).

  20. Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil.

    Science.gov (United States)

    Pennacchio, Angela; Sannino, Vincenzo; Sorrentino, Giosuè; Rossi, Mosè; Raia, Carlo A; Esposito, Luciana

    2013-05-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.

  1. Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site.

    Science.gov (United States)

    Kim, Han-Woo; Wang, Ji-Yeon; Lee, Ji-Yeon; Park, Ae-Kyung; Park, Hyun; Jeon, Sung-Jong

    2016-10-15

    The gene encoding a quinoprotein aldose sugar dehydrogenase (ASD) from Thermus thermophilus HJ6 (Tt_ASD) was cloned and sequenced; it comprised 1059 nucleotides encoding a protein containing 352 amino acids that had a predicted molecular mass of 38.9 kDa. The deduced amino acid sequence showed 42.9% and 33.9% identities to the ASD proteins from Pyrobaculum aerophilum and Escherichia coli, respectively. The biochemical properties of Tt_ASD were characterized. The optimum pH for the oxidation of glucose was 7.0-7.5 and the optimum temperature was 70 °C. The half-life of heat inactivation for the apoenzyme was about 25 min at 85 °C. The enzyme was highly thermostable, and the activity of the pyrroloquinoline quinone-bound holoenzyme was not lost after incubation at 85 °C for 100 min. Tt_ASD could oxidize various sugars, including hexoses, pentoses, disaccharides, and polysaccharides, in addition to alcohols. Structural analysis suggested that Tyr156 would be the substrate-binding residue. Two mutants, Y156A and Y156K, had impaired activities and affinities for all substrates and completely lost their activities for alcohols. This structural and mutational analysis of Tt_ASD demonstrates the crucial role of Tyr156 in determining substrate specificity.

  2. Heterologous overexpression and biochemical characterization of the (galactophospho)lipase from Fusarium solani in Pichia pastoris that is expressed in planta.

    Science.gov (United States)

    Jallouli, Raida; Ali, Madiha Bou; Charfeddine, Mariam; Gargouri-Bouzid, Radhia; Gargouri, Youssef; Bezzine, Sofiane

    2016-03-01

    High-level extracellular production of Fusarium solani (galactophospho)lipase, named FSL, was achieved using a Pichia pastoris X33 expression system. The (galactophospho) lipase encoding gene was cloned into pGAPZαA with the Saccharomyces cerevisiae α-factor signal sequence by two different ways. The two constructs consist of an additional sequence of a (His)6-tag of the vector fused to the N-terminus of this enzyme (tFSL) while the other expression vector was constructed without any additional sequence (rFSL). Compared to the native enzyme (nFSL) (18.75 mg/L), a high level secretion of rFSL (310 mg/L) and tFSL (240 mg/L) was achieved providing an important improvement in enzyme production. Biochemical characterization showed that pure recombinant proteins (rFSL and tFSL) presented similar behaviour towards triglycerides, phospholipid and galactolipid. Like the nFSL, rFSL and tFSL are active at high concentration of bile salts (4mM) and calcium ions enhanced lipase activity. During plant infection, transcripts of this fungal lipase gene were detected 3, 7 and 10 days post infection.

  3. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  4. The Effect of Mitochondrial Supplements on Mitochondrial Activity in Children with Autism Spectrum Disorder

    Science.gov (United States)

    Delhey, Leanna M.; Nur Kilinc, Ekim; Yin, Li; Slattery, John C.; Tippett, Marie L.; Rose, Shannon; Bennuri, Sirish C.; Kahler, Stephen G.; Damle, Shirish; Legido, Agustin; Goldenthal, Michael J.; Frye, Richard E.

    2017-01-01

    Treatment for mitochondrial dysfunction is typically guided by expert opinion with a paucity of empirical evidence of the effect of treatment on mitochondrial activity. We examined citrate synthase and Complex I and IV activities using a validated buccal swab method in 127 children with autism spectrum disorder with and without mitochondrial disease, a portion of which were on common mitochondrial supplements. Mixed-model linear regression determined whether specific supplements altered the absolute mitochondrial activity as well as the relationship between the activities of mitochondrial components. Complex I activity was increased by fatty acid and folate supplementation, but folate only effected those with mitochondrial disease. Citrate synthase activity was increased by antioxidant supplementation but only for the mitochondrial disease subgroup. The relationship between Complex I and IV was modulated by folate while the relationship between Complex I and Citrate Synthase was modulated by both folate and B12. This study provides empirical support for common mitochondrial treatments and demonstrates that the relationship between activities of mitochondrial components might be a marker to follow in addition to absolute activities. Measurements of mitochondrial activity that can be practically repeated over time may be very useful to monitor the biochemical effects of treatments. PMID:28208802

  5. Mitochondrial dysfunction and organophosphorus compounds

    Energy Technology Data Exchange (ETDEWEB)

    Karami-Mohajeri, Somayyeh [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Abdollahi, Mohammad, E-mail: Mohammad.Abdollahi@UToronto.Ca [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2013-07-01

    Organophosphorous (OPs) pesticides are the most widely used pesticides in the agriculture and home. However, many acute or chronic poisoning reports about OPs have been published in the recent years. Mitochondria as a site of cellular oxygen consumption and energy production can be a target for OPs poisoning as a non-cholinergic mechanism of toxicity of OPs. In the present review, we have reviewed and criticized all the evidences about the mitochondrial dysfunctions as a mechanism of toxicity of OPs. For this purpose, all biochemical, molecular, and morphological data were retrieved from various studies. Some toxicities of OPs are arisen from dysfunction of mitochondrial oxidative phosphorylation through alteration of complexes I, II, III, IV and V activities and disruption of mitochondrial membrane. Reductions of adenosine triphosphate (ATP) synthesis or induction of its hydrolysis can impair the cellular energy. The OPs disrupt cellular and mitochondrial antioxidant defense, reactive oxygen species generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome C released from mitochondria and consequent activation of caspases. The mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, electron donors, and through increasing the cytosolic ATP level. However, to elucidate many aspect of mitochondrial toxicity of Ops, further studies should be performed. - Highlights: • As a non-cholinergic mechanism of toxicity, mitochondria is a target for OPs. • OPs affect action of complexes I, II, III, IV and V in the mitochondria. • OPs reduce mitochondrial ATP. • OPs promote oxidative and genotoxic damage via release of cytochrome C from mitochondria. • OP-induced mitochondrial dysfunction can be restored by increasing the cytosolic ATP.

  6. Mitochondrial Plasticity With Exercise Training and Extreme Environments

    DEFF Research Database (Denmark)

    Boushel, Robert; Lundby, Carsten; Qvortrup, Klaus;

    2014-01-01

    Mitochondria form a reticulum in skeletal muscle. Exercise training stimulates mitochondrial biogenesis, yet an emerging hypothesis is that training also induces qualitative regulatory changes. Substrate oxidation, oxygen affinity and biochemical coupling efficiency may be differentially regulated...... with training and exposure to extreme environments. Threshold training doses inducing mitochondrial up-regulation remain to be elucidated considering fitness level. SUMMARY: Muscle mitochondrial are responsive to training and environment, yet thresholds for volume vs. regulatory changes and their physiological...

  7. Congenital and acquired mitochondrial disorders of the central nervous system

    OpenAIRE

    V. V. Nikitina; A. N. Pravdina

    2014-01-01

    Clinical presentations of disorders of the nervous system manifest in young and middle-aged patients with congenital and acquired mitochondrial dysfunctions and cognitive disorders manifest in patients with mitochondrial diseases more often. Nowadays the effective methods of initial diagnosing of these conditions are neurological and neuropsychological examination of patients, using of biochemical markers of mitochondrial diseases: the indices of lactate, total homocysteine in plasma and liqu...

  8. Sulfite disrupts brain mitochondrial energy homeostasis and induces mitochondrial permeability transition pore opening via thiol group modification.

    Science.gov (United States)

    Grings, Mateus; Moura, Alana P; Amaral, Alexandre U; Parmeggiani, Belisa; Gasparotto, Juciano; Moreira, José C F; Gelain, Daniel P; Wyse, Angela T S; Wajner, Moacir; Leipnitz, Guilhian

    2014-09-01

    Sulfite oxidase (SO) deficiency is biochemically characterized by the accumulation of sulfite, thiosulfate and S-sulfocysteine in tissues and biological fluids of the affected patients. The main clinical symptoms include severe neurological dysfunction and brain abnormalities, whose pathophysiology is still unknown. The present study investigated the in vitro effects of sulfite and thiosulfate on mitochondrial homeostasis in rat brain mitochondria. It was verified that sulfite per se, but not thiosulfate, decreased state 3, CCCP-stimulated state and respiratory control ratio in mitochondria respiring with glutamate plus malate. In line with this, we found that sulfite inhibited the activities of glutamate and malate (MDH) dehydrogenases. In addition, sulfite decreased the activity of a commercial solution of MDH, that was prevented by antioxidants and dithiothreitol. Sulfite also induced mitochondrial swelling and reduced mitochondrial membrane potential, Ca(2+) retention capacity, NAD(P)H pool and cytochrome c immunocontent when Ca(2+) was present in the medium. These alterations were prevented by ruthenium red, cyclosporine A (CsA) and ADP, supporting the involvement of mitochondrial permeability transition (MPT) in these effects. We further observed that N-ethylmaleimide prevented the sulfite-elicited swelling and that sulfite decreased free thiol group content in brain mitochondria. These findings indicate that sulfite acts directly on MPT pore containing thiol groups. Finally, we verified that sulfite reduced cell viability in cerebral cortex slices and that this effect was prevented by CsA. Therefore, it may be presumed that disturbance of mitochondrial energy homeostasis and MPT induced by sulfite could be involved in the neuronal damage characteristic of SO deficiency.

  9. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    Science.gov (United States)

    Kandel, Judith; Angelin, Alessia A; Wallace, Douglas C; Eckmann, David M

    2016-11-07

    An abundance of research suggests that cellular mitochondrial and cytoskeletal disruption are related, but few studies have directly investigated causative connections between the two. We previously demonstrated that inhibiting microtubule and microfilament polymerization affects mitochondrial motility on the whole-cell level in fibroblasts. Since mitochondrial motility can be indicative of mitochondrial function, we now further characterize the effects of these cytoskeletal inhibitors on mitochondrial potential, morphology and respiration. We found that although they did not reduce mitochondrial inner membrane potential, cytoskeletal toxins induced significant decreases in basal mitochondrial respiration. In some cases, basal respiration was only affected after cells were pretreated with the calcium ionophore A23187 in order to stress mitochondrial function. In most cases, mitochondrial morphology remained unaffected, but extreme microfilament depolymerization or combined intermediate doses of microtubule and microfilament toxins resulted in decreased mitochondrial lengths. Interestingly, these two particular exposures did not affect mitochondrial respiration in cells not sensitized with A23187, indicating an interplay between mitochondrial morphology and respiration. In all cases, inducing maximal respiration diminished differences between control and experimental groups, suggesting that reduced basal respiration originates as a largely elective rather than pathological symptom of cytoskeletal impairment. However, viability experiments suggest that even this type of respiration decrease may be associated with cell death.

  10. Biochemical and Thermodynamic Characterization of a Novel, Low Molecular Weight Xylanase from Bacillus Methylotrophicus CSB40 Isolated from Traditional Korean Food.

    Science.gov (United States)

    Panthi, Sandesh; Choi, Yoon Seok; Choi, Yun Hee; Kim, MiRi; Yoo, Jin Cheol

    2016-04-01

    A low molecular weight xylanase from Bacillus strain CSB40, isolated from traditional Korean food and produced in beechwood xylan, was biochemically and thermodynamically characterized. It was purified 8.12-fold with a 15.88 % yield using DEAE sepharose fast flow, and it was determined to have a mass of ∼27 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and xylan zymography. The purified xylanase was optimally active at 50 °C and pH 6 and stable over a wide range of pH (4.5-12.5). The N-terminal amino acid sequence of xylanase was GIQQGDDGKL. The activation energy for beechwood xylan hydrolysis was 29.39 kJmol(-1) with k cat value of 927.582 × 10(2) s(-1). K m and V max were 0.080 mg/ml and 794.63 mmol min(-1) mg(-1). The analysis of other thermodynamic parameters like ∆H, ∆G, ∆S, Q10, ∆GE-S, and ∆GE-T also supported the spontaneous formation of products, greater hydrolytic efficiency, and feasibility of enzymatic reaction, which also ratifies the novelty of this xylanase. The enzyme was strongly activated by Zn(2+) and inhibited by Cu(2+). The principal hydrolyzed end-products of this xylanase are xylobiose, xylotriose, and xylotetrose, which can be used in the pharmaceutical industry and as prebiotic in food.

  11. Clinical and biochemical characterization of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH deficiency that causes Leigh-like disease and ketoacidosis

    Directory of Open Access Journals (Sweden)

    Kenichiro Yamada

    2014-01-01

    Full Text Available 3-Hydroxyisobutyryl-CoA hydrolase (HIBCH deficiency is an autosomal recessive disorder characterized by episodes of ketoacidosis and a Leigh-like basal ganglia disease, without high concentrations of pyruvate and lactate in the cerebrospinal fluid. Only 4 cases of HIBCH deficiency have been reported. However, clinical–biochemical correlation in HIBCH deficiency by determining the detailed residual enzyme activities has not yet been elucidated. Here, we report a case of two Japanese siblings with HIBCH deficiency carrying a new homozygous missense mutation (c.287C > A, [p.A96D] at the substrate-binding site. A transfection study using HIBCH expression vectors harboring wild type or 4 reported mutations, including the newly identified mutation (p.A96D, p.Y122C, p.G317E, and p.K74Lfs*13, revealed a correlation between residual HIBCH activities and the severity of the disease. All HIBCH mutants, except p.K74Lfs*13, showed residual enzyme activity and only the patient with p.K74Lfs*13 had congenital anomalies. p.G317E showed only low enzyme activity (~3% of that of wild-type HIBCH. Although p.A96D had approximately 7 times higher enzyme activity than p.G317E, patients with p.A96D died during childhood. These findings are essential for clinical management, genetic counseling, and specific meal and concomitant drug considerations as part of the treatment for patients with HIBCH deficiency.

  12. Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers

    Science.gov (United States)

    Bai, Yuxiang; van der Kaaij, Rachel Maria; Leemhuis, Hans; Pijning, Tjaard; van Leeuwen, Sander Sebastiaan; Jin, Zhengyu

    2015-01-01

    4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application. PMID:26253678

  13. An alpha-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum. Cloning and biochemical characterization of the enzyme.

    Science.gov (United States)

    Tripathi, Abhai K; Desai, Prashant V; Pradhan, Anupam; Khan, Shabana I; Avery, Mitchell A; Walker, Larry A; Tekwani, Babu L

    2004-09-01

    Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites. The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli. The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD(+)(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate. Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD. The enzyme exhibited strict substrate and cofactor specificity. The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts. A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets. The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in alpha-proteobacterial MDHs. Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also. Treatment of a synchronized culture of P. falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged. Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites. Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs.

  14. Biochemical and pharmacological characterization of a toxic fraction and its cytotoxin-like component isolated from Russell's viper (Daboia russelii russelii) venom.

    Science.gov (United States)

    Thakur, Rupamoni; Chattopadhyay, Pronobesh; Mukherjee, Ashis K

    2015-02-01

    The pathophysiological significance of a toxic fraction (GF-VI DEAE-II) isolated from Russell's viper venom (RVV) is characterized. GF-VI DEAE-II represents 1.6% of the total RVV protein and it comprises of a 27.6kDa minor component (RP-I) (0.04%, w/w) and a major 6.6kDa non-enzymatic peptide (1.11%, w/w), named Rusvitoxin. The LC-MS/MS analysis of RP-I showed its identity to snake venom serine proteases, whereas Rusvitoxin demonstrated its close identity with snake venom three finger toxins, cytotoxins and cardiotoxins particularly from Naja sp. GF-VI DEAE-II was found to be non-cytotoxic to the tested mammalian cancer cells and non-hemolytic; nevertheless, it demonstrated α-fibrin(ogen)ase activity and in vivo toxicity in BALB/c mice with an LD50 (i.p.) of 2.3mg/kg. GF-VI DEAE-II induced lethargy and hind-leg paralysis in mice within 10min of i.p. injection. GF-VI DEAE-II induced hyperfibrinogenomia, and significantly altered (p<0.05) the plasma levels of factor X, pro- and anti-inflammatory cytokines viz. TNF-α, IL-6 and IL-10 in treated mice. Histological observations of tissues and biochemical properties of serum from GF-VI DEAE-II-treated mice suggested multiple organ dysfunctions. Conversely, Rusvitoxin at a dose of 5mg/kg did not induce toxicity in BALB/c mice. At 1:15 (antigen: antivenom, w/w) ratio, commercially polyvalent and monovalent antivenoms neutralized more than 80% of the fibrinolytic and anticoagulant activities of GF-VI DEAE-II. The present study suggests the significant role of GF-VI DEAE-II in RVV-induced pathogenesis in victim/prey.

  15. Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers.

    Science.gov (United States)

    Farjallah, Sarra; Ben Slimane, Badreddine; Piras, Cristina Maria; Amor, Nabil; Garippa, Giovanni; Merella, Paolo

    2013-11-01

    The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n=66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus

  16. Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157.

    Science.gov (United States)

    Gao, Yin; Liu, Bin; Strum, Scott; Schutzbach, John S; Druzhinina, Tatyana N; Utkina, Natalia S; Torgov, Vladimir I; Danilov, Leonid L; Veselovsky, Vladimir V; Vlahakis, Jason Z; Szarek, Walter A; Wang, Lei; Brockhausen, Inka

    2012-08-01

    The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.

  17. Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene.

    Science.gov (United States)

    Sánchez, Elizabeth; Cáceres, Omar; Náquira, César; Garcia, David; Patiño, Gladys; Silvia, Herrera; Volotão, Aline C; Fernandes, Octavio

    2010-09-01

    Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  18. Characterization of population structure from the mitochondrial DNA vis-à-vis language and geography in Papua New Guinea.

    Science.gov (United States)

    Lee, Esther J; Koki, George; Merriwether, D Andrew

    2010-08-01

    Situated along a corridor linking the Asian continent with the outer islands of the Pacific, Papua New Guinea has long played a key role in understanding the initial peopling of Oceania. The vast diversity in languages and unique geographical environments in the region have been central to the debates on human migration and the degree of interaction between the Pleistocene settlers and newer migrants. To better understand the role of Papua New Guinea in shaping the region's prehistory, we sequenced the mitochondrial DNA (mtDNA) control region of three populations, a total of 94 individuals, located in the East Sepik Province of Papua New Guinea. We analyzed these samples with a large data set of Oceania populations to examine the role of geography and language in shaping population structure within New Guinea and between the region and Island Melanesia. Our results from median-joining networks, star-cluster age estimates, and population genetic analyses show that while highland New Guinea populations seem to be the oldest settlers, there has been significant gene flow within New Guinea with little influence from geography or language. The highest genetic division is between Papuan speakers of New Guinea versus East Papuan speakers located outside of mainland New Guinea. Our study supports the weak language barriers to genetic structuring among populations in close contact and highlights the complexity of understanding the genetic histories of Papua New Guinea in association with language and geography.

  19. Characterization of the Complete Mitochondrial Genome of Leucoma salicis (Lepidoptera: Lymantriidae) and Comparison with Other Lepidopteran Insects

    Science.gov (United States)

    Sun, Yu-Xuan; Wang, Lei; Wei, Guo-Qing; Qian, Cen; Dai, Li-Shang; Sun, Yu; Abbas, Muhammad Nadeem; Zhu, Bao-Jian; Liu, Chao-Liang

    2016-01-01

    The complete mitochondrial genome (mitogenome) of Leucoma salicis (Lepidoptera: Lymantriidae) was sequenced and annotated. It is a circular molecule of 15,334 bp, containing the 37 genes usually present in insect mitogenomes. All protein-coding genes (PCGs) are initiated by ATN codons, other than cox1, which is initiated by CGA. Three of the 13 PCGs had an incomplete termination codon, T or TA, while the others terminated with TAA. The relative synonymous codon usage of the 13 protein-coding genes (PCGs) was consistent with those of published lepidopteran sequences. All tRNA genes had typical clover-leaf secondary structures, except for the tRNASer (AGN), in which the dihydrouridine (DHU) arm could not form a stable stem-loop structure. The A + T-rich region of 325 bp had several distinctive features, including the motif ‘ATAGA’ followed by an 18 bp poly-T stretch, a microsatellite-like (AT)7 element, and an 11-bp poly-A present immediately upstream of tRNAMet. Relationships among 32 insect species were determined using Maximum Likelihood (ML), Neighbor Joining (NJ) and Bayesian Inference (BI) phylogenetic methods. These analyses confirm that L. salicis belongs to the Lymantriidae; and that Lymantriidae is a member of Noctuoidea, and is a sister taxon to Erebidae, Nolidae and Noctuidae, most closely related to Erebidae. PMID:27974854

  20. Genetic characterization of the Pacific sheath-tailed bat (Emballonura semicaudata rotensis) using mitochondrial DNA sequence data

    Science.gov (United States)

    Oyler-McCance, Sara J.; Valdez, Ernest W.; O'Shea, Thomas; Fike, Jennifer A.

    2013-01-01

    Emballonura semicaudata occurs in the southwestern Pacific and populations on many islands have declined or disappeared. One subspecies (E. semicaudata rotensis) occurs in the Northern Mariana Islands, where it has been extirpated from all but 1 island (Aguiguan). We assessed genetic similarity between the last population of E. s. rotensis and 2 other subspecies, and examined genetic diversity on Aguiguan. We sampled 12 E. s. rotensis, sequenced them at 3 mitochondrial loci, and compared them with published sequences from 2 other subspecies. All 12 E. s. rotensis had identical sequences in each of the 3 regions. Using cytochrome-b (Cytb) data E. s. rotensis was sister to E. s. palauensis in a clade separate from E. s. semicaudata. 12S ribosomal RNA (12S) sequences grouped all E. s. semicaudata in 1 clade with E. s. rotensis in a clade by itself. Genetic distances among the 3 subspecies at Cytb were smallest between E. s. palauensis and E. s. rotensis. Distance between E. s. semicaudata and the other 2 subspecies was not different from the distance between E. s. semicaudata and the full species E. raffrayana. A similar relationship was found using the 12S data. These distances are larger than those typically reported for mammalian subspecies using Cytb sequence and within the range of sister species.

  1. Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I.

    Science.gov (United States)

    Smeitink, J; Loeffen, J; Smeets, R; Triepels, R; Ruitenbeek, W; Trijbels, F; van den Heuvel, L

    1998-08-01

    Bovine NADH:ubiquinone oxidoreductase (complex 1) of the mitochondrial respiratory chain consists of about 36 nuclear-encoded subunits. We review the current knowledge of the 15 human complex I subunits cloned so far, and report the 598-bp cDNA sequence, the chromosomal localization and the tissue expression of an additional subunit, the B17 subunit. The cDNA open reading frame of B17 comprises 387 bp and encodes a protein of 128 amino acids (calculated Mr 15.5 kDa). There is 82.7% and 78.1% homology, respectively, at the cDNA and amino acid level with the bovine counterpart. The gene of the B17 subunit has been mapped to chromosome 2. Multiple-tissue dot-blots showed ubiquitous expression of the mRNA with relatively higher expression in tissues known for their high energy demand. Of these, kidney showed the highest expression. Mutational analysis of the subunit revealed no mutations or polymorphisms in 20 patients with isolated enzymatic complex I deficiency in cultured skin fibroblasts.

  2. Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

    Directory of Open Access Journals (Sweden)

    Elizabeth Sánchez

    2010-09-01

    Full Text Available Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10, have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44, sheep (41 and humans (14 from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4. The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  3. Characterization of mitochondrial control region, two intergenic spacers and tRNAs of Zaprionus indianus (Diptera: Drosophilidae).

    Science.gov (United States)

    da Silva, Norma Machado; de Souza Dias, Aline; da Silva Valente, Vera Lúcia; Valiati, Victor Hugo

    2009-12-01

    The control region in insects is the major noncoding region in animal mitochondrial DNA (mtDNA), and is responsible for a large part of the variation in the DNA sequence and size of the genome of this organelle. In this study, the mtDNA control region, two intergenic spacers and tRNA genes of a Zaprionus indianus strain were cloned, sequenced and compared with other Drosophila species. The overall A+T content in the Z. indianus control region is 94.3%, and a comparison with other Drosophila species demonstrated that the most conserved region appears to be the 420 base pairs nearest to the tRNA(ile), similar to the findings of other authors. We also describe conserved sequence blocks, including a poly-T involved in the replication process of Drosophila mtDNA; a putative secondary structure also involved in the replication process and repeated sequences. tRNA(ile) sequence demonstrated the greatest variability when the tRNA sequences of species were compared.

  4. Molecular characterization of Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola sp. in China based on nuclear and mitochondrial DNA.

    Science.gov (United States)

    Peng, Mao; Ichinomiya, Mie; Ohtori, Maiko; Ichikawa, Madoka; Shibahara, Toshiyuki; Itagaki, Tadashi

    2009-09-01

    Parthenogenic Fasciola forms as well as bisexual Fasciola hepatica and Fasciola gigantica in mainland China have been identified on the basis of their spermatogenesis and genotypes in nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase I (NDI). The Chinese aspermic Fasciola would include forms originating in interspecific hybrids between F. hepatica and F. gigantica, since they showed the genotype of ITS1-Fh/Fg that had mixed sequences of the two Fasciola species or heterogeneous genotypes in ITS1 and NDI. Additionally, there were Chinese aspermic flukes in which the sequences of ITS1 and NDI genotypes completely coincided with those in aspermic forms from Japan, Korea, and Vietnam, suggesting that the aspermic forms from these four countries are offspring with a common provenance. The Fh-C4 haplotype in NDI was detected in both aspermic specimens and F. hepatica, indicating that aspermic forms showing the haplotype might come into existence in China. The ratio of body length and width in aspermic Fasciola specimens showed intermediate values between those of F. hepatica and F. gigantica.

  5. Mitochondrial Myopathy

    Science.gov (United States)

    ... diseases are caused by CoQ10 deficiency, and CoQ10 supplementation is clearly beneficial in these cases. It might provide some relief from other mitochondrial diseases. Creatine, L-carnitine, and CoQ10 supplements often are combined into a “ ...

  6. Mitochondrial oxidative function and type 2 diabetes

    DEFF Research Database (Denmark)

    Rabøl, Rasmus; Boushel, Robert; Dela, Flemming

    2006-01-01

    The cause of insulin resistance and type 2 diabetes is unknown. The major part of insulin-mediated glucose disposal takes place in the skeletal muscle, and increased amounts of intramyocellular lipid has been associated with insulin resistance and linked to decreased activity of mitochondrial...... oxidative phosphorylation. This review will cover the present knowledge and literature on the topics of the activity of oxidative enzymes and the electron transport chain (ETC) in skeletal muscle of patients with type 2 diabetes. Different methods of studying mitochondrial function are described, including...... biochemical measurements of oxidative enzyme and electron transport activity, isolation of mitochondria for measurements of respiration, and ATP production and indirect measurements of ATP production using nuclear magnetic resonance (NMR) - spectroscopy. Biochemical markers of mitochondrial content are also...

  7. Parkin suppresses Drp1-independent mitochondrial division.

    Science.gov (United States)

    Roy, Madhuparna; Itoh, Kie; Iijima, Miho; Sesaki, Hiromi

    2016-07-01

    The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson's disease-associated protein-parkin, which biochemically and genetically interacts with Drp1-in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division.

  8. Purification and partial biochemical characterization of a membrane-bound type II-like α-glucosidase from the yeast morphotype of Sporothrix schenckii.

    Science.gov (United States)

    Torres-Rodríguez, Blanca I; Flores-Berrout, Karina; Villagómez-Castro, Julio C; López-Romero, Everardo

    2012-02-01

    The early steps of glycoprotein biosynthesis involve processing of the N-glycan core by endoplasmic reticulum α-glucosidases I and II which sequentially trim the outermost α1,2-linked and the two more internal α1,3-linked glucose units, respectively. We have demonstrated the presence of some components of the enzymic machinery required for glycoprotein synthesis in Sporothrix schenckii, the etiological agent of human and animal sporotrichosis. However, information on this process is still very limited. Here, a distribution analysis of α-glucosidase revealed that 38 and 50% of total enzyme activity were present in a soluble and in a mixed membrane fraction, respectively. From the latter, the enzyme was solubilized, purified to apparent homogeneity and biochemically characterized. Analysis of the enzyme by denaturing electrophoresis and size exclusion chromatography revealed molecular masses of 75.4 and 152.7 kDa, respectively, suggesting a homodimeric structure. Purified α-glucosidase cleaved the fluorogenic substrate 4-methylumbelliferyl-α-D: -glucopyranoside with high affinity as judged from K(m) and V(max) values of 0.3 μM and 250 nmol of MU/min/mg protein, respectively. Analysis of linkage specificity using a number of glucose α-disaccharides as substrates demonstrated a clear preference of the enzyme for nigerose, an α1,3-linked disaccharide, over other substrates such as kojibiose (α1,2), trehalose (α1,1) and isomaltose (α1,6). Use of selective inhibitors of processing α-glucosidases such as 1-deoxynojirimycin, castanospermine and australine provided further evidence of the possible type of α-glucosidase. Accordingly, 1-deoxynojirimycin, a more specific inhibitor of α-glucosidase II than I, was a stronger inhibitor of hydrolysis of 4-methylumbelliferyl-α-D: -glucopyranoside and nigerose than castanospermine, a preferential inhibitor of α-glucosidase I. Inhibition of hydrolysis of kojibiose and maltose by 1-deoxynojirimycin and

  9. Elevated mitochondrial oxidative stress impairs metabolic adaptations to exercise in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Justin D Crane

    Full Text Available Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in Sod2 (+/- mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training Sod2 (+/- mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised Sod2 (+/- mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.

  10. Multivariate analysis of fatty acid and biochemical constitutes of seaweeds to characterize their potential as bioresource for biofuel and fine chemicals.

    Science.gov (United States)

    Verma, Priyanka; Kumar, Manoj; Mishra, Girish; Sahoo, Dinabandhu

    2017-02-01

    In the present study bio prospecting of thirty seaweeds from Indian coasts was analyzed for their biochemical components including pigments, fatty acid and ash content. Multivariate analysis of biochemical components and fatty acids was done using Principal Component Analysis (PCA) and Agglomerative hierarchical clustering (AHC) to manifest chemotaxonomic relationship among various seaweeds. The overall analysis suggests that these seaweeds have multi-functional properties and can be utilized as promising bioresource for proteins, lipids, pigments and carbohydrates for the food/feed and biofuel industry.

  11. Genetic counseling in mitochondrial disease.

    Science.gov (United States)

    Vento, Jodie M; Pappa, Belen

    2013-04-01

    Mitochondrial diseases are a genetically and clinically diverse group of disorders that arise as a result of dysfunction of the mitochondria. Mitochondrial disorders can be caused by alterations in nuclear DNA and/or mitochondrial DNA. Although some mitochondrial syndromes have been described clearly in the literature many others present as challenging clinical cases with multisystemic involvement at variable ages of onset. Given the clinical variability and genetic heterogeneity of these conditions, patients and their families often experience a lengthy and complicated diagnostic process. The diagnostic journey may be characterized by heightened levels of uncertainty due to the delayed diagnosis and the absence of a clear prognosis, among other factors. Uncertainty surrounding issues of family planning and genetic testing may also affect the patient. The role of the genetic counselor is particularly important to help explain these complexities and support the patient and family's ability to achieve effective coping strategies in dealing with increased levels of uncertainty.

  12. Characterization of the mitochondrial genome of the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) and phylogenetic analysis of advanced moths and butterflies.

    Science.gov (United States)

    Wei, Shu-Jun; Shi, Bao-Cai; Gong, Ya-Jun; Li, Qian; Chen, Xue-Xin

    2013-04-01

    Here we determined the mitochondrial genome sequence of a notorious pest, the diamondback moth Plutella xylostella (Lepidoptera: Yponomeutoidea: Plutellidae). The mitochondrial genome contains 37 typical animal mitochondrial genes and an A+T-rich region. The gene arrangement is identical to that of other ditrysian lepidopteran mitochondrial genomes, but different from the ancestral gene arrangement in the non-ditrysian Hepialidae of Lepidoptera. The start codon of the cox1 gene is CGA, which is dissimilar to its homologs in most other insects. In Lepidoptera, cox1 and cox2 have low nucleotide diversities, while the nad6, nad2, and nad3 genes are highly variable. Phylogenetic analyses uncovered the reciprocal monophyly of Ditrysia, Apoditrysia, Obtectomera, and Macrolepidoptera, and the placement of the Hesperiidae within Papilionoidea. Our analyses suggest that the complete mitochondrial genome sequences are a promising marker toward fully resolving the phylogenetic relationships within Lepidoptera.

  13. Disruption of mitochondrial DNA replication in Drosophila increases mitochondrial fast axonal transport in vivo.

    Directory of Open Access Journals (Sweden)

    Rehan M Baqri

    Full Text Available Mutations in mitochondrial DNA polymerase (pol gamma cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol gamma leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol gamma deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol gamma-alpha mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.

  14. Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor-Independent Pathway Characterized by Mitochondrial Stress.

    Science.gov (United States)

    Baker, Simon C; Shabir, Saqib; Georgopoulos, Nikolaos T; Southgate, Jennifer

    2016-05-01

    Recreational abuse of ketamine has been associated with the emergence of a new bladder pain syndrome, ketamine-induced cystitis, characterized by chronic inflammation and urothelial ulceration. We investigated the direct effects of ketamine on normal human urothelium maintained in organ culture or as finite cell lines in vitro. Exposure of urothelium to ketamine resulted in apoptosis, with cytochrome c release from mitochondria and significant subsequent caspase 9 and 3/7 activation. The anesthetic mode-of-action for ketamine is mediated primarily through N-methyl d-aspartate receptor (NMDAR) antagonism; however, normal (nonimmortalized) human urothelial cells were unresponsive to NMDAR agonists or antagonists, and no expression of NMDAR transcript was detected. Exposure to noncytotoxic concentrations of ketamine (≤1 mmol/L) induced rapid release of ATP, which activated purinergic P2Y receptors and stimulated the inositol trisphosphate receptor to provoke transient release of calcium from the endoplasmic reticulum into the cytosol. Ketamine concentrations >1 mmol/L were cytotoxic and provoked a larger-amplitude increase in cytosolic Ca(2+) concentration that was unresolved. The sustained elevation in cytosolic Ca(2+) concentration was associated with pathological mitochondrial oxygen consumption and ATP deficiency. Damage to the urinary barrier initiates bladder pain and, in ketamine-induced cystitis, loss of urothelium from large areas of the bladder wall is a reported feature. This study offers first evidence for a mechanism of direct toxicity of ketamine to urothelial cells by activating the intrinsic apoptotic pathway.

  15. Characterization of radish mitochondrial atpA: influence of nuclear background on transcription of atpA-associated sequences and relationship with male sterility.

    Science.gov (United States)

    Makaroff, C A; Apel, I J; Palmer, J D

    1990-11-01

    We have previously shown that the mitochondrial gene atpA, encoding the alpha subunit of F1 ATP synthase, is associated with DNA rearrangements and nuclear-specific transcript patterns in the male-sterile cytoplasm of Ogura radish. Here we present a detailed characterization of this gene from both the normal (fertile) and Ogura (male-sterile) cytoplasms of radish to determine if it is involved in Ogura cytoplasmic male sterility. The normal and Ogura radish atpA loci are virtually identical for 3.8 kb, including a 507 codon open reading frame whose product is approximately 92% identical to other plant ATPA polypeptides. Rearrangement breakpoints have been identified 613 bp 5' and 1663 bp 3' to the atpA coding region. The 5' rearrangement breakpoint is located within a repeated sequence that has been associated with other rearrangement events in radish mitochondria. The previously identified transcript difference results from transcription originating upstream of this rearrangement site. Although the presence of this transcript is affected by nuclear background, analyses in several different sterile and fertile nuclear backgrounds indicate that the presence of this transcript is not strictly correlated with male sterility. In addition, normal levels of ATPA polypeptide are present in sterile plants containing the Ogura cytoplasm.

  16. Deregulation of mitochondrial functions provoked by long-chain fatty acid accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial permeability transition deficiencies in rat heart--mitochondrial permeability transition pore opening as a potential contributing pathomechanism of cardiac alterations in these disorders.

    Science.gov (United States)

    Cecatto, Cristiane; Hickmann, Fernanda H; Rodrigues, Marília D N; Amaral, Alexandre U; Wajner, Moacir

    2015-12-01

    Mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies are fatty acid oxidation disorders biochemically characterized by tissue accumulation of long-chain fatty acids and derivatives, including the monocarboxylic long-chain 3-hydroxy fatty acids (LCHFAs) 3-hydroxytetradecanoic acid (3HTA) and 3-hydroxypalmitic acid (3HPA). Patients commonly present severe cardiomyopathy for which the pathogenesis is still poorly established. We investigated the effects of 3HTA and 3HPA, the major metabolites accumulating in these disorders, on important parameters of mitochondrial homeostasis in Ca(2+) -loaded heart mitochondria. 3HTA and 3HPA significantly decreased mitochondrial membrane potential, the matrix NAD(P)H pool and Ca(2+) retention capacity, and also induced mitochondrial swelling. These fatty acids also provoked a marked decrease of ATP production reflecting severe energy dysfunction. Furthermore, 3HTA-induced mitochondrial alterations were completely prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca(2+) uptake blocker, indicating that LCHFAs induced Ca(2+)-dependent mPT pore opening. Milder effects only achieved at higher doses of LCHFAs were observed in brain mitochondria, implying a higher vulnerability of heart to these fatty acids. By contrast, 3HTA and docosanoic acids did not change mitochondrial homeostasis, indicating selective effects for monocarboxylic LCHFAs. The present data indicate that the major LCHFAs accumulating in mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies induce mPT pore opening, compromising Ca(2+) homeostasis and oxidative phosphorylation more intensely in the heart. It is proposed that these pathomechanisms may contribute at least in part to the severe cardiac alterations characteristic of patients affected by these diseases.

  17. Barth Syndrome:From mitochondrial dysfunctions associated with aberrant production of reactive oxygen species to pluripotent stem cell studies

    Directory of Open Access Journals (Sweden)

    Ana eSaric

    2016-01-01

    Full Text Available Mutations in the gene encoding the enzyme tafazzin, TAZ, cause Barth syndrome (BTHS. Individuals with this X-linked multisystem disorder present cardiomyopathy (often dilated, skeletal muscle weakness, neutropenia, growth retardation and 3-methylglutaconic aciduria. Biopsies of the heart, liver and skeletal muscle of patients have revealed mitochondrial malformations and dysfunctions. It is the purpose of this review to summarize recent results of studies on various animal or cell models of Barth syndrome, which have characterized biochemically the strong cellular defects associated with TAZ mutations. Tafazzin is a mitochondrial phospholipid-lysophospholipid transacylase that shuttles acyl groups between phospholipids and regulates the remodeling of cardiolipin (CL, a unique inner mitochondrial membrane phospholipid dimer consisting of two phosphatidyl residues linked by a glycerol bridge. After their biosynthesis, the acyl chains of CLs may be modified in remodeling processes involving up to three different enzymes. Their characteristic acyl chain composition depends on the function of tafazzin, although the enzyme itself surprisingly lacks acyl specificity. CLs are crucial for correct mitochondrial structure and function. In addition to their function in the basic mitochondrial function of ATP production, CLs play essential roles in cardiac function, apoptosis, autophagy, cell cycle regulation and Fe-S cluster biosynthesis. Recent developments in tafazzin research have provided strong insights into the link between mitochondrial dysfunction and the production of reactive oxygen species (ROS. An important tool has been the generation of BTHS-specific induced pluripotent stem cells (iPSCs from BTHS patients. In a complementary approach, disease-specific mutations have been introduced into wild-type iPSC lines enabling direct comparison with isogenic controls. iPSC-derived cardiomyocytes were then characterized using biochemical and classical

  18. Mitochondria: impaired mitochondrial translation in human disease.

    Science.gov (United States)

    Boczonadi, Veronika; Horvath, Rita

    2014-03-01

    Defects of the mitochondrial protein synthesis cause a subgroup of mitochondrial diseases, which are usually associated with decreased activities of multiple respiratory chain (RC) enzymes. The clinical presentations of these disorders are often disabling, progressive or fatal, affecting the brain, liver, skeletal muscle, heart and other organs. Currently there are no effective cures for these disorders and treatment is at best symptomatic. The diagnosis in patients with multiple respiratory chain complex defects is particularly difficult because of the massive number of nuclear genes potentially involved in intra-mitochondrial protein synthesis. Many of these genes are not yet linked to human disease. Whole exome sequencing rapidly changed the diagnosis of these patients by identifying the primary defect in DNA, and preventing the need for invasive and complex biochemical testing. Better understanding of the mitochondrial protein synthesis apparatus will help us to explore disease mechanisms and will provide clues for developing novel therapies.

  19. Three dimensional reconstruction of the human skeletal muscle mitochondrial network as a tool to assess mitochondrial content and structural organization

    DEFF Research Database (Denmark)

    Dahl, Rannvá; Larsen, Steen; Dohlmann, Tine L

    2015-01-01

    with 3D reconstruction was used as a tool to analyze mitochondrial morphology and measure mitochondrial fractional volume. Results: Most type I and type II muscle fibers have tubular highly interconnected profusion mitochondria, which are thicker and more structured in type I muscle fibers (Figure 1......). In some muscle fibers, profission isolated ellipsoid-shaped mitochondria were observed. Mitochondrial volume was significantly higher in type I muscle fibers and showed no correlation with any of the investigated molecular and biochemical mitochondrial measurements (Figure 2). Three dimensional...

  20. Mitochondrial NAD+-dependent malic enzyme from Anopheles stephensi: a possible novel target for malaria mosquito control

    Directory of Open Access Journals (Sweden)

    Pon Jennifer

    2011-10-01

    Full Text Available Abstract Background Anopheles stephensi mitochondrial malic enzyme (ME emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development. Methods To characterize the mitochondrial ME from immortalized ASE cells (Mos. 43; ASE, mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference. Results The encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from Anopheles gambiae (98% and 59% homologous to the mitochondrial NADP+-dependent ME isoform from Homo sapiens. Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that (i Vmax with NAD+ was 3-fold higher than that with NADP+, (ii addition of Mg2+ or Mn2+ increased the Vmax by 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, (iii succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, (iv among the analogs of L-malate tested as inhibitors of the NAD+-dependent ME catalyzed reaction, small (2- to 3-carbons organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity (e.g., oxaloacetate, tartronic acid and oxalate. Conclusions The biochemical characterization of Anopheles stephensi ME is of critical relevance given its important role in

  1. Oxidative stress, mitochondrial damage and neurodegenerative diseases****

    Institute of Scientific and Technical Information of China (English)

    Chunyan Guo; Li Sun; Xueping Chen; Danshen Zhang

    2013-01-01

    Oxidative stress and mitochondrial damage have been implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Oxidative stress is characterized by the overproduction of reactive oxygen species, which can induce mitochondrial DNA mutations, damage the mitochondrial respiratory chain, alter membrane permeability, and influence Ca2+ homeostasis and mitochondrial defense systems. Al these changes are implicated in the development of these neurodegenerative diseases, mediating or amplifying neuronal dysfunction and triggering neurodegeneration. This paper summarizes the contribution of oxidative stress and mitochondrial damage to the onset of neurodegenerative eases and discusses strategies to modify mitochondrial dysfunction that may be attractive thera-peutic interventions for the treatment of various neurodegenerative diseases.

  2. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  3. Characterization of water quality and simulation of temperature, nutrients, biochemical oxygen demand, and dissolved oxygen in the Wateree River, South Carolina, 1996-98

    Science.gov (United States)

    Feaster, Toby D.; Conrads, Paul A.

    2000-01-01

    In May 1996, the U.S. Geological Survey entered into a cooperative agreement with the Kershaw County Water and Sewer Authority to characterize and simulate the water quality in the Wateree River, South Carolina. Longitudinal profiling of dissolved-oxygen concentrations during the spring and summer of 1996 revealed dissolved-oxygen minimums occurring upstream from the point-source discharges. The mean dissolved-oxygen decrease upstream from the effluent discharges was 2.0 milligrams per liter, and the decrease downstream from the effluent discharges was 0.2 milligram per liter. Several theories were investigated to obtain an improved understanding of the dissolved-oxygen dynamics in the upper Wateree River. Data suggest that the dissolved-oxygen concentration decrease is associated with elevated levels of oxygen-consuming nutrients and metals that are flowing into the Wateree River from Lake Wateree. Analysis of long-term streamflow and water-quality data collected at two U.S. Geological Survey gaging stations suggests that no strong correlation exists between streamflow and dissolved-oxygen concentrations in the Wateree River. However, a strong negative correlation does exist between dissolved-oxygen concentrations and water temperature. Analysis of data from six South Carolina Department of Health and Environmental Control monitoring stations for 1980.95 revealed decreasing trends in ammonia nitrogen at all stations where data were available and decreasing trends in 5-day biochemical oxygen demand at three river stations. The influence of various hydrologic and point-source loading conditions on dissolved-oxygen concentrations in the Wateree River were determined by using results from water-quality simulations by the Branched Lagrangian Transport Model. The effects of five tributaries and four point-source discharges were included in the model. Data collected during two synoptic water-quality samplings on June 23.25 and August 11.13, 1997, were used to calibrate

  4. Mitochondrial biogenesis in plants during seed germination.

    Science.gov (United States)

    Law, Simon R; Narsai, Reena; Whelan, James

    2014-11-01

    Mitochondria occupy a central role in the eukaryotic cell. In addition to being major sources of cellular energy, mitochondria are also involved in a diverse range of functions including signalling, the synthesis of many essential organic compounds and a role in programmed cell death. The active proliferation and differentiation of mitochondria is termed mitochondrial biogenesis and necessitates the coordinated communication of mitochondrial status within an integrated cellular network. Two models of mitochondrial biogenesis have been defined previously, the growth and division model and the maturation model. The former describes the growth and division of pre-existing mature organelles through a form of binary fission, while the latter describes the propagation of mitochondria from structurally and biochemically simple promitochondrial structures that upon appropriate stimuli, mature into fully functional mitochondria. In the last decade, a number of studies have utilised seed germination in plants as a platform for the examination of the processes occurring during mitochondrial biogenesis. These studies have revealed many new aspects of the tightly regulated procession of events that define mitochondrial biogenesis during this period of rapid development. A model for mitochondrial biogenesis that supports the maturation of mitochondria from promitochondrial structures has emerged, where mitochondrial signalling plays a crucial role in the early steps of seed germination.

  5. The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

    Science.gov (United States)

    Lyonnais, Sébastien; Tarrés-Soler, Aleix; Rubio-Cosials, Anna; Cuppari, Anna; Brito, Reicy; Jaumot, Joaquim; Gargallo, Raimundo; Vilaseca, Marta; Silva, Cristina; Granzhan, Anton; Teulade-Fichou, Marie-Paule; Eritja, Ramon; Solà, Maria

    2017-01-01

    The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. PMID:28276514

  6. Characterization of rice (Oryza sativa L.) genotypes on the basis of morpho-physiological and biochemical traits grown under aerobic situation in rainfed ecosystem .

    Science.gov (United States)

    Kumar, Santosh; Dwivedi, Sharad Kumar; Singh, S S; Kumar, Sanjeev; Sundaram, R K; Shivani; Mall, A K

    2015-07-01

    The objective of the present study was to examine the effect of aerobic situation on yield, physiological and biochemical traits of advanced breeding lines of rice. Experiment was conducted with two set of rice genotypes under two water regimes (aerobic and irrigated), during three consecutive wet seasons 2010-2012. Significant decrease in yield was observed in rice genotypes grown under aerobic situation as compared to the irrigated ones. Promising rice genotypes having the ability to maintain high plant biomass, harvest index, early vegetative vigour, improved physiological and biochemical traits in terms of relative water content (RWC), leaf area index (LAI), total soluble sugar, starch, protien and proline content help to sustain higher grain yield under aerobic situation. The yield gap between aerobic and irrigated rice ranged between 24% to 68%. Grain yield showed positive correlation with harvest index (0.434), test weight (0.647), plant biomass (0.411) and effective tiller numbers (0.473), whereas spikelet sterility was negative associated (-0.380). The current study suggested that promising genotypes viz., IR77298-14-1-2-130-2, IR84899-B-182-3-1-1-2, IR84887-B-157-38-1-1-3 and IR 84899-B-179-1-1-1-2 for aerobic situation, showing yield advantage due to better performance of physiological and biochemical traits, might be adopted in large area of rainfed ecosystem as well as in irrigated areas where water scarcity was a major problem.

  7. Mitochondrial Energy-Deficient Endophenotype in Autism

    Directory of Open Access Journals (Sweden)

    J. J. Gargus

    2008-01-01

    Full Text Available While evidence points to a multigenic etiology of most autism, the pathophysiology of the disorder has yet to be defined and the underlying genes and biochemical pathways they subserve remain unknown. Autism is considered to be influenced by a combination of various genetic, environmental and immunological factors; more recently, evidence has suggested that increased vulnerability to oxidative stress may be involved in the etiology of this multifactorial disorder. Furthermore, recent studies have pointed to a subset of autism associated with the biochemical endophenotype of mitochondrial energy deficiency, identified as a subtle impairment in fat and carbohydrate oxidation. This phenotype is similar, but more subtle than those seen in classic mitochondrial defects. In some cases the beginnings of the genetic underpinnings of these mitochondrial defects are emerging, such as mild mitochondrial dysfunction and secondary carnitine deficiency observed in the subset of autistic patients with an inverted duplication of chromosome 15q11-q13. In addition, rare cases of familial autism associated with sudden infant death syndrome (SIDS or associated with abnormalities in cellular calcium homeostasis, such as malignant hyperthermia or cardiac arrhythmia, are beginning to emerge. Such special cases suggest that the pathophysiology of autism may comprise pathways that are directly or indirectly involved in mitochondrial energy production and to further probe this connection three new avenues seem worthy of exploration: 1 metabolomic clinical studies provoking controlled aerobic exercise stress to expand the biochemical phenotype, 2 high-throughput expression arrays to directly survey activity of the genes underlying these biochemical pathways and 3 model systems, either based upon neuronal stem cells or model genetic organisms, to discover novel genetic and environmental inputs into these pathways.

  8. Mitochondrial DNA mutations and male infertility

    Directory of Open Access Journals (Sweden)

    Kumar D

    2009-01-01

    Full Text Available Infertility can be defined as difficulty in conceiving a child after 1 year of unprotected intercourse. Infertility can arise either because of the male factor or female factor or both. According to the current estimates, 15% of couples attempting their first pregnancy could not succeed. Infertility is either primary or secondary. Mitochondria have profound effect on all biochemical pathways, including the one that drivessperm motility. Sperm motility is heavily dependent on the ATP generated by oxidative phosphorylation in the mitochondrial sheath. In this review, the very positive role of mitochondrial genome′s association with infertility is discussed

  9. Mitochondrial dysfunction in autism.

    Science.gov (United States)

    Legido, Agustín; Jethva, Reena; Goldenthal, Michael J

    2013-09-01

    Using data of the current prevalence of autism as 200:10,000 and a 1:2000 incidence of definite mitochondrial (mt) disease, if there was no linkage of autism spectrum disorder (ASD) and mt disease, it would be expected that 1 in 110 subjects with mt disease would have ASD and 1 in 2000 individuals with ASD would have mt disease. The co-occurrence of autism and mt disease is much higher than these figures, suggesting a possible pathogenetic relationship. Such hypothesis was initially suggested by the presence of biochemical markers of abnormal mt metabolic function in patients with ASD, including elevation of lactate, pyruvate, or alanine levels in blood, cerebrospinal fluid, or brain; carnitine level in plasma; and level of organic acids in urine, and by demonstrating impaired mt fatty acid β-oxidation. More recently, mtDNA genetic mutations or deletions or mutations of nuclear genes regulating mt function have been associated with ASD in patients or in neuropathologic studies on the brains of patients with autism. In addition, the presence of dysfunction of the complexes of the mt respiratory chain or electron transport chain, indicating abnormal oxidative phosphorylation, has been reported in patients with ASD and in the autopsy samples of brains. Possible pathogenetic mechanisms linking mt dysfunction and ASD include mt activation of the immune system, abnormal mt Ca(2+) handling, and mt-induced oxidative stress. Genetic and epigenetic regulation of brain development may also be disrupted by mt dysfunction, including mt-induced oxidative stress. The role of the purinergic system linking mt dysfunction and ASD is currently under investigation. In summary, there is genetic and biochemical evidence for a mitochondria (mt) role in the pathogenesis of ASD in a subset of children. To determine the prevalence and type of genetic and biochemical mt defects in ASD, there is a need for further research using the latest genetic technology such as next

  10. Mitochondrial rejuvenation after induced pluripotency.

    Directory of Open Access Journals (Sweden)

    Steven T Suhr

    Full Text Available BACKGROUND: As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion "resets" some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1 that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2 the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal. CONCLUSIONS/SIGNIFICANCE: These results - coupled with earlier data from our laboratory - suggest that IPSC conversion not only resets the "biological clock", but can also rejuvenate the energetic capacity of derived cells.

  11. Mitochondrial DNA variants of respiratory complex I that uniquely characterize haplogroup T2 are associated with increased risk of age-related macular degeneration.

    Directory of Open Access Journals (Sweden)

    John Paul SanGiovanni

    Full Text Available BACKGROUND: Age-related macular degeneration (AMD, a chronic neurodegenerative and neovascular retinal disease, is the leading cause of blindness in elderly people of western European origin. While structural and functional alterations in mitochondria (mt and their metabolites have been implicated in the pathogenesis of chronic neurodegenerative and vascular diseases, the relationship of inherited variants in the mitochondrial genome and mt haplogroup subtypes with advanced AMD has not been reported in large prospective cohorts. METHODOLOGY/PRINICIPAL FINDINGS: We examined the relationship of inherited mtDNA variants with advanced AMD in 1168 people using a three-stage design on samples from 12-year and 10-year prospective studies on the natural history of age-related eye disease. In Stage I we resequenced the entire genome in 99 elderly AMD-free controls and 215 people with advanced AMD from the 12-year study. A consistent association with AMD in 14 of 17 SNPs characterizing the mtDNA T haplogroup emerged. Further analysis revealed these associations were driven entirely by the T2 haplogroup, and characterized by two variants in Complex I genes (A11812G of MT-ND4 and A14233G of MT-ND6. We genotyped T haplogroups in an independent sample of 490 cases and 61 controls from the same study (Stage II and in 56 cases and 246 controls from the 10-year study (Stage III. People in the T2 haplogroup were approximately 2.5 times more likely to have advanced AMD than their peers (odds ratio [OR] = 2.54, 95%CI 1.36-4.80, P

  12. Mitochondrial DNA Alterations and Reduced Mitochondrial Function in Aging

    OpenAIRE

    Hebert, Sadie L.; Lanza, Ian R.; Nair, K. Sreekumaran

    2010-01-01

    Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed.

  13. Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

    Science.gov (United States)

    Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

    2009-10-01

    Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

  14. cDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins.

    Science.gov (United States)

    Nakai, Y; Yoshihara, Y; Hayashi, H; Kagamiyama, H

    1998-08-14

    We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y-NFS1), and termed it m-Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence. m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria. The m-Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the gamma-NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.

  15. Mitochondrial preconditioning: a potential neuroprotective strategy.

    Science.gov (United States)

    Correia, Sónia C; Carvalho, Cristina; Cardoso, Susana; Santos, Renato X; Santos, Maria S; Oliveira, Catarina R; Perry, George; Zhu, Xiongwei; Smith, Mark A; Moreira, Paula I

    2010-01-01

    Mitochondria have long been known as the powerhouse of the cell. However, these organelles are also pivotal players in neuronal cell death. Mitochondrial dysfunction is a prominent feature of chronic brain disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), and cerebral ischemic stroke. Data derived from morphologic, biochemical, and molecular genetic studies indicate that mitochondria constitute a convergence point for neurodegeneration. Conversely, mitochondria have also been implicated in the neuroprotective signaling processes of preconditioning. Despite the precise molecular mechanisms underlying preconditioning-induced brain tolerance are still unclear, mitochondrial reactive oxygen species generation and mitochondrial ATP-sensitive potassium channels activation have been shown to be involved in the preconditioning phenomenon. This review intends to discuss how mitochondrial malfunction contributes to the onset and progression of cerebral ischemic stroke and AD and PD, two major neurodegenerative disorders. The role of mitochondrial mechanisms involved in the preconditioning-mediated neuroprotective events will be also discussed. Mitochondrial targeted preconditioning may represent a promising therapeutic weapon to fight neurodegeneration.

  16. Mitochondrial preconditioning: a potential neuroprotective strategy

    Directory of Open Access Journals (Sweden)

    Sónia C Correia

    2010-08-01

    Full Text Available Mitochondria have long been known as the powerhouse of the cell. However, these organelles are also pivotal players in neuronal cell death. Mitochondrial dysfunction is a prominent feature of chronic brain disorders, including Alzheimer's and Parkinson's diseases, and cerebral ischemic stroke. Data derived from morphologic, biochemical and molecular genetic studies indicate that mitochondria constitute a convergence point for neurodegeneration. Conversely, mitochondria have also been implicated in the neuroprotective signaling processes of preconditioning. Despite the precise molecular mechanisms underlying preconditioning-induced brain tolerance are still unclear, mitochondrial reactive oxygen species generation and mitochondrial ATP-sensitive potassium channels activation have been shown to be involved in the preconditioning phenomenon. This review intends to discuss how mitochondrial malfunction contributes to the onset and progression of cerebral ischemic stroke and Alzheimer’s and Parkinson’s diseases, two major neurodegenerative disorders. The role of mitochondrial mechanisms involved in the preconditioning-mediated neuroprotective events will be also discussed. Mitochondrial targeted preconditioning may represent a promising therapeutic weapon to fight neurodegeneration.

  17. Modeling mitochondrial bioenergetics with integrated volume dynamics.

    Directory of Open Access Journals (Sweden)

    Jason N Bazil

    2010-01-01

    Full Text Available Mathematical models of mitochondrial bioenergetics provide powerful analytical tools to help interpret experimental data and facilitate experimental design for elucidating the supporting biochemical and physical processes. As a next step towards constructing a complete physiologically faithful mitochondrial bioenergetics model, a mathematical model was developed targeting the cardiac mitochondrial bioenergetic based upon previous efforts, and corroborated using both transient and steady state data. The model consists of several modified rate functions of mitochondrial bioenergetics, integrated calcium dynamics and a detailed description of the K(+-cycle and its effect on mitochondrial bioenergetics and matrix volume regulation. Model simulations were used to fit 42 adjustable parameters to four independent experimental data sets consisting of 32 data curves. During the model development, a certain network topology had to be in place and some assumptions about uncertain or unobserved experimental factors and conditions were explicitly constrained in order to faithfully reproduce all the data sets. These realizations are discussed, and their necessity helps contribute to the collective understanding of the mitochondrial bioenergetics.

  18. Modeling mitochondrial bioenergetics with integrated volume dynamics.

    Science.gov (United States)

    Bazil, Jason N; Buzzard, Gregery T; Rundell, Ann E

    2010-01-01

    Mathematical models of mitochondrial bioenergetics provide powerful analytical tools to help interpret experimental data and facilitate experimental design for elucidating the supporting biochemical and physical processes. As a next step towards constructing a complete physiologically faithful mitochondrial bioenergetics model, a mathematical model was developed targeting the cardiac mitochondrial bioenergetic based upon previous efforts, and corroborated using both transient and steady state data. The model consists of several modified rate functions of mitochondrial bioenergetics, integrated calcium dynamics and a detailed description of the K(+)-cycle and its effect on mitochondrial bioenergetics and matrix volume regulation. Model simulations were used to fit 42 adjustable parameters to four independent experimental data sets consisting of 32 data curves. During the model development, a certain network topology had to be in place and some assumptions about uncertain or unobserved experimental factors and conditions were explicitly constrained in order to faithfully reproduce all the data sets. These realizations are discussed, and their necessity helps contribute to the collective understanding of the mitochondrial bioenergetics.

  19. Centella asiatica Attenuates D-Galactose-Induced Cognitive Impairment, Oxidative and Mitochondrial Dysfunction in Mice

    Directory of Open Access Journals (Sweden)

    Anil Kumar

    2011-01-01

    l (D-galactose. Centella asiatica also attenuated enhanced acetylcholine esterase enzyme level in D-galactose senescence mice. Present study highlights the protective effect of Centella asiatica against D-galactose induced behavioral, biochemical and mitochondrial dysfunction in mice.

  20. LHON/MELAS overlap syndrome associated with a mitochondrial MTND1 gene mutation.

    Science.gov (United States)

    Blakely, Emma L; de Silva, Rajith; King, Andrew; Schwarzer, Verena; Harrower, Tim; Dawidek, Gervase; Turnbull, Douglass M; Taylor, Robert W

    2005-05-01

    Pathogenic point mutations in the mitochondrial MTND1 gene have previously been described in association with two distinct clinical phenotypes -- Leber hereditary optic neuropathy (LHON) and mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Here we report the first heteroplasmic mitochondrial DNA (mtDNA) point mutation (3376G>A) in the MTND1 gene associated with an overlap syndrome comprising the clinical features of both LHON and MELAS. Muscle histochemistry revealed subtle mitochondrial abnormalities, while biochemical analysis showed an isolated complex I deficiency. Our findings serve to highlight the growing importance of mutations in mitochondrial complex I structural genes in MELAS and its associated overlap syndromes.

  1. Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea.

    Science.gov (United States)

    Syam Prakash, S R; Jayabaskaran, Chelliah

    2006-11-01

    In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as Ca(2+)-dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of Ca(2+). The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including Ca(2+) sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8-8.6 and 35-42 degrees C, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2-9 and 35-42 degrees C, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in Ca(2+)-mediated signaling in chickpea plants.

  2. A role of taurine in mitochondrial function

    DEFF Research Database (Denmark)

    Hansen, Svend Høime; Andersen, Mogens Larsen; Cornett, Claus;

    2010-01-01

    The mitochondrial pH gradient across the inner-membrane is stabilised by buffering of the matrix. A low-molecular mass buffer compound has to be localised in the matrix to maintain its alkaline pH value. Taurine is found ubiquitously in animal cells with concentrations in the millimolar range...... and its pKa value is determined to 9.0 (25 degrees C) and 8.6 (37 degrees C), respectively. Localisation of such a low-molecular buffer in the mitochondrial matrix, transforms the matrix into a biochemical reaction chamber for the important matrix-localised enzyme systems. Three acyl-CoA dehydrogenase...... enzymes, which are pivotal for beta-oxidation of fatty acids, are demonstrated to have optimal activity in a taurine buffer. By application of the model presented, taurine depletion caused by hyperglycemia could provide a link between mitochondrial dysfunction and diabetes....

  3. The mitochondrial connection in auditory neuropathy.

    Science.gov (United States)

    Cacace, Anthony T; Pinheiro, Joaquim M B

    2011-01-01

    'Auditory neuropathy' (AN), the term used to codify a primary degeneration of the auditory nerve, can be linked directly or indirectly to mitochondrial dysfunction. These observations are based on the expression of AN in known mitochondrial-based neurological diseases (Friedreich's ataxia, Mohr-Tranebjærg syndrome), in conditions where defects in axonal transport, protein trafficking, and fusion processes perturb and/or disrupt mitochondrial dynamics (Charcot-Marie-Tooth disease, autosomal dominant optic atrophy), in a common neonatal condition known to be toxic to mitochondria (hyperbilirubinemia), and where respiratory chain deficiencies produce reductions in oxidative phosphorylation that adversely affect peripheral auditory mechanisms. This body of evidence is solidified by data derived from temporal bone and genetic studies, biochemical, molecular biologic, behavioral, electroacoustic, and electrophysiological investigations.

  4. Mitochondrial network energetics in the heart.

    Science.gov (United States)

    Aon, Miguel A; Cortassa, Sonia

    2012-01-01

    At the core of eukaryotic aerobic life, mitochondrial function like 'hubs' in the web of energetic and redox processes in cells. In the heart, these networks-extending beyond the complex connectivity of biochemical circuit diagrams and apparent morphology-exhibit collective dynamics spanning several spatiotemporal levels of organization, from the cell, to the tissue, and the organ. The network function of mitochondria, i.e., mitochondrial network energetics, represents an advantageous behavior. Its coordinated action, under normal physiology, provides robustness despite failure in a few nodes, and improves energy supply toward a swiftly changing demand. Extensive diffuse loops, encompassing mitochondrial-cytoplasmic reaction/transport networks, control and regulate energy supply and demand in the heart. Under severe energy crises, the network behavior of mitochondria and associated glycolytic and other metabolic networks collapse, thereby triggering fatal arrhythmias.

  5. Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form.

    Science.gov (United States)

    Maldonado, Edio; Rojas, Diego A; Moreira-Ramos, Sandra; Urbina, Fabiola; Miralles, Vicente J; Solari, Aldo; Venegas, Juan

    2015-04-01

    Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.

  6. Human papillomaviruses associated with epidermodysplasia verruciformis. II. Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes.

    OpenAIRE

    Kremsdorf, D; Jablonska, S.; Favre, M.; Orth, G

    1983-01-01

    The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to t...

  7. Metagenomic Characterization and Biochemical Analysis of Cellulose-Degrading Bacterial Communities from Sheep Rumen, Termite Hindgut, Decaying Plant Materials, and Soil

    Science.gov (United States)

    2016-01-04

    characterized from the phylum to the genus level 48 22 Microbiome of the termite colony from the Appomattox River bank characterized from the phylum...localized long-term renewable energy sources. Currently, fossil fuels account for 84% of the country’s energy consumption (The World Bank 2015), which... liquid mixture of water and ethanol. Ethanol vapors are separated from the liquid portion because ethanol has a lower boiling point compared to water

  8. Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Héctor Villalobos-Duno

    Full Text Available α-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade α-1,3-glucan and allow remodeling of the cell wall, α-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal α-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (α-1,3-glucan synthase-encoding gene increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall α-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl α-1,3-glucan (SCMG, chemically modified from P. brasiliensis α-1,3-glucan, used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose, showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1Δ strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71, showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal

  9. LHON and other optic nerve atrophies: the mitochondrial connection.

    Science.gov (United States)

    Howell, Neil

    2003-01-01

    The clinical, biochemical and genetic features of Leber's hereditary optic neuropathy (LHON) are reviewed. The etiology of LHON is complex, but the primary risk factor is a mutation in one of the seven mitochondrial genes that encode subunits of respiratory chain complex I. The pathogenesis of LHON is not yet understood, but one plausible model is that increased or altered mitochondrial ROS production renders the retinal ganglion cells vulnerable to apoptotic cell death. In addition to LHON, there are a large number of other optic nerve degenerative disorders including autosomal dominant optic atrophy, the toxic/nutritional optic neuropathies and glaucoma. A review of the recent scientific literature suggests that these disorders also involve mitochondrial dysfunction or altered mitochondrial signaling pathways in their pathogenesis. This mitochondrial link provides new avenues of experimental investigation to these major causes of loss of vision.

  10. Structural, biochemical, and physiological characterization of C4 photosynthesis in species having two vastly different types of kranz anatomy in genus Suaeda (Chenopodiaceae).

    Science.gov (United States)

    Voznesenskaya, E V; Chuong, S D X; Koteyeva, N K; Franceschi, V R; Freitag, H; Edwards, G E

    2007-11-01

    C (4) species of family Chenopodiaceae, subfamily Suaedoideae have two types of Kranz anatomy in genus Suaeda, sections Salsina and Schoberia, both of which have an outer (palisade mesophyll) and an inner (Kranz) layer of chlorenchyma cells in usually semi-terete leaves. Features of Salsina (S. AEGYPTIACA, S. arcuata, S. taxifolia) and Schoberia type (S. acuminata, S. Eltonica, S. cochlearifoliA) were compared to C (3) type S. Heterophylla. In Salsina type, two layers of chlorenchyma at the leaf periphery surround water-storage tissue in which the vascular bundles are embedded. In leaves of the Schoberia type, enlarged water-storage hypodermal cells surround two layers of chlorenchyma tissue, with the latter surrounding the vascular bundles. The chloroplasts in Kranz cells are located in the centripetal position in Salsina type and in the centrifugal position in the Schoberia type. Western blots on C (4) acid decarboxylases show that both Kranz forms are NAD-malic enzyme (NAD-ME) type C (4) species. Transmission electron microscopy shows that mesophyll cells have chloroplasts with reduced grana, while Kranz cells have chloroplasts with well-developed grana and large, specialized mitochondria, characteristic of NAD-ME type C (4) chenopods. In both C (4) types, phosphoenolpyruvate carboxylase is localized in the palisade mesophyll, and Rubisco and mitochondrial NAD-ME are localized in Kranz cells, where starch is mainly stored. The C (3) species S. heterophylla has Brezia type isolateral leaf structure, with several layers of Rubisco-containing chlorenchyma. Photosynthetic response curv