WorldWideScience

Sample records for biochemical characterization mitochondrial

  1. Biochemical characterization of the deafness-associated mitochondrial tRNASer(UCN) A7445G mutation in osteosarcoma cell cybrids

    International Nuclear Information System (INIS)

    The deafness-associated A7445G mutation in the precursor of mitochondrial tRNASer(UCN) has been identified in several pedigrees from different ethnic backgrounds. To determine the role of nuclear background in the biochemical manifestation associated with the A7445G mutation, we performed a biochemical characterization of this mutation using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a New Zealand family into human osteosarcoma mtDNA-less (ρ0) cells. Compared with three control cybrids, three cybrids derived from an affected matrilineal relative carrying the homoplasmic A7445G mutation exhibited ∼38-57% decrease in the steady-state level of tRNASer(UCN), which is less reduced levels than in lymphoblastoid cells in the previous study. Furthermore, ∼22% reduction in the level of aminoacylation of tRNASer(UCN) was observed in the mutant cybrid cells. Interestingly, ∼60-63% decrease of steady-state level of ND6 gene, which belongs to the same precursor as that of tRNASer(UCN), in cybrid cell lines carrying the A7445G mutation, is more than that observed in lymphoblastoid cells. These observations strongly point out a mechanistic link between the processing defect of the tRNASer(UCN) precursor and decreased stability of ND6 mRNA precursor. These results also imply the influence of nuclear background on the biochemical phenotype associated with the A7445G mutation

  2. Biochemical diagnosis of mitochondrial disorders

    NARCIS (Netherlands)

    Rodenburg, R.J.T.

    2011-01-01

    Establishing a diagnosis in patients with a suspected mitochondrial disorder is often a challenge. Both knowledge of the clinical spectrum of mitochondrial disorders and the number of identified disease-causing molecular genetic defects are continuously expanding. The diagnostic examination of patie

  3. The mitochondrial 60-kDa heat shock protein in marine invertebrates: biochemical purification and molecular characterization

    Science.gov (United States)

    Choresh, Omer; Loya, Yossi; Müller, Werner E.G.; Wiedenmann, Jörg; Azem, Abdussalam

    2004-01-01

    Sessile marine invertebrates undergo constant direct exposure to the surrounding environmental conditions, including local and global environmental fluctuations that may lead to fatal protein damage. Induction of heat shock proteins (Hsps) constitutes an important defense mechanism that protects these organisms from deleterious stress conditions. In a previous study, we reported the immunological detection of a 60-kDa Hsp (Hsp60) in the sea anemone Anemonia viridis (formerly called Anemonia sulcata) and studied its expression under a variety of stress conditions. In the present study, we show that the sponge Tetilla sp. from tidal habitats with a highly variable temperature regime is characterized by an increased level of Hsp60. Moreover, we show the expression of Hsp60 in various species among Porifera and Cnidaria, suggesting a general importance of this protein among marine invertebrates. We further cloned the hsp60 gene from A viridis, using a combination of conventional protein isolation methods and screening of a complementary deoxyribonucleic acid library by polymerase chain reaction. The cloned sequence (1764 bp) encodes for a protein of 62.8 kDa (588 amino acids). The 62.8-kDa protein, which contains an amino terminal extension that may serve as a mitochondrial targeting signal, shares a significant identity with mitochondrial Hsp60s from several animals but less identity with Hsp60s from either bacteria or plants. PMID:15270076

  4. Unraveling biochemical pathways affected by mitochondrial dysfunctions using metabolomic approaches.

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  6. [Mitochondrial diseases in children including Leigh syndrome--biochemical and molecular background].

    Science.gov (United States)

    Pronicka, Ewa; Piekutowska-Abramczuk, Dorota; Pronicki, Maciej

    2008-01-01

    Mitochondrial diseases in children are more frequently caused by mutations in nuclear DNA then in mtDNA. Special clinical phenotypes are associated with the mutations in SURF1 gene, in SCO2 gene and with mtDNA depletion syndromes. Leigh syndrome is the most common clinical presentation of various mitochondrial disorders during childhood. Elevation of lactate in blood, cerebrospinal fluid and urine is a simple biochemical marker of mitochondrial disorders but its specificity and sensitivity are low. Biochemical investigation of muscle biopsy and search for mitochondrial mutations remain a gold standard in the diagnosis. The standarized diagnostic criteria to establish level of diagnostic certainty (possible, probable, definite) are proposed to be used in practice; these include clinical features, neuroimaging and muscle biopsy investigations. Further research directions to improve our understanding of mitochondrial pathologies in children are suggested. PMID:18807927

  7. Stroke-like episodes in familial mitochondrial encephalomyopathy: clinical and biochemical aspects.

    Science.gov (United States)

    Damian, M S; Reichmann, H; Schütz, H J; Dorndorf, W; Schachenmayr, W

    1991-04-01

    Acute episodes of focal neurological dysfunction are a well-recognized complication of the mitochondrial encephalomyopathies. Because of rapid remission, biochemical tests and other diagnostic procedures are mostly performed after the acute phase. We report the case of a patient suffering from mitochondrial disease manifesting primarily with seizures, progressive deafness and dementia, who experienced multiple stroke-like episodes. Other members of the family with evidence of mitochondrial dysfunction are presented briefly. EEG and biochemical findings in the acute stage are correlated with clinical symptoms, showing characteristics distinct from the chronic illness. The possible involvement of dietary factors in the provocation of stroke-like episodes is discussed and regulation of glucose intake suggested as a strategy in the prevention of stroke-like episodes. PMID:1906933

  8. Biochemical and proteomic characterization of alkaptonuric chondrocytes.

    Science.gov (United States)

    Braconi, Daniela; Bernardini, Giulia; Bianchini, Claretta; Laschi, Marcella; Millucci, Lia; Amato, Loredana; Tinti, Laura; Serchi, Tommaso; Chellini, Federico; Spreafico, Adriano; Santucci, Annalisa

    2012-09-01

    Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as "black" AKU chondrocytes, while those coming from the white portion were referred to as "white" AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both "white" and "black" AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in "black" AKU chondrocytes. PMID:22213341

  9. Leigh syndrome in Drosophila melanogaster: morphological and biochemical characterization of Surf1 post-transcriptional silencing.

    Science.gov (United States)

    Da-Rè, Caterina; von Stockum, Sophia; Biscontin, Alberto; Millino, Caterina; Cisotto, Paola; Zordan, Mauro A; Zeviani, Massimo; Bernardi, Paolo; De Pittà, Cristiano; Costa, Rodolfo

    2014-10-17

    Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LS(Surf1) patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R(+) cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS. PMID:25164807

  10. Biochemical and histological characterization of tomato mutants

    Directory of Open Access Journals (Sweden)

    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

  11. Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency

    DEFF Research Database (Denmark)

    Olpin, S E; Clark, S; Andresen, B S;

    2005-01-01

    General mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl...... from an early age. We present biochemical, clinical and mutation data in 9 patients spanning the full spectrum of disease. Fibroblast acylcarnitine profiling shows good correlation with clinical phenotype using the ratio C18(OH)/(C14(OH)+C12(OH)). This ratio shows a gradation of values, from high in...... four patients with severe neonatal disease (2.5+/-0.8), to low in two neuromyopathic patients (0.35, 0.2). Fibroblast fatty acid oxidation flux assays also show correlation with the patient phenotype, when expressed either as percentage residual activity with palmitate or as a ratio of percentage...

  12. Murine interleukin 2 receptor. IV. Biochemical characterization

    International Nuclear Information System (INIS)

    The IL 2 receptor isolated from the IL 2-dependent CTL-L cell line was subjected to biochemical analysis. Pulse-chase and tunicamycin studies, as well as digestion with the endoglycosidases, Endo-F and Endo-H, of 35S-methionine-labeled IL 2 receptors suggested a single protein pecursor of 32,000 (p32) daltons. The p32 precursor was rapidly processed by addition of high-mannose-containing core N-linked sugars to intracytoplasmic precursor intermediates of 38,000 (p38) and 40,000 (p40) daltons, which undergo further processing to yield a mature surface receptor with heterogeneous apparent m.w. of 52,000 to 65,000 (p58). Two-dimensional gel studies indicated that p58 exhibited broad charge heterogeneity between pH 4.6 and 6.3. Endo-F digestions of p58 shifted the isoelectric focus point to a more basic 5.5 to 7.4. This considerable charge heterogeneity is consistent with the possibility that other post-translational modifications to the mouse IL 2 receptor occur besides addition of complex N-linked glycans. Immunoprecipitations of the IL 2 receptor from surface iodinated cells also revealed an additional band at 110,000 (p110) daltons. IEF vs SDS-PAGE two-dimensional gel studies demonstrated that p110 also had an isoelectric focus point identical to p58. Western blot studies with an anti-IL 2 receptor monoclonal antibody (7D4) demonstrates that p38, p40, p58, and p110 each expressed the epitope recognized by this antibody. Thus, it is likely that p110 is not a unique molecule that coprecipitates with IL 2 receptor. Western blot analysis of mitogen-stimulated T and B lymphocytes also revealed bands similar to p58 and p110, although these bands had an average apparent m.w. 3000 and 6000 less than those seen for CTL-L cells

  13. Medicinal plants: production and biochemical characterization

    International Nuclear Information System (INIS)

    Recent advances in the area of biotechnology offer some possibility for the development of new technologies for the conservation, characterization and mass production of medicinal plant species, (i.e. in vitro cell culture techniques for the mass production of sterile, consistent, standardized medicinal plant materials). This paper discussed the following subjects - plant tissue culture, de novo shoot organogenesis, de novo root organogenesis, somatic embryogenesis, large scale propagation in bioreactors and discovery of unique biomolecules

  14. Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Han-Pil Choi

    Full Text Available The functional characterization of Open Reading Frames (ORFs from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP cyclohydrolase (HP0959, an ATPase (HP1079, an adenosine deaminase (HP0267, a phosphodiesterase (HP1042, an aminopeptidase (HP1037, and new substrates were characterized for a peptidoglycan deacetylase (HP0310. Generally, characterized enzymes were active at acidic to neutral pH (4.0-7.5 with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.

  15. Biochemical characterization of the maltokinase from Mycobacterium bovis BCG

    Directory of Open Access Journals (Sweden)

    Lamosa Pedro

    2010-05-01

    Full Text Available Abstract Background Maltose-1-phosphate was detected in Mycobacterium bovis BCG extracts in the 1960's but a maltose-1-phosphate synthetase (maltokinase, Mak was only much later purified from Actinoplanes missouriensis, allowing the identification of the mak gene. Recently, this metabolite was proposed to be the intermediate in a pathway linking trehalose with the synthesis of glycogen in M. smegmatis. Although the M. tuberculosis H37Rv mak gene (Rv0127 was considered essential for growth, no mycobacterial Mak has, to date, been characterized. Results The sequence of the Mak from M. bovis BCG was identical to that from M. tuberculosis strains (99-100% amino acid identity. The enzyme was dependent on maltose and ATP, although GTP and UTP could be used to produce maltose-1-phosphate, which we identified by TLC and characterized by NMR. The Km for maltose was 2.52 ± 0.40 mM and 0.74 ± 0.12 mM for ATP; the Vmax was 21.05 ± 0.89 μmol/min.mg-1. Divalent cations were required for activity and Mg2+ was the best activator. The enzyme was a monomer in solution, had maximal activity at 60°C, between pH 7 and 9 (at 37°C and was unstable on ice and upon freeze/thawing. The addition of 50 mM NaCl markedly enhanced Mak stability. Conclusions The unknown role of maltokinases in mycobacterial metabolism and the lack of biochemical data led us to express the mak gene from M. bovis BCG for biochemical characterization. This is the first mycobacterial Mak to be characterized and its properties represent essential knowledge towards deeper understanding of mycobacterial physiology. Since Mak may be a potential drug target in M. tuberculosis, its high-level production and purification in bioactive form provide important tools for further functional and structural studies.

  16. Biochemical and mechanical characterization of Nereis worm jaws

    Science.gov (United States)

    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

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

    OpenAIRE

    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; L. P. van den Heuvel

    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...

  18. Biochemical characterization of the GM2 gangliosidosis B1 variant

    Directory of Open Access Journals (Sweden)

    J.C. Tutor

    2004-06-01

    Full Text Available The deficiency of the A isoenzyme of ß-hexosaminidase (Hex produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-ß-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphaß heterodimer Hex A means that its activation energy (41.8 kJ/mol is significantly lower than that of the ßß homodimer Hex B (75.1 kJ/mol; however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

  19. 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

  20. Biochemical Characterization of Prion Strains in Bank Voles

    Directory of Open Access Journals (Sweden)

    Romolo Nonno

    2013-07-01

    Full Text Available Prions exist as different strains exhibiting distinct disease phenotypes. Currently, the identification of prion strains is still based on biological strain typing in rodents. However, it has been shown that prion strains may be associated with distinct PrPSc biochemical types. Taking advantage of the availability of several prion strains adapted to a novel rodent model, the bank vole, we investigated if any prion strain was actually associated with distinctive PrPSc biochemical characteristics and if it was possible to univocally identify strains through PrPSc biochemical phenotypes. We selected six different vole-adapted strains (three human-derived and three animal-derived and analyzed PrPSc from individual voles by epitope mapping of protease resistant core of PrPSc (PrPres and by conformational stability and solubility assay. Overall, we discriminated five out of six prion strains, while two different scrapie strains showed identical PrPSc types. Our results suggest that the biochemical strain typing approach here proposed was highly discriminative, although by itself it did not allow us to identify all prion strains analyzed.

  1. Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei

    OpenAIRE

    Singha, Ujjal K; PEPRAH, EMMANUEL; Williams, Shuntae; Walker, Robert; Saha, Lipi; Chaudhuri, Minu

    2008-01-01

    Mitochondrial protein translocation machinery in the kinetoplastid parasites, like Trypanosoma brucei, has been characterized poorly. In T. brucei genome data base, one homolog for a protein translocator of mitochondrial inner membrane (Tim) has been found, which is closely related to Tim17 from other species. The T. brucei Tim17 (TbTim17) has a molecular mass 16.2 kDa and it possesses four characteristic transmembrane domains. The protein is localized in the mitochondrial inner membrane. The...

  2. Mitochondrial cofactors in experimental Huntington's disease: behavioral, biochemical and histological evaluation.

    Science.gov (United States)

    Mehrotra, Arpit; Sandhir, Rajat

    2014-03-15

    The present study was carried out to evaluate the beneficial effect of mitochondrial cofactors; alpha-lipoic acid (ALA) and acetyl-l-carnitine (ALCAR) in 3-nitropropionic acid (3-NP) induced experimental model of Huntington's disease (HD). HD was developed by administering sub-chronic doses of 3-NP, intraperitoneally, twice daily for 17 days. The animals were assessed for their behavioral performance in terms of motor (spontaneous locomotor activity, narrow beam walk test, footprint analysis and rotarod test) and cognitive (elevated plus maze and T-maze tests) functions. 3-NP treated animals showed impairment in motor coordination such as decreased stride length, increased distance between inner toes, and increased gait angle. Increased transfer latency on elevated plus maze and T-maze tasks revealed cognition deficits in 3-NP treated animals. Increased lipid peroxidation and concomitant decrease in thiol levels were also observed. 3-NP administration also induced histopathological changes in terms of enhanced striatal lesion volume, presence of pyknotic nuclei and astrogliosis. However, combined supplementation with ALA+ALCAR to 3-NP administered animals for 21 days was able to efficiently improve behavioral deficits, attenuate oxidative stress and histological changes, suggesting a putative role of these two supplements if given together in ameliorating 3-NP induced impairments and thus could be engaged in managing HD. PMID:24393741

  3. Biochemical Characterization of Rice Somaclones Resistant to Blast

    OpenAIRE

    Antar N. El-Banna; Ismael A. Khatab

    2012-01-01

    The aim of the present study was to produce rice somaclones resistant to P. oryza that could be useful to improve rice plants against blast disease and use them in the future breeding programs. In addition, to detect biochemical changes among somaclones and their original cultivars. Mature embryo-derived calli of the three susceptible rice varieties, Sakha 101, Sakha 104 and Riho were stressed with different concentrations of fungal toxin filtrate. Blast-resistant lines of rice were developed...

  4. [The study of biochemical mechanisms of mitochondrial dysfunction in rats' hepatocytes during experimental hyperhomocysteinemia].

    Science.gov (United States)

    Medvedev, D V; Zvyagina, V I

    2016-01-01

    Methionine is an essential proteinogenic amino acid found in many foods. During its metabolism homocysteine is formed. With elevated level of homocysteine in the blood--hyperhomocysteinemia--increased risk of developing certain diseases, such as non-alcoholic fatty liver disease, is associated. There is evidence that the homocysteine is able to reduce the effect of nitric oxide and induce mitochondrial dysfunction. The present study investigates the relationship of the functional state of the liver cells mitochondria and the level of nitric oxide metabolites in them in experimental hyperhomocysteinemia caused by excessive intake of methionine. The experiment was conducted on 17 male Wistar rats with an initial weight of 220-270 g, rats were divided into 2 groups. A 25%. suspension of methionine was administered (in a dose of 1.5 g of methionine per kg body weight) two times a day for 21 days intragastrically (by gavage) to rats of the first group (n=9) while instead of drinking water animals received a 1% aqueous solution of methionine. Drinks daily volume of methionine solution was 17.2 [15.5; 18.1] ml. In the experiment 8 animals were used, in which severe hyperhomocysteinemia (> 100 mmol/l) was developed. The second group (n = 8) served as a control. These rats were administered suspension base containing no methionine (10% Tween-80, 1% starch, 89% water). The total homocysteine concentration was measured in blood serum by ELISA. In the suspension of liver mitochondria total protein was measured by Lowry method; the concentration of NO metabolites by screening method; succinate dehydrogenase activity--under the reaction of hexacyanoferrate (III) potassium reduction; lactate dehydrogenase activity--by decrease of NADH concentration in the reaction of pyruvate's reduction; activity of H(+)-ATPase--by measuring the inorganic phosphate; superoxide dismutase--by inhibition of quercetin auto-oxidation, the level of Ca(2+)--by reaction with Arsenazo III. Oxidative

  5. Leveraging genomic approaches to characterize mitochondrial RNA biology

    OpenAIRE

    Wolf, Ashley Robin

    2014-01-01

    Transcription and translation of mammalian mitochondrial DNA (mtDNA) occurs within the mitochondrial matrix to produce oxidative phosphorylation subunits required for efficient energy production. These mtDNA-encoded subunits complex with mitochondrial-localized, nuclear-encoded subunits to form the respiratory chain, and aberrant production or function of these subunits can cause devastating human disease. In addition to 13 oxidative phosphorylation subunits, mtDNA encodes 2 rRNAs and 22 tRNA...

  6. Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis

    Directory of Open Access Journals (Sweden)

    Cherkaoui-Malki Mustapha

    2008-09-01

    Full Text Available Abstract Background The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies, is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30, a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis, a hibernating rodent adapted to extreme diet and environmental conditions. Results Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced. Conclusion This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

  7. Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori

    OpenAIRE

    Han-Pil Choi; Silvia Juarez; Sergio Ciordia; Marisol Fernandez; Rafael Bargiela; Albar, Juan P; Varun Mazumdar; Anton, Brian P.; Simon Kasif; Manuel Ferrer; Martin Steffen

    2013-01-01

    The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem....

  8. Enzymatic and biochemical characterization of Bungarus sindanus snake venom acetylcholinesterase

    Directory of Open Access Journals (Sweden)

    M Ahmed

    2012-01-01

    Full Text Available This study analyses venom from the elapid krait snake Bungarus sindanus, which contains a high level of acetylcholinesterase (AChE activity. The enzyme showed optimum activity at alkaline pH (8.5 and 45ºC. Krait venom AChE was inhibited by substrate. Inhibition was significantly reduced by using a high ionic strength buffer; low ionic strength buffer (10 mM PO4 pH 7.5 inhibited the enzyme by 1. 5mM AcSCh, while high ionic strength buffer (62 mM PO4 pH 7.5 inhibited it by 1 mM AcSCh. Venom acetylcholinesterase was also found to be thermally stable at 45ºC; it only lost 5% of its activity after incubation at 45ºC for 40 minutes. The Michaelis-Menten constant (Km for acetylthiocholine iodide hydrolysis was found to be 0.068 mM. Krait venom acetylcholinesterase was also inhibited by ZnCl2, CdCl2, and HgCl2 in a concentrationdependent manner. Due to the elevated levels of AChE with high catalytic activity and because it is more stable than any other sources, Bungarus sindanus venom is highly valuable for biochemical studies of this enzyme.

  9. 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.

  10. Myoclonus in mitochondrial disorders.

    Science.gov (United States)

    Mancuso, Michelangelo; Orsucci, Daniele; Angelini, Corrado; Bertini, Enrico; Catteruccia, Michela; Pegoraro, Elena; Carelli, Valerio; Valentino, Maria L; Comi, Giacomo P; Minetti, Carlo; Bruno, Claudio; Moggio, Maurizio; Ienco, Elena Caldarazzo; Mongini, Tiziana; Vercelli, Liliana; Primiano, Guido; Servidei, Serenella; Tonin, Paola; Scarpelli, Mauro; Toscano, Antonio; Musumeci, Olimpia; Moroni, Isabella; Uziel, Graziella; Santorelli, Filippo M; Nesti, Claudia; Filosto, Massimiliano; Lamperti, Costanza; Zeviani, Massimo; Siciliano, Gabriele

    2014-05-01

    Myoclonus is a possible manifestation of mitochondrial disorders, and its presence is considered, in association with epilepsy and the ragged red fibers, pivotal for the syndromic diagnosis of MERRF (myoclonic epilepsy with ragged red fibers). However, its prevalence in mitochondrial diseases is not known. The aims of this study are the evaluation of the prevalence of myoclonus in a big cohort of mitochondrial patients and the clinical characterization of these subjects. Based on the database of the "Nation-wide Italian Collaborative Network of Mitochondrial Diseases," we reviewed the clinical and molecular data of mitochondrial patients with myoclonus among their clinical features. Myoclonus is a rather uncommon clinical feature of mitochondrial diseases (3.6% of 1,086 patients registered in our database). It is not strictly linked to a specific genotype or phenotype, and only 1 of 3 patients with MERRF harbors the 8344A>G mutation (frequently labeled as "the MERRF mutation"). Finally, myoclonus is not inextricably linked to epilepsy in MERRF patients, but more to cerebellar ataxia. In a myoclonic patient, evidences of mitochondrial dysfunction must be investigated, even though myoclonus is not a common sign of mitochondriopathy. Clinical, histological, and biochemical data may predict the finding of a mitochondrial or nuclear DNA mutation. Finally, this study reinforces the notion that myoclonus is not inextricably linked to epilepsy in MERRF patients, and therefore the term "myoclonic epilepsy" seems inadequate and potentially misleading. PMID:24510442

  11. Biochemical characterization of rab proteins from Bombyx mori.

    Science.gov (United States)

    Uno, Tomohide; Moriwaki, Tsubasa; Nakamura, Masahiko; Matsubara, Mamoru; Yamagata, Hiroshi; Kanamaru, Kengo; Takagi, Michihiro

    2009-02-01

    The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc. PMID:18949803

  12. 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

  13. 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. PMID:27146408

  14. GENETIC CHARACTERIZATION OF ROMANIAN CATTLE BREEDS USING BIOCHEMICAL MARKERS

    Directory of Open Access Journals (Sweden)

    REBEDEA MARIANA

    2007-01-01

    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.

  15. 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.

  16. Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K m of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K i was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 deg C and pH 9.0, and did not require mono- or divalent cations for its activity

  17. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    Science.gov (United States)

    Tomazett, Mariana Vieira; Zanoelo, Fabiana Fonseca; Bailão, Elisa Flávia Cardoso; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

    2016-01-01

    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. PMID:27560991

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

    OpenAIRE

    Janiffe Peres de Oliveira; Jonny Everson Scherwinski-Pereira

    2016-01-01

    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 ...

  19. 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...... intake' 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...

  20. 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.

  1. 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.

  2. Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures

    Directory of Open Access Journals (Sweden)

    Mainil Jacques

    2005-12-01

    Full Text Available Abstract Background Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. Results Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein as well as an epithelial cytoskeleton component (cytokeratin 18. However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker. Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1–2 mM or using a glucose-deprived culture medium. Conclusion The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.

  3. ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF PHOSPHATE SOLUBILIZING MICROBES

    Directory of Open Access Journals (Sweden)

    MAHANTESH P AND PATIL CS

    2011-07-01

    Full Text Available Phosphorus (P is one of the essential macronutrients for plant growth and reproduction. Plants acquire Pfrom the soil solution as phosphate anions. However, phosphate anions are extremely reactive and may be immobilizedthrough precipitation with cations such as Ca2+, Mg2+, Fe3+ and Al3+, depending on the particular properties of thesoil and as a result, the phosphate is highly insoluble and unavailable to plants. Application of phosphate-solubilizingfungi (PSF has been added as fertilizer to increase P uptake and plant growth. The improvement of soil fertility is oneof the most common strategies to increase agricultural production. Maintaining high levels of available nitrogen (N andphosphorus (P, the two most limiting nutrients in soil, remains being a challenge.Major researches on biofertilizer have concentrated on understanding and improving N2 fixation. However, it is knownthat every aspect of the process of nodule formation is limited by the availability of P. Legumes like alfalfa and clovershow a high positive response to P supplementation (Gyaneshwar et al., 2002, but most of the supplemented Pbecome unavailable when its reacts with soil components.Many soil microorganisms are able to solubilize this unavailable P through their metabolic activities exudating organicacids, which directly dissolve the rock phosphate, or chelating calcium ions that release P to the solution. Production ofmicrobial metabolites results in a decrease in soil pH, which probably plays an important role in the solubilization (Abd-Alla, 1994.Phosphate solubilizing bacteria (PSB were isolated from the area around Bidar region and screened on the basis oftheir solubilization of inorganic tricalcium phosphate in liquid cultures. Ten strains that had higher solubilization potentialwere selected, and they characterized

  4. 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.

  5. 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.

  6. Biochemical characterization of Cdk2-Speedy/Ringo A2

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2005-09-01

    Full Text Available Abstract Background Normal cell cycle progression requires the precise activation and inactivation of cyclin-dependent protein kinases (CDKs, which consist of a CDK and a cyclin subunit. A novel cell cycle regulator called Speedy/Ringo shows no sequence similarity to cyclins, yet can directly bind to and activate CDKs. Speedy/Ringo proteins, which bind to and activate Cdc2 and Cdk2 in vitro, are required for the G2 to M transition during Xenopus oocyte maturation and for normal S-phase entry in cultured human cells. Results We have characterized the substrate specificity and enzymatic activity of human Cdk2-Speedy/Ringo A2 in order to gain insights into the possible functions of this complex. In contrast to Cdk2-cyclin A, which has a well-defined consensus target site ((S/TPX(K/R that strongly favors substrates containing a lysine at the +3 position of substrates, Cdk2-Speedy/Ringo A2 displayed a broad substrate specificity at this position. Consequently, Cdk2-Ringo/Speedy A2 phosphorylated optimal Cdk2 substrates such as histone H1 and a KSPRK peptide poorly, only ~0.08% as well as Cdk2-cyclin A, but non-canonical Cdk2 substrates such as a KSPRY peptide relatively well, with an efficiency of ~80% compared to Cdk2-cyclin A. Cdk2-Speedy/Ringo A2 also phosphorylated authentic Cdk2 substrates, such as Cdc25 proteins, which contain non-canonical CDK phosphorylation sites, nearly as well as Cdk2-cyclin A. Phosphopeptide mapping indicated that Cdk2-Speedy/Ringo A2 and Cdk2-cyclin A phosphorylate distinct subsets of sites on Cdc25 proteins. Thus, the low activity that Cdk2-Speedy/Ringo A2 displays when assayed on conventional Cdk2 substrates may significantly underestimate the potential physiological importance of Cdk2-Speedy/Ringo A2 in phosphorylating key subsets of Cdk2 substrates. Unlike Cdk2-cyclin A, whose activity depends strongly on activating phosphorylation of Cdk2 on Thr-160, neither the overall catalytic activity nor the substrate

  7. Induction and Characterization of Mitochondrial DNA Mutants in Chlamydomonas Reinhardtii

    OpenAIRE

    Matagne, René-Fernand; Michel-Wolwertz, M.R.; Munaut, Carine; Duyckaerts, Claire; Sluse, Francis

    1989-01-01

    In addition to lethal minute colony mutations which correspond to loss of mitochondrial DNA, acriflavin induces in Chlamydomonas reinhardtii a low percentage of cells that grow in the light but do not divide under heterotrophic conditions. Two such obligate photoautotrophic mutants were shown to lack the cyanide-sensitive cytochrome pathway of the respiration and to have a reduced cytochrome c oxidase activity. In crosses to wild type, the mutations are transmitted almost exclusively from the...

  8. Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

    OpenAIRE

    Bonvin, Julie; Aponte, Raphael A.; Marcantonio, Maria; Singh, Sasha; Christendat, Dinesh; Turnbull, Joanne L.

    2006-01-01

    A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full-length PD are compared with one of these products, an N-terminally truncated protein variant (Δ19PD) also expressed recombinantly in E. coli. Bo...

  9. Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis

    OpenAIRE

    Bouacem, K.; Bouanane-Darenfed, A.; Laribi-Habchi, H.; Ben Elhoul, M.; Hmida-Sayari, A.; Hacene, H.; Ollivier, Bernard; Fardeau, Marie-Laure; Jaouadi, B.; Bejar, S.

    2015-01-01

    Caldicoprobacter guelmensis isolated from the hydrothermal hot spring of Guelma (Algeria) produced high amounts of extracellular thermostable serine alkaline protease (called SAPCG) (23,000 U/mL). The latter was purified by ammonium sulphate precipitation, UNO Q-6 FPLC and Zorbex PSM 300 HPLC, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer...

  10. Raman Spectroscopy Enables Noninvasive Biochemical Characterization and Identification of the Stage of Healing of a Wound

    OpenAIRE

    Jain, Rishabh; Calderon, Diego; Kierski, Patricia R.; Schurr, Michael J.; Czuprynski, Charles J.; Murphy, Christopher J.; McAnulty, Jonathan F.; Abbott, Nicholas L.

    2014-01-01

    Accurate and rapid assessment of the healing status of a wound in a simple and noninvasive manner would enable clinicians to diagnose wounds in real time and promptly adjust treatments to hasten the resolution of nonhealing wounds. Histologic and biochemical characterization of biopsied wound tissue, which is currently the only reliable method for wound assessment, is invasive, complex to interpret, and slow. Here we demonstrate the use of Raman microspectroscopy coupled with multivariate spe...

  11. Biochemical characterization of Extracellular Polymeric Substances extracted from an intertidal mudflat using a cation exchange resin.

    OpenAIRE

    Pierre, Guillaume; Graber, Marianne; Orvain, Francis; Dupuy, Christine; Maugard, Thierry

    2010-01-01

    The biochemical characterization of Extracellular Polymeric Substances (EPS) excreted in a European intertidal mudflat (Marennes-Oléron Bay) was performed. Experiments were carried out for the first time in situ, by using an improved extraction recently developed. This innovative procedure, using a cation exchange resin (Dowex), allows separating precisely different fractions of EPS, especially pure bound EPS. Moreover, it avoids the contamination of EPS fractions by residual and intracellula...

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

    OpenAIRE

    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 shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal a-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the ...

  13. Characterization of the mitochondrial genome of Amolops tuberodepressus (Anura: Ranidae).

    Science.gov (United States)

    Zhang, Chaohua; Xia, Yun; Zeng, Xiaomao

    2016-07-01

    Amolops tuberodepressus is a vulnerable torrent frog, and only know distributed in the Wuliang Mountain in southwestern China. In the present study, the mitochondrial DNA (mtDNA) sequence of A. tuberodepressus was determined. The genome was 18 348 bp in length, and it contained 37 genes (13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs), one partial control region and one light strand replication origin. The gene rearrangement was observed within the WANCY tRNA gene cluster region, which similar to other Amolops species. In this paper, we utilized 13 protein-coding genes of A. tuberodepressus and other 10 closely ranid species to construct the species phylogenetic tree to verify the A. tuberodepressus was accuracy. PMID:26153745

  14. Mitochondrial DNA characterization of two Partamona species (Hymenoptera, Apidae, Meliponini) by PCR+RFLP and sequencing

    OpenAIRE

    Magalhães Brito, Rute; Cristina Arias, Maria

    2005-01-01

    We characterized the mitochondrial DNA of two stingless bee species of the genus Partamona. Partial restriction maps were obtained based on digestion of PCR amplified fragments with 8 restriction enzymes. Using Melipona bicolor mtDNA sequence as a model, we were able to amplify 12120 bp of P. mulata and 10300 bp of P. helleri, about 65.5% and 55.7% of their mitochondrial genome, respectively. The digestion assays showed 16 restriction sites for P. mulata and 20 for P. helleri, some of which w...

  15. Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase.

    Science.gov (United States)

    Jackson, R G; Lim, E K; Li, Y; Kowalczyk, M; Sandberg, G; Hoggett, J; Ashford, D A; Bowles, D J

    2001-02-01

    Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDP-glucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDP-glucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis. PMID:11042207

  16. Molecular and biochemical evidence on the protection of cardiomyocytes from phosphine-induced oxidative stress, mitochondrial dysfunction and apoptosis by acetyl-l-carnitine.

    Science.gov (United States)

    Baghaei, Amir; Solgi, Reza; Jafari, Abbas; Abdolghaffari, Amir Hossein; Golaghaei, Alireza; Asghari, Mohammad Hossein; Baeeri, Maryam; Ostad, Seyed Nasser; Sharifzadeh, Mohammad; Abdollahi, Mohammad

    2016-03-01

    The aim of the present study was to investigate the efficacy of acetyl-l-carnitine (ALCAR) on pathologic changes of mitochondrial respiratory chain activity, ATP production, oxidative stress, and cellular apoptosis/necrosis induced by aluminum phosphide (AlP) poisoning. The study groups included: the Sham that received almond oil only; the AlP that received oral LD50 dose of aluminum; the AC-100, AC-200, and AC-300 which received concurrent oral LD50 dose of AlP and single 100, 200, and 300mg/kg of ALCAR by intraperitoneal injection. After 24h, the rats were sacrificed; the heart and blood sample were taken for measurement of biochemical and mitochondrial factors. The results specified that ALCAR significantly attenuated the oxidative stress (elevated ROS and plasma iron levels) caused by AlP poisoning. ALCAR also increased the activity of cytochrome oxidase, which in turn amplified ATP production. Furthermore, flow cytometric assays and caspase activity indicated that ALCAR prohibited AlP-induced apoptosis in cardiomyocytes. PMID:26773361

  17. Isolation, molecular and biochemical characterization of oil degrading bacteria from contaminated soil at an oil refinery

    International Nuclear Information System (INIS)

    Biodegradation using microorganisms is considered to be cost-effective and environmentally friendly treatment of oil-contaminated sites. Oil-biodegrading bacterial strains were isolated, identified and characterized from oil contaminated soil samples at oil refinery in Zarqa (Jordan). Thirty four bacterial isolates were grown on mineral salt media supplemented with crude oil, but 16 showed positive biodegradation of diesel. All the 34 bacterial isolates were characterized at the molecular and bio-chemical levels, and showed positive polymerase chain reaction (PCR) amplification product size of 1500 bp when 16s rDNA bacterial universal primers were used. Eighteen bacterial isolates showed positive PCR amplification product size of 150 bp specific for the genus Pseudomonas and 3 bacterial isolates showed positive amplification product size of 1500 bp specific for the genus Acinetobacter. Biochemical and physiological characterization performed on the 34 bacterial isolates revealed the presence of oil biodegrading bacterial genera and species of Pseudomonas Acidovorans, P. aeruginosa, P. vesicularis, Acinetobacter calcoaceticus, Ac. lowffii, Micro-ococcus luteus, M. varians, M. lylae, M. roseus, Alcaligenes denitrificians, Bacillus megaterium, Comamonas sp., Moralxella sp., Bordetella sp., P. putida, P. stutzeri and P. mallei. (au)

  18. Cyto-palynological, Biochemical and Molecular Characterization of Original and Induced Mutants of Garden Chrysanthemum

    International Nuclear Information System (INIS)

    A large number of new somatic flower color/type mutants have been evolved by induced mutations in different ornamental plants. Few reports are available on the systematic work being done on the comparative analysis of the original and mutant cultivars. This paper reports the result of comparative analysis on cyto-palynological, biochemical and molecular characters of original and mutant cultivars for a better understanding of the exact mechanism involved in the origin and evolution of flower color mutations. Cultivar identification and cultivar relatedness are important issues for horticultural breeders. Proper characterization and identification of new mutant cultivars is extremely important to protect plant breeder's rights for commercial exploitation. (author)

  19. Alterations in mitochondrial number and function in Alzheimer's disease fibroblasts.

    Science.gov (United States)

    Gray, Nora E; Quinn, Joseph F

    2015-10-01

    Mitochondrial dysfunction is observed in brains of Alzheimer's Disease patients as well as many rodent model systems including those modeling mutations in preseinilin 1 (PSEN1). The aim of our study was to characterize mitochondrial function and number in fibroblasts from AD patients with PSEN1 mutations. We used biochemical assays, metabolic profiling and fluorescent labeling to assess mitochondrial number and function in fibroblasts from three AD patients compared to fibroblasts from three controls. The mutant AD fibroblasts had increased Aβ42 relative to controls along with reduction in ATP, basal and maximal mitochondrial respiration as well as impaired spare mitochondrial respiratory capacity. Fluorescent staining and expression of genes encoding electron transport chain enzymes showed diminished mitochondrial content in the AD fibroblasts. This study demonstrates that mitochondrial dysfunction is observable in AD fibroblasts and provides evidence that this model system could be useful as a tool to screen disease-modifying compounds. PMID:25862550

  20. A New Phospholipase A₂ from Lachesis muta rhombeata: Purification, Biochemical and Comparative Characterization with Crotoxin B.

    Science.gov (United States)

    Cordeiro, Francielle A; Perini, Tibério G K; Bregge-Silva, Cristiane; Cremonez, Caroline M; Rodrigues, Renata S; Boldrini-França, Johara; Bordon, Karla de C F; De Souza, Dayane L N; Ache, David C; de M Rodrigues, Veridiana; Dos Santos, Wagner F; Rosa, Jose C; Arantesa, Eliane C

    2015-01-01

    Phospholipases A2 (PLA2s) are enzymes responsible for inflammatory effects, edema formation, myotoxicity, neurotoxicity and other manifestations from envenoming. In this paper we report the isolation and biochemical characterization of Lmr-PLA2, the first acidic PLA2 found in Lachesis muta rhombeata venom. Furthermore, this study compared biological effects of Lmr-PLA2 and crotoxin B (CB), a PLA2 from Crotalus durissus terrificus venom. Lmr-PLA2 was isolated by molecular exclusion and reversed phase chromatography. The purified enzyme showed a molecular mass of 13,975 Da, pI of 5.46 and its partial amino acid sequence showed a high identity with PLA2s already described in the literature. In addition, this enzyme possesses the residue D49 in its amino acid sequence, indicating that it is a catalytically active PLA2. Lmr-PLA2 presented high phospholipase activity and was able to inhibit platelet aggregation. Studies of biochemical characterization of new PLA2s, as Lmr-PLA2, are relevant since they help to clarify the structure-function relationship of this important class of toxins. PMID:26145564

  1. Biochemical characterization of plant Rad52 protein from rice (Oryza sativa).

    Science.gov (United States)

    Nair, Anuradha; Agarwal, Rachna; Chittela, Rajani Kant

    2016-09-01

    DNA damage in living cells is repaired by two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Of all the genes promoting HR, Rad52 (Radiation sensitive 52) is an important gene which is found to be highly conserved across different species. It was believed that RAD52 is absent in plant systems until lately. However, recent genetic studies have shown the presence of RAD52 homologues in plants. Rad52 homologues in plant systems have not yet been characterized biochemically. In the current study, we bring out the biochemical properties of rice Rad52-2a protein. OsRad52-2a was over-expressed in Escherichia coli BL21 (DE3) cells and the protein was purified. The identity of purified OsRad52-2a protein was confirmed via peptide mass fingerprinting. Gel filtration and native PAGE analysis indicated that the OsRad52-2a protein in its native state probably formed an undecameric structure. Purified OsRad52-2a protein showed binding to single stranded DNA, double stranded DNA. Protein also mediated the renaturation of complementary single strands into duplex DNA in both agarose gel and FRET based assays. Put together, OsRad52-2a forms oligomeric structures and binds to ssDNA/dsDNA for mediating an important function like renaturation during homologous recombination. This study represents the first report on biochemical properties of OsRad52-2a protein from important crop like rice. This information will help in dissecting the recombination and repair machinery in plant systems. PMID:27156135

  2. Biochemical characterization of the eelgrass Zostera marina at its southern distribution limit in the North Pacific

    Science.gov (United States)

    Cabello-Pasini, Alejandro; Munoz-Salazar, R.; Ward, D.H.

    2004-01-01

    The eelgrass Zostera marina L. is distributed along the Baja California Peninsula (Mexico) where it is exposed to a wide range of irradiances and temperatures that could promote changes in its biochemical composition. Consequently, the objective of this study was to characterize the variations in the levels of chlorophyll, carbohydrates, proteins, fiber, ash and calories in the shoots of Z. marina from the north (San Quintin) and south (Ojo de Liebre and San Ignacio lagoons) of the peninsula. Temperature in the southern lagoons was 5-6??C higher than in the northern lagoon; likewise, in situ irradiance was two-fold greater in the south than in the north. As a result of the lower irradiance levels, the concentration of chlorophyll in the shoots of Z. marina was twice as high (1.7 mg gWW-1) in the northern lagoon than in the southern ones (0.8 mg gWW-1). Similar to chlorophyll levels, the concentration of soluble carbohydrates in the shoots was greater in the northern lagoon than in the southern ones, suggesting that the high levels of chlorophyll are enough to compensate for the low irradiance levels and to maintain a positive carbon balance at San Quintin. On the other hand, the levels of proteins in the shoots from the north of the peninsula were slightly lower than those from the southern populations. In general, these results suggest that the different environmental conditions to which Z. marina is exposed along the peninsula impact its biochemical composition.

  3. 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.

  4. Characterization of Biochemical Composition for Different Types of Spent Mushroom Substrate in Malaysia

    International Nuclear Information System (INIS)

    A preliminary study was conducted to identify the amount and changes of biochemical composition of different types of Malaysian spent mushroom substrate (SMS) before and after several cycle of mushroom cultivation. The characterization of SMS involved the analysis of crude protein, carbohydrate, fat, lignin and ash for selected mushrooms namely as white oyster (Pleuratos oestrous), grey oyster (Pleuratos sajor-caju), abalone (Pleuratos cystidiosus), ganoderma (Ganoderma lucidium) and black jelly (Auricularia polytricha). Overall trend showed that there were increment for crude protein and fat, whereas carbohydrate and lignin showed reduction in the content. Significant results were showed on protein increment where ganoderma attained the highest value, 36.6 g, followed by black jelly, white oyster, grey oyster and abalone. Contradictory, lowest carbohydrate reduction was observed in ganoderma at 70.42 g and the most was in black jelly. Increment in fat and reduction in lignin was almost similar for each SMS. There was an increment in the ash percentage resulted from sterilization process. Clearly cultivation by mushroom had changed biochemical value especially in increasing the protein content that might be useful in protein required industry such as animal feeding. (author)

  5. Biochemical characterization of novel bioactive protein from silkworm (Bombyx mori L) fecal matter.

    Science.gov (United States)

    Raghavendra, R; Neelagund, S E

    2012-07-01

    In this study, complete purification and biochemical characterization of protein is presented. The protein was purified by using Sephadex G-75 gel filtration column followed by reverse-phase high-performance liquid chromatography in a C18 column. The molecular weight of the protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, mass spectrum matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization tandem mass spectrometry. Protein was fragmented by trypsin based on the m/z values obtained by MALDI-TOF-MS analysis. The peptide fragments sequence showed homology with DEAD-box-ATP-dependent RNA helicase 45, present in a public domain, National Centre for Biotechnology Information. The protein exhibited antibacterial activity against selected Gram +/- bacteria. The analgesic activity was determined by conducting acetic-acid-induced writhing test in mice. PMID:22328263

  6. Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis.

    Science.gov (United States)

    Batten, Laura E; Parnell, Alice E; Wells, Neil J; Murch, Amber L; Oyston, Petra C F; Roach, Peter L

    2016-01-01

    The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors. PMID:26582818

  7. A Multiscale Vibrational Spectroscopic Approach for Identification and Biochemical Characterization of Pollen.

    Directory of Open Access Journals (Sweden)

    Murat Bağcıoğlu

    Full Text Available Analysis of pollen grains reveals valuable information on biology, ecology, forensics, climate change, insect migration, food sources and aeroallergens. Vibrational (infrared and Raman spectroscopies offer chemical characterization of pollen via identifiable spectral features without any sample pretreatment. We have compared the level of chemical information that can be obtained by different multiscale vibrational spectroscopic techniques.Pollen from 15 different species of Pinales (conifers were measured by seven infrared and Raman methodologies. In order to obtain infrared spectra, both reflectance and transmission measurements were performed on ground and intact pollen grains (bulk measurements, in addition, infrared spectra were obtained by microspectroscopy of multigrain and single pollen grain measurements. For Raman microspectroscopy measurements, spectra were obtained from the same pollen grains by focusing two different substructures of pollen grain. The spectral data from the seven methodologies were integrated into one data model by the Consensus Principal Component Analysis, in order to obtain the relations between the molecular signatures traced by different techniques.The vibrational spectroscopy enabled biochemical characterization of pollen and detection of phylogenetic variation. The spectral differences were clearly connected to specific chemical constituents, such as lipids, carbohydrates, carotenoids and sporopollenins. The extensive differences between pollen of Cedrus and the rest of Pinaceae family were unambiguously connected with molecular composition of sporopollenins in pollen grain wall, while pollen of Picea has apparently higher concentration of carotenoids than the rest of the family. It is shown that vibrational methodologies have great potential for systematic collection of data on ecosystems and that the obtained phylogenetic variation can be well explained by the biochemical composition of pollen. Out of the

  8. Biochemical and cytological characterization of DROP-1: a widely distributed proteoglycan in Drosophila.

    Science.gov (United States)

    Graner, M; Stupka, K; Karr, T L

    1994-06-01

    Using Drosophila testis as a source of antigen, 12 monoclonal antibodies were isolated that all recognize a set of three high molecular weight molecules present on Drosophila sperm and also in the fertilized egg. Among these antibodies, one is highly specific for sperm, while the remaining 11 detect epitopes present not only on sperm, but also in yolk spheres or in a punctate distribution in the egg. Here we cytologically and biochemically characterize the (common) antigens to five of these antibodies. Several biochemical properties suggest that these antibodies recognize a family of glycosaminoglycan-containing proteoglycans: (1) three diffuse, poorly focused high molecular weight bands, all in excess of 200,000 Da were observed on Western blots of denaturing SDS gels; (2) all three bands have a pI in the range of 3.0-3.5; (3) the molecules are strongly resistant to proteolysis; (4) mild periodate oxidation renders the molecules reactive towards the derivatizing agent digoxygenin-hydrazide, indicating the likely presence of saccharide moieties; (5) trifluoromethyl sulfonic acid treatment, which removes saccharide moieties, shifts the pI to 7.0; (6) beta-elimination increases electrophoretic mobility of the antigens on SDS gels; (7) nitrous acid treatment, which cleaves N-sulfated glycosaminoglycans, also increases the electrophoretic mobility of the antigens on SDS gels. We conclude that the antigens recognized by these antibodies are likely to be heparan sulfate proteoglycans. These results indicate that DROP-1 may represent a family of proteoglycans present during embryogenesis and later stages of development in Drosophila. DROP-1 represents the third proteoglycan to be characterized in Drosophila. PMID:8044173

  9. Protease from Aspergillus oryzae: Biochemical Characterization and Application as a Potential Biocatalyst for Production of Protein Hydrolysates with Antioxidant Activities

    OpenAIRE

    Ruann Janser Soares de Castro; Helia Harumi Sato

    2014-01-01

    This study reports the biochemical characterization of a protease from Aspergillus oryzae LBA 01 and the study of the antioxidant properties of protein hydrolysates produced with this protease. The biochemical characterization showed that the enzyme was most active over the pH range 5.0–5.5 and was stable from pH 4.5 to 5.5. The optimum temperature range for activity was 55–60°C, and the enzyme was stable at temperatures below 45°C. The activation energy (Ea) for azocasein hydrolysis and temp...

  10. Association of 5-methylcytosine and 5-hydroxymethylcytosine with mitochondrial DNA content and clinical and biochemical parameters in hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Fan Shen

    Full Text Available Increasing epidemiological evidence has indicated that inherited variations of mitochondrial DNA (mtDNA copy number affect the genetic susceptibility of many malignancies in a tumour-specific manner and that DNA methylation also plays an important role in controlling gene expression during the differentiation and development of hepatocellular carcinoma (HCC. Our previous study demonstrated that HCC tissues showed a lower 5-hydroxymethylcytosine (5-hmC content when compared to tumour-adjacent tissues, but the relationship among 5-hmC, 5-methylcytosine (5-mC and mtDNA content in HCC patients is still unknown. This study aimed to clarify the correlation among mtDNA content, 5-mC and 5-hmC by quantitative real-time PCR and liquid chromatography tandem mass spectrometry analysis. We demonstrated that 5-hmC correlated with tumour size [odds ratio (OR 0.847, 95% confidence interval (CI 0.746-0.962, P = 0.011], and HCC patients with a tumour size ≥ 5.0 cm showed a lower 5-hmC content and higher levels of fasting plasma aspartate aminotransferase, the ratio of alanine aminotransferase to aspartate aminotransferase, γ-glutamyltransferase, alpha-fetoprotein than those with a tumour size <5 cm (all P<0.05. We further revealed that the mtDNA content of HCC tumour tissues was 225.97(105.42, 430.54 [median (25th Percentile, 75th Percentile] and was negatively correlated with 5-mC content (P = 0.035, but not 5-hmC content, in genomic DNA from HCC tumour tissues.

  11. Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16).

    Science.gov (United States)

    Meng, Fan; Yang, Hao; Aitha, Mahesh; George, Sam; Tierney, David L; Crowder, Michael W

    2016-07-01

    Membrane-bound matrix metalloproteinase 16 (MMP16/MT3-MMP) is considered a drug target due to its role(s) in disease processes such as cancer and inflammation. Biochemical characterization of MMP16 is critical for developing new generation MMP inhibitors (MMPi), which exhibit high efficacies and selectivities. Herein, a modified over-expression and purification protocol was used to prepare the catalytic domain of MMP16 (cdMMP16). The resulting recombinant enzyme exhibited steady-state kinetic constants of K m = 10.6 ± 0.7 μM and k cat = 1.14 ± 0.02 s(-1), when using FS-6 as substrate, and the enzyme bound 1.8 ± 0.1 eq of Zn(II). The enzymatic activity of cdMMP16 is salt concentration-dependent, and cdMMP16 exhibits autoproteolytic activity under certain conditions, which may be related to an in vivo regulatory mechanism of MMP16 and of other membrane-type MMPs (MT-MMPs). Co(II)-substituted analogs (Co2- and ZnCo) of cdMMP16 were prepared and characterized using several spectroscopic techniques, such as UV-Vis, (1)H NMR, and EXAFS spectroscopies. A well-characterized cdMMP16 is now available for future inhibitor screening efforts. PMID:27229514

  12. 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. PMID:26822593

  13. Mitochondrial DNA characterization of five species of Plebeia (Apidae: Meliponini): RFLP and restriction maps

    OpenAIRE

    Flávio De Oliveira Francisco,; Silvestre, Daniela; Arias, Maria

    2001-01-01

    The present work characterized the mitochondrial DNA (mtDNA) of five species of Plebeia ( Plebeia droryana, P. emerina, P. remota, P. saiqui and P. sp.) and generate a data set to be used in further populational, phylogenetic, and biogeographic studies. The mtDNA of each species was analyzed using 17 restriction enzymes and restriction maps were built. A high level of interspecific variability was found. The total size of the mtDNA was estimated to be 18500 bp. Through a combination of PCR an...

  14. 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

  15. Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2.

    Science.gov (United States)

    Zhang, Likui; Huang, Yanchao; Xu, Dandan; Yang, Lixiang; Qian, Kaicheng; Chang, Guozhu; Gong, Yong; Zhou, Xiaojian; Ma, Kesen

    2016-09-01

    His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60-65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0-9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology. PMID:27131500

  16. Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target.

    Science.gov (United States)

    Munkhjargal, Tserendorj; Ishizaki, Takahiro; Guswanto, Azirwan; Takemae, Hitoshi; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-05-15

    Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies are required to confirm this. PMID:27084466

  17. Genetic, physiological and biochemical characterization of multiple methanol methyltransferase isozymes in Methanosarcina acetivorans C2A.

    Science.gov (United States)

    Pritchett, Matthew A; Metcalf, William W

    2005-06-01

    Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes. PMID:15882413

  18. 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.

  19. 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.

  20. Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi.

    Science.gov (United States)

    Manchola, Nubia C; Rapado, Ludmila N; Barisón, María J; Silber, Ariel M

    2016-05-01

    Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H(+) gradient, but not on a Na(+) or K(+) gradient at the cytoplasmic membrane level. PMID:26496801

  1. 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. PMID:18051297

  2. 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.

  3. 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.

  4. Mitochondrial cytopathies.

    Science.gov (United States)

    El-Hattab, Ayman W; Scaglia, Fernando

    2016-09-01

    Mitochondria are found in all nucleated human cells and perform a variety of essential functions, including the generation of cellular energy. Most of mitochondrial proteins are encoded by the nuclear DNA (nDNA) whereas a very small fraction is encoded by the mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction which leads to a wide range of cellular perturbations including aberrant calcium homeostasis, excessive reactive oxygen species production, dysregulated apoptosis, and insufficient energy generation to meet the needs of various organs, particularly those with high energy demand. Impaired mitochondrial function in various tissues and organs results in the multi-organ manifestations of mitochondrial diseases including epilepsy, intellectual disability, skeletal and cardiac myopathies, hepatopathies, endocrinopathies, and nephropathies. Defects in nDNA genes can be inherited in an autosomal or X-linked manners, whereas, mtDNA is maternally inherited. Mitochondrial diseases can result from mutations of nDNA genes encoding subunits of the electron transport chain complexes or their assembly factors, proteins associated with the mitochondrial import or networking, mitochondrial translation factors, or proteins involved in mtDNA maintenance. MtDNA defects can be either point mutations or rearrangements. The diagnosis of mitochondrial disorders can be challenging in many cases and is based on clinical recognition, biochemical screening, histopathological studies, functional studies, and molecular genetic testing. Currently, there are no satisfactory therapies available for mitochondrial disorders that significantly alter the course of the disease. Therapeutic options include symptomatic treatment, cofactor supplementation, and exercise. PMID:26996063

  5. Isolation and biochemical and molecular characterization of Listeria monocytogenes in food

    International Nuclear Information System (INIS)

    monocytogenes is a Gram-positive bacteria, saprophytic, non-spore. This is an extremely resistant seeds to environmental conditions outside, especially since the cold psychrotrophic. It can contaminate raw vegetables, cooked meals ready for consumption or foods to be stored in the refrigerator, such as cheese or meat. It is the bacteria responsible for listeriosis. It threatens first unborn children, infants, pregnant women, the elderly and people whose immune system is weakened. Strains of Listeria spp isolated from foods (seafood, meat, meat) were first identified at the stage of the genus by classical tests (Gram staining, catalase test, oxidase test and mobility) and stage of the test case by hemolysis, CAMP test and the gallery Api Listeria. Biochemical characterization allowed after a numerical analysis, to assign 100% of isolates to the genus Listeria. Molecular characterization was performed by PCR amplification of genes coding for protein p60 (iap), the listeriolysine O (hly), the Phosphatidylinositol Phospholipase C (PI-PLC plca) Phosphatidylcholine Phospholipase C (plcB). The result showed an amplification of the iap gene of 100% of the hly gene, plca, plcB of 31.81%. This characterization represents an identification of the collection on the genetic level and shows that 31.81% of isolates, is likely to express the genes responsible for virulence factors of L. monocytogenes, to produce listeriolysine O, phospholipase C and Lecithinase. The molecular identification was performed by microarray technique and identified isolates L. September monocytogenes (five original clinical isolates and two food-borne), fourteen L. innocua (of food) and a strain not identified by DNA chip.. (Author)

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

    Directory of Open Access Journals (Sweden)

    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

  7. Cyto-palynological, biochemical and molecular characterization of original and induced mutants of garden chrysanthemum

    International Nuclear Information System (INIS)

    A large number of new somatic flower color/type mutants have been evolved by induced mutations in different ornamentals. Few reports are available on the systematic work being done on comparative analysis of the original and the mutant cultivars. Attempt has been made for the comparative analysis of original cultivars and their respective induced mutants on cyto-palynological, biochemical and molecular characters for better and clear understanding of the exact mechanism involved in the origin and evolution of flower color mutations. Proper characterization and identification of new mutant cultivars is extremely important to protect plant breeder's rights for commercial exploitation. Chrysanthemum original varieties and their gamma ray induced mutants were selected as the materials for the present analysis. Critical cytological analysis with special reference to chromosome number, chromosomal aberrations, ICV, INV and DNA content showed no differences. The karyotypes were reasonably symmetrical. No mutant specific chromosomal aberrations could be detected. Thin layer chromatographic and spectrophotometric analysis of floret pigments indicated that somatic flower color changes in chrysanthemum are due to both qualitative and/or quantitative changes in pigments as a result of mutation induced by gamma rays in pigment biosynthesis pathway. Significant increase in pollen grain sterility was found in all the mutants. The pollen grains of all the cultivars and their mutants are basically 3(-4) zonocolporate with tectate spinose exine having perforations. No appreciable variation in pollen apertural character was noticed in any of the mutants. Significant changes in pollen exine surface pattern were found in 4 mutant varieties. The changes are inconsistent and do not correspond to the intensity of radiation. The similarity among the cultivars and mutants varied from 0.17 to 0.90 using RAPD analyses. Cultivars with different flower colors could be clearly distinguished. But

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

    Directory of Open Access Journals (Sweden)

    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

  9. Mitochondrial dysfunction in inherited renal disease and acute kidney injury.

    Science.gov (United States)

    Emma, Francesco; Montini, Giovanni; Parikh, Samir M; Salviati, Leonardo

    2016-05-01

    Mitochondria are increasingly recognized as key players in genetic and acquired renal diseases. Most mitochondrial cytopathies that cause renal symptoms are characterized by tubular defects, but glomerular, tubulointerstitial and cystic diseases have also been described. For example, defects in coenzyme Q10 (CoQ10) biosynthesis and the mitochondrial DNA 3243 A>G mutation are important causes of focal segmental glomerulosclerosis in children and in adults, respectively. Although they sometimes present with isolated renal findings, mitochondrial diseases are frequently associated with symptoms related to central nervous system and neuromuscular involvement. They can result from mutations in nuclear genes that are inherited according to classic Mendelian rules or from mutations in mitochondrial DNA, which are transmitted according to more complex rules of mitochondrial genetics. Diagnosis of mitochondrial disorders involves clinical characterization of patients in combination with biochemical and genetic analyses. In particular, prompt diagnosis of CoQ10 biosynthesis defects is imperative because of their potentially reversible nature. In acute kidney injury (AKI), mitochondrial dysfunction contributes to the physiopathology of tissue injury, whereas mitochondrial biogenesis has an important role in the recovery of renal function. Potential therapies that target mitochondrial dysfunction or promote mitochondrial regeneration are being developed to limit renal damage during AKI and promote repair of injured tissue. PMID:26804019

  10. 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.

  11. Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham.

    Science.gov (United States)

    Howcroft, C F; Gravato, C; Amorim, M J B; Novais, S C; Soares, A M V M; Guilhermino, L

    2011-01-01

    Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour. PMID:21080225

  12. 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.

    2011-12-01

    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)], 10.1063/1.2987701. 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.

  13. Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis.

    Science.gov (United States)

    Bouacem, Khelifa; Bouanane-Darenfed, Amel; Laribi-Habchi, Hassiba; Elhoul, Mouna Ben; Hmida-Sayari, Aïda; Hacene, Hocine; Ollivier, Bernard; Fardeau, Marie-Laure; Jaouadi, Bassem; Bejar, Samir

    2015-11-01

    Caldicoprobacter guelmensis isolated from the hydrothermal hot spring of Guelma (Algeria) produced high amounts of extracellular thermostable serine alkaline protease (called SAPCG) (23,000U/mL). The latter was purified by ammonium sulphate precipitation, UNO Q-6 FPLC and Zorbex PSM 300 HPLC, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 55,824.19Da. The 19 N-terminal residue sequence of SAPCG showed high homology with those of microbial proteases. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine protease family. It showed optimum protease activity at pH 10 and 70°C with casein as a substrate. The thermoactivity and thermostability of SAPCG were enhanced in the presence of 2mM Ca(2+). Its half-life times at 80 and 90°C were 180 and 60min, respectively. Interestingly, the SAPCG protease exhibited significant compatibility with iSiS and Persil, and wash performance analysis revealed that it could remove blood-stains effectively. Overall, SAPCG displayed a number of attractive properties that make it a promising candidate for future applications as an additive in detergent formulations. PMID:26261082

  14. 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

    2016-01-01

    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. PMID:26927087

  15. 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.

  16. Understanding tenderness variability and ageing changes in buffalo meat: biochemical, ultrastructural and proteome characterization.

    Science.gov (United States)

    Kiran, M; Naveena, B M; Reddy, K S; Shahikumar, M; Reddy, V R; Kulkarni, V V; Rapole, S; More, T H

    2016-06-01

    Understanding of biological impact of proteome profile on meat quality is vital for developing different approaches to improve meat quality. Present study was conducted to unravel the differences in biochemical, ultrastructural and proteome profile of longissimus dorsi muscle between buffaloes (Bubalus bubalis) of different age groups (young v. old). Higher (Pspace in young compared with old buffalo meat. Transmission electron microscopy results revealed longer sarcomeres in young buffalo meat relative to meat from old buffaloes. Proteomic characterization using two-dimensional gel electrophoresis (2DE) found 93 differentially expressed proteins between old and young buffalo meat. Proteome analysis using 2DE revealed 191 and 95 differentially expressed protein spots after 6 days of ageing in young and old buffalo meat, respectively. The matrix assisted laser desorption ionization time-of flight/time-of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of selected gel spots helped in identifying molecular markers of tenderness mainly consisting of structural proteins. Protein biomarkers identified in the present study have the potential to differentiate meat from young and old buffaloes and pave the way for optimizing strategies for improved buffalo meat quality. PMID:27076348

  17. 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. PMID:24209354

  18. Biochemical Characterization of Bovine Brain Myristoyl-CoA:Protein N-Myristoyltransferase Type 2

    Directory of Open Access Journals (Sweden)

    Ponniah Selvakumar

    2009-01-01

    Full Text Available Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT from Bos tarus brain. The open reading frame codes for a 410-amino-acid protein and overexpressed in Escherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ion Ca2+ had stimulatory effects on NMT2 activity while Mn2+ and Zn2+ inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

  19. Biochemical and antigenic characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus.

    Science.gov (United States)

    Beauvais, A; Monod, M; Debeaupuis, J P; Diaquin, M; Kobayashi, H; Latgé, J P

    1997-03-01

    A novel dipeptidyl-peptidase (DPP V) was purified from the culture medium of Aspergillus fumigatus. This is the first report of a secreted dipeptidyl-peptidase. The enzyme had a molecular mass of 88 kDa and contained approximately 9 kDa of N-linked carbohydrate. The expression and secretion of dipeptidyl-peptidase varied with the growth conditions; maximal intra- and extracellular levels were detected when the culture medium contained only proteins or protein hydrolysates in the absence of sugars. The gene of DPP V was cloned and showed significant sequence homology to other eukaryotic dipeptidyl-peptidase genes. Unlike the other dipeptidyl-peptidases, which are all intracellular, DPP V contained a signal peptide. Like the genes of other dipeptidyl-peptidases, that of DPP V displayed the consensus sequences of the catalytic site of the nonclassical serine proteases. The biochemical properties of native and recombinant DPP V obtained in Pichia pastoris were unique and were characterized by a substrate specificity limited to the hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral pH optimum. In addition, we showed that DPP V is identical to one of the two major antigens used for the diagnosis of aspergillosis. PMID:9045640

  20. Biochemical and immunological characterization of kafirins in lysine rich cultivars of sorghum

    International Nuclear Information System (INIS)

    Seed storage proteins from naturally occurring lysine rich cultivars selected for their resistance to (S)-2-aminoethyl-L-cystein were analyzed biochemically and immunologically and compared with the cultivar White Martin (low lysine content) and a chemically induced high lysine mutant P721o. Quantitative analysis of these cultivars indicated a 10-25% decrease in synthesis of the alcohol soluble proteins (kafirisn and alcohol soluble reduced glutelins) and a two times increase in albumins and globulins without affecting the total protein content. SDS-PAGE and two-dimensional gel electrophoresis indicated that the kafirins consist of at least six to eight polypeptides, which were immunologically characterized as α-kafirins (25kD and 27kD), β-kafirins (18 kD and 19 kD) and γ-kafirins (28 kD). All these kafirin polypeptides are represented in White Martin, while in the case of the high lysine mutant P721o, a general decrease in alcohol soluble proteins has been reported. 4 refs

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

    Directory of Open Access Journals (Sweden)

    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.

  2. PHENOTYPIC AND BIOCHEMICAL CHARACTERIZATION OF BRADYRHIZOBIUM AND ENSIFER SPP. ISOLATED FROM SOYBEAN RHIZOSPHERE

    Directory of Open Access Journals (Sweden)

    Harpreet Kaur

    2012-01-01

    Full Text Available A total of 15 rhizobia were isolated from soybean rhizosphere on yeast extract mannitol agar (YEMA medium. The isolates were further subjected to morphological, cultural and biochemical characterization along with two reference culture (DS-1, National check and SB271(Local check. Out of 15 isolates, 10 were selected as rhizobia on ketolactose medium (circular, light pink colonies, further 6 (LSER4,LSER5,LSER6,LSER7,LSER8,LSER9 were selected as fast and 4 (LSBR1, LSBR2, LSBR3, LSBR10 as slow growing isolates based on bromothymol blue (BTB test. Fast and slow growing rhizobia were found to be positive for oxidase, urease, citrate utilization and catalase whereas negative reactions for methyl red and voges-proskauer test. The optimum physical condition for growth of fast and slow growing rhizobia was 28˚C at neutral pH (7.0. Four bradyrhizobial spp. and one Ensifer spp. showed hydrogen uptake (Hup+ positive system with 0.01% TTC dye.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core

    International Nuclear Information System (INIS)

    Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host

  5. Expression and partial biochemical characterization of a recombinant serine protease from Bothrops pauloensis snake venom.

    Science.gov (United States)

    Isabel, Thais F; Costa, Guilherme Nunes Moreira; Pacheco, Isabela B; Barbosa, Luana G; Santos-Junior, Célio D; Fonseca, Fernando P P; Boldrini França, Johara; Henrique-Silva, Flávio; Yoneyama, Kelly A G; Rodrigues, Renata S; Rodrigues, Veridiana de Melo

    2016-06-01

    Snake venom serine proteases (SVSPs) are enzymes capable of interfering at several points of hemostasis. Some serine proteases present thrombin-like activity, which makes them targets for the development of therapeutics agents in the treatment of many hemostatic disorders. In this study, a recombinant thrombin-like serine protease, denominated rBpSP-II, was obtained from cDNA of the Bothrops pauloensis venom gland and was characterized enzymatically and biochemically. The enzyme rBpSP-II showed clotting activity on bovine plasma and proteolytic activity on fibrinogen, cleaving exclusively the Aα chain. The evaluation of rBpSP-II activity on chromogenic substrates demonstrated thrombin-like activity of the enzyme due to its capacity to hydrolyze the thrombin substrate. These characteristics make rBpSP-II an attractive molecule for additional studies. Further research is needed to verify whether rBpSP-II can serve as a template for the synthesis of therapeutic agents to treat hemostatic disorders. PMID:26965926

  6. Structural and biochemical characterization of the Bacillus cereus 3-hydroxyisobutyrate dehydrogenase.

    Science.gov (United States)

    Park, Sun Cheol; Kim, Pyeung-Hyeun; Lee, Geun-Shik; Kang, Seung Goo; Ko, Hyun-Jeong; Yoon, Sung-Il

    2016-06-01

    The 3-hydroxyisobutyrate dehydrogenase (HIBADH) family catalyzes the NAD(+)- or NADP(+)-dependent oxidation of various β-hydroxyacid substrates into their cognate semialdehydes for diverse metabolic pathways. Because HIBADH group members exhibit different substrate specificities, the substrate-recognition mode of each enzyme should be individually characterized. In the current study, we report the biochemical and structural analysis of a HIBADH group enzyme from Bacillus cereus (bcHIBADH). bcHIBADH mediates a dehydrogenation reaction on S-3-hydroxyisobutyrate substrate with high catalytic efficiency in an NAD(+)-dependent manner; it also oxidizes l-serine and 3-hydroxypropionate with lower activity. bcHIBADH consists of two domains and is further assembled into a functional dimer rather than a tetramer that has been commonly observed in other prokaryotic HIBADH group members. In the bcHIBADH structure, the interdomain cleft forms a putative active site and simultaneously accommodates both an NAD(+) cofactor and a substrate mimic. Our structure-based comparative analysis highlights structural motifs that are important in the cofactor and substrate recognition of the HIBADH group. PMID:27120461

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

    Directory of Open Access Journals (Sweden)

    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

  8. 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. PMID:26703456

  9. Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Mikael Crona

    Full Text Available The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR: the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb, a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related γ-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  12. Lung damage induced by butylated hydroxytoluene in mice. Biochemical, cellular, and morphologic characterization.

    Science.gov (United States)

    Smith, L J

    1984-11-01

    This study was designed to characterize the biochemical, cellular, and morphologic events produced in mice by butylated hydroxytoluene (BHT) and to relate these events to changes in extracellular angiotensin-converting enzyme (ACE) activity. On Day 1 after the administration of BHT, bronchoalveolar lavage (BAL) ACE activity increased 4-fold (p less than 0.001), its specific activity relative to BAL protein increased 3-fold (p less than 0.001), and both type 1 cell damage and endothelial cell damage were detected by electron microscopy. The early increase in BAL ACE activity preceded changes in plasma ACE levels, BAL cell number, protein, lactate, and lactate dehydrogenase (LDH) activity in both plasma and BAL, and the ACE content of alveolar macrophages. On Day 2, BAL ACE activity increased 9-fold, BAL protein increased 4-fold (p less than 0.001), BAL LDH activity increased 34% (p less than 0.05), and the BAL cell count doubled (p less than 0.01). Changes in each animal's appearance, body weight, wet and dry lung weights, and plasma ACE levels occurred between Days 3 and 5. The BAL differential cell count, which consisted of greater than 95% macrophages in uninjured mice, did not change until Day 5 when there was a small increase in polymorphonuclear leukocytes (PMN). On Day 7, the number of PMN peaked, and some of the other measures of lung injury began returning toward normal. These results indicate that BAL ACE activity is a sensitive, early marker of BHT-induced lung injury, which appears to reflect damage to the cells of the alveolar-capillary barrier. In addition, PMN do not appear to play a major role in this model of lung injury. Because of its effects on angiotensin, bradykinin, and prostaglandins, the early release of ACE from damaged cells may modulate the subsequent injury. PMID:6093659

  13. Site-specific isotope fractionation in the characterization of biochemical mechanisms: the glycolytic pathway

    International Nuclear Information System (INIS)

    For a given biochemical transformation, such as the fermentation reaction, the redistribution coefficients, which relate the natural site-specific isotope contents in end products to those of their precursors, are a source of mechanistic information. These coefficients characterize the traceability of specific hydrogens in the products (ethanol and water) to their parent hydrogens in the starting materials (glucose and water). In conditions of complete transformation, they also enable intermolecular exchanges with the water medium to be estimated. Thus it is directly confirmed that hydrogens 1, 2, 6, and 6' of glucose are strongly connected to the methyl site I of ethanol obtained by fermentation by Saccharomyces cerevisiae. However, whereas hydrogens 6 and 6' are transferred to a great extent, transfer is only partial for hydrogen 2, and it is even less for hydrogen 1. Because the two moieties of glucose corresponding to carbons 1-2-3 and 4-5-6 are scrambled by the aldolase and triosephosphate isomerase reactions, additional exchange of hydrogens at positions 1 and 2 must have occurred before these steps. The value of the coefficient that relates site 2 of glucose to site I of ethanol in particular can be used to quantify the contribution of intermolecular exchange occurring in the course of the transfer from site 2 of glucose 6-phosphate to site 1 of fructose 6-phosphate mediated by phosphoglucoisomerase. The average hydrogen isotope effects associated with the transfer of hydrogen from the water pool to the methyl or methylene site of ethanol are estimated. In contrast to conventional experiments carried out in strongly deuterium-enriched media where metabolic switching may occur, the NMR investigation of site-specific natural isotope fractionation, which operates at tracer isotopic abundance, faithfully describes the unperturbed metabolic pathways

  14. 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. PMID:21605585

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

    Science.gov (United States)

    De Santi, Concetta; Willassen, Nils Peder

    2016-01-01

    Background 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. Results 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. PMID:27433797

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Biochemical Characterization of a Protease Involved in the Processing of a Streptomyces reticuli Cellulase (Avicelase).

    Science.gov (United States)

    Moormann, M; Schlochtermeier, A; Schrempf, H

    1993-05-01

    A 36-kDa protease from Streptomyces reticuli had recently been shown to be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from S. reticuli to a 42-kDa truncated enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55 degrees C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor O glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55 degrees C, pH 7.5, in the presence of 2 mM dithiothreitol. PMID:16348937

  1. General biochemical and immunological characterization of the venom from the scorpion Tityus trivittatus of Argentina.

    Science.gov (United States)

    de Roodt, Adolfo R; Coronas, Fredy I V; Lago, Nestor; González, María E; Laskowicz, Rodrigo D; Beltramino, Juan C; Saavedra, Silvina; López, Raúl A; Reati, Gustavo J; Vucharchuk, Miriam G; Bazán, Eduardo; Varni, Liliana; Salomón, Oscar D; Possani, Lourival D

    2010-01-01

    Tityus trivittatus is the Argentinean scorpion reported to cause the majority of human fatalities in the country, however no systematic studies have been conducted with the venom of this species. This communication describes a general biochemical and immunological characterization of the venom obtained from T. trivittatus scorpions collected in the city of Buenos Aires and various provinces of Argentina: Catamarca, Cordoba, Entre Rios, La Rioja, Santa Fe and Santiago del Estero. These are places where human accidents were reported to occur due to this scorpion. For comparative purposes two types of samples were assayed: whole soluble venom obtained by electrical stimulation and supernatant from homogenized venomous glands. Two strains of mice (NIH and CF-1) were used for LD(50) determinations by two distinct routes of administration (intravenously and intraperitoneally). Important variations were found that goes from 0.5 to 12 mg/kg mouse body weight. Samples of soluble venom were always more potent than Telson homogenates. More complex pattern was observed in homogenates compared to soluble venom, as expected. This was supported by gel electrophoretic analysis and high performance liquid chromatographic (HPLC) separations. Additionally, the HPLC profile was enriched in proteins resolved at similar elution times as other known toxins from scorpion venoms studied. Immune enzymatic assays were also conducted comparatively, using four different anti-venoms commercially available for treatment of scorpion stings (Argentinean antidote from INPB, two anti-venoms from Butantan Institute of Brazil and Alacramyn from the Mexican Bioclon Institute). Cross-reactivities were observed and are reported among the various venoms and anti-venoms used. Lung, heart, liver and pancreas pathological modifications were observed on tissues of intoxicated mice. It seems that there are important variations on the venom compositions of the various samples studied and reported here

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

    OpenAIRE

    Yurany Dayana Rivera; Andrés Leonardo Moreno Chacón; Hernán Mauricio Romero

    2013-01-01

    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 photo...

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

    OpenAIRE

    Mejdoub Hafedh; Ben Bacha Abir G

    2011-01-01

    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 S...

  4. Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91

    OpenAIRE

    Wladyka, Benedykt; Wielebska, Katarzyna; Wloka, Marcin; Bochenska, Oliwia; Dubin, Grzegorz; Dubin, Adam; Mak, Pawel

    2012-01-01

    Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is...

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  7. Biochemical characterization of nuclear receptors for vitamin D3 and glucocorticoids in prostate stroma cell microenvironment

    International Nuclear Information System (INIS)

    Highlights: → Fibroblasts from benign and carcinoma-associated stroma were biochemically characterized for VDR and GR function as transcription factors in prostate stroma cell microenvironment. → Decreased SRC-1/CBP coactivators recruitment to VDR and GR may result in hormone resistance to 1,25D3 in stromal cell microenvironment prostate cancer. → 1a,25-Dyhidroxyvitamin D3 (1,25D3) 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. 1α,25-Dihydroxyvitamin D3 (1,25D3) 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 1,25D3 is mediated by the 1,25D3 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 1,25D3 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 1,25D3 action. Conversely, VDR-mediated transcriptional activity was more efficient in 4 out of 13 CAS (30%), as compared

  8. Mitochondrial Dysfunction and Psychiatric Disorders

    OpenAIRE

    Shaw-Hwa Jou; Nan-Yin Chiu; Chin-San Liu

    2009-01-01

    Mitochondria are intracellular organelles crucial in the production of cellular energy.Mitochondrial diseases may result from malfunctions in this biochemical cascade. Severalinvestigators have proposed that mitochondrial dysfunction is related to the pathophysiologyof bipolar disorder (BD), major depressive disorder (MDD) and schizophrenia (SZ). Theauthors reviewed recent study findings and tried to delineate the current understanding of thecorrelation between mitochondrial dysfunction and p...

  9. Biochemical Characterization of Compatible Plant Virus Interaction: A Case Study with Bunchy Top Virus-Banana Host-Pathosystem

    Directory of Open Access Journals (Sweden)

    C. Anuradha

    2015-01-01

    Full Text Available Virus infection can result in the alteration of physiological, biochemical and metabolic processes within plants leading to symptom development. Banana bunchy top virus (BBTV is one of the most destructive viral diseases in Tropical Asia, Pacific Indian Oceania (PIO regions and Africa leading to 100% yield loss in banana and plantains. Though molecular characterization and their diversity were studied in depth in recent years, information on physiological and biochemical changes during banana-BBTV interaction is still not convincingly explained. Therefore, the present investigation was conducted to find out the quantifiable changes in physiological and biochemical parameters such as proteins, pigment and carbohydrate content, phenolic compounds, polyphenol oxidase (PPO, peroxidase (POX, ascorbate peroxidase (APX, guaiacol peroxidase (GPX, catalase (CAT and superoxide dismutase (SOD activities in leaves of banana cultivars Grand Nain (AAA and Virupakshi (AAB. The amount of carbohydrate contents, phenolic compounds, PPO, POX, APX, GPX, CAT were significantly higher in BBTV infected leaves of both the cultivars over the healthy, whereas total protein content, pigments and SOD activity showed an opposite trend. Overall the results suggest that BBTV infection induces significant changes in enzyme levels leading to irreversible symptom development. Further studies would lead to identification of biochemical markers for studying plant-virus compatible and incompatible interactions.

  10. Bedside diagnosis of mitochondrial dysfunction in aneurysmal subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Jacobsen, A.; Nielsen, T. H.; Nilsson, O.; Schalen, W.; Nordstrom, C. H.

    2014-01-01

    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...

  11. Genotypic, physiological, and biochemical characterization of potentially pathogenic Acanthamoeba isolated from the environment in Cairo, Egypt.

    Science.gov (United States)

    Tawfeek, Gihan Mostafa; Bishara, Sawsan Abdel-Hamid; Sarhan, Rania Mohammad; ElShabrawi Taher, Eman; ElSaady Khayyal, Amira

    2016-05-01

    Acanthamoebae are the most common opportunistic amphizoic protozoa that cause life-threatening granulomatous amoebic encephalitis in immunocompromised individuals and sight-threatening amoebic keratitis (AK) in contact lens wearers. The present work aimed to determine the presence of Acanthamoeba isolates in different environmental sources: water, soil, and dust in Cairo, Egypt and to characterize the pathogenic potential of the isolated Acanthamoeba using physiological and biochemical assays as well as determination of the genotypes in an attempt to correlate pathogenicity with certain genotypes. The study included the collection of 22 corneal scrapings from patients complaining of symptoms and signs indicative of acanthamoeba keratitis (AK) and 75 environmental samples followed by cultivation on non-nutrient agar plates preseeded with E. coli. Positive samples for Acanthamoeba were subjected to osmo- and thermo-tolerance assays and zymography analysis. Potentially pathogenic isolates were subjected to PCR amplification using genus-specific primer pair. Isolates were classified at the genotype level based on the sequence analysis of Acanthamoeba 18S rRNA gene (diagnostic fragment 3). The total detection rate for Acanthamoeba in environmental samples was 33.3 %, 31.4 % in water, 40 % in soil, and 20 % in dust samples. Three and two Acanthamoeba isolates from water and soil sources, respectively, had the potential for pathogenicity as they exhibited full range of pathogenic traits. Other 12 isolates were designated as weak potential pathogens. Only ten of the environmental isolates were positive in PCR and were classified by genotype analysis into T4 genotype (70 %), T3 (10 %) and T5 (20 %). Potential pathogens belonged to genotypes T4 (from water) and T5 (from soil) while weak potential pathogens belonged to genotypes T3 (from water) and T4 (from water and soil). Additionally, T7 genotype was isolated from keratitis patients. There is a considerable

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zambare, Vasudeo; Zambare, Archana; Christopher, Lew P. [Center for Bioprocessing Research & Development, South Dakota School of Mines and Technology, Rapid City 57701, SD (United States); Muthukumarappan, Kasiviswanath [Center for Bioprocessing Research & Development, South Dakota State University, Brookings 57007, SD (United States)

    2011-07-01

    . 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.

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

    Directory of Open Access Journals (Sweden)

    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

  15. Characterization of a Functional Hydrogel Layer on a Silicon-Based Grating Waveguide for a Biochemical Sensor

    Science.gov (United States)

    Hong, Yoo-Seung; Kim, Jongseong; Sung, Hyuk-Kee

    2016-01-01

    We numerically demonstrated the characteristics of a functional hydrogel layer on a silicon-based grating waveguide for a simple, cost-effective refractive index (RI) biochemical sensor. The RI of the functional hydrogel layer changes when a specific biochemical interaction occurs between the hydrogel-linked receptors and injected ligand molecules. The transmission spectral profile of the grating waveguide shifts depends on the amount of RI change caused by the functional layer. Our characterization includes the effective RI change caused by the thickness, functional volume ratio, and functional strength of the hydrogel layer. The results confirm the feasibility of, and set design rules for, hydrogel-assisted silicon-based grating waveguides. PMID:27322286

  16. Characterization of the reaction of decoupling ubiquinone with bovine mitochondrial respiratory complex I.

    Science.gov (United States)

    Masuya, Takahiro; Okuda, Kenji; Murai, Masatoshi; Miyoshi, Hideto

    2016-08-01

    We previously produced the unique ubiquinone QT ("decoupling" quinone), the catalytic reduction of which in NADH-quinone oxidoreduction with bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) is completely decoupled from proton translocation across the membrane domain. This feature is markedly distinct from those of typical short-chain quinones such as ubiquinone-1. To further characterize the features of the QT reaction with complex I, we herein synthesized three QT analogs, QT2-QT4, and characterized their electron transfer reactions. We found that all aspects of electron transfer (e.g. electron-accepting activity and membrane potential formation) vary significantly among these analogs. The features of QT2 as decoupling quinone were slightly superior to those of original QT. Based on these results, we conclude that the bound positions of QTs within the quinone binding cavity susceptibly change depending on their side-chain structures, and the positions, in turn, govern the behavior of QTs as electron acceptors. PMID:27140857

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

    OpenAIRE

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

    2014-01-01

    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...

  18. Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium

    DEFF Research Database (Denmark)

    Larsen, Thomas Ostenfeld; Svendsen, Anne; Smedsgaard, Jørn

    2001-01-01

    In order to explore the biochemical scope of ochratoxin A- producing penicillia, we screened 48 Penicillium verrucosum isolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spec...

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

    OpenAIRE

    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; S. Kolker; 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 alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by propionyl-CoA in purified porcine enzyme and in submitochondrial ...

  20. Characterization of the complete mitochondrial genome of the Rhinolophus sinicus sinicus (Chiroptera: Rhinolophidae) from Central China.

    Science.gov (United States)

    Xie, Lifen; Sun, Keping; Feng, Jiang

    2016-07-01

    We present a complete mitochondrial genome sequence of Rhinolophus sinicus sinicus from Central China and provide its annotation, as well as showed the phylogenetic relationship and mitogenomic variation with other published mitochondrial genomes of congeneric bat species. Our results revealed a relatively high mitogenomic variation between two R. s. sinucus from Central and East China, which is similar to interspecific divergence level. PMID:26057010

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Contribution to the biochemical characterization of ripeness of olives of different varieties

    OpenAIRE

    Ramalheiro, João Pedro da Silva Canas

    2009-01-01

    Mestrado em Engenharia Alimentar - Qualidade e segurança Alimentar - Instituto Superior de Agronomia The changes in the activities of the enzymatic antioxidant system (POD, PPO and APX) of several varieties of olives, namely, Azeiteira, Cordovil, Maçanilha and Picual in three different ripening stages, have been carried out. The characterisation of the endogenous enzymatic antioxidant system can contribute to a biochemical description of the ripening process, because the conditions, in whi...

  3. Transcriptomic, biochemical and individual markers in transplanted Daphnia magna to characterize impacts in the field

    OpenAIRE

    Campos, Bruno; Malik, Amrita; Tauler, Romà; Barata, Carlos

    2014-01-01

    Daphnia magna individuals were transplanted across 12 sites from three Spanish river basins (Llobregat, Ebro, Jucar) showing different sources of pollution. Gene transcription, feeding and biochemical responses in the field were assessed and compared with those obtained in re-constituted water treatments spiked with organic eluates obtained from water samples collected at the same locations and sampling periods. Up to 166 trace contaminants were detected in water and classified by their mode ...

  4. Biochemical Characterization of Phosphate Degrading Pseudomonas Cichorii Isolated From Forest Soils In Seshachalam Hills

    OpenAIRE

    G Prasada Babu; D Chakravarthy; K Jaya Kumar; Chinthala Paramageetham

    2013-01-01

    Pseudomonas cichoriiisolates were obtained from forest soils in Seshachalm hills using selective medium. The isolates were screened for phosphate activity and all the isolates were gram negative and showed bright fluorescence under UV light. The cultural and biochemical characteristics confirmed that the isolates were P.cichorii. Carbohydrates utilization profiles confirmed that the isolates were able to utilize Lactose, Xylose, Fructose, Glycerol, Trehalose, Mannitol and Ribose. However, the...

  5. Biochemical characterization of resistance against Alternaria helianthi in cultivated and wild sunflowers

    OpenAIRE

    Madhavi K.J.; Sujatha M; Reddy Ram Raja P.; Rao Chander

    2005-01-01

    The biochemical basis of resistance to the leaf spot/blight pathogen A. helianthi was compared in six wild Helianthus species, possessing three ploidy levels (dlploid, telrapiold and hexaplotd) and different degrees of resistance to helianthi, and cultivated sunflower (H. annuus cv. CO-4 susceptible check) in terms of sugar, phenols and isozymes of peroxidase. polyhenol oxidase and chitinase. The resistant species of wild sunflowers (H. tuberosus. H. occidentalis) possessed higher levels of c...

  6. Y chromosome and mitochondrial DNA characterization of Pasiegos, a human isolate from Cantabria (Spain).

    Science.gov (United States)

    Maca-Meyer, N; Sánchez-Velasco, P; Flores, C; Larruga, J-M; González, A-M; Oterino, A; Leyva-Cobián, F

    2003-07-01

    Mitochondrial DNA sequences and Y chromosome haplotypes were characterized in Pasiegos, a human isolate from Cantabria, and compared with those of other Cantabrian and neighbouring Northern Spain populations. Cantabria appears to be a genetically heterogeneous community. Whereas Lebaniegos do not differ from their eastern Basque and western Asturian and Galician neighbours, Pasiegos and other non-Lebaniego Cantabrians show significant differences with all of them. Pasiegos are peculiar for their high frequencies of Y chromosomal markers (E-M81) with North African assignation, and Y chromosomal (R-SRY2627) and mtDNA (V, I, U5) markers related to northern European populations. This dual geographic contribution is more in agreement with the complex demographic history of this isolate, as opposed to recent drift effects. The high incidence in Cantabrians with pre-V and V mtDNA haplotypes, considered as a signal of Postglacial recolonization in Europe from south-western refugees, points to such refugees as a better candidate population than Basques for this expansion. However, this does not discount a conjoint recolonization. PMID:12914567

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Biochemical Characterization of Phosphate Degrading Pseudomonas Cichorii Isolated From Forest Soils In Seshachalam Hills

    Directory of Open Access Journals (Sweden)

    G Prasada Babu

    2013-03-01

    Full Text Available Pseudomonas cichoriiisolates were obtained from forest soils in Seshachalm hills using selective medium. The isolates were screened for phosphate activity and all the isolates were gram negative and showed bright fluorescence under UV light. The cultural and biochemical characteristics confirmed that the isolates were P.cichorii. Carbohydrates utilization profiles confirmed that the isolates were able to utilize Lactose, Xylose, Fructose, Glycerol, Trehalose, Mannitol and Ribose. However, the isolates were unable to utilize glucose and sucrose. The isolates showed positive results for levan production, oxidase and HCN. However they were negative for citrate utilization, ONPG and Gelatin hydrolysis.

  9. Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed Pichia pastoris cells.

    Science.gov (United States)

    Montesino, R; Cremata, J; Rodríguez, M; Besada, V; Falcón, V; de la Fuente, J

    1996-02-01

    In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris. The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated, forming particles ranging between 17 and 40 nm. The rBm86 was N-glycosylated, having at least two non-glycosylated sequons (Asn-329 and Asn-363) and a ratio of only 0.4/65 (free Cys/total Cys)/mol of protein. PMID:8867893

  10. 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.

  11. 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).

  12. Characterization of mitochondrial ATPase 6/8 genes in wild Labeo calbasu (Hamilton, 1822) and mapping of natural genetic diversity.

    Science.gov (United States)

    Singh, Rajeev K; Lal, Kuldeep K; Mohindra, Vindhya; Sah, Rama S; Kumar, Rajesh; Jena, J K

    2016-09-01

    We characterized mitochondrial ATP synthase (ATPase) 6 and 8 genes in Labeo calbasu (Hamilton, 1822) and determined genetic variation in wild populations across the natural distribution in Indian rivers. A total of 206 individuals were sampled from 11 riverine sites belonging to distinct geographical locations covering five major river basins. Sequencing of 842 base pairs of ATPase 6/8 revealed 21 haplotypes with haplotype diversity ranging from 0.1250 (River Satluj) to 0.8846 (River Bhagirathi). Analysis of molecular variance (AMOVA) of mitochondrial DNA (mtDNA) data revealed significant genetic differentiation among sites (FST = 0.192, p analysis of data demonstrated the potential of ATPase 6/8 genes in determining the genetic diversity and indicated considerable sub-structuring in wild calbasu populations present in different rivers. PMID:25630739

  13. 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

  14. Biochemical and biophysical characterization of sugarcane proteins involved in thermal stress

    International Nuclear Information System (INIS)

    Sugarcane has gained worldwide interest due to its potential to produce ethanol as an alternative energy source. 2 Therefore, it is of general interest to study the physiological and biochemical aspects of sugarcane, especially those involved in stress tolerance, which may be important to understand survival during exposure to high temperatures. In plants, sHsps rank among the most important heat shock proteins expressed during heat stress. They can accumulate up to 1% in leaves and are correlated with the acquisition of thermotolerance. In this context, the study of sHsps proteins may lead to strategies for increasing sugarcane survival and fitness, allowing for its growth in regions where sugarcane has not been produced before. (author)

  15. Biochemical and pharmacological characterization of the human lymphocyte antigen B-associated transcript 5 (BAT5/ABHD16A.

    Directory of Open Access Journals (Sweden)

    Juha R Savinainen

    Full Text Available Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A is a poorly characterized 63 kDa protein belonging to the α/β-hydrolase domain (ABHD containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown.Amino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (≥95% conserved. Activity-based protein profiling (ABPP confirmed successful generation of catalytically active human (h and mouse (m BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14:0, long-chain unsaturated (C18:1, C18:2, C20:4 monoacylglycerols (MAGs and 15-deoxy-Δ12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G. In contrast, hBAT5 possessed only marginal diacylglycerol (DAG, triacylglycerol (TAG, or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3- vs. 2-isomers of MAGs C18:1, C18:2 and C20:4. Inhibitor profiling revealed that β-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A. Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl-3H-[1], [3], [4]oxadiazol-2-one was identified as a highly potent inhibitor (IC50 8.3 nM. Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions.This study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and inhibitor profiles. Utilization of glycerolipid substrates and sensitivity to

  16. Biochemical and Pharmacological Characterization of the Human Lymphocyte Antigen B-Associated Transcript 5 (BAT5/ABHD16A)

    Science.gov (United States)

    Savinainen, Juha R.; Patel, Jayendra Z.; Parkkari, Teija; Navia-Paldanius, Dina; Marjamaa, Joona J. T.; Laitinen, Tuomo; Nevalainen, Tapio; Laitinen, Jarmo T.

    2014-01-01

    Background Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the α/β-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown. Methodology/Principal Findings Amino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (≥95%) conserved. Activity-based protein profiling (ABPP) confirmed successful generation of catalytically active human (h) and mouse (m) BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14∶0), long-chain unsaturated (C18∶1, C18∶2, C20∶4) monoacylglycerols (MAGs) and 15-deoxy-Δ12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G). In contrast, hBAT5 possessed only marginal diacylglycerol (DAG), triacylglycerol (TAG), or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG) and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3)- vs. 2-isomers of MAGs C18∶1, C18∶2 and C20∶4. Inhibitor profiling revealed that β-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A). Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1], [3], [4]oxadiazol-2-one) was identified as a highly potent inhibitor (IC50 8.3 nM). Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions. Conclusions/Significance This study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and

  17. Initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa related to the human cytosolic 5'-nucleotidase I.

    Science.gov (United States)

    Santos, Clelton A; Saraiva, Antonio M; Toledo, Marcelo A S; Beloti, Lilian L; Crucello, Aline; Favaro, Marianna T P; Horta, Maria A C; Santiago, André S; Mendes, Juliano S; Souza, Alessandra A; Souza, Anete P

    2013-01-01

    The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity. PMID:23474016

  18. Biochemical Characterization of 3-Methyl-4-nitrophenol Degradation in Burkholderia sp. Strain SJ98

    Science.gov (United States)

    Min, Jun; Lu, Yang; Hu, Xiaoke; Zhou, Ning-Yi

    2016-01-01

    Several strains have been reported to grow on 3-methyl-4-nitrophenol (3M4NP), the primary breakdown product of the excessively used insecticide fenitrothion. However, the microbial degradation of 3M4NP at molecular and biochemical levels remains unknown. Here, methyl-1,4-benzoquinone (MBQ) and methylhydroquinone (MHQ), rather than catechol proposed previously, were identified as the intermediates before ring cleavage during 3M4NP degradation by Burkholderia sp. strain SJ98. Real-time quantitative PCR analysis indicated that the pnpABA1CDEF cluster involved in para-nitrophenol (PNP) and 2-chloro-4-nitrophenol (2C4NP) catabolism was also likely responsible for 3M4NP degradation in this strain. Purified PNP 4-monooxygenase (PnpA) is able to catalyze the monooxygenation of 3M4NP to MBQ and exhibited an apparent Km value of 20.3 ± 2.54 μM for 3M4NP, and pnpA is absolutely necessary for the catabolism of 3M4NP by gene knock-out and complementation. PnpB, a 1,4-benzoquinone reductase catalyzes the reduction of MBQ to MHQ, and also found to enhance PnpA activity in vitro in the conversion of 3M4NP to MBQ. By sequential catalysis assays, PnpCD, PnpE, and PnpF were likely involved in the lower pathway of 3M4NP catabolism. Although NpcCD, NpcE, and NpcF are able to catalyze the sequential conversion of MHQ in vitro, these enzymes are unlikely involved in 3M4NP catabolism because their coding genes were not upregulated by 3M4NP induction in vivo. These results revealed that the enzymes involved in PNP and 2C4NP catabolism were also responsible for 3M4NP degradation in strain SJ98. This fills a gap in our understanding of the microbial degradation of 3M4NP at molecular and biochemical levels and also provides another example to illustrate the adaptive flexibility in microbial catabolism for structurally similar compounds. PMID:27252697

  19. Molecular and biochemical characterization of the tetralin degradation pathway in Rhodococcus sp. strain TFB

    OpenAIRE

    Tomás‐Gallardo, Laura; Santero, Eduardo; Camafeita, Emilio; Calvo, Enrique; Schlömann, Michael; Floriano, Belén

    2009-01-01

    Summary The tetralin biodegradation pathway in Rhodococcus sp. strain TFB, a Gram‐positive bacterium resistant to genetic manipulation, was characterized using a proteomic approach. Relative protein expression in cell free extracts from tetralin‐ and glucose‐grown cells was compared using the 2D‐DIGE technique. Identification of proteins specifically expressed in tetralin‐grown cells was used to characterize a complete set of genes involved in tetralin degradation by reverse genetics. We prop...

  20. 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.

  1. Biochemical and genetic characterization of the multidrug resistance phenotype in murine macrophage-like J774.2 cells.

    Science.gov (United States)

    Kirschner, L S; Greenberger, L M; Hsu, S I; Yang, C P; Cohen, D; Piekarz, R L; Castillo, G; Han, E K; Yu, L J; Horwitz, S B

    1992-01-01

    The development of multidrug resistance (MDR) in malignant tumors is a major obstacle to the treatment of many cancers. MDR sublines have been derived from the J774.2 mouse macrophage-like cell line and utilized to characterize the phenotype at the biochemical and genetic level. Two isoforms of the drug resistance-associated P-glycoprotein are present and distinguishable both electrophoretically and pharmacologically. Genetic analysis has revealed the presence of a three-member gene family; expression of two of these genes, mdr1a and mdr1b, is associated with MDR whereas the expression of the third, mdr2, is not. Studies of these three genes have revealed similarities and differences in the manner in which they are regulated at the transcriptional level, and have suggested that post-transcriptional effects may also be important. PMID:1346495

  2. 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. PMID:25648922

  3. 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.

  4. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    Science.gov (United States)

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

  5. Characterization of the biochemical-pathway of uranium (VI) reduction in facultative anaerobic bacteria.

    Science.gov (United States)

    Mtimunye, Phalazane J; Chirwa, Evans M N

    2014-10-01

    Cultures of U(VI) reducing bacteria sourced from abandoned uranium mine tailing dam were evaluated for their ability to reduce U(VI) to U(IV). The species in the cultures reduced U(VI) in solutions with initial U(VI) concentration up to 400mgL(-)(1) under a near neutral pH of 6.5. The electron flow pathway and fate of reduced species was also analysed in the individual species in order to evaluate the potential for control and optimisation of the reduction potential at the biochemical level. The results showed that U(VI) reduction in live cells was completely blocked by the NADH-dehydrogenase inhibitor, rotenone (C23H22O6), and thioredoxin inhibitor, cadmium chloride (CdCl2), showing that U(VI) reduction involves the electron flow through NADH-dehydrogenase, a primary electron donor to the electron transport respiratory (ETR) system. Mass balance analysis of uranium species aided by visual and electron microscopy suggest that most U(VI) reduction occurred on the cell surface of the isolated species. This finding indicates the possibility of easy uranium recovery for beneficial use through biological remediation. Should the U(VI) be reduced inside the cell, recovery would require complete disruption of the cells and therefore would be difficult. The study contributes new knowledge on the underlying mechanisms in the U(VI) reduction in facultative anaerobes. PMID:25065785

  6. Biochemical characterization and subcellular localization of the red kidney bean purple acid phosphatase

    International Nuclear Information System (INIS)

    Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the nonspecificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination

  7. Characterization of collagen fibrils after equine suspensory ligament injury: an ultrastructural and biochemical approach.

    Science.gov (United States)

    Shikh Alsook, M K; Gabriel, A; Salouci, M; Piret, J; Alzamel, N; Moula, N; Denoix, J-M; Antoine, N; Baise, E

    2015-04-01

    Suspensory ligament (SL) injuries are an important cause of lameness in horses. The mechanical properties of connective tissue in normal and pathological ligaments are mainly related to fibril morphology, as well as collagen content and types. The purpose of this study was to evaluate, using biochemical and ultrastructural approaches, the alterations in collagen fibrils after injury. Eight Warmblood horses with visible signs of injury in only one forelimb SL were selected and specimens were examined by transmission electron microscope (TEM). Collagen types I, III and V were purified by differential salt precipitation after collagen extraction with acetic acid containing pepsin. TEM revealed abnormal organization as well as alterations in the diameter and shape of fibrils after SL injury. The bands corresponding to types I, III and V collagen were assessed by densitometry after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis indicated that the proportions of type III and type V collagen were higher (P < 0.001) in damaged tissues compared with normal tissues with a mean increase of 20.9% and 17.3%, respectively. Concurrently, a decrease (P < 0.001) in type I collagen within damaged tissues was recorded with a mean decrease of 15.2%. These alterations could be the hallmark of a decrease in the tissue quality and mechanical properties of the ligament. The findings provide new insight for subsequent research on tissue regeneration that may lead to the development of future treatment strategies for SL injury. PMID:25795168

  8. Biochemical and physiological characterization of three rice cultivars under different daytime temperature conditions

    Directory of Open Access Journals (Sweden)

    Alefsi David Sanchez-Reinoso

    2014-12-01

    Full Text Available Heat stress due to high daytime temperatures is one of the main limiting factors in rice (Oryza sativa L. yield in Colombia. Thus, the objective of the present research was to analyze the effect of three different daytime temperatures (25, 35, and 40 °C on the physiological responses of three Colombian rice cultivars (F60, F733, and F473, thereby contributing to the knowledge of rice acclimation mechanisms. For 10 d, eight plants of each of the three cultivars were subjected daily to 5 h periods of 35 and 40 °C. The control treatment corresponded to normal growth conditions (25 °C. Thermal stress was assessed based on a series of physiological and biochemical parameters. The 35 °C treatment produced photosynthetic and respiratory differences in all three cultivars. At 40 °C, 'F60' displayed the lowest photosynthetic rate and the highest respiratory rate. Although this cultivar experienced particularly strong electrolyte leakage and changes in proline when subjected to the high-temperature treatments, similar trends were observed in 'F733' and 'F473'. At 40 °C, the concentration of malondialdehyde (MDA was lower in 'F473' than in the other cultivars. These results may explain the poor agronomic performance of 'F60' in the field under daytime heat stress. The methodologies employed in the present work may be useful in Colombian rice breeding programs, particularly for the selection of heat-tolerant breeding stocks.

  9. 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. PMID:25138314

  10. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

    Science.gov (United States)

    Jourdain, Alexis A; Boehm, Erik; Maundrell, Kinsey; Martinou, Jean-Claude

    2016-03-14

    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  11. 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.

  12. 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.

  13. Behavioral and biochemical characterization of elevated “I-maze” as animal model of anxiety

    Directory of Open Access Journals (Sweden)

    Ritu Gilhotra

    2015-09-01

    Full Text Available The elevated I-maze is a modification of the elevated plus-maze model of anxiety in mice. The design of I-maze comprises a straight wooden passage, resembling the English letter “I,” divided equally into three areas; two enclosed areas (close arms at both ends of the “maze” and an open area in the center of two enclosed areas. The I-maze completely avoids the central platform of elevated plus-maze, removing any ambiguity in time spent on central platform and allowing uninterrupted animal exploration. In this model, diazepam (1 mg/kg and gabapentin (10 mg/kg significantly increased the percentage of time spent in the open areas (%TO and the number of unprotected head dips (uHDIPS, and reduced the number of protected head dips (pHDIPS and stretch attend postures (SAP from close to open arm. Similarly, fluoxetine (5 mg/kg significantly increased %TO and uHDIPS, and significantly decreased SAP from close to open arm, but it did not have any significant effect on pHDIPS. The 5-HT3 receptor antagonist, ondansetron (0.1 mg/kg, did not produce any significant change in all the behaviors, observed, as compared to vehicle-treated control mice. On the other hand, the anxiogenic agent, caffeine (15 mg/kg, did produce a significant decrease in %TO and uHDIPS, and significantly increased pHDIPS and SAP from close to open arm. Mice confined in open area of I-maze bring the relevant biochemical changes associated with anxiety behavior, showing significant increase in the levels of plasma nitrate and plasma corticosterone. These data indicate that a combination of novel design of elevated I-maze and a detailed behavioral analysis provides a sensitive model for the measurement of anxiety.

  14. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii.

    Science.gov (United States)

    Hortua Triana, Miryam Andrea; Huynh, My-Hang; Garavito, Manuel F; Fox, Barbara A; Bzik, David J; Carruthers, Vern B; Löffler, Monika; Zimmermann, Barbara H

    2012-08-01

    The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60μM for l-dihydroorotate, and a K(m)=29μM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91μM, 96μM, and 60μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs. PMID:22580100

  15. 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.

  16. 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

  17. A Multiscale Vibrational Spectroscopic Approach for Identification and Biochemical Characterization of Pollen

    OpenAIRE

    Bagcioglu, Murat; Zimmermann, Boris; Kohler, Achim

    2015-01-01

    Background Analysis of pollen grains reveals valuable information on biology, ecology, forensics, climate change, insect migration, food sources and aeroallergens. Vibrational (infrared and Raman) spectroscopies offer chemical characterization of pollen via identifiable spectral features without any sample pretreatment. We have compared the level of chemical information that can be obtained by different multiscale vibrational spectroscopic techniques. Methodology Pollen from 15 different spec...

  18. BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF SALMONELLA-ENTERICA SEROVAR BERTA, AND COMPARISON OF METHODS FOR TYPING

    DEFF Research Database (Denmark)

    Olsen, J. E.; Brown, D. J.; Baggesen, Dorte Lau; Bisgaard, M.

    1992-01-01

    Strains of Salmonella enterica serovar berta (S. berta) from Denmark and seven other countries have been characterized with the aim of developing a rational typing strategy in connection with outbreak investigations, Biotyping divided the strains into H2S-positive (90 %) and H2S-negative (10...

  19. Characterization of the complete mitochondrial genome of the firefly, Luciola substriata (Coleoptera: Lampyridae).

    Science.gov (United States)

    Mu, Feng-Juan; Ao, Liang; Zhao, Hua-Bin; Wang, Kai

    2016-09-01

    The firefly, Luciola substriata (Coleoptera: Lampyridae), is an aquatic firefly species, whose larvae inhabit ponds or lakes. Here we present the complete mitochondrial (mt) genome of the firefly (GenBank accession number KP313820) and provide its annotation. This circular genome is 16,248 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a non-coding AT-rich region. Similar to other firefly species, the base composition of this mitochondrial genome is also biased toward A and T (44.09% A, 34.00% T, 12.89% C, and 9.01% G). All 13 protein-coding genes start with a typical mitochondrial start codon, and terminate with a usual stop codon TAA, or TAG or a single T. The non-coding AT-rich region (1636 bp in length) include one (A)20, and two (T)15 tandem repeats, and one (AAT)5 element. This mitochondrial genome sequence will promote a better understanding for firefly evolution in the future. PMID:25714154

  20. Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus.

    Science.gov (United States)

    Chen, Y Y; Clancy, K A; Burne, R A

    1996-01-01

    The hydrolysis of urea by urease enzyme of oral bacteria is believed to have a major impact on oral microbial ecology and to be intimately involved in oral health and diseases. To begin to understand the biochemistry and genetics of oral ureolysis, a study of the urease of Streptococcus salivarius, a highly ureolytic organism which is present in large numbers on the soft tissues of the oral cavity, has been initiated. By using as a probe a 0.6-kpb internal fragment of the S. salivarius 57.I ureC gene, two clones from subgenomic libraries of S. salivarius 57.I in an Escherichia coli plasmid vector were identified. Nucleotide sequence analysis revealed the presence of one partial and six complete open reading frames which were most homologous to ureIAB-CEFGD of other ureolytic bacteria. Plasmid clones were generated to construct a complete gene cluster and used to transform E. coli and Streptococcus gordonii DL1, a nonureolytic, dental plaque microorganism. The recombinant organisms expressed high levels of urease activity when the growth medium was supplemented with NiCl2. The urease enzyme was purified from E. coli, and its biochemical properties were compared with those of the urease produced by S. salivarius and those of the urease produced by S. gordonii carrying the plasmid-borne ure genes. In all cases, the enzyme had a Km of 3.5 to 4.1 mM, a pH optimum near 7.0, and a temperature optimum near 60 degrees C. S. gordonii carrying the urease genes was then demonstrated to have a significant capacity to temper glycolytic acidification in vitro in the presence of concentrations of urea commonly found in the oral cavity. The ability to genetically engineer plaque bacteria that can modulate environmental pH through ureolysis will open the way to using recombinant ureolytic organisms to test hypotheses regarding the role of oral ureolysis in dental caries, calculus formation, and periodontal diseases. Such recombinant organisms may eventually prove useful for

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

    OpenAIRE

    Claudia Schillings; Mikael Sunnåker; Jörg Stelling; Christoph Schwab

    2015-01-01

    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 alternat...

  2. 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...

  3. Clinical, Biochemical, and Molecular Characterization of Novel Mutations in ABCA1 in Families with Tangier Disease

    OpenAIRE

    Brunham, Liam R.; Kang, Martin H.; Van Karnebeek, Clara; Sadananda, Singh N.; Collins, Jennifer A.; Zhang, Lin-Hua; Sayson, Bryan; Miao, Fudan; Stockler, Sylvia; Frohlich, Jiri; Cassiman, David; Rabkin, Simon W; Hayden, Michael R

    2014-01-01

    Tangier disease is a rare, autosomal recessive disorder caused by mutations in the ABCA1 gene and is characterized by near absence of plasma high-density lipoprotein cholesterol, accumulation of cholesterol in multiple tissues, peripheral neuropathy, and accelerated atherosclerosis. Here we report three new kindreds with Tangier disease harboring both known and novel mutations in ABCA1. One patient was identified to be homozygous for a nonsense mutation, p.Gln1038*. In a remarkably large Tang...

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

    OpenAIRE

    Claudia Schillings; Mikael Sunnåker; Jörg Stelling; Christoph Schwab

    2015-01-01

    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 alternat...

  5. Production, purification and biochemical characterization of lactate oxidase wild type and double mutant

    OpenAIRE

    García Grillasca, Gustavo

    2010-01-01

    Mutations N212S and S178N of Lactate Oxidase (LOX) from Aeorcoccus viridanswere prepared by site directed mutagenesis and confirmed by sequencing. Once these two mutations were created, the mutated protein was expressed, purified and characterized along with the Wild Type (WT). A protocol was established for purifying the Double Mutant (DM) and the WT, consisting ofan ammonium sulfide precipitation before two subsequent FPLC steps,the former one with a hydrophobic interactio...

  6. Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase inhibition.

    Directory of Open Access Journals (Sweden)

    Jay M West

    Full Text Available The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA, with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. 1-Isothiocyanatopentadecane (AM9023 was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-ylmethyl-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701 and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone, inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126 of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.

  7. Mitochondrial and Cell Death Mechanisms in Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    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.

  8. The Porcine Mitochondrial Transcription Factor a Gene: Molecular Characterization, Radiation Hybrid Mapping and Genetic Diversity among 12 Pig Breeds

    Directory of Open Access Journals (Sweden)

    Tanja Kunej

    2009-01-01

    Full Text Available Problem statement: Mitochondrial transcription factor A (TFAM is a nucleus-encoded protein that is a key activator of mitochondrial transcription as well as a major participant in mitochondrial genome replication. Genomic characterization of the porcine TFAM gene is, therefore, necessary to determine its involvement in regulation of fat depots and meat quality traits in pigs. Approach: Genomic DNA sequence was determined using a comparative in silico annotation approach. RT-PCR was used for analysis of alternative splicing. Genome location was determined using Radiation Hybrid (RH mapping. Genetic marker was identified by sequencing and genotyped by the PCR-RFLP method with SacI. GENEPOP version 3.3 software was used for statistic analysis. Results: We determined both full-length cDNA and genomic DNA sequences of the porcine TFAM gene. Gene expression analysis revealed an alternative 5’ splice site, which excludes exon 4 of the pig gene. We assigned this gene to porcine chromosome 14 (SSC14. A G/A substitution was detected in intron 1 of porcine TFAM gene and genotyped on a total of 252 animals, including 165 from seven Chinese and 87 from five Western pig breeds. The Bayesian analysis via MCMC (Markov chain Monte Carlo revealed that these two groups of pigs were well separated at this locus during the breed history; 95% of the posterior difference of TFAM allelic frequency between these two pig groups was greater than zero. Conclusion/Recommendations: All these data provided basic genomic information needed for further functional characterization of the porcine TFAM gene. Because marked differences in fat and lean tissue deposition exist between Western and Chinese pig breeds, the G557A mutation in the TFAM gene deserves further evaluation to determine its phenotypic effect on fattening and carcass traits in commercial pig populations.

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

    Directory of Open Access Journals (Sweden)

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Biochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3.

    Science.gov (United States)

    Chen, Chao; Liu, Bin; Xu, Yongchang; Utkina, Natalia; Zhou, Dawei; Danilov, Leonid; Torgov, Vladimir; Veselovsky, Vladimir; Feng, Lu

    2016-07-22

    Glycosyltransferases (GTs) catalyze the formation of regio- and stereo-specific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the gene wclR from the Escherichia coli O3 O-antigen gene cluster that encodes an α 1, 3-galactosyltransferase (GalT) that acts on the linkage Gal α 1, 3-GlcNAc was biochemically characterized. WclR was expressed in E. coli BL21 (DE3), and the enzymatic product was identified by liquid chromatography-mass spectrometry (LC-MS), collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)) and galactosidase digestion, using UDP-Gal as the donor substrate and the synthetic acceptor substrate GlcNAc-PP-De (decyl diphosphate N-acetylglucosamine). The physiochemical properties and the substrate specificity of WclR were investigated. WclR is the first bacterial GalT characterized that acts on the linkage Gal α 1, 3-GlcNAc. This study enhanced our knowledge of the diversified functions of GTs and provided a novel enzyme source for possible pharmaceutical application. PMID:27196310

  12. 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. PMID:25618244

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

    OpenAIRE

    Gargouri Youssef; Bouchaala Emna; Karray Aida; Bacha Abir; Ali Yassine

    2011-01-01

    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 fo...

  14. Biochemical and biophysical characterization of humanized IgG1 produced in Pichia pastoris

    OpenAIRE

    Ha, Sha; Wang, Yang; Richard R. Rustandi

    2011-01-01

    The first full length IgG produced in Pichia pastoris was reported in late 1980. However, use of a wild-type Pichia expression system to produce IgGs with human-like N-linked glycans was not possible until recently. Advances in glycoengineering have enabled organisms such as Pichia to mimic human N-glycan biosynthesis and produce IgGs with human glycans on an industrial scale. Since there are only a few reports of the analytical characterization of Pichia-produced IgG, we summarize the result...

  15. PRODUCTION, PARTIAL PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF POLYPHENOL OXIDASE FROM TRICOTHECIUMSP (ENDOPHYTIC FUNGUS OF COCOA PODHUSK)

    OpenAIRE

    Sartini; Patong, Abd. Rauf; Harlim, Tjodi; Pirman

    2013-01-01

    Fungal strains are considered to be excellent sources for industrial polyphenol oxidase production. Polyphenol oxidase could be produced by endophytic fungus Trichotheciumsp from podhusk of Theobroma cacaoL. The aim of this research is to partial purify and characterize of the enzyme. The enzyme was produced by submerged fermentation using media with composition, i.e: 1 % yeast extract, 25 ppm CuSO 4.5H 2O, 0.034 % gallic acid, 0.1 % KH2PO 4, 0.05 % MgSO 4.7H 2 0. The steps of purification we...

  16. 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...

  17. 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.

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

    Science.gov (United States)

    Marín, C; Fernández-Ramos, C; Entrala, E; Quesada, J M; Sánchez-Moreno, M

    2000-01-01

    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. PMID:10998214

  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 characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

    DEFF Research Database (Denmark)

    Zameitat, E.; Gojkovic, Zoran; Knecht, Wolfgang; Piskur, Jure; Loffler, M.

    2006-01-01

    Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine...... lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine...... derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in...

  1. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Biochemical and pharmacological characterization of irradiated crotamine by gamma rays of 60Co

    International Nuclear Information System (INIS)

    The serum production in Brazil, the only effective treatment in cases of snakebites, uses horses that although large size, have reduced l lifespan compared with horses not immunized. Ionizing radiation has been shown as an excellent tool in reducing the toxicity of venoms and toxins isolated, and promote the achievement of better immunogens for serum production, and contributing to the welfare of serum-producing animals. It is known, however, that the effects of ionizing radiation on protein are characterized by various chemical modifications, such as fragmentation, cross-linking due to aggregation and oxidation products generated by water radiolysis. However, the action of gamma radiation on toxins is not yet fully understood structurally and pharmacologically, a fact that prevents the application of this methodology in the serum production process. So we proposed in this paper the characterization of crotamine, an important protein from the venom of Crotalus durissus terrificus species, irradiated with 60Co gamma rays. After isolating the toxin by chromatographic techniques and testing to prove the obtaining of pure crotamine, it was irradiated with gamma rays and subjected to structural analysis, Fluorescence and Circular Dichroism. Using high hydrostatic pressure tests were also conducted in order to verify that the conformational changes caused by radiation suffer modifications under high pressures. From the pharmacological point of view, muscle contraction tests were conducted with the objective of limiting the action of crotamine in smooth muscle as well as the change in the action of toxin caused structural changes to the front. Analysis of Circular Dichroism and Fluorescence showed changes in structural conformation of crotamine when subjected to gamma radiation and that such changes possibly occurring in the secondary and tertiary structure of the protein. The observed in pharmacological tests showed that the irradiated crotamine was less effective in

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

    International Nuclear Information System (INIS)

    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 km 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 oC 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 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.

  6. Molecular and biochemical characterizations of the monoacylglycerol lipase gene family of Arabidopsis thaliana.

    Science.gov (United States)

    Kim, Ryeo Jin; Kim, Hae Jin; Shim, Donghwan; Suh, Mi Chung

    2016-03-01

    Monoacylglycerol lipase (MAGL) catalyzes the last step of triacylglycerol breakdown, which is the hydrolysis of monoacylglycerol (MAG) to fatty acid and glycerol. Arabidopsis harbors over 270 genes annotated as 'lipase', the largest class of acyl lipid metabolism genes that have not been characterized experimentally. In this study, computational modeling suggested that 16 Arabidopsis putative MAGLs (AtMAGLs) have a three-dimensional structure that is similar to a human MAGL. Heterologous expression and enzyme assays indicated that 11 of the 16 encoded proteins indeed possess MAG lipase activity. Additionally, AtMAGL4 displayed hydrolase activity with lysophosphatidylcholine and lysophosphatidylethanolamine (LPE) substrates and AtMAGL1 and 2 utilized LPE as a substrate. All recombinant AtMAGLs preferred MAG substrates with unsaturated fatty acids over saturated fatty acids and AtMAGL8 exhibited the highest hydrolase activities with MAG containing 20:1 fatty acids. Except for AtMAGL4, -14 and -16, all AtMAGLs showed similar activity with both sn-1 and sn-2 MAG isomers. Spatial, temporal and stress-induced expression of the 16 AtMAGL genes was analyzed by transcriptome analyses. AtMAGL:eYFP fusion proteins provided initial evidence that AtMAGL1, -3, -6, -7, -8, -11, -13, -14 and -16 are targeted to the endoplasmic reticulum and/or Golgi network, AtMAGL10, -12 and -15 to the cytosol and AtMAGL2, -4 and -5 to the chloroplasts. Furthermore, AtMAGL8 was associated with the surface of oil bodies in germinating seeds and leaves accumulating oil bodies. This study provides the broad characterization of one of the least well-understood groups of Arabidopsis lipid-related enzymes and will be useful for better understanding their roles in planta. PMID:26932457

  7. 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.

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

    International Nuclear Information System (INIS)

    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 99mTc-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.)

  9. Biochemical characterization of the nucleic acids of some human and animal viruses

    International Nuclear Information System (INIS)

    The isolation and partial characterization of human polyomaviruses from a number of renal transplant patients is described. These isolates proved refractory to cell culture propagation by the methods used, and were thus extracted directly from large volumes of patient's urine. This approach has the advantage that the virus cannot undergo any possible genomic modification. The quantity of DNA obtained directly from urine was asually very limited. In order to produce adequate DNA for complete analysis, viral DNA was recombined with a bacterial vector and then cloned. This clone was used to prepare radioactively-labelled DNA probes for the detection of BK-specific sequences in urine isolates and in subsequent recombinants with patient material. The genomes of four rotaviruses were also studied. Experiments were performed to confirm the double-strainded RNA (dsRNA) nature of the Simian agents II genome. The difficulties in obtaining precise molecular weight values for rotavirus genome segments are also discussed. Gel systems were developed to improve on the resolution obtained in co-electrophoresis experiments. During attempts to culture Simian agents II and offal agent viruses in cell culture, it was observed that treatment of the cells and/or virus with versene-trypsin solution during infection gave a marked increase in virus yield

  10. Biochemical characterization and cellular effects of CADASIL mutants of NOTCH3.

    Directory of Open Access Journals (Sweden)

    He Meng

    Full Text Available 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 muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells.

  11. Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria.

    Science.gov (United States)

    Ban, L; Scaloni, A; D'Ambrosio, C; Zhang, L; Yahn, Y; Pelosi, P

    2003-02-01

    Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera. PMID:12678502

  12. 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. PMID:25623240

  13. Pyranose 2-oxidase from Phanerochaete chrysosporium--expression in E. coli and biochemical characterization.

    Science.gov (United States)

    Pisanelli, Ines; Kujawa, Magdalena; Spadiut, Oliver; Kittl, Roman; Halada, Petr; Volc, Jindrich; Mozuch, Michael D; Kersten, Philip; Haltrich, Dietmar; Peterbauer, Clemens

    2009-06-15

    The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in yields of approximately 270 mg/l medium. The recombinant enzyme was provided with an N-terminal T7-tag and a C-terminal His(6)-tag to facilitate simple one-step purification. We obtained an apparently homogenous enzyme preparation with a specific activity of 16.5 U/mg. Recombinant P2Ox from P. chrysosporium was characterized in some detail with respect to its physical and catalytic properties, both for electron donor (sugar substrates) and - for the first time - alternative electron acceptors (1,4-benzoquinone, substituted quinones, 2,6-dichloroindophenol and ferricenium ion). As judged from the catalytic efficiencies k(cat)/K(m), some of these alternative electron acceptors are better substrates than oxygen, which might have implications for the proposed in vivo function of pyranose 2-oxidase. PMID:19501263

  14. 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. PMID:21501629

  15. Physiological, biochemical and molecular characterization of an induced mutation conferring imidazolinone resistance in wheat.

    Science.gov (United States)

    Jimenez, Francisco; Rojano-Delgado, Antonia M; Fernández, Pablo Tomas; Rodríguez-Suárez, Cristina; Atienza, Sergio G; De Prado, Rafael

    2016-09-01

    The Clearfield(®) wheat cultivars possessing imidazolinone (IMI)-resistant traits provide an efficient option for controlling weeds. The imazamox-resistant cultivar Pantera (Clearfield(®) ) was compared with a susceptible cultivar (Gazul). Target and non-target mechanisms of resistance were studied to characterize the resistance of Pantera and to identify the importance of each mechanism involved in this resistance. Pantera is resistant to imazamox as was determined in previous experiments. The molecular study confirmed that it carries a mutation Ser-Asn627 conferring resistance to imazamox in two out of three acetolactate synthase (ALS) genes (imi1 and imi2), located in wheat on chromosomes 6B and 6D, respectively. However, the last gene (imi3) located on chromosome 6A does not carry any mutation conferring resistance. As a result, photosynthetic activity and chlorophyll content were reduced after imazamox treatment. Detoxification was higher in the resistant biotype as shown by metabolomic study while imazamox translocation was higher in the susceptible cultivar. Interestingly, imazamox metabolism was higher at higher doses of herbicide, which suggests that the detoxification process is an inducible mechanism in which the upregulation of key gene coding for detoxification enzymes could play an important role. Thus, the identification of cultivars with a higher detoxification potential would allow the development of more resistant varieties. PMID:26991509

  16. 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.

  17. Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Tuininga, J E; Verhees, C H; van der Oost, J; Kengen, S W; Stams, A J; de Vos, W M

    1999-07-23

    Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively. PMID:10409652

  18. 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. PMID:26317784

  19. 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.

  20. 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. PMID:22170098

  1. Assessment of inactivated human rabies vaccines: biochemical characterization and genetic identification of virus strains.

    Science.gov (United States)

    Finke, Stefan; Karger, Axel; Freuling, Conrad; Müller, Thomas

    2012-05-21

    The World Health Organization (WHO) recommends the periodic evaluation of the purity of the cell lines used in the production of rabies vaccines, as well as the antigenic identity of the virus strains. Here, we analyzed seventeen marketed inactivated human rabies virus vaccines for viral and non-viral proteins by SDS-PAGE and Coomassie/silver staining. Mass spectrometric analysis of an abundant 60-70 kDa signal indicated that in most vaccines serum albumin of human origin (HSA) was the major component. Quantification of HSA in the vaccines revealed a mean concentration of 22 mg HSA/dose in all tested PVRV (purified vero cell rabies vaccine), HDCV (human diploid cell rabies vaccine) and PHK (primary hamster kidney) vaccines. In contrast, 1000-fold lower HSA levels and no HSA were detected in PCECV (purified chick embryo cell-culture vaccine) and PDEV (duck embryo rabies vaccine), respectively. Western blot analyses further confirmed a high bias in the HSA content, whereas the virus protein levels were rather similar in all tested vaccines. In addition, the vaccine viruses were sequenced within the N- and G-genes to identify the strain. In the majority of sequenced vaccines, the declared vaccine strain was confirmed. However, some discrepancies in the genetic identification were observed, supporting WHO's recommendation for the molecular characterization of vaccine seed strains. This research highlights the variation in purity found between different human rabies virus vaccines, and suggests that further research is needed to establish the impact non-active components have on the potency of such vaccines. PMID:22469862

  2. Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes.

    Science.gov (United States)

    Lufrano, Daniela; Cotabarren, Juliana; Garcia-Pardo, Javier; Fernandez-Alvarez, Roberto; Tort, Olivia; Tanco, Sebastián; Avilés, Francesc Xavier; Lorenzo, Julia; Obregón, Walter D

    2015-12-01

    Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B

  3. Structural and biochemical characterization of peroxiredoxin Qbeta from Xylella fastidiosa: catalytic mechanism and high reactivity.

    Science.gov (United States)

    Horta, Bruno Brasil; de Oliveira, Marcos Antonio; Discola, Karen Fulan; Cussiol, José Renato Rosa; Netto, Luis Eduardo Soares

    2010-05-21

    The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 10(7) and 10(6) M(-1) S(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie approximately 12.3 A apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account. PMID:20335172

  4. Immunological and biochemical characterization of coxsackie virus A16 viral particles.

    Directory of Open Access Journals (Sweden)

    Pele Chong

    Full Text Available BACKGROUND: Coxsackie virus A16 (CVA16 infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD. There are currently no vaccine or effective medical treatments available. PRINCIPAL FINDING: In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10(6 the tissue culture's infectious dose (TCID(50 per mL within 7 days post-infection when a multiplicity of infection (MOI of 10(-5 was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24-28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35-38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4, as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176-190. CONCLUSION: These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.

  5. Biochemical characterization of a halotolerant feruloyl esterase from Actinomyces spp.: refolding and activity following thermal deactivation.

    Science.gov (United States)

    Hunt, Cameron J; Tanksale, Akshat; Haritos, Victoria S

    2016-02-01

    Ferulic acid esterases (FAE, EC. 3.1.1.73) hydrolyse the linkage between hemicellulose and lignin and thus have potential for use in mild enzymatic pretreatment of biomass as an alternative to thermochemical approaches. Here, we report the characterization of a novel FAE (ActOFaeI) obtained from the bacterium, Actinomyces sp. oral which was recombinantly expressed in Escherichia coli BL21 in two forms: with and without its putative signal peptide. The truncated form was found to have purification. The enzyme with retained peptide demonstrated 2 to 4-fold higher activity against methyl caffeate and methyl p-coumarate, with specific activities of 477.6 and 174.4 U mg(-1) respectively, than the equivalent activities of the benchmark FAE from Aspergillus niger A and B. ActOFaeI retained activity over a broad pH range with a maximum at 9 but >90 % relative activity at pH 6.5 and an optimum reaction temperature of 30 °C. ActOFaeI increased activity by 15% in high salt conditions (1000 mMNaCl) and its thermal unfolding temperature improved from 41.5 °C in standard buffer to 74 °C in the presence of 2500 mM sodium malonate. ActOFaeI also released ferulic acid from destarched wheat bran when combined with a xylanase preparation. After treatment above the thermal denaturation temperature followed by cooling to room temperature, ActOFaeI demonstrated spontaneous refolding into an active state. ActOFaeI displays many useful characteristics for enzymatic pretreatment of lignocellulose and contributes to our understanding of this important family. PMID:26497017

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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. PMID:25818323

  8. 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.

  9. 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. PMID:24856135

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

    Directory of Open Access Journals (Sweden)

    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

  12. Ultrasound and Biochemical Diagnostic Tools for the Characterization of Vulnerable Carotid Atherosclerotic Plaque.

    Science.gov (United States)

    Lechareas, Simeon; Yanni, Amalia E; Golemati, Spyretta; Chatziioannou, Achilles; Perrea, Despoina

    2016-01-01

    Stroke is a leading cause of morbidity and mortality worldwide, and characterization of vulnerable carotid plaque remains the spearhead of scientific research. Plaque destabilization, the key factor that induces the series of events leading to the clinical symptoms of carotid artery disease, is a consequence of complex mechanical, structural and biochemical processes. Novel imaging and molecular markers have been studied as predictors of disease outcome with promising results. The aim of this review is to present the current state of research on the association between ultrasound-derived echogenicity indices and blood parameters indicative of carotid plaque stability and activity. Bibliographic research revealed that there are limited available data. Among the biomarkers studied, those related to oxidative stress, lipoproteins and diabetes/insulin resistance are associated with echolucent plaques, whereas adipokines are associated with echogenic plaques. Biomarkers of inflammation and coagulation have not exhibited any conclusive relationship with plaque echogenicity, and it is not possible to come to any conclusion regarding calcification-, apoptosis- and neo-angiogenesis-related parameters because of the extremely limited bibliographic data. PMID:26493239

  13. Growth Parameters, Photosynthetic Performance, and Biochemical Characterization of Newly Isolated Green Microalgae in Response to Culture Condition Variations.

    Science.gov (United States)

    Jazzar, Souhir; Berrejeb, Nadia; Messaoud, Chokri; Marzouki, Mohamed Néjib; Smaali, Issam

    2016-08-01

    This work aimed to characterize two native microalgal strains newly isolated from South Mediterranean areas and identified as Chlorella sorokiniana ES3 and Neochloris sp. AM2. The growth properties and biochemical composition of these microalgae were evaluated in different culture media (Algal, BG-11, f/2, and Conway). Among the tested media, nitrate- and phosphate-rich Algal medium provided the maximum biomass productivities (85.5 and 111.5 mg l(-1) day(-1) for C. sorokiniana and Neochloris sp., respectively), while the nitrate- and phosphate-deficient f/2 medium resulted in the highest lipid productivities (24.1 and 35.8 mg l(-1) day(-1) for C. sorokiniana and Neochloris sp., respectively). The physiological state of both microalgae was investigated under different light and temperature levels using the pulse amplitude-modulated fluorometry. The better photosynthetic efficiency of C. sorokiniana was obtained at 23 °C with a light saturation of 156 μE m(-2) s(-1), while that of Neochloris sp. was achieved at 15 °C with a light saturation of 151 μE m(-2) s(-1). The analysis of fatty acid profile and biodiesel parameters revealed that C. sorokiniana, cultivated in Algal and f/2 media, can be considered as a suitable candidate for high-quality biodiesel production. PMID:27052210

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Structural and Biochemical Characterization of Xylella fastidiosa DsbA Family Members: New insightsinto the Enzyme-Substrate Interaction

    Energy Technology Data Exchange (ETDEWEB)

    Rinaldi, F.; Meza, A; Gulmarges, B

    2009-01-01

    Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.

  16. 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 (ADHES77). Interestingly, the ADHES77 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 (NH4)2SO4 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. PMID:26216639

  17. A new lipase as a pharmaceutical target for battling infections caused by Staphylococcus aureus: Gene cloning and biochemical characterization.

    Science.gov (United States)

    Ünlü, Aişe; Tanriseven, Aziz; Sezen, I Yavuz; Çelik, Ayhan

    2015-01-01

    Staphylococcus aureus lipases along with other cell-wall-associated proteins and enzymes (i.e., catalase, coagulase, protease, hyaluronidase, and β-lactamase) play important roles in the pathogenesis of S. aureus and are important subject of drug targeting. The appearance of antibiotic-resistant types of pathogenic S. aureus (e.g., methicillin-resistant S. aureus, MRSA) is a worldwide medical problem. In the present work, a novel lipase from a newly isolated MRSA strain from a cow with subclinical mastitis was cloned and biochemically characterized. The mature part of the lipase was expressed in Escherichia coli and purified by nickel affinity chromatography. It displays a high lipase activity at pH 8.0 and 25 °C using p-nitrophenyl palmitate and has a preference for medium-long-chain substrates of p-nitrophenyl esters (pNPC10-C16). Furthermore, in search of inhibitors, the effect of farnesol on the growth of S. aureus and the lipase activity was also studied. Farnesol inhibits the growth of S. aureus and is a mixed-type inhibitor with Ki and Ki (') values of 0.2 and 1.2 mmol L(-1), respectively. A lipase with known properties could not only serve as a template for developing inhibitors for S. aureus but also a valuable addition to enzyme toolbox of biocatalysis. The discovery of this lipase can be potentially important and could provide a new target for pharmaceutical intervention against S. aureus infection. PMID:25385356

  18. Anaerobic co-digestion of commercial food waste and dairy manure: Characterizing biochemical parameters and synergistic effects.

    Science.gov (United States)

    Ebner, Jacqueline H; Labatut, Rodrigo A; Lodge, Jeffrey S; Williamson, Anahita A; Trabold, Thomas A

    2016-06-01

    Anaerobic digestion of commercial food waste is a promising alternative to landfilling commercial food waste. This study characterized 11 types of commercial food wastes and 12 co-digestion blends. Bio-methane potential, biodegradable fraction, and apparent first-order hydrolysis rate coefficients were reported based upon biochemical methane potential (BMP) assays. Food waste bio-methane potentials ranged from 165 to 496 mL CH4/g VS. Substrates high in lipids or readily degradable carbohydrates showed the highest methane production. Average bio-methane potential observed for co-digested substrates was -5% to +20% that of the bio-methane potential of the individual substrates weighted by VS content. Apparent hydrolysis rate coefficients ranged from 0.19d(-1) to 0.65d(-1). Co-digested substrates showed an accelerated apparent hydrolysis rate relative to the weighted average of individual substrate rates. These results provide a database of key bio-digestion parameters to advance modeling and utilization of commercial food waste in anaerobic digestion. PMID:27090232

  19. Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti.

    Science.gov (United States)

    Chauhan, Nikhil; Sharma, Rohit; Hoti, S L

    2015-02-01

    Homologues of human macrophage migration inhibitory factor (hMIF) have been reported from vertebrates, invertebrates and prokaryotes, as well as plants. Filarial parasites produce two homologues of hMIF viz., MIF-1 and MIF-2, which play important role in the host immune modulation. Earlier, we have characterized MIF-1 (Wba-mif-1) from Wuchereria bancrofti, the major causal organism of human lymphatic filariasis. Here, we are reporting the molecular and biochemical characterization of MIF-2 from this parasite (Wba-mif-2). The complete Wba-mif-2 gene and its cDNA were amplified, cloned and sequenced. The size of Wba-mif-2 gene and cDNA were found to be 4.275 kb and 363 bp, respectively. The gene annotation revealed the presence of a large intron of 3.912 kb interspersed with two exons of 183 bp and 180 bp. The alignment of derived amino acid sequences of Wba-MIF-2 with Wba-MIF-1 showed 44% homology. The conserved CXXC oxido-reductase catalytic site present in Wba-mif-1 was found absent in Wba-mif-2 coding sequence. The amplified Wba-mif-2 cDNA was cloned into an expression vector pRSET-B and transformed into salt inducible Escherichia coli strain GJ1158. The expressed recombinant Wba-MIF-2 protein showed tautomerase activity against L-dopachrome methyl ester and the specific activity was determined to be 18.57±0.77 μmol/mg/min. Three known inhibitors of hMIF tautomerase activity significantly inhibited the tautomerase activity of recombinant Wba-MIF-2. Although the conserved CXXC oxido-reductase motif is absent in Wba-mif-2, the recombinant protein showed significant oxido-reductase activity in the insulin reduction assay, possibly because of the presence of vicinal cysteine residues. PMID:25446175

  20. Identification and biochemical characterization of polyamine oxidases in amphioxus: Implications for emergence of vertebrate-specific spermine and acetylpolyamine oxidases.

    Science.gov (United States)

    Wang, Huihui; Liu, Baobao; Li, Hongyan; Zhang, Shicui

    2016-01-10

    Polyamine oxidases (PAOs) have been identified in a wide variety of animals, as well as in fungi and plant. Generally, plant PAOs oxidize spermine (Spm), spermidine (Spd) and their acetylated derivatives, N(1)-acetylspermine (N(1)-Aspm) and N(1)-acetylspermidine (N(1)-Aspd), while yeast PAOs oxidize Spm, N(1)-Aspm and N(1)-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N(1)-Aspm/N(1)-Aspd, respectively. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic. In this study, two amphioxus (Branchiostoma japonicum) PAO genes, named Bjpao1 and Bjpao2, were cloned and characterized. Both Bjpao1 and Bjpao2 displayed distinct tissue-specific expression patterns. Notably, rBjPAO1 oxidized both spermine and spermidine, but not N(1)-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N(1)-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAOs that were generated by site-directed mutagenesis and expressed in E. coli were examined, The results indicate that the residues H64, K301 and T460 in rBjPAO1, and H69, K315 and T467 in rBjPAO2 were all involved in substrate binding and enzyme catalytic activity to some extent. Based on our results and those of others, a model depicting the divergent evolution and functional specialization of vertebrate SMO and APAO genes is proposed. PMID:26367330

  1. The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy.

    Science.gov (United States)

    Vincent, Amy E; Ng, Yi Shiau; White, Kathryn; Davey, Tracey; Mannella, Carmen; Falkous, Gavin; Feeney, Catherine; Schaefer, Andrew M; McFarland, Robert; Gorman, Grainne S; Taylor, Robert W; Turnbull, Doug M; Picard, Martin

    2016-01-01

    Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease. PMID:27506553

  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. "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.

  4. 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.

  5. Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Hatanaka, Rie; Furuki, Takao; Shimizu, Tempei; Takezawa, Daisuke; Kikawada, Takahiro; Sakurai, Minoru; Sugawara, Yasutake

    2014-11-28

    Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation-rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a "molecular shield". Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins. PMID:25450698

  6. Structural and biochemical characterization of bacterial YpgQ protein reveals a metal-dependent nucleotide pyrophosphohydrolase.

    Science.gov (United States)

    Jeon, Ye Ji; Park, Sun Cheol; Song, Wan Seok; Kim, Ok-Hee; Oh, Byung-Chul; Yoon, Sung-Il

    2016-07-01

    The optimal balance of cellular nucleotides and the efficient elimination of non-canonical nucleotides are critical to avoiding erroneous mutation during DNA replication. One such mechanism involves the degradation of excessive or abnormal nucleotides by nucleotide-hydrolyzing enzymes. YpgQ contains the histidine-aspartate (HD) domain that is involved in the hydrolysis of nucleotides or nucleic acids, but the enzymatic activity and substrate specificity of YpgQ have never been characterized. Here, we unravel the catalytic activity and structural features of YpgQ to report the first Mn(2+)-dependent pyrophosphohydrolase that hydrolyzes (deoxy)ribonucleoside triphosphate [(d)NTP] to (deoxy)ribonucleoside monophosphate and pyrophosphate using the HD domain. YpgQ from Bacillus subtilis (bsYpgQ) displays a helical structure and assembles into a unique dimeric architecture that has not been observed in other HD domain-containing proteins. Each bsYpgQ monomer accommodates a metal ion and a nucleotide substrate in a cavity located between the N- and C-terminal lobes. The metal cofactor is coordinated by the canonical residues of the HD domain, namely, two histidine residues and two aspartate residues, and is positioned in close proximity to the β-phosphate group of the nucleotide, allowing us to propose a nucleophilic attack mechanism for the nucleotide hydrolysis reaction. YpgQ enzymes from other bacterial species also catalyze pyrophosphohydrolysis but exhibit different substrate specificity. Comparative structural and mutational studies demonstrated that residues outside the major substrate-binding site of bsYpgQ are responsible for the species-specific substrate preference. Taken together, our structural and biochemical analyses highlight the substrate-recognition mode and catalysis mechanism of YpgQ in pyrophosphohydrolysis. PMID:27062940

  7. Homologous expression and biochemical characterization of the arylsulfatase from Kluyveromyces lactis and its relevance in milk processing.

    Science.gov (United States)

    Stressler, Timo; Leisibach, Desirée; Lutz-Wahl, Sabine; Kuhn, Andreas; Fischer, Lutz

    2016-06-01

    The industrial manufacturing process of lactose-free milk products depends on the application of commercial β-galactosidase (lactase) preparations. These preparations are often obtained from Kluyveromyces lactis. There is a gene present in the genome of K. lactis which should encode for an enzyme called arylsulfatase (EC 3.1.6.1). Therefore, this enzyme could also be present in β-galactosidase preparations. The arylsulfatase is suspected of being responsible for an unpleasant "cowshed-like" off-flavor resulting from the release of p-cresol from milk endogenous alkylphenol sulfuric esters. So far, no gene/functionality relationship is described. In addition, no study is available which has shown that arylsulfatase from K. lactis is truly responsible for the flavor generation. In this study, we cloned the putative arylsulfatase gene from K. lactis GG799 into the commercially available vector pKLAC2. The cloning strategy chosen resulted in a homologous, secretory expression of the arylsulfatase. We showed that the heretofore putative arylsulfatase has the desired activity with the synthetic substrate p-nitrophenyl sulfate and with the natural substrate p-cresol sulfate. The enzyme was biochemically characterized and showed an optimum temperature of 45-50 °C and an optimum pH of 9-10. Additionally, the arylsulfatase was activated by Ca(2+) ions and was inactivated by Zn(2+) ions. Moreover, the arylsulfatase was inhibited by p-cresol and sulfate ions. Finally, the enzyme was added to ultra-heat treated (UHT) milk and a sensory triangle test verified that the arylsulfatase from K. lactis can cause an unpleasant "cowshed-like" off-flavor. PMID:26875879

  8. Cell wall-bound cationic and anionic class III isoperoxidases of pea root: biochemical characterization and function in root growth.

    Science.gov (United States)

    Kukavica, Biljana M; Veljovicc-Jovanovicc, Sonja D; Menckhoff, Ljiljana; Lüthje, Sabine

    2012-07-01

    Cell wall isolated from pea roots was used to separate and characterize two fractions possessing class III peroxidase activity: (i) ionically bound proteins and (ii) covalently bound proteins. Modified SDS-PAGE separated peroxidase isoforms by their apparent molecular weights: four bands of 56, 46, 44, and 41kDa were found in the ionically bound fraction (iPOD) and one band (70kDa) was resolved after treatment of the cell wall with cellulase and pectinase (cPOD). Isoelectric focusing (IEF) patterns for iPODs and cPODs were significantly different: five iPODs with highly cationic pI (9.5-9.2) were detected, whereas the nine cPODs were anionic with pI values between pH 3.7 and 5. iPODs and cPODs showed rather specific substrate affinity and different sensitivity to inhibitors, heat, and deglycosylation treatments. Peroxidase and oxidase activities and their IEF patterns for both fractions were determined in different zones along the root and in roots of different ages. New iPODs with pI 9.34 and 9.5 were induced with root growth, while the activity of cPODs was more related to the formation of the cell wall in non-elongating tissue. Treatment with auxin that inhibits root growth led to suppression of iPOD and induction of cPOD. A similar effect was obtained with the widely used elicitor, chitosan, which also induced cPODs with pI 5.3 and 5.7, which may be specifically related to pathogen defence. The differences reported here between biochemical properties of cPOD and iPOD and their differential induction during development and under specific treatments implicate that they are involved in specific and different physiological processes. PMID:22760472

  9. Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers.

    Directory of Open Access Journals (Sweden)

    Laura Civiero

    Full Text Available Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2 are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD. Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.

  10. Characterization of a Functional Hydrogel Layer on a Silicon-Based Grating Waveguide for a Biochemical Sensor

    OpenAIRE

    Hong, Yoo-Seung; Kim, Jongseong; Sung, Hyuk-Kee

    2016-01-01

    We numerically demonstrated the characteristics of a functional hydrogel layer on a silicon-based grating waveguide for a simple, cost-effective refractive index (RI) biochemical sensor. The RI of the functional hydrogel layer changes when a specific biochemical interaction occurs between the hydrogel-linked receptors and injected ligand molecules. The transmission spectral profile of the grating waveguide shifts depends on the amount of RI change caused by the functional layer. Our character...

  11. Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications†,‡

    OpenAIRE

    Cao, Heping

    2004-01-01

    Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding act...

  12. Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization

    OpenAIRE

    Bakonyi, Tamás; Derakhshifar, Irmgard; Grabensteiner, Elvira; Nowotny, Norbert

    2003-01-01

    PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by anal...

  13. 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

  14. 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.

  15. 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

  16. Molecular characterization of six sub population Indonesian local goats based on mitochondrial DNA D-loop

    Directory of Open Access Journals (Sweden)

    Aron Batubara

    2013-03-01

    Full Text Available Indonesian local goats were spread in some region, but there was still limited data’s known about the characteristics of its genetic diversity and origin. The Mitochondrial DNA D-loop sequences were used to study the genetic diversity and relationships of six sub population Indonesian local goats, namely, Kacang, Marica, Samosir, Jawarandu, Muara and Bengali goats. From 539 blood samples and DNA extraction collections were selected about 60 samples (10 samples each sub populations analyzed by PCR-RFLP methods, followed sequence analyzed about 5 PCR products each sub population. The results of the sequence analyses were edited and acquired about 957 bp of nucleotides length. After the alignment analyses were found 50 polymorphic sites which divided into 19 haplotype groups of mtDNA D-loop region. The value of nucleotide diversity was 0.014 ± 0.002. Analysis of Neighbour Joining with Kimura 2 Parameter methods and bootstrap test with 1000 replication indicated that each sub population groups was significantly different between one groups to the others. The maternal lineages origin of six breeds of Indonesian local goats was included to the group of lineage B. The Lineage B was the maternal origin of the haplogroup of goats in the region of East Asia, South Asia, China, Mongolia, North and South Africa, Malaysia, Indonesia, Pakistan and India.

  17. Characterization of the complete mitochondrial genome of the Scarlet Tiger moth Callimorpha dominula (Insecta: Lepidoptera: Arctiidae).

    Science.gov (United States)

    Peng, Xiao-Yi; Duan, Xiao-Yu; Qiang, Yi

    2016-09-01

    The complete mitochondrial genome of the Scarlet Tiger moth Callimorpha dominula (Insecta: Lepidoptera: Arctiidae) has been reconstructed from the whole-genome Illumina sequencing data. This circular genome is 15 496 bp in size, and contains 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and one A + T-rich D-loop or control region. Most PCGs are initiated with the ATN codons, except for COX1 with the unusual CGA as its initiation codon. Four PCGs (COX1, COX2, ND3, and ND4) are terminated with incomplete codon T, ND4L uses TAG as its termination codon, while all the other eight PCGs employ the usual ATN codons. The nucleotide composition is highly asymmetric (40.1% A, 40.9% T, 7.6% G, and 11.4% C) with an overall A + T content of 81.0%. The phylogenetic analysis based on the neighbor-joining (NJ) method suggests that C. dominula is more phylogenetically related to its confamilial counterparts than to those from other families. PMID:26329289

  18. Characterization of the complete mitochondrial genome of the red crayfish, Procambarus clarkii (Decapoda: Cambaridae).

    Science.gov (United States)

    Liu, Qiu-Ning; Chai, Xin-Yue; Jiang, Sen-Hao; Zhou, Chun-Lin; Xuan, Fu-Jun; Tang, Bo-Ping

    2016-09-01

    Here we present the complete mitochondrial (mt) genome of Procambarus clarkii (Decapoda: Cambaridae) and provide its annotation. The complete mt genome was determined to be 15 929 bp and contains 22 tRNA genes, 13 protein-coding genes (PCGs), two rRNA genes, and a D-loop region. The nucleotide composition was biased toward A + T nucleotides (72.91%) and the AT skew of this mt genome was slightly negative. All the 22 tRNA genes displayed a typical clover-leaf structure, with the exception of trnS1 (AGN). About 13 PCGs were initiated by ATN codons, except for cox1 and nad2 genes which were initiated by ACG and GTG, respectively. Six of the 13 PCGs harbor the incomplete termination codon by T or TA. The D-loop region of the mt genome was 1188 bp in length and the A + T content was 81.08%. Phylogenetic analysis showed that the placement of P. clarkii was within the Cambaridae. This mt genome sequence will provide a better understanding for crayfish evolution in the future. PMID:26258501

  19. The complete mitochondrial genome of rabbit pinworm Passalurus ambiguus: genome characterization and phylogenetic analysis.

    Science.gov (United States)

    Liu, Guo-Hua; Li, Sheng; Zou, Feng-Cai; Wang, Chun-Ren; Zhu, Xing-Quan

    2016-01-01

    Passalurus ambiguus (Nematda: Oxyuridae) is a common pinworm which parasitizes in the caecum and colon of rabbits. Despite its significance as a pathogen, the epidemiology, genetics, systematics, and biology of this pinworm remain poorly understood. In the present study, we sequenced the complete mitochondrial (mt) genome of P. ambiguus. The circular mt genome is 14,023 bp in size and encodes of 36 genes, including 12 protein-coding, two ribosomal RNA, and 22 transfer RNA genes. The mt gene order of P. ambiguus is the same as that of Wellcomia siamensis, but distinct from that of Enterobius vermicularis. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference (BI) showed that P. ambiguus was more closely related to W. siamensis than to E. vermicularis. This mt genome provides novel genetic markers for studying the molecular epidemiology, population genetics, systematics of pinworm of animals and humans, and should have implications for the diagnosis, prevention, and control of passaluriasis in rabbits and other animals. PMID:26472717

  20. Purification and characterization of pyrroline-5-carboxylate dehydrogenase from rat liver mitochondrial matrix

    International Nuclear Information System (INIS)

    Pyrroline-5-carboxylate (P5C) dehydrogenase catalyzes the second step of the irreversible two-step oxidation of proline to glutamate or the oxidative second step of the two-step conversion of ornithine to glutamate in mitochondria. Activity was assayed by monitoring directly the conversion of (3H) L-P5C to (3H) L-glutamate. Using this assay, the authors find P5C dehydrogenase most prevalent in liver in rat, with kidney having 71%, heart 51%, and and spleen 15% of the specific activity of liver. Starting with a subcellular fraction enriched for mitochondria, they have isolated a protein fraction enriched in this activity. The soluble protein fraction of the mitochondrial isolate was subjected to (NH4)2SO4 precipitation and successive chromatography on DE 52 anion exchange and Brown 10 dye ligand affinity resins. This procedure yielded a fraction purified more than 500-fold over whole liver homogenate. HPLC and 5'-AMP agarose fractionation experiments now in progress to achieve further purification show promise. Physical studies show a M/sub r/ of 105,000 upon sucrose density gradient centrifugation and 94,000 on molecular sieve HPLC for the activity. Flat bed gel isoelectric focusing of the protein indicates a pI of 5.7. The purified protein exhibits an apparent K/sub m/ of 0.1 mM for L-P5C

  1. Complete sequence and characterization of the Silurus lanzhouensis (Siluriformes: Siluridae) mitochondrial genome.

    Science.gov (United States)

    Lian, Zong-Qiang; Wu, Xu-Dong; Xiao, Wei; Sai, Qing-Yun; Gun, Shuang-Bao

    2016-07-01

    The complete mitochondrial DNA (mtDNA) sequence of Silurus lanzhouensis was constructed from whole-genome Illumina sequencing data. The 16 523 bp circular genome comprises typical mtDNA components. All 13 protein-coding genes (PCGs) are initiated by an ATG except for COX1, which uses GTG. Some PCGs harbor TAG (ND2 and ND3) or an incomplete stop codon T (COX2, ND4, and CYTB), while others use TAA as their stop codon. 12S rRNA and 16S rRNA secondary structures are composed of four domains with 45 helices and six domains with 54 helices, respectively. All tRNAs are predicted to fold into the expected typical cloverleaf secondary structure except tRNA-Ser((AGN)). The largest intergenic spacer sequence was predicted to be the origin of light-strand replication. Eight conserved sequences were identified in the control region (CR). This complete S. lanzhouensis mitogenome provides useful data for further studies on molecular systematics, taxonomic status, stock evaluation, and conservation genetics. PMID:26171872

  2. Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake.

    Science.gov (United States)

    Santos-Filho, Norival A; Boldrini-França, Johara; Santos-Silva, Ludier K; Menaldo, Danilo L; Henrique-Silva, Flávio; Sousa, Tiago S; Cintra, Adélia C O; Mamede, Carla C N; Oliveira, Fábio; Arantes, Eliane C; Antunes, Lusânia M Greggi; Cilli, Eduardo M; Sampaio, Suely V

    2014-10-01

    Venomous and non-venomous snakes possess phospholipase A2 (PLA2) inhibitory proteins (PLIs) in their blood serum. This study shows the expression and biochemical and functional characterization of a recombinant alpha inhibitor from Bothrops alternatus snake, named rBaltMIP. Its expression was performed in Pichia pastoris heterologous system, resulting in an active recombinant protein. The expressed inhibitor was tested regarding its ability to inhibit the phospholipase activity of different PLA2s, showing slight inhibitions especially at the molar ratios of 1:1 and 1:3 (PLA2:PLI). rBaltMIP was also effective in decreasing the myotoxic activity of the tested toxins at molar ratios greater than 1:0.4 (myotoxin:PLI). The inhibition of the myotoxic activity of different Asp49 (BthTX-II and PrTX-III) and Lys49 (BthTX-I and PrTX-I) myotoxins was also performed without the prior incubation of myotoxins/inhibitor in order to analyze the real possibility of using snake plasma inhibitors or recombinant inhibitors as therapeutic agents for treating envenomations. As a result, rBaltMIP was able to significantly inhibit the myotoxicity of Lys49 myotoxins. Histopathological analysis of the gastrocnemius muscles of mice showed that the myotoxins are able to induce severe damage to the muscle fibers of experimental animals by recruiting a large number of leukocyte infiltrates, besides forming an intense accumulation of intercellular fluid, leading to local edema. When those myotoxins were incubated with rBaltMIP, a reduction of the damage site could be observed. Furthermore, the cytotoxic activity of Asp49 PLA2s and Lys49 PLA2-like enzymes on C2C12 cell lines was decreased, as shown by the higher cell viabilities after preincubation with rBaltMIP. Heterologous expression would enable large-scale obtainment of rBaltMIP, thus allowing further investigations for the elucidation of possible mechanisms of inhibition of snake PLA2s, which have not yet been fully clarified. PMID:25047442

  3. 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

  4. Characterization and evaluation of metformin-loaded solid lipid nanoparticles for celluar and mitochondrial uptake.

    Science.gov (United States)

    Xu, Qiang; Zhu, Tao; Yi, Chaoli; Shen, Qi

    2016-01-01

    Considered a popular drug for diabetes in recent years, metformin was determined to have a moderate anti-tumor effect, particularly in breast cancer. In this study, the anticancer mechanism of metformin was verified by preparing solid lipid nanoparticles (SLNs) and chitosan-modified solid lipid nanoparticles (CSLNs) containing metformin and then estimating the potential of these SLNs for uptake in cells and mitochondria. Metformin-SLNs were prepared using an emulsification and low-temperature solidification method. The mean particle size, zeta potential, entrapment efficiency, and loading efficiency of metformin-SLNs and metformin chitosan-modified SLNs were 102.3 ± 4.16 and 200.1 ± 17.69 nm, -21.25 ± 4.89 and 50.6 ± 4.09 mv, 26.25 ± 2.59% and 33.6 ± 2.21%, and 1.74 ± 0.16% and 1.46 ± 0.10%, respectively. TEM images showed that both the nanoparticles had spherical morphologies with no aggregation. Results of cellular and mitochondrial uptake showed that the metformin-SLNs were easier to uptake in cells and mitochondria than the pure drug group (that was the control group without SLN structure modification). The findings of this research provide a basis for conducting further studies on the anticancer mechanism of metformin. PMID:26288997

  5. 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.

  6. Cucumber: a model angiosperm for mitochondrial transformation?

    Science.gov (United States)

    Havey, Michael J; Lilly, Jason W; Bohanec, Borut; Bartoszewski, Grzegorz; Malepszy, Stefan

    2002-01-01

    Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants. PMID:12084966

  7. 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.

  8. Molecular characterization of Opisthorchis noverca (Digenea: Opisthorchiidae) based on nuclear ribosomal ITS2 and mitochondrial COI genes.

    Science.gov (United States)

    Sahu, R; Biswal, D K; Roy, B; Tandon, V

    2016-09-01

    Opisthorchiasis is a public health problem in South-East Asian countries and Eastern Europe. The infection implicates mainly two species of Opisthorchis, namely O. viverrini and O. felineus, that occur mostly in fish-eating mammals and humans, although there are rare reports of human cases involving two other species, O. noverca and O. guayaquilensis. Opisthorchis noverca has been reported frequently in dogs and pigs from the Indian subcontinent, with rare reports from cattle and human subjects. With a view to supplementing morphology-based identification of this species, the present study aimed to provide molecular characterization of O. noverca, using rDNA internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase I (mt COI) markers so as to determine its genetic correlation with other species of Opisthorchiidae, and also to generate a taxon-specific molecular marker based on the ITS2 region. The phylogenetic relationship between O. noverca and other species of the genus was determined using molecular sequence data. To strengthen the result, secondary structure sequence analyses of ITS2 with hemi-compensatory base changes (hCBCs), and amino acid sequence analyses, were also evaluated. Our results confirm that O. noverca is a distinct and valid species. PMID:26467395

  9. Mitochondrial Dysfunction and Psychiatric Disorders

    Directory of Open Access Journals (Sweden)

    Shaw-Hwa Jou

    2009-10-01

    Full Text Available Mitochondria are intracellular organelles crucial in the production of cellular energy.Mitochondrial diseases may result from malfunctions in this biochemical cascade. Severalinvestigators have proposed that mitochondrial dysfunction is related to the pathophysiologyof bipolar disorder (BD, major depressive disorder (MDD and schizophrenia (SZ. Theauthors reviewed recent study findings and tried to delineate the current understanding of thecorrelation between mitochondrial dysfunction and psychiatric disorders. A growing body ofevidence suggests that mitochondrial dysfunction is important in patients with psychiatricdisorders. The evidence include impaired energy metabolism in the brain detected usingresults of magnetic resonance spectroscopy, electron microscopy, co-morbidity with mitochondrialdiseases, the effects of psychotropics on mitochondria, increased mitochondrialDNA (mtDNA deletion in the brain, and association with mtDNA mutations/polymorphismsor nuclear-encoded mitochondrial genes. It is possible that the new information willlead to a focus on psychiatric disorder as a metabolic disease. Treatment with psychotropicsmight ultimately enhance energy metabolism and reduce the damage of oxidative stress. Thenext step in the study of mitochondrial dysfunction in patients with psychiatric disordersshould be clarification of how mitochondrial dysfunction, a nonspecific risk factor, causesspecific symptoms. Further study of mitochondrial dysfunction in patients with psychiatricdisorder is expected to be useful for the development of cellular disease markers and newpsychotropics.

  10. Microbiological and Biochemical Characterization of Cassava Retting, a Traditional Lactic Acid Fermentation for Foo-Foo (Cassava Flour) Production

    OpenAIRE

    Brauman, A.; Keleke, S.; Malonga, M.; Miambi, E.; Ampe, F

    1996-01-01

    The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters. During the traditional process, endogenous cyanogens were almost totally degraded, plant cell walls were lysed by the simultaneous action of pectin methylesterase and pectate lysate, and organic acids (C2 to C4) were produced. Most microorganisms identified were found to be facultat...

  11. Genetic and biochemical characterization of FRI-1, a carbapenem-hydrolyzing class A β-Lactamase from Enterobacter cloacae

    OpenAIRE

    Dortet, Laurent; Poirel , Laurent; Abbas, Samia; Oueslati, Saoussen; Nordmann, Patrice

    2016-01-01

    An Enterobacter cloacae isolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed ...

  12. Variants of mitochondrial autophagy: Types 1 and 2 mitophagy and micromitophagy (Type 3

    Directory of Open Access Journals (Sweden)

    John J. Lemasters

    2014-01-01

    Full Text Available Mitophagy (mitochondrial autophagy, which removes damaged, effete and superfluous mitochondria, has several distinct variants. In Type 1 mitophagy occurring during nutrient deprivation, preautophagic structures (PAS grow into cup-shaped phagophores that surround and sequester individual mitochondria into mitophagosomes, a process requiring phosphatidylinositol-3-kinase (PI3K and often occurring in coordination with mitochondrial fission. After sequestration, the outer compartment of the mitophagosome acidifies, followed by mitochondrial depolarization and ultimately hydrolytic digestion in lysosomes. Mitochondrial damage stimulates Type 2 mitophagy. After photodamage to single mitochondria, depolarization occurs followed by decoration and then coalescence of autophagic LC3-containing structures on mitochondrial surfaces. Vesicular acidification then occurs. By contrast to Type 1 mitophagy, PI3K inhibition does not block Type 2 mitophagy. Further, Type 2 mitophagy is not associated with phagophore formation or mitochondrial fission. A third form of self-eating of mitochondria is formation of mitochondria-derived vesicles (MDVs enriched in oxidized mitochondrial proteins that bud off and transit into multivesicular bodies. Topologically, the internalization of MDV by invagination of the surface of multivesicular bodies followed by vesicle scission into the lumen is a form of microautophagy, or micromitophagy (Type 3 mitophagy. Cell biological distinctions are the basis for these three types of mitophagy. Future studies are needed to better characterize the molecular and biochemical differences between Types 1, 2 and 3 mitophagy.

  13. 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...

  14. Potentially diagnostic electron paramagnetic resonance spectra elucidate the underlying mechanism of mitochondrial dysfunction in the deoxyguanosine kinase deficient rat model of a genetic mitochondrial DNA depletion syndrome.

    Science.gov (United States)

    Bennett, Brian; Helbling, Daniel; Meng, Hui; Jarzembowski, Jason; Geurts, Aron M; Friederich, Marisa W; Van Hove, Johan L K; Lawlor, Michael W; Dimmock, David P

    2016-03-01

    A novel rat model for a well-characterized human mitochondrial disease, mitochondrial DNA depletion syndrome with associated deoxyguanosine kinase (DGUOK) deficiency, is described. The rat model recapitulates the pathologic and biochemical signatures of the human disease. The application of electron paramagnetic (spin) resonance (EPR) spectroscopy to the identification and characterization of respiratory chain abnormalities in the mitochondria from freshly frozen tissue of the mitochondrial disease model rat is introduced. EPR is shown to be a sensitive technique for detecting mitochondrial functional abnormalities in situ and, here, is particularly useful in characterizing the redox state changes and oxidative stress that can result from depressed expression and/or diminished specific activity of the distinct respiratory chain complexes. As EPR requires no sample preparation or non-physiological reagents, it provides information on the status of the mitochondrion as it was in the functioning state. On its own, this information is of use in identifying respiratory chain dysfunction; in conjunction with other techniques, the information from EPR shows how the respiratory chain is affected at the molecular level by the dysfunction. It is proposed that EPR has a role in mechanistic pathophysiological studies of mitochondrial disease and could be used to study the impact of new treatment modalities or as an additional diagnostic tool. PMID:26773591

  15. 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.

  16. 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.

  17. Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching

    Science.gov (United States)

    Chiruvella, Kishore K.; Rajaei, Naghmeh; Jonna, Venkateswara Rao; Hofer, Anders; Åström, Stefan U.

    2016-01-01

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MATα). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons. PMID:26902909

  18. 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

  19. Biochemical characterization of protein composition of high-lysine mutant ML-109 of winter barley (Hordeum vulgare L.)

    International Nuclear Information System (INIS)

    The objective of the present studies was to determine the biochemical basis of a high lysine content in the mutant ML-109. The protein composition of kernels of high-lysine mutant of winter barley and its initial breeding line were analysed. The portion of individual fractions differing by their solubility in total proteins as well as amino acid composition of kernel protein and separated main protein fractions were also determined. The performed studies showed that a high lysine content in the kernels of the analysed mutant results from a decrease in the hordein level and from an increase in the portion of salt soluble proteins - albumins and globulins. Besides that, the obtained results indicate that the amino acid composition of the main protein fractions of the analysed mutant was advantageously changed; these fractions contained more lysine and other exogenic amino acids. (author). 15 refs, 6 tabs

  20. 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.

  1. 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.

  2. Biochemical characterization and molecular dynamic simulation of β-sitosterol as a tubulin-binding anticancer agent.

    Science.gov (United States)

    Mahaddalkar, Tejashree; Suri, Charu; Naik, Pradeep Kumar; Lopus, Manu

    2015-08-01

    Βeta-sitosterol (β-SITO), a phytosterol present in pomegranate, peanut, corn oil, almond, and avocado, has been recognized to offer health benefits and potential clinical uses. β-SITO is orally bioavailable and, as a constituent of edible natural products, is considered to have no undesired side effects. It has also been considered as a potent anticancer agent. However, the molecular mechanism of action of β-SITO as a tubulin-binding anticancer agent and its binding site on tubulin are poorly understood. Using a combination of biochemical analyses and molecular dynamic simulation, we investigated the molecular details of the binding interactions of β-SITO with tubulin. A polymer mass assay comparing the effects of β-SITO and of taxol and vinblastine on tubulin assembly showed that this phytosterol stabilized microtubule assembly in a manner similar to taxol. An 8-anilino-1-naphthalenesulfonic acid assay confirmed the direct interaction of β-SITO with tubulin. Although β-SITO did not show direct binding to the colchicine site on tubulin, it stabilized the colchicine binding. Interestingly, no sulfhydryl groups of tubulin were involved in the binding interaction of β-SITO with tubulin. Based on the results from the biochemical assays, we computationally modeled the binding of β-SITO with tubulin. Using molecular docking followed by molecular dynamic simulations, we found that β-SITO binds tubulin at a novel site (which we call the 'SITO site') adjacent to the colchicine and noscapine sites. Our data suggest that β-SITO is a potent anticancer compound that interferes with microtubule assembly dynamics by binding to a novel site on tubulin. PMID:25912799

  3. Mitochondrial vasculopathy

    Science.gov (United States)

    Finsterer, Josef; Zarrouk-Mahjoub, Sinda

    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 also the arteries may be affected (mitochondrial arteriopathy). This review focuses on the type, diagnosis, and treatment 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 spontaneous 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. PMID:27231520

  4. 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...

  5. 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

  6. Some biochemical properties of melatonin and the characterization of a relevant metabolite arising from its interaction with H2O2.

    Science.gov (United States)

    Carampin, Paolo; Rosan, Stefania; Dalzoppo, Daniele; Zagotto, Giuseppe; Zatta, Paolo

    2003-03-01

    Melatonin is an efficient protector against hydrogen peroxide(H2O2)-induced lipid peroxidation and acts in a concentration-dependent manner. Hydrogen peroxide is rather a water stable molecule which is able to cross the cell membrane much better than some important free radicals such as superoxide anion, and consequently its local production can lead to significant spread by diffusion. In this paper we report data regarding some biochemical properties of melatonin as well as the chemical characterization of the major product formed from the interaction between melatonin and H2O2 (N1-acetyl-N2-formyl-5-methoxykynuramine) that are consistent with previous data reported by other authors. The effect of melatonin on catalase, glutathione peroxidase and superoxide dismutase in in vitro and in vivo experiments is also reported. PMID:12562505

  7. Biochemical characterization, molecular cloning and localization of a putative odorant-binding protein in the honey bee Apis mellifera L. (Hymenoptera : Apidea).

    Science.gov (United States)

    Danty, E; Michard-Vanhée, C; Huet, J C; Genecque, E; Pernollet, J C; Masson, C

    1997-09-15

    A honey bee antennal water-soluble protein, APS2, was purified and characterized as the first Hymenoptera putative odorant-binding protein. Comparison of its measured Mr (13695.2+/-1.6) to that of the corresponding cDNA clone shows it does not undergo any post-translational modification other than a 19-residue signal peptide cleavage and formation of three disulfide bridges. These biochemical features are close to those of Lepidoptera odorant-binding proteins. In situ hybridization experiments demonstrated its specific expression in olfactory areas. Based on its higher expression in the worker than in the drone, ASP2 might be more involved in general odorant than in sex pheromone detection. PMID:9323043

  8. 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

  9. Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta.

    Science.gov (United States)

    Dash, Rupesh; Ghosh, Sudip K; Kaplan, David L; Kundu, S C

    2007-05-01

    Sericin isolated from the cocoon of the tropical tasar silkmoth Antheraea mylitta showed three major bands, with the lowest 70 kDa. This band was purified by anion exchange chromatography. Immunoblotting with concanavalin-A suggests a glycoprotein and CD analysis of secondary structure includes beta-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of these two amino acids are different from sericin of mulberry silkworm. An anti A. mylitta sericin antibody was able to cross-react with sericin from A. assamensis but not the sericin of Bombyx mori and Philosamia ricini. Immunoblot analysis with proteins isolated from middle silk gland of A. mylitta at different developmental stages of larva showed that the 70 kDa sericin is developmentally regulated. These data extend the range of biochemical features found in this unusual family of proteins and may help in developing an improved understanding of their role in forming environmentally stable fibroin fiber-sericin composite structures (cocoons). PMID:17350301

  10. Microbiological and biochemical characterization of cassava retting, a traditional lactic Acid fermentation for foo-foo (cassava flour) production.

    Science.gov (United States)

    Brauman, A; Keleke, S; Malonga, M; Miambi, E; Ampe, F

    1996-08-01

    The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters. During the traditional process, endogenous cyanogens were almost totally degraded, plant cell walls were lysed by the simultaneous action of pectin methylesterase and pectate lyase, and organic acids (C(inf2) to C(inf4)) were produced. Most microorganisms identified were found to be facultative anaerobes which used the sugars (sucrose, glucose, and fructose) present in the roots as carbon sources. After 24 h of retting, the fermentation reached an equilibrium that was reproducible in all the spontaneous fermentations studied. Lactic acid bacteria were largely predominant (over 99% of the total flora after 48 h) and governed the fermentation. The epiphytic flora was first replaced by Lactococcus lactis, then by Leuconostoc mesenteroides, and finally, at the end of the process, by Lactobacillus plantarum. These organisms produced ethanol and high concentrations of lactate, which strongly acidified the retting juice. In addition, the rapid decrease in partial oxygen pressure rendered the process anaerobic. Strict anaerobes, such as Clostridium spp., developed and produced the volatile fatty acids (mainly butyrate) responsible, together with lactate, for the typical flavor of retted cassava. Yeasts (mostly Candida spp.) did not seem to play a significant role in the process, but their increasing numbers in the last stage of the process might influence the flavor and the preservation of the end products. PMID:16535378

  11. 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%. PMID:25795061

  12. 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.

  13. Biological and Biochemical Potential of Sea Snake Venom and Characterization of Phospholipase A2 and Anticoagulation Activity.

    Science.gov (United States)

    Damotharan, Palani; Veeruraj, Anguchamy; Arumugam, Muthuvel; Balasubramanian, Thangavel

    2016-03-01

    This study is designed to isolate and purify a novel anti-clotting protein component from the venom of Enhydrina schistosa, and explore its biochemical and biological activities. The active protein was purified from the venom of E. schistosa by ion-exchange chromatography using DEAE-cellulose. The venom protein was tested by various parameters such as, proteolytic, haemolytic, phospholipase and anti-coagulant activities. 80 % purity was obtained in the final stage of purification and the purity level of venom was revealed as a single protein band of about 44 kDa in SDS-polyacrylamide electrophoresis under reducing conditions. The results showed that the Potent hemolytic activity was observed against cow, goat, chicken and human (A, B and O positive) erythrocytes. Furthermore, the clotting assays showed that the venom of E. schistosa significantly prolonged in activated partial thromboplastin time, thrombin time, and prothrombin time. Venomous enzymes which hydrolyzed casein and gelatin substrate were found in this venom protein. Gelatinolytic activity was optimal at pH 5-9 and (1)H NMR analysis of purified venom was the base line information for the structural determination. These results suggested that the E. schistosa venom holds good promise for the development of novel lead compounds for pharmacological applications in near future. PMID:26855489

  14. Preparation and characterization of Pd/Al2O3 and Pd nanoparticles as standardized test material for chemical and biochemical studies of traffic related emissions.

    Science.gov (United States)

    Leopold, K; Maier, M; Schuster, M

    2008-05-01

    Palladium model particles similar to those emitted from catalytic car exhaust converters were prepared and characterized with the intention of providing a standardized material for investigations of the chemical behavior and bioavailability of traffic related Pd emissions. Two series of Pd particles were prepared and characterized in detail: Pd nanoparticles (2-4 nm) dispersed on aluminum oxide particles of a diameter range between 0.1 to 30 microm and "Pd-only" nanoparticles of 5-10 nm in diameter. The Pd/alpha-Al2O3 particles are very similar to particles emitted from catalytic converters by mechanical abrasion. The Pd-only particles are useful e.g. for exposure studies in which the presence of aluminum could lead to interferences when studying biological and biochemical effects. The sample preparation procedure of both series was optimized in order to achieve elemental particles with proper sizes and a narrow size distribution. The obtained particles were characterized by transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), selective area diffraction (SAD), laser granulometry and graphite furnace atomic absorption spectrometry (GFAAS) for the measurement of Pd concentrations. PMID:18279916

  15. Transcriptional and biochemical markers in transplanted Perca flavescens to characterize cadmium- and copper-induced oxidative stress in the field

    International Nuclear Information System (INIS)

    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

  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. 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. PMID:25156536

  18. Biochemical characterization of protein quality control mechanisms during disease progression in the C22 mouse model of CMT1A

    Directory of Open Access Journals (Sweden)

    Vinita G. Chittoor

    2013-12-01

    Full Text Available Charcot–Marie–Tooth disease type 1A (CMT1A is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22 gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin–proteasome system (UPS, autophagosomes and heat-shock proteins (HSPs. Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins.

  19. Evaluation of Antioxidant Enzyme Activity and Biochemical Characterization from Static and Suspension Culture of Withania somnifera L.

    Directory of Open Access Journals (Sweden)

    Satyajit Kanungo

    2015-03-01

    Full Text Available Withania somnifera (L. Dunal, is an erect evergreen shrub commonly known as Ashwagandha. It is widely used in Ayurvedic and in the traditional pharmacopeia system of India. It is one of the major ingredients in many formulations prescribed for a variety of musculoskeletal conditions including arthritis and rheumatism. In the present study the variation in quality and quantity of protein and antioxidant enzymes were evaluated biochemically and enzymatically from the static and suspension cultures. The nodal segments had provided maximum callusing of 90.25±0.06 % with (1mg/l of BAP and Kn with (2mg/l of 2, 4-D. The static and suspension cultures were taken for the analysis of total soluble protein and screened for antioxidant enzyme activity [catalase (CAT, superoxide dismutase (SOD and guaiacol peroxidase (GPX]. The protein content (1.2016 µg/µl was found to be higher in static culture samples (0.870 µg/µl than the protein obtained from the suspension culture. The antioxidant enzyme activity (CAT, SOD and GPX was higher in static culture samples (301.01± 0.42, 198.92 ± 0.29, 103.75 ± 0.11 nkat/ mg of protein than that of suspension culture. Specific activity staining of isozyme pattern exhibited three isoforms (CAT 1, CAT 2 and CAT 3 in static culture samples but CAT 1 was absent in the sample extracted from suspension cultures.  In case of SOD, four bands (SOD 1, SOD 2, SOD 3 and SOD 4 were found in both the samples whereas intensity of GPX activity was found to be more in static culture but both the samples exhibited three isoforms such as  (GPX 1, GPX 2 and GPX 3. The supplementation of required nutrients along with the phytohormones under in vitro condition might be an enhancing factor to yield antioxidant enzymes in the static culture samples. 

  20. 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.

  1. 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...

  2. Genetic, physiological and biochemical characterization of Bacillus sp. strain RMB7 exhibiting plant growth promoting and broad spectrum antifungal activities

    OpenAIRE

    Ali, Saira; Hameed, Sohail; Imran, Asma; Iqbal, Mazhar; Lazarovits, George

    2014-01-01

    Background Plant growth promoting rhizobacteria (PGPR) are functionally diverse group of bacteria having immense potential as biofertilizers and biopesticides. Depending upon their function, they may serve as partial replacements for chemical fertilizer or pesticides as an eco-friendly and cost-effective alternatives as compared to their synthetic counterparts. Therefore, isolation, characterization and practical evaluation of PGPRs having the aforementioned multifaceted beneficial characteri...

  3. 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

  4. Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome.

    Science.gov (United States)

    Grose, Julianne H; Langston, Kelsey; Wang, Xiaohui; Squires, Shayne; Mustafi, Soumyajit Banerjee; Hayes, Whitney; Neubert, Jonathan; Fischer, Susan K; Fasano, Matthew; Saunders, Gina Moore; Dai, Qiang; Christians, Elisabeth; Lewandowski, E Douglas; Ping, Peipei; Benjamin, Ivor J

    2015-01-01

    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 tempting to

  5. Mitochondrial Myopathy

    Science.gov (United States)

    ... NINDS supports research focused on effective treatments and cures for mitochondrial myopathies and other mitochondrial diseases. Scientists are investigating the possible benefits of exercise programs and nutritional supplements, primarily natural and synthetic versions of CoQ10. While CoQ10 has ...

  6. Characterization of the complete mitochondrial genome and a set of polymorphic microsatellite markers through next-generation sequencing for the brown brocket deer Mazama gouazoubira

    Science.gov (United States)

    Caparroz, Renato; Mantellatto, Aline M.B.; Bertioli, David J.; Figueiredo, Marina G.; Duarte, José Maurício B.

    2015-01-01

    The complete mitochondrial genome of the brown brocket deer Mazama gouazoubira and a set of polymorphic microsatellite markers were identified by 454-pyrosequencing. De novo genome assembly recovered 98% of the mitochondrial genome with a mean coverage of 9-fold. The mitogenome consisted of 16,356 base pairs that included 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and the control region, as found in other deer. The genetic divergence between the mitogenome described here and a previously published report was ∼0.5%, with the control region and ND5 gene showing the highest intraspecific variation. Seven polymorphic loci were characterized using 15 unrelated individuals; there was moderate genetic variation across most loci (mean of 5.6 alleles/locus, mean expected heterozygosity = 0.70), with only one locus deviating significantly from Hardy-Weinberg equilibrium, probably because of null alleles. Marker independence was confirmed with tests for linkage disequilibrium. The genetic variation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the phylogeography and population genetic patterns in M. gouazoubira, particularly in the context of habitat fragmentation in South America. PMID:26500438

  7. 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.

  8. Biochemical characterization of a Ca2+/NAD(P)H-dependent H2O2 generator in human thyroid tissue.

    Science.gov (United States)

    Leseney, A M; Dème, D; Legué, O; Ohayon, R; Chanson, P; Sales, J P; Carvalho, D P; Dupuy, C; Virion, A

    1999-04-01

    An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca2+-dependent and activated by inorganic phosphate anions in the same range of concentrations. Both are flavoproteins using FAD as cofactor, but the human enzyme was also able to utilize FMN. The apparent Km for NADPH of the human enzyme (100 microM) was 5-10 times higher than that of porcine enzyme. Vm was 3 to 10 times higher in pig (150 nmol x h(-1) x mg(-1)) than in man (14 to 45). Total content in human tissue was 7 to 9% of that in porcine tissue. An unidentified inhibitor has been detected in the 3000 g particulate fraction from most patients, which could account for this apparently low enzyme content. An NADH-dependent H2O2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca2+-dependent (man, 50-70%; pig, 80-90%) and presented similar apparent Km values for NADH (man, 100 microM; pig, 200 microM). In pig thyrocytes, the expression of the Ca2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFbeta, as was the NADPH oxidase activity. Furthermore, NADPH and NADH-dependent activities were not additive. We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NADH as substrate to catalyse H2O2 formation, and that human and porcine NAD(P)H-oxidases are highly similar. Differences observed could be attributed to minor differences in enzyme structure and/or in membrane microenvironment. The NADH-dependent Ca2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme. PMID:10401672

  9. Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094.

    Science.gov (United States)

    Ehteshami, Maryam; Tao, Sijia; Ozturk, Tugba; Zhou, Longhu; Cho, Jong Hyun; Zhang, Hongwang; Amiralaei, Sheida; Shelton, Jadd R; Lu, Xiao; Khalil, Ahmed; Domaoal, Robert A; Stanton, Richard A; Suesserman, Justin E; Lin, Biing; Lee, Sam S; Amblard, Franck; Whitaker, Tony; Coats, Steven J; Schinazi, Raymond F

    2016-08-01

    Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on β-d-2'-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2'-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2'-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo Finally, we found that although both 2'-C-methyl-GTP and 2'-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2'-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2'-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites. PMID:27216050

  10. Characterization of the complete mitochondrial genome of Cynoglossus gracilis and a comparative analysis with other Cynoglossinae fishes.

    Science.gov (United States)

    Wei, Min; Liu, Yang; Guo, Hua; Zhao, Fazhen; Chen, Songlin

    2016-10-15

    Mitochondrial genomes can provide basic information for phylogenetic analysis and evolutionary studies. We present here the mitochondrial genome of Cynoglossus gracilis, which is 16,565bp in length. Numerous distinct regions were identified, including 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, a light-strand replication origin, and a control region. Interestingly, we detected rearrangement of genes in C. gracilis, including a control region translocation, tRNA(Gln) gene inversion, and tRNA(Ile) gene shuffling. Additionally, a phylogenetic analysis based on the nucleotide sequences of the 13 PCGs using maximum likelihood and Bayesian inference methods reveals that C. gracilis is closely related to Cynoglossus semilaevis. This study provides important mitogenomic data for analyzing phylogenetic relationships in the Cynoglossinae. PMID:27312953

  11. Synthesis and biological characterization of new amino-phosphonates for mitochondrial pH determination by 31P NMR spectroscopy

    International Nuclear Information System (INIS)

    A series of mitochondria targeted α-amino-phosphonates combining a diethoxy-phosphoryl group and an alkyl chain-connected triphenylphosphonium bromide tail were designed and synthesized, and their pH-sensitive 31P NMR properties and biological activities in vitro and in vivo were evaluated. The results showed a number of these mitoaminophosphonates exhibiting pKa values fitting the mitochondrial pH range, short relaxation, and chemical shift parameters compatible with sensitive 31P NMR detection, and low cytotoxicity on green algae and murine fibroblasts cell cultures. Of these, two selected compounds demonstrated to distribute at NMR detectable levels within the cytosolic and mitochondrial sites following their perfusion to isolated rat livers, with no detrimental effects on cell energetics and aerobic respiration. This study provided a new molecular scaffold for further development of in situ spectroscopic real-time monitoring of mitochondrion/cytosol pH gradients. (authors)

  12. Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion

    OpenAIRE

    Wilson-Welder, Jennifer H.; Elliott, Margaret K.; Zuerner, Richard L.; Bayles, Darrell O.; Alt, David P.; Stanton, Thad B.

    2013-01-01

    Background 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 papillomatous lesion frequently located near the interdigital cleft and above the bulbs of the heel. While the exact etiology is unknown, several spirochete species have been isolated from lesion m...

  13. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

    OpenAIRE

    Saúl Gómez-Manzo; Jaime Marcial-Quino; America Vanoye-Carlo; Hugo Serrano-Posada; Abigail González-Valdez; Víctor Martínez-Rosas; Beatriz Hernández-Ochoa; Edgar Sierra-Palacios; Rosa Angélica Castillo-Rodríguez; Miguel Cuevas-Cruz; Eduardo Rodríguez-Bustamante; Roberto Arreguin-Espinosa

    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 clinic...

  14. Gestational diabetes is characterized by reduced mitochondrial protein expression and altered calcium signaling proteins in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    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.

  15. Genetic characterization of the honeybee (Apis mellifera) population of Rodrigues Island, based on microsatellite and mitochondrial DNA

    OpenAIRE

    Techer, Maéva Angélique; Clémencet, Johanna; Turpin, Patrick; Volbert, Nicolas; Reynaud, Bernard; Delatte, Hélène

    2015-01-01

    International audience AbstractApis mellifera is present in Rodrigues, an island in the South-West Indian Ocean. The history of the established honeybee population is poorly known, and its biodiversity has never been studied. In this study, maternal origins of A. mellifera in Rodrigues have been assessed with the DraI test and sequencing of the mitochondrial COI-COII region. Nuclear genetic diversity was investigated with 18 microsatellite markers. A total of 524 colonies were sampled from...

  16. Identification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship with clinical phenotype in MPS-VI patients

    Energy Technology Data Exchange (ETDEWEB)

    Litjens, T.; Brooks, D.A.; Hopwood, J.J. [Women`s and Children`s Hospital, North Adelaide (Australia)] [and others

    1996-06-01

    Maroteaux-Lamy syndrome, or mucopolysaccharidosis type VI (MPS-VI), is a lysosomal storage disorder characterized by the defective degradation of dermatan sulfate due to the deficiency of N-acetylgalactosamine-4-sulfatase (4S). The clinical severity of MPS-VI ranges in a continuum from mildly affected to severely affected patients. Mutations in MPS-VI patient samples were identified by chemical cleavage and direct DNA sequencing of PCR products derived from patient cDNA. Five amino acid substitutions were identified (T92M, R95Q, Y210C, H393P, and L498P), individually introduced into the wild-type 4S cDNA by site-directed in vitro mutagenesis, and transfected into Chinese hamster ovary cells. Three of the five mutations (R95Q, Y210C, and H393P) were observed in > 1 of 25 unrelated MPS-VI patients; however, the mutations were not found in 20 control individuals. The residual 4S activity and protein (biochemical phenotype) were determined for each mutant in order to confirm their identity as mutations and to dissect the contribution of each mutant allele to the overall clinical phenotype of the patient. For each patient, the combined biochemical phenotypes of the two 4S mutant alleles demonstrated a good correspondence with the observed clinical phenotype (with the possible exception of a patient who was a compound heterozygote for T92M and L498P). This preliminary correspondence between genotype and the phenotype in MPS-VI may, with further refinement, contribute to the assessment of therapeutic approaches for MPS-VI patients. 30 refs., 4 tabs.

  17. 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

  18. Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation

    OpenAIRE

    Yang, Che-Chuan; Wang, Yi-Ting; Hsiao, Yu-Yuan; Doudeva, Lyudmila G.; Kuo, Pan-Hsien; Chow, Sih Yao; Yuan, Hanna S.

    2010-01-01

    Rrp46 was first identified as a protein component of the eukaryotic exosome, a protein complex involved in 3′ processing of RNA during RNA turnover and surveillance. The Rrp46 homolog, CRN-5, was subsequently characterized as a cell death-related nuclease, participating in DNA fragmentation during apoptosis in Caenorhabditis elegans. Here we report the crystal structures of CRN-5 and rice Rrp46 (oRrp46) at a resolution of 3.9 Å and 2.0 Å, respectively. We found that recombinant human Rrp46 (h...

  19. Purification and biochemical characterization of the complete structure of a proteolytically modified beta-2-microglobulin with biological activity

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Thim, L; Christensen, M

    1987-01-01

    . (1985) Clin. Chem. 31, 1411-1412; Nissen et al. (1984) Clin. Chim. Acta 141, 41-50]. In the present study we describe the purification and characterization of this modified human serum beta-2-m from patients with small-cell lung cancer. Purified urinary beta-2-m was added to the serum samples incubated...... analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and analytical isoelectric focusing respectively. Amino acid analysis of m-beta-2-m revealed that the protein is missing one lysine residue compared to the composition deduced from the cDNA sequence of beta-2-m. Amino acid sequence...

  20. Cobalt(ll) Coordination Compounds of Ethyl 4-Methyl-5-Imidazolecarboxylate: Chemical and Biochemical Characterization on Photosynthesis and Seed Germination

    OpenAIRE

    Beatriz King-Díaz; Josefina Montes-Ayala; Concepción Escartín-Guzmán; Silvia E. Castillo-Blum; Roberto Iglesias-Prieto; Blas Lotina-Hennsen; Norah Barba-Behrens

    2005-01-01

    In this work we present the synthesis, structural and spectroscopic characterization of Co2+ coordination compounds with ethyl 4-methyl-5-imidazolecarboxylate (emizco). The effects of emizco, the metal salts CoCl2.6H2O, CoBr2, Co(NO3)2.6H2O and their metal coordination compounds [Co(emizco)2Cl2], [Co(emizco)2 Br2].H2O, [Co(emizco)2 (H2O)2(NO2)2.2H2O were evaluated on photosynthesis in spinach chloroplasts. Seed germination and seedling growth of the monocotyledonous species Lolium multiflorum...

  1. 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

  2. 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

  3. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

    Directory of Open Access Journals (Sweden)

    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.

  4. Biochemical characterization, molecular cloning, and structural modeling of an interesting β-1,4-glucanase from Sclerotinia sclerotiorum.

    Science.gov (United States)

    Chahed, Haifa; Ezzine, Aymen; Mlouka, Amine Ben; Hardouin, Julie; Jouenne, Thierry; Marzouki, Mohamed Najib

    2014-04-01

    The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes needed for efficient solubilization of native cellulose, the major component of plants. In this work, we reported the molecular characterization of an important glycosyl-hydrolase enzyme classified as endo-β-1,4-glucanase. The importance of this enzyme was revealed with the in-gel activity staining, showing a high degradation capacity of cellulose. When purified from native gel and ran in denaturing polyacrylamide gel, the polypeptide has an apparent molecular mass of about 34 kDa called Endo2. For further characterization of this protein, a mass spectrometry approach was carried out. The LC-MS/MS analysis revealed two peptides belonging to this enzyme. The genomic DNA and cDNA sequences were resolved by PCR amplification and sequencing, revealing a gene with two intron sequences. The open reading frame of 987 bp encoded a putative polypeptide of 328 amino acids having a calculated molecular mass of 33,297 Da. Yet, the molecular modeling and comparative investigation of different 3D cellulase structures showed that this endoglucanase isoform has probably two domains. A core domain having a high similarity with endoglucanases family 5 and a cellulose-binding domain having similarities with those of exo-type cellulases of family 1, linked together by a serine-threonine-rich region. These results are with great interests and show new characteristics of S. sclerotiorum glucanase. PMID:24146430

  5. MOLECULAR NEUROGENETICS OF MITOCHONDRIAL DISEASES

    Directory of Open Access Journals (Sweden)

    E. Cardaioli

    2012-01-01

    Full Text Available Mitochondrial diseases are an expanding group of clinically heterogeneous disorders associated with mitochondrial DNA (mtDNA mutations or nuclear gene defects. Whatever the mechanism, the final common step in mitochondrial disorders is a defect of energy production resulting from respiratory chain impairment. The complexity of the biochemical and genetic features of the respiratory chain accounts for the extraordinarily wide range of clinical presentations of mitochondrial disorders. In general, organs with high aerobic demand, such as skeletal muscle, brain and heart, are the most affected. However, virtually any organ or tissue in the body may be affected and the disorders can be multisystemic (mitochondrial encephalomyopathiesor confined to a single tissue. Moreover, mitochondrial diseases can be sporadic or transmitted by mendelian (nuclear genes or maternal inheritance (mutations in mtDNA. Precise diagnosis is often a challenge; we go through the traditional steps of the diagnostic process, starting with study of inheritance in the family, clinical manifestations in the individual,electrophysiology and imaging techniques at organ level, down to biochemistry, pathology and molecular genetics at tissue, cell and DNA level, respectively. In fact the ultimate goal is to reach, whenever possible, a definitive molecular diagnosis, which can permit rational therapeutic approach and a genetic counseling.

  6. Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production.

    Science.gov (United States)

    Díaz, Rosario; Saparrat, Mario C N; Jurado, Miguel; García-Romera, Inmaculada; Ocampo, Juan Antonio; Martínez, María Jesús

    2010-09-01

    Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of "alpeorujo" (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues. PMID:20607234

  7. 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. PMID:27297992

  8. Dysregulation of cellular iron metabolism in Friedreich ataxia: from primary iron-sulfur cluster deficit to mitochondrial iron accumulation

    Directory of Open Access Journals (Sweden)

    HelenePuccio

    2014-06-01

    Full Text Available Friedreich ataxia (FRDA is the most common recessive ataxia in the Caucasian population and is characterized by a mixed spinocerebellar and sensory ataxia frequently associating cardiomyopathy. The disease results from decreased expression of the FXN gene coding for the mitochondrial protein frataxin. Early histological and biochemical study of the pathophysiology in patient’s samples revealed that dysregulation of iron metabolism is a key feature of the disease, mainly characterized by mitochondrial iron accumulation and by decreased activity of iron-sulfur cluster enzymes. In the recent past years, considerable progress in understanding the function of frataxin has been provided through cellular and biochemical approaches, pointing to the primary role of frataxin in iron-sulfur cluster biogenesis. However, why and how the impact of frataxin deficiency on this essential biosynthetic pathway leads to mitochondrial iron accumulation is still poorly understood. Herein, we review data on both the primary function of frataxin and the nature of the iron metabolism dysregulation in FRDA. To date, the pathophysiological implication of the mitochondrial iron overload in FRDA remains to be clarified.

  9. 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.

  10. Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307

    Directory of Open Access Journals (Sweden)

    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

  11. Mitochondrial Diseases

    Science.gov (United States)

    ... in your body tissues. If you have a metabolic disorder, something goes wrong with this process. Mitochondrial diseases are a group of metabolic disorders. Mitochondria are small structures that produce energy in ...

  12. 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.

  13. Characterization of a novel plasmid-like element in Neurospora crassa derived mostly from the mitochondrial DNA.

    OpenAIRE

    Almasan, A.; Mishra, N. C.

    1990-01-01

    We have identified a plasmid-like element within mitochondria of Neurospora crassa strain stp-B1. It is derived from the EcoRI-4 and EcoRI-6 regions of the mitochondrial DNA, and an additional 124 bp DNA segment of unknown origin. The plasmid DNA consists of an oligomeric series of circular molecules of monomer length 2.2 kbp. The abundance of the plasmid suggests its autonomous replication and the presence of an efficient origin of replication. An unusually large number of palindromes capabl...

  14. Biochemical and Molecular Characterization of a Novel Cu/Zn Superoxide Dismutase from Amaranthus hypochondriacus L.: an Intrinsically Disordered Protein.

    Science.gov (United States)

    Montero-Morán, Gabriela M; Sampedro, José G; Saab-Rincón, Gloria; Cervantes-González, Miguel A; Huerta-Ocampo, José Á; De León-Rodríguez, Antonio; Barba de la Rosa, Ana P

    2015-08-01

    A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456 bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50 % of catalytic activity after incubation at 100 °C for 30 min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability. PMID:26129702

  15. Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

    Directory of Open Access Journals (Sweden)

    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.

  16. Isolation and characterization of beta-glucan synthase: A potential biochemical regulator of gravistimulated differential cell wall loosening

    Science.gov (United States)

    Kuzmanoff, K. M.

    1984-01-01

    In plants, gravity stimulates differential growth in the upper and lower halves of horizontally oriented organs. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the activity of Golgi-localized Beta-1,4-glucan synthase, an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. The primary objective is to determine if auxin induces de novo formation of Golgi glucan synthase and increases the level of this glucan synthase mRNA. This shall be accomplished by (a) preparation of a monoclonal antibody to the synthase, (b) isolation, and characterization of the glucan synthase, and (c) examination for cross reactivity between the antibody and translation products of auxin induced mRNAs in pea tissue. The antibody will also be used to localize the glucan synthase in upper and lower halves of pea stem tissue before, during and after the response to gravity.

  17. Biophysical and Biochemical Characterization of Avian Secretory Component Provides Structural Insights into the Evolution of the Polymeric Ig Receptor.

    Science.gov (United States)

    Stadtmueller, Beth M; Yang, Zhongyu; Huey-Tubman, Kathryn E; Roberts-Mataric, Helena; Hubbell, Wayne L; Bjorkman, Pamela J

    2016-08-15

    The polymeric Ig receptor (pIgR) transports polymeric Abs across epithelia to the mucosa, where proteolytic cleavage releases the ectodomain (secretory component [SC]) as an integral component of secretory Abs, or as an unliganded protein that can mediate interactions with bacteria. SC is conserved among vertebrates, but domain organization is variable: mammalian SC has five domains (D1-D5), whereas avian, amphibian, and reptilian SC lack the D2 domain, and fish SC lacks domains D2-D4. In this study, we used double electron-electron resonance spectroscopy and surface plasmon resonance binding studies to characterize the structure, dynamics, and ligand binding properties of avian SC, avian SC domain variants, and a human SC (hSC) variant lacking the D2 domain. These experiments demonstrated that, unlike hSC, which adopts a compact or "closed" domain arrangement, unliganded avian SC is flexible and exists in both closed and open states, suggesting that the mammalian SC D2 domain stabilizes the closed conformation observed for hSC D1-D5. Experiments also demonstrated that avian and mammalian pIgR share related, but distinct, mechanisms of ligand binding. Together, our data reveal differences in the molecular recognition mechanisms associated with evolutionary changes in the pIgR protein. PMID:27412418

  18. Mitochondrial dysfunction and organophosphorus compounds

    International Nuclear Information System (INIS)

    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

  19. 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.

  20. Teaching the Role of Mitochondrial Transport in Energy Metabolism

    Science.gov (United States)

    Passarella, Salvatore; Atlante, Anna

    2007-01-01

    Studies from our laboratories over recent years have uncovered the existence, and established the properties of a variety of mitochondrial transporters. The properties of these transporters throw light on a variety of biochemical phenomena that were previously poorly understood. In particular the role of mitochondrial transport in energy…

  1. Biochemical characterization and functional analysis of the POU transcription factor POU-M2 of Bombyx mori.

    Science.gov (United States)

    Liu, Lina; Li, Yu; Wang, Yejing; Zhao, Peng; Wei, Shuguang; Li, Zhenzhen; Chang, Huaipu; He, Huawei

    2016-05-01

    POU-M2 is a homeodomain transcription factor which plays important roles in the development and silk synthesis of Bombyx mori. In this study, we expressed, purified and characterized POU-M2 and studied its transcription regulation on fibroin heavy chain gene of Bombyx mori. Gel filtration showed POU-M2 existed as a dimer in solution. Far-UV circular dichroism spectra indicated POU-M2 had a well-defined α-helix structure and the α-helix content was about 26.4%. The thermal unfolding transition of POU-M2 was a cooperative process. Tm, ΔH and ΔS were 45.15±0.2°C, 138.4±0.5KJ/mol and 0.4349±0.04KJ/(mol·K), respectively. Western blotting analysis indicated the expression level of POU-M2 increased slightly from day 3 to day 7 of the fifth instar larvae in the posterior silk gland. POU-M2 was positioned in the nucleus of cells. The luciferase reporter assay demonstrated POU-M2 could stimulate the promoter activity of fibroin heavy chain gene, and the activation effect was dependent on the amount of POU-M2. Our study suggested POU-M2 may be involved in the transcriptional regulation of fibroin heavy chain gene. These findings expand toward a better understanding of the structure of POU-M2 and its function in silk synthesis of Bombyx mori. PMID:26854886

  2. 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...

  3. Complete mitochondrial DNA sequences of the Victoria tilapia (Oreochromis variabilis) and Redbelly Tilapia (Tilapia zilli): genome characterization and phylogeny analysis.

    Science.gov (United States)

    Kinaro, Zachary Omambia; Xue, Liangyi; Volatiana, Josies Ancella

    2016-07-01

    The Cichlid fishes have played an important role in evolutionary biology, population studies and aquaculture industry with East African species representing a model suited for studying adaptive radiation and speciation for cichlid genome projects in which closely related genomes are fast emerging presenting questions on phenotype-genotype relations. The complete mitochondrial genomes presented here are for two closely related but eco-morphologically distinct Lake Victoria basin cichlids, Oreochromis variabilis, an endangered native species and Tilapia zilli, an invasive species, both of which are important economic fishes in local areas. The complete mitochondrial genomes determined for O. variabilis and T. zilli are 16 626 and 16,619 bp, respectively. Both the mitogenomes contain 13 protein-coding genes, 22 tRNAs, 2 rRNAs and a non-coding control region, which are typical of vertebrate mitogenomes. Phylogenetic analyses of the two species revealed that though both lie within family Cichlidae, they are remotely related. PMID:27158785

  4. Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-D-glucoside

    Directory of Open Access Journals (Sweden)

    Herbert Michlmayr

    2015-07-01

    Full Text Available Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON, deoxynivalenol-3-O-β-D-glucoside (D3G is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79 and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON, and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins.

  5. Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major.

    Science.gov (United States)

    Belaz, Sorya; Rattier, Thibault; Lafite, Pierre; Moreau, Philippe; Routier, Françoise H; Robert-Gangneux, Florence; Gangneux, Jean-Pierre; Daniellou, Richard

    2015-10-13

    The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism. PMID:26279524

  6. The mitochondrial quality control protein Yme1 is necessary to prevent defective mitophagy in a yeast model of Barth syndrome.

    Science.gov (United States)

    Gaspard, Gerard J; McMaster, Christopher R

    2015-04-01

    The Saccharomyces cerevisiae TAZ1 gene is an orthologue of human TAZ; both encode the protein tafazzin. Tafazzin is a transacylase that transfers acyl chains with unsaturated fatty acids from phospholipids to monolysocardiolipin to generate cardiolipin with unsaturated fatty acids. Mutations in human TAZ cause Barth syndrome, a fatal childhood cardiomyopathy biochemically characterized by reduced cardiolipin mass and increased monolysocardiolipin levels. To uncover cellular processes that require tafazzin to maintain cell health, we performed a synthetic genetic array screen using taz1Δ yeast cells to identify genes whose deletion aggravated its fitness. The synthetic genetic array screen uncovered several mitochondrial cellular processes that require tafazzin. Focusing on the i-AAA protease Yme1, a mitochondrial quality control protein that degrades misfolded proteins, we determined that in cells lacking both Yme1 and Taz1 function, there were substantive mitochondrial ultrastructural defects, ineffective superoxide scavenging, and a severe defect in mitophagy. We identify an important role for the mitochondrial protease Yme1 in the ability of cells that lack tafazzin function to maintain mitochondrial structural integrity and mitochondrial quality control and to undergo mitophagy. PMID:25688091

  7. Complete sequence and characterization of mitochondrial genome in the swimming crab Portunus sanguinolentus (Herbst, 1783) (Decapoda, Brachyura, Portunidae).

    Science.gov (United States)

    Meng, Xianliang; Jia, Fulong; Zhang, Xiaohui; Liu, Ping; Li, Jian

    2016-07-01

    The three-spot swimming crab Portunus sanguinolentus (Herbst, 1783) is a commercially important fishery species, widely distributed in the Indo-Pacific region. In this study, we present the complete mitochondrial genome of P. sanguinolentus. The genome is 16 027 bp in length with circular organization, encoding the standard set of 13 protein-coding genes, 22 tRNA genes, and two rRNA genes. The overall A + T content is 65.60%, which is lower than that of its congeneric species Portunus pelagicus and Portunus trituberculatus. The mitogenome carries 1254 bp of intergenic region constituting 7.82% of the genome, and six pairs of overlapping genes with the overlap size from 1 to 7 bp. The complete mitogenome sequence information of P. sanguinolentus would provide useful data for further studies on population genetics and molecular systematics. PMID:26153754

  8. The mitochondrial DNA makeup of Romanians: A forensic mtDNA control region database and phylogenetic characterization.

    Science.gov (United States)

    Turchi, Chiara; Stanciu, Florin; Paselli, Giorgia; Buscemi, Loredana; Parson, Walther; Tagliabracci, Adriano

    2016-09-01

    To evaluate the pattern of Romanian population from a mitochondrial perspective and to establish an appropriate mtDNA forensic database, we generated a high-quality mtDNA control region dataset from 407 Romanian subjects belonging to four major historical regions: Moldavia, Transylvania, Wallachia and Dobruja. The entire control region (CR) was analyzed by Sanger-type sequencing assays and the resulting 306 different haplotypes were classified into haplogroups according to the most updated mtDNA phylogeny. The Romanian gene pool is mainly composed of West Eurasian lineages H (31.7%), U (12.8%), J (10.8%), R (10.1%), T (9.1%), N (8.1%), HV (5.4%),K (3.7%), HV0 (4.2%), with exceptions of East Asian haplogroup M (3.4%) and African haplogroup L (0.7%). The pattern of mtDNA variation observed in this study indicates that the mitochondrial DNA pool is geographically homogeneous across Romania and that the haplogroup composition reveals signals of admixture of populations of different origin. The PCA scatterplot supported this scenario, with Romania located in southeastern Europe area, close to Bulgaria and Hungary, and as a borderland with respect to east Mediterranean and other eastern European countries. High haplotype diversity (0.993) and nucleotide diversity indices (0.00838±0.00426), together with low random match probability (0.0087) suggest the usefulness of this control region dataset as a forensic database in routine forensic mtDNA analysis and in the investigation of maternal genetic lineages in the Romanian population. PMID:27414754

  9. Characterization of uranium effects on the zebra fish Danio rerio. Stress mechanisms, neuro-toxicity and mitochondrial metabolism

    International Nuclear Information System (INIS)

    This research explored several biological effects of uranium (U) in zebra fish exposed to low waterborne uranium concentrations (20 and 100 microgram/L). In tissue specific study (brain, liver, skeletal muscles and gills) of transcriptional responses in 20 genes identified the nature of the potential U effects during 28 days of exposure followed by an 8-day depuration phase in connection with U bioaccumulation. Liver and gills accumulate high concentrations of U and the depuration is efficient contrary to the brain and muscles. U exposure induced a later response in liver (inflammatory process, apoptosis and detoxification) and gills (oxidative balance) and an early one in brain (neuronal response) and muscles (mitochondrial metabolism). Brain and muscles appear sensitive since defence mechanisms are inefficient above low concentrations. A further study on these two organs examined the function and protein content of the respiratory mitochondrial chain following U exposure. An inhibition of the respiratory control ratio for the lowest concentration, variation in the protein synthesis of the complex IV (induction of cytochrome c oxidase sub-unit I and IV) and histological damage (dilatation in brain and vacuolisation in muscles) were observed. Another study focused on the early effects on the brain and was accomplished through a large transcriptional analysis coupled with examinations of the olfactory bulb ultrastructure. A depression of genes encoding olfactory receptor or111-7 and or102-5 was observed as rapidly as 3 days post-exposure to the lowest concentration of U. These responses and histological injuries suggest that the olfactory system could be sensitive to U exposure. (author)

  10. Mitochondrial Dysfunction in Neurodegenerative Diseases

    OpenAIRE

    Johri, Ashu; Beal, M. Flint

    2012-01-01

    Neurodegenerative diseases are a large group of disabling disorders of the nervous system, characterized by the relative selective death of neuronal subtypes. In most cases, there is overwhelming evidence of impaired mitochondrial function as a causative factor in these diseases. More recently, evidence has emerged for impaired mitochondrial dynamics (shape, size, fission-fusion, distribution, movement etc.) in neurodegenerative diseases such as Parkinson's disease, Huntington's disease, amyo...

  11. Neuroradiologic findings in children with mitochondrial disorder: correlation with mitochondrial respiratory chain defects

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jinna; Lee, Seung-Koo; Kim, Dong Ik [Yonsei University College of Medicine, Department of Radiology, Research Institute of Radiological Science, Seoul (Korea); Kim, Eung Yeop [Yonsei University College of Medicine, Department of Radiology, Research Institute of Radiological Science, Brain Korea 21 Project for Medical Science, Seoul (Korea); Lee, Young-Mock; Lee, Joon Soo [Yonsei University College of Medicine, Department of Pediatrics, Pediatric Epilepsy Clinics, Severance Children' s Hospital, Brain Research Institute, Seoul (Korea); Kim, Heung Dong [Yonsei University College of Medicine, Department of Pediatrics, Pediatric Epilepsy Clinics, Severance Children' s Hospital, Brain Research Institute, Seoul (Korea); Yonsei University College of Medicine, Department of Pediatrics, Seoul (Korea)

    2008-08-15

    Mitochondrial disorders are a heterogeneous group of disorders affecting energy metabolism that can present at any age with a wide variety of clinical symptoms. We investigated brain magnetic resonance (MR) findings in 40 children with defects of the mitochondrial respiratory chain (MRC) complex and correlated them with the type of MRC defects. Enrolled were 40 children with MRC defects in biochemical enzyme assay of the muscle specimen. Twenty-one children were found to have classical syndromes of mitochondrial disorders and 19 children presented nonspecific mitochondrial encephalomyopathies. Their brain MR imaging findings were retrospectively reviewed and correlated with the biochemical defect in the MRC complex. Children with MRC defects showed various neuroradiologic features on brain MR imaging that resulted from a complex genetic background and a heterogeneous phenotype. Rapid progression of atrophy involving all structures of the brain with variable involvement of deep gray and white matter are the most frequent MR findings in children with MRC defects in both classical syndromes of mitochondrial disorder and nonspecific mitochondrial encephalomyopathies. The type of biochemical defect in the MRC complex enzyme did not correlate with brain MR findings in child patients. (orig.)

  12. Neuroradiologic findings in children with mitochondrial disorder: correlation with mitochondrial respiratory chain defects

    International Nuclear Information System (INIS)

    Mitochondrial disorders are a heterogeneous group of disorders affecting energy metabolism that can present at any age with a wide variety of clinical symptoms. We investigated brain magnetic resonance (MR) findings in 40 children with defects of the mitochondrial respiratory chain (MRC) complex and correlated them with the type of MRC defects. Enrolled were 40 children with MRC defects in biochemical enzyme assay of the muscle specimen. Twenty-one children were found to have classical syndromes of mitochondrial disorders and 19 children presented nonspecific mitochondrial encephalomyopathies. Their brain MR imaging findings were retrospectively reviewed and correlated with the biochemical defect in the MRC complex. Children with MRC defects showed various neuroradiologic features on brain MR imaging that resulted from a complex genetic background and a heterogeneous phenotype. Rapid progression of atrophy involving all structures of the brain with variable involvement of deep gray and white matter are the most frequent MR findings in children with MRC defects in both classical syndromes of mitochondrial disorder and nonspecific mitochondrial encephalomyopathies. The type of biochemical defect in the MRC complex enzyme did not correlate with brain MR findings in child patients. (orig.)

  13. Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae.

    Directory of Open Access Journals (Sweden)

    Gaurab Sircar

    Full Text Available Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation 'Rhi o 1'.The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies.The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross

  14. 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. PMID:26780766

  15. Biochemical and structural characterization of Pseudomonas aeruginosa Bfd and FPR: ferredoxin NADP+ reductase and not ferredoxin is the redox partner of heme oxygenase under iron-starvation conditions.

    Science.gov (United States)

    Wang, An; Zeng, Yuhong; Han, Huijong; Weeratunga, Saroja; Morgan, Bailey N; Moënne-Loccoz, Pierre; Schönbrunn, Ernst; Rivera, Mario

    2007-10-30

    Among the 118 genes upregulated by Pseudomonas aeruginosa in response to iron starvation [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287], we focused on the products of the two genes encoding electron transfer proteins, as a means of identifying the redox partners of the heme oxygenase (pa-HO) expressed under low-iron stress conditions. Biochemical and spectroscopic investigations demonstrated that the bfd gene encodes a 73-amino acid protein (pa-Bfd) that incorporates a [2Fe-2S]2+/+ center, whereas the fpr gene encodes a 258-residue NADPH-dependent ferredoxin reductase (pa-FPR) that utilizes FAD as a cofactor. In vitro reconstitution of pa-HO catalytic activity with the newly characterized proteins led to the surprising observation that pa-FPR efficiently supports the catalytic cycle of pa-HO, without the need of a ferredoxin. In comparison, electron transfer from pa-Bfd to pa-HO is sluggish, which strongly argues against the possibility that the seven electrons needed by pa-HO to degrade biliverdin are transferred from NADPH to pa-HO in a ferredoxin (Bfd)-dependent manner. Given that pa-HO functions to release iron from exogenous heme acquired under iron-starvation conditions, the use of a flavoenzyme rather than an iron-sulfur center-containing protein to support heme degradation is an efficient use of resources in the cell. The crystal structure of pa-FPR (1.6 A resolution) showed that its fold is comparable that of the superfamily of ferredoxin reductases and most similar to the structure of Azotobacter vinelandii FPR and Escherichia coli flavodoxin reductase. The latter two enzymes interact with distinct redox partners, a ferredoxin and a flavodoxin, respectively. Hence, findings reported herein extend the range of redox partners recognized by the fold of pa-FPR to include a heme oxygenase (pa-HO). PMID:17915950

  16. Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4.

    Science.gov (United States)

    Wang, X C; Zhao, H Y; Liu, G; Cheng, X J; Feng, H

    2016-01-01

    The feather is a valuable by-product with a huge annual yield produced by the poultry industry. Degradation of feathers by microorganisms is a prerequisite to utilize this insoluble protein resource. To improve the degrading efficiency of feathers, mutagenesis of the bacterium Bacillus subtilis S1-4 was performed. By combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine treatment for mutagenesis, a high protease-producing mutant (UMU4) of B. subtilis S1-4 was selected, which exhibited 2.5-fold higher extracellular caseinolytic activity than did the wild-type strain. UMU4 degraded chicken feathers more efficiently, particularly for the release of soluble proteins from the feathers, compared to the wild-type strain. Furthermore, an extracellular protease with a molecular weight of 45 kDa, as determined by SDS-PAGE, was purified from UMU4. Biochemical characterization indicated that the caseinolytic activity of the protease was largely inhibited by phenylmethanesulfonyl fluoride, suggesting that the purified enzyme is a serine protease. This protease was highly active over a wide range of pHs (6.0 to 12.0) and temperatures (50° to 75°C) with an optimal pH and temperature of 8.0 and 65°C, respectively. The purified enzyme exhibited good thermostability with a 72.2 min half-life of thermal denaturation at 60°C. In addition, this protease was not sensitive to heavy metal ions, surfactants, or oxidative reagents. In conclusion, strain improvement for protease production can serve as an alternative strategy to promote feather degradation. The UMU4 mutant of B. subtilis and its serine protease could be potentially used in various industries. PMID:27323184

  17. 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. PMID:27181353

  18. Characterization of a mitochondrial manganese superoxide dismutase gene from Apis cerana cerana and its role in oxidative stress.

    Science.gov (United States)

    Jia, Haihong; Sun, Rujiang; Shi, Weina; Yan, Yan; Li, Han; Guo, Xingqi; Xu, Baohua

    2014-01-01

    Mitochondrial manganese superoxide dismutase (mMnSOD) plays a vital role in the defense against reactive oxygen species (ROS) in eukaryotic mitochondria. In this study, we isolated and identified a mMnSOD gene from Apis cerana cerana, which we named AccSOD2. Several putative transcription factor-binding sites were identified within the 5'-flanking region of AccSOD2, which suggests that AccSOD2 may be involved in organismal development and/or environmental stress responses. Quantitative real-time PCR analysis showed that AccSOD2 is highly expressed in larva and pupae during different developmental stages. In addition, the expression of AccSOD2 could be induced by cold (4 °C), heat (42 °C), H2O2, ultraviolet light (UV), HgCl2, and pesticide treatment. Using a disc diffusion assay, we provide evidence that recombinant AccSOD2 protein can play a functional role in protecting cells from oxidative stress. Finally, the in vivo activities of AccSOD2 were measured under a variety of stressful conditions. Taken together, our results indicate that AccSOD2 plays an important role in cellular stress responses and anti-oxidative processes and that it may be of critical importance to honeybee survival. PMID:24269344

  19. 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.

  20. Barth Syndrome: From Mitochondrial Dysfunctions Associated with Aberrant Production of Reactive Oxygen Species to Pluripotent Stem Cell Studies.

    Science.gov (United States)

    Saric, Ana; Andreau, Karine; Armand, Anne-Sophie; Møller, Ian M; Petit, Patrice X

    2015-01-01

    Mutations in the gene encoding the enzyme tafazzin, TAZ, cause Barth syndrome (BTHS). Individuals with this X-linked multisystem disorder present cardiomyopathy (CM) (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 phospholipidlysophospholipid 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 bioenergetic

  1. The mitochondrial carnitine/acylcarnitine carrier: function, structure and physiopathology.

    Science.gov (United States)

    Indiveri, Cesare; Iacobazzi, Vito; Tonazzi, Annamaria; Giangregorio, Nicola; Infantino, Vittoria; Convertini, Paolo; Console, Lara; Palmieri, Ferdinando

    2011-08-01

    The carnitine/acylcarnitine carrier (CAC) is a transport protein of the inner mitochondrial membrane that belongs to the mitochondrial carrier protein family. In its cytosolic conformation the carrier consists of a bundle of six transmembrane α-helices, which delimit a water filled cavity opened towards the cytosol and closed towards the matrix by a network of interacting charged residues. Most of the functional data on this transporter come from studies performed with the protein purified from rat liver mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The carnitine/acylcarnitine carrier transports carnitine and acylcarnitines with acyl chains of various lengths from 2 to 18 carbon atoms. The mammalian transporter exhibits higher affinity for acylcarnitines with longer carbon chains. The functional data indicate that CAC plays the important function of catalyzing transport of acylcarnitines into the mitochondria in exchange for intramitochondrial free carnitine. This results in net transport of fatty acyl units into the mitochondrial matrix where they are oxidized by the β-oxidation enzymes. The essential role of the transporter in cell metabolism is demonstrated by the fact that alterations of the human gene SLC25A20 coding for CAC are associated with a severe disease known as carnitine carrier deficiency. This autosomal recessive disorder is characterized by life-threatening episodes of coma induced by fasting, cardiomyopathy, liver dysfunction, muscle weakness, respiratory distress and seizures. Until now 35 different mutations of CAC gene have been identified in carnitine carrier deficient patients. Some missense mutations concern residues of the signature motif present in all mitochondrial carriers. Diagnosis of carnitine carrier deficiency requires biochemical and genetic tests; treatment is essentially limited to important dietetic measures. Recently, a pharmacological approach based on the

  2. Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

    Science.gov (United States)

    Arreola, Sheryl Lozel; Intanon, Montira; Suljic, Jasmina; Kittl, Roman; Pham, Ngoc Hung; Kosma, Paul; Haltrich, Dietmar; Nguyen, Thu-Ha

    2014-01-01

    Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for β-gal I, and 543 and 25 mM-1s-1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide. PMID:25089712

  3. Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

    Directory of Open Access Journals (Sweden)

    Sheryl Lozel Arreola

    Full Text Available Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for β-gal I, and 543 and 25 mM-1s-1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6-D-Glc (allolactose and β-D-Galp-(1→3-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6 and β-(1→3-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.

  4. Mitochondrial Evolution

    OpenAIRE

    Gray, Michael W

    2012-01-01

    Viewed through the lens of the genome it contains, the mitochondrion is of unquestioned bacterial ancestry, originating from within the bacterial phylum α-Proteobacteria (Alphaproteobacteria). Accordingly, the endosymbiont hypothesis—the idea that the mitochondrion evolved from a bacterial progenitor via symbiosis within an essentially eukaryotic host cell—has assumed the status of a theory. Yet mitochondrial genome evolution has taken radically different pathways in diverse eukaryotic lineag...

  5. 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.

  6. 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. PMID:27001627

  7. 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 ...

  8. Toxicological and Biochemical Characterizations of AChE in Phosalone-Susceptible and Resistant Populations of the Common Pistachio Psyllid, Agonoscena pistaciae

    OpenAIRE

    Alizadeh, Ali; Talebi-Jahromi, Khalil; Hosseininaveh, Vahid; Ghadamyari, Mohammad

    2014-01-01

    The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioa...

  9. 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.

  10. Effects of mitochondrial dysfunction on the immunological properties of microglia

    OpenAIRE

    Ferger Annette I; Campanelli Loretta; Reimer Valentina; Muth Katharina N; Merdian Irma; Ludolph Albert C; Witting Anke

    2010-01-01

    Abstract Background Neurodegenerative diseases are characterized by both mitochondrial dysfunction and activation of microglia, the macrophages of the brain. Here, we investigate the effects of mitochondrial dysfunction on the activation profile of microglial cells. Methods We incubated primary mouse microglia with the mitochondrial toxins 3-nitropropionic acid (3-NP) or rotenone. These mitochondrial toxins are known to induce neurodegeneration in humans and in experimental animals. We charac...

  11. 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

  12. Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans

    Directory of Open Access Journals (Sweden)

    Selvin Joseph

    2012-06-01

    Full Text Available Abstract Background Lipases (EC 3.1.1.3 catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome. Results A metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C β-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16 at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3–12 and temperature (4 -60°C ranges and high levels of stability in the presence of various solvents at NaCl concentrations

  13. Physicochemical And Biochemical Characterization Of Non-Biodegradable Cellulose In Miocene Gymnosperm Wood From The Entre-Sambre-Et-Meuse, Southern Belgium

    OpenAIRE

    Lechien, Valérie; Rodriguez, Christian; Ongena, Marc; Hiligsmann, Serge; Rulmont, André; Thonart, Philippe

    2006-01-01

    Specimens of Miocene fossil wood from the Entre-Sambre-et-Meuse karsts (southern Belgium) were examined using physicochemical and biochemical techniques in order to understand the reasons for the exceptional preservation of these fossilized remains after 15 million years. Structural and chemical changes were assessed by comparing the structural features of the fossil samples with those of their modern counterpart, Metaseguoia. Solid state C-13 nuclear magnetic resonance (NMR) and microscopic ...

  14. Purification, biochemical characterization and structural modelling of alkali-stable β-1,4-xylan xylanohydrolase from Aspergillus fumigatus R1 isolated from soil

    OpenAIRE

    Deshmukh, Rehan Ahmed; Jagtap, Sharmili; Mandal, Madan Kumar; Mandal, Suraj Kumar

    2016-01-01

    Background Aspergillus fumigatus R1 produced xylanase under submerged fermentation which degrades the complex hemicelluloses contained in agricultural substrates. Xylanases have gained considerable attention because of their tremendous applications in industries. The purpose of our study was to purify xylanase and study its biochemical properties. We have predicted the secondary structure of purified xylanase and evaluated its active site residues and substrate binding sites based on the glob...

  15. Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis

    OpenAIRE

    Ma, Liang; Huang, Da-Wei; Cuomo, Christina A.; Sykes, Sean; Fantoni, Giovanna; Das, Biswajit; Sherman, Brad T.; Yang, Jun; Huber, Charles; Xia, Yun; Davey, Emma; Kutty, Geetha; Bishop, Lisa; Sassi, Monica; Lempicki, Richard A.

    2013-01-01

    Pneumocystis jirovecii is an important opportunistic pathogen associated with AIDS and other immunodeficient conditions. Currently, very little is known about its nuclear and mitochondrial genomes. In this study, we sequenced the complete mitochondrial genome (mtDNA) of this organism and its closely related species Pneumocystis carinii and Pneumocystis murina by a combination of sequencing technologies. Our study shows that P. carinii and P. murina mtDNA share a nearly identical number and or...

  16. Drosophila Porin/VDAC affects mitochondrial morphology.

    Directory of Open Access Journals (Sweden)

    Jeehye Park

    Full Text Available Voltage-dependent anion channel (VDAC has been suggested to be a mediator of mitochondrial-dependent cell death induced by Ca(2+ overload, oxidative stress and Bax-Bid activation. To confirm this hypothesis in vivo, we generated and characterized Drosophila VDAC (porin mutants and found that Porin is not required for mitochondrial apoptosis, which is consistent with the previous mouse studies. We also reported a novel physiological role of Porin. Loss of porin resulted in locomotive defects and male sterility. Intriguingly, porin mutants exhibited elongated mitochondria in indirect flight muscle, whereas Porin overexpression produced fragmented mitochondria. Through genetic analysis with the components of mitochondrial fission and fusion, we found that the elongated mitochondria phenotype in porin mutants were suppressed by increased mitochondrial fission, but enhanced by increased mitochondrial fusion. Furthermore, increased mitochondrial fission by Drp1 expression suppressed the flight defects in the porin mutants. Collectively, our study showed that loss of Drosophila Porin results in mitochondrial morphological defects and suggested that the defective mitochondrial function by Porin deficiency affects the mitochondrial remodeling process.

  17. Increased intrinsic mitochondrial function in humans with mitochondrial haplogroup H

    DEFF Research Database (Denmark)

    Larsen, Steen; Díez-Sánchez, Carmen; Rabøl, Rasmus;

    2014-01-01

    determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30......% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present....

  18. Organization of the human mitochondrial transcription initiation complex

    OpenAIRE

    Yakubovskaya, Elena; Guja, Kip E.; Eng, Edward T.; Choi, Woo Suk; Mejia, Edison; Beglov, Dmitri; Lukin, Mark; Kozakov, Dima; Garcia-Diaz, Miguel

    2014-01-01

    Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human mitochondrial transcription initiation complex on the light-strand promoter (LSP) through solution X-ray scattering, electron microscopy (EM) and biochemical studies. Our EM results demonstrate a compact organization of the initiation complex, suggesting that protein–protein interactions might help m...

  19. Mitochondrial Dynamics and Mitochondrial Dysfunction in Diabetes.

    Science.gov (United States)

    Wada, Jun; Nakatsuka, Atsuko

    2016-06-01

    The mitochondria are involved in active and dynamic processes, such as mitochondrial biogenesis, fission, fusion and mitophagy to maintain mitochondrial and cellular functions. In obesity and type 2 diabetes, impaired oxidation, reduced mitochondrial contents, lowered rates of oxidative phosphorylation and excessive reactive oxygen species (ROS) production have been reported. Mitochondrial biogenesis is regulated by various transcription factors such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and nuclear respiratory factors (NRFs). Mitochondrial fusion is promoted by mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy 1 (OPA1), while fission is governed by the recruitment of dynamin-related protein 1 (DRP1) by adaptor proteins such as mitochondrial fission factor (MFF), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51), and fission 1 (FIS1). Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARKIN promote DRP1-dependent mitochondrial fission, and the outer mitochondrial adaptor MiD51 is required in DRP1 recruitment and PARKIN-dependent mitophagy. This review describes the molecular mechanism of mitochondrial dynamics, its abnormality in diabetes and obesity, and pharmaceuticals targeting mitochondrial biogenesis, fission, fusion and mitophagy. PMID:27339203

  20. Emerging aspects of treatment in mitochondrial disorders.

    Science.gov (United States)

    Rahman, Shamima

    2015-07-01

    Mitochondrial diseases are clinically, biochemically and genetically heterogeneous disorders of two genomes, for which effective curative therapies are currently lacking. With the exception of a few rare vitamin/cofactor responsive conditions (including ACAD9 deficiency, disorders of coenzyme Q(10) biosynthesis, and Leigh syndrome caused by mutations in the SLC19A3 transporter), the mainstay of treatment for the vast majority of patients involves supportive measures. The search for a cure for mitochondrial disease is the subject of intensive research efforts by many investigators across the globe, but the goal remains elusive. The clinical and genetic heterogeneity, multisystemic nature of many of these disorders, unpredictable natural course, relative inaccessibility of the mitochondrion and lack of validated, clinically meaningful outcome measures, have all presented great challenges to the design of rigorous clinical trials. This review discusses barriers to developing effective therapies for mitochondrial disease, models for evaluating the efficacy of novel treatments and summarises the most promising emerging therapies in six key areas: 1) antioxidant approaches; 2) stimulating mitochondrial biogenesis; 3) targeting mitochondrial membrane lipids, dynamics and mitophagy; 4) replacement therapy; 5) cell-based therapies; and 6) gene therapy approaches for both mtDNA and nuclear-encoded defects of mitochondrial metabolism. PMID:25962587

  1. Mitochondrial preconditioning: a potential neuroprotective strategy

    Directory of Open Access Journals (Sweden)

    Cristina Carvalho

    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.

  2. POS5 Gene of Saccharomyces cerevisiae Encodes a Mitochondrial NADH Kinase Required for Stability of Mitochondrial DNA

    OpenAIRE

    Strand, Micheline K.; Stuart, Gregory R.; Longley, Matthew J.; Graziewicz, Maria A.; Dominick, Olivia C.; Copeland, William C.

    2003-01-01

    In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD+, with a twofold preference for NADH. Disruption of POS5 inc...

  3. Biochemical, histologic, and biomechanical characterization of native and decellularized flexor tendon specimens harvested from the pelvic limbs of orthopedically normal dogs.

    Science.gov (United States)

    Balogh, Daniel G; Biskup, Jeffery J; O'Sullivan, M Gerard; Scott, Ruth M; Groschen, Donna; Evans, Richard B; Conzemius, Michael G

    2016-04-01

    OBJECTIVE To evaluate the biochemical and biomechanical properties of native and decellularized superficial digital flexor tendons (SDFTs) and deep digital flexor tendons (DDFTs) harvested from the pelvic limbs of orthopedically normal dogs. SAMPLE 22 commercially supplied tendon specimens (10 SDFT and 12 DDFT) harvested from the pelvic limbs of 13 canine cadavers. PROCEDURES DNA, glycosaminoglycan, collagen, and protein content were measured to biochemically compare native and decellularized SDFT and DDFT specimens. Mechanical testing was performed on 4 groups consisting of native tendons (5 SDFTs and 6 DDFTs) and decellularized tendons (5 SDFTs and 6 DDFTs). All tendons were preconditioned, and tension was applied to failure at 0.5 mm/s. Failure mode was video recorded for each tendon. Load-deformation and stress-strain curves were generated; calculations were performed to determine the Young modulus and stiffness. Biochemical and biomechanical data were statistically compared by use of the Wilcoxon rank sum test. RESULTS Decellularized SDFT and DDFT specimens had significantly less DNA content than did native tendons. No significant differences were identified between native and decellularized specimens with respect to glycosaminoglycan, collagen, or protein content. Biomechanical comparison yielded no significant intra- or intergroup differences. All DDFT constructs failed at the tendon-clamp interface, whereas nearly half (4/10) of the SDFT constructs failed at midsubstance. CONCLUSIONS AND CLINICAL RELEVANCE Decellularized commercial canine SDFT and DDFT specimens had similar biomechanical properties, compared with each other and with native tendons. The decellularization process significantly decreased DNA content while minimizing loss of extracellular matrix components. Decellularized canine flexor tendons may provide suitable, biocompatible graft scaffolds for bioengineering applications such as tendon or ligament repair. PMID:27027838

  4. Biochemical Characterization and 16S rRNA Sequencing of Few Lipase-Producing Thermophilic Bacteria from Taptapani Hot Water Spring, Orissa, India

    OpenAIRE

    Satpal S. Bisht; Amrita K. Panda

    2011-01-01

    Three lipase-producing thermophilic bacteria (AK-P1, AK-P2, and AK-P3) were isolated from the Taptapani hot water spring in Orissa, India. The crude extra cellular lipases from cell-free culture supernatant were reacted in an olive oil mixture, and their lipolytic activities were compared. Identification of the bacteria was carried out using biochemical tests, 16SrRNA sequencing and sequences submitted to NCBI GenBank. Strain AK-P3, exhibited the highest lipolytic activity of 5.5 U/mL was ide...

  5. Mitochondrial network energetics in the heart

    OpenAIRE

    Aon, Miguel A.; Cortassa, Sonia

    2012-01-01

    At the core of eukaryotic aerobic life, mitochondria 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 spatio-temporal 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 beha...

  6. Transport of proteins across mitochondrial membranes

    OpenAIRE

    Neupert, Walter

    1994-01-01

    The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this...

  7. A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy.

    NARCIS (Netherlands)

    Jonckheere, A.I.; Hogeveen, M.; Nijtmans, L.G.J.; Brand, M.A.M. van den; Janssen, A.J.M.; Diepstra, J.H.S.; Brandt, FC van den; Heuvel, L.P.W.J. van den; Hol, F.A.; Hofste, T.G.; Kapusta, L.; Dillmann, U.; Shamdeen, M.G.; Smeitink, J.A.M.; Rodenburg, R.J.T.

    2008-01-01

    PURPOSE: To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder. METHODS: Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme

  8. Mitochondrial DNA disease—molecular insights and potential routes to a cure

    OpenAIRE

    Russell, Oliver; Turnbull, Doug

    2014-01-01

    Mitochondrial DNA diseases are common neurological conditions caused by mutations in the mitochondrial genome or nuclear genes responsible for its maintenance. Current treatments for these disorders are focussed on the management of the symptoms, rather than the correction of biochemical defects caused by the mutation. This review focuses on the molecular effects of mutations, the symptoms they cause and current work focusing on the development of targeted treatments for mitochondrial DNA dis...

  9. Inherited mitochondrial disorders.

    Science.gov (United States)

    Finsterer, Josef

    2012-01-01

    Though inherited mitochondrial disorders (MIDs) are most well known for their syndromic forms, for which widely known acronyms (MELAS, MERRF, NARP, LHON etc.) have been coined, the vast majority of inherited MIDs presents in a non-syndromic form. Since MIDs are most frequently multisystem disorders already at onset or during the disease course, a MID should be suspected if there is a combination of neurological and non-neurological abnormalities. Neurological abnormalities occurring as a part of a MID include stroke-like episodes, epilepsy, migraine-like headache, movement disorders, cerebellar ataxia, visual impairment, encephalopathy, cognitive impairment, dementia, psychosis, hypopituitarism, aneurysms, or peripheral nervous system disease, such as myopathy, neuropathy, or neuronopathy. Non-neurological manifestations concern the ears, the endocrine organs, the heart, the gastrointestinal tract, the kidneys, the bone marrow, and the skin. Whenever there is an unexplained combination of neurological and non-neurological disease in a patient or kindred, a MID should be suspected and appropriate diagnostic measures initiated. Genetic testing should be guided by the phenotype, the biopsy findings, and the biochemical results. PMID:22399423

  10. Mitochondrial disease and epilepsy.

    Science.gov (United States)

    Rahman, Shamima

    2012-05-01

    Mitochondrial respiratory chain disorders are relatively common inborn errors of energy metabolism, with a combined prevalence of one in 5000. These disorders typically affect tissues with high energy requirements, and cerebral involvement occurs frequently in childhood, often manifesting in seizures. Mitochondrial diseases are genetically heterogeneous; to date, mutations have been reported in all 37 mitochondrially encoded genes and more than 80 nuclear genes. The major genetic causes of mitochondrial epilepsy are mitochondrial DNA mutations (including those typically associated with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes [MELAS] and myoclonic epilepsy with ragged red fibres [MERRF] syndromes); mutations in POLG (classically associated with Alpers syndrome but also presenting as the mitochondrial recessive ataxia syndrome [MIRAS], spinocerebellar ataxia with epilepsy [SCAE], and myoclonus, epilepsy, myopathy, sensory ataxia [MEMSA] syndromes in older individuals) and other disorders of mitochondrial DNA maintenance; complex I deficiency; disorders of coenzyme Q(10) biosynthesis; and disorders of mitochondrial translation such as RARS2 mutations. It is not clear why some genetic defects, but not others, are particularly associated with seizures. Epilepsy may be the presenting feature of mitochondrial disease but is often part of a multisystem clinical presentation. Mitochondrial epilepsy may be very difficult to manage, and is often a poor prognostic feature. At present there are no curative treatments for mitochondrial disease. Individuals with mitochondrial epilepsy are frequently prescribed multiple anticonvulsants, and the role of vitamins and other nutritional supplements and the ketogenic diet remain unproven. PMID:22283595

  11. Recent abstracts in biochemical technology

    OpenAIRE

    R R Siva Kiran; Brijesh P

    2008-01-01

    “Recent abstracts in biochemical technology” is a collection of interesting research articles published in “List of biochemical technology journals” (Table 1). The abstracts are most likely to report significant results in biochemical technology.

  12. Interaction of glutaric aciduria type 1-related glutaryl-CoA dehydrogenase with mitochondrial matrix proteins.

    Directory of Open Access Journals (Sweden)

    Jessica Schmiesing

    Full Text Available Glutaric aciduria type 1 (GA1 is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH, which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1.

  13. Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction

    International Nuclear Information System (INIS)

    Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10μM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25μM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function

  14. 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...

  15. Mitochondrial dysfunction in liver failure requiring transplantation.

    Science.gov (United States)

    Lane, Maria; Boczonadi, Veronika; Bachtari, Sahar; Gomez-Duran, Aurora; Langer, Thorsten; Griffiths, Alexandra; Kleinle, Stephanie; Dineiger, Christine; Abicht, Angela; Holinski-Feder, Elke; Schara, Ulrike; Gerner, Patrick; Horvath, Rita

    2016-05-01

    Liver failure is a heterogeneous condition which may be fatal and the primary cause is frequently unknown. We investigated mitochondrial oxidative phosphorylation in patients undergoing liver transplantation. We studied 45 patients who had liver transplantation due to a variety of clinical presentations. Blue native polyacrylamide gel electrophoresis with immunodetection of respiratory chain complexes I-V, biochemical activity of respiratory chain complexes II and IV and quantification of mitochondrial DNA (mtDNA) copy number were investigated in liver tissue collected from the explanted liver during transplantation. Abnormal mitochondrial function was frequently present in this cohort: ten of 40 patients (25 %) had a defect of one or more respiratory chain enzyme complexes on blue native gels, 20 patients (44 %) had low activity of complex II and/or IV and ten (22 %) had a reduced mtDNA copy number. Combined respiratory chain deficiency and reduced numbers of mitochondria were detected in all three patients with acute liver failure. Low complex IV activity in biliary atresia and complex II defects in cirrhosis were common findings. All six patients diagnosed with liver tumours showed variable alterations in mitochondrial function, probably due to the heterogeneity of the presenting tumour. In conclusion, mitochondrial dysfunction is common in severe liver failure in non-mitochondrial conditions. Therefore, in contrast to the common practice detection of respiratory chain abnormalities in liver should not restrict the inclusion of patients for liver transplantation. Furthermore, improving mitochondrial function may be targeted as part of a complex therapy approach in different forms of liver diseases. PMID:27053192

  16. Mitochondrial disorders: disease mechanisms and therapeutic approaches.

    Science.gov (United States)

    Poole, Olivia V; Hanna, Michael G; Pitceathly, Robert D S

    2015-11-01

    Mitochondrial disorders are now well recognized as an important cause of genetic disease. They exhibit remarkable phenotypic, biochemical, and molecular heterogeneity, and frequently involve multiple organ systems. Their complexity partly relates to the dual expression of mitochondrial proteins by both mitochondrial and nuclear genomic DNA. Multiple copies of mitochondrial DNA (mtDNA) are present in a single human mitochondrion. Each molecule exists as a double-stranded, circular, helical structure containing 37 genes: 13 encode polypeptide subunits, whilst the remaining 24 encode 22 transfer and 2 ribosomal RNAs necessary for their synthesis. These protein subunits contribute towards four of five multimeric enzymes (so-called complex I/III/IV/V, with complex II entirely nuclear-encoded) embedded in the inner mitochondrial membrane. The enzymes catalyze a sequence of redox reactions which ultimately generates adenine triphosphate, the cellular unit of energy, during oxidative phosphorylation (OXPHOS). The remaining OXPHOS subunits (more than 70 in total), in addition to the apparatus required for their transcription, translation, post-translational modification and assembly, are nuclear-encoded. The mitochondrion's dependence on nuclear DNA extends further to include the machinery required for the maintenance, replication, and repair of mtDNA molecules, the proteins for which are synthesized in the cell cytoplasm prior to transport across mitochondrial membrane for replication. Recent advancements in DNA analysis using next generation sequencing technology have provided an unprecedented expansion in the depth of knowledge concerning both molecular mechanisms and biological pathways which underpin many mitochondrial diseases. This understanding has led to the emergence of many potential targets and treatment strategies for these disorders for which there is currently no cure. This review highlights the challenges to therapy development and clinical trial design and

  17. Miro, MCU, and calcium: bridging our understanding of mitochondrial movement in axons

    Directory of Open Access Journals (Sweden)

    Karen Chang

    2013-09-01

    Full Text Available Neurons are extremely polarized structures with long axons and dendrites, which require proper distribution of mitochondria and maintenance of mitochondrial dynamics for neuronal functions and survival. Indeed, recent studies show that various neurological disorders are linked to mitochondrial transport in neurons. Mitochondrial anterograde transport is believed to deliver metabolic energy to synaptic terminals where energy demands are high, while mitochondrial retrograde transport is required to repair or remove damaged mitochondria in axons. It has been suggested that Ca2+ plays a key role in regulating mitochondrial transport by altering the configuration of mitochondrial protein, miro. However, molecular mechanisms that regulate mitochondrial transport in neurons still are not well characterized. In this review, we will discuss the roles of miro in mitochondrial transport and how the recently identified components of the mitochondrial calcium uniporter add to our current model of mitochondrial mobility regulation.

  18. Epidemiological, clinical and biochemical characterization of the p.(Ala359Asp) SMPD1 variant causing Niemann-Pick disease type B.

    Science.gov (United States)

    Acuña, Mariana; Martínez, Pablo; Moraga, Carol; He, Xingxuan; Moraga, Mauricio; Hunter, Bessie; Nuernberg, Peter; Gutiérrez, Rodrigo A; González, Mauricio; Schuchman, Edward H; Santos, José Luis; Miquel, Juan Francisco; Mabe, Paulina; Zanlungo, Silvana

    2016-02-01

    Niemann-Pick disease type B (NPDB) is a rare, inherited lysosomal storage disorder that occurs due to variants in the sphingomyelin phosphodiesterase 1 (SMPD1) gene and the resultant deficiency of acid sphingomyelinase (ASM) activity. While numerous variants causing NPDB have been described, only a small number have been studied in any detail. Herein, we describe the frequency of the p.(Ala359Asp) variant in the healthy Chilean population, and determine the haplotype background of homozygous patients to establish if this variant originated from a common founder. Genomic DNA samples from 1691 healthy individuals were analyzed for the p.(Ala359Asp) variant. The frequency of p.(Ala359Asp) was found to be 1/105.7, predicting a disease incidence of 1/44 960 in Chile, higher than the incidence estimated by the number of confirmed NPDB cases. We also describe the clinical characteristics of 13 patients homozygous for p.(Ala359Asp) and all of them had moderate to severe NPDB disease. In addition, a conserved haplotype and shared 280 Kb region around the SMPD1 gene was observed in the patients analyzed, indicating that the variant originated from a common ancestor. The haplotype frequency and mitochondrial DNA analysis suggest an Amerindian origin for the variant. To assess the effect of the p.(Ala359Asp) variant, we transfected cells with the ASM-p.(Ala359Asp) cDNA and the activity was only 4.2% compared with the wild-type cDNA, definitively demonstrating the causative effect of the variant on ASM function. Information on common variants such as p.(Ala359Asp) is essential to guide the successful implementation for future therapies and benefit to patients. PMID:25920558

  19. Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

    Directory of Open Access Journals (Sweden)

    Colin A Cooper

    2010-02-01

    Full Text Available Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2 is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2 and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  20. Physiological and biochemical characterization of the assai palm (Euterpe oleracea Mart. during seed germination and seedling growth under aerobic and anaerobic conditions

    Directory of Open Access Journals (Sweden)

    José Francisco de Carvalho Gonçalves

    2010-12-01

    Full Text Available Physiological and biochemical aspects of assai palm during seed germination and early seedling growth were investigated. Seeds collected from plants growing in flooded and upland forests were used to determine the influence of normoxic (aerobic and anoxic (anaerobic conditions in germination and the initial and average time of development in the roots and shoots. After 75 days, seedlings germinated under normoxia were transferred to trays and submitted to flooding. Seed reserves (lipids, proteins, soluble sugars and starch were monitored for quiescent and germinated seeds maintained under normoxic and anoxic conditions, as well as after 5, 10 and 20 days of seedling growth. Alcohol dehydrogenase (ADH activity was quantified in roots and leaves of seedlings without or with flooding (partial and total. Seeds were not able to germinate under anoxia. Different strategies of storage mobilization of lipids, proteins, soluble sugars and starch were observed in seeds of each environment. ADH activity was induced by anoxia, with the highest level observed in the leaves. This study showed that, under normoxic conditions, the best developmental performance of assai palm seeds, from flooded or upland forest areas, during germination was associated with primary metabolites mobilization and seedling flooding tolerance with increased ADH activity. We conclude that the assai palm is well adapted to the anoxic conditions provoked by flooding.

  1. Biochemical Characterization and 16S rRNA Sequencing of Few Lipase-Producing Thermophilic Bacteria from Taptapani Hot Water Spring, Orissa, India

    Directory of Open Access Journals (Sweden)

    Satpal S. Bisht

    2011-01-01

    Full Text Available Three lipase-producing thermophilic bacteria (AK-P1, AK-P2, and AK-P3 were isolated from the Taptapani hot water spring in Orissa, India. The crude extra cellular lipases from cell-free culture supernatant were reacted in an olive oil mixture, and their lipolytic activities were compared. Identification of the bacteria was carried out using biochemical tests, 16SrRNA sequencing and sequences submitted to NCBI GenBank. Strain AK-P3, exhibited the highest lipolytic activity of 5.5 U/mL was identified as Porphyrobacter sp. The lipolytic activities of strains AK-P1 and AK-P 2 were 4.5 U/mL and 3.5 U/mL, respectively. Strains AK-P1 and AK-P2 were identified as Acinetobacter sp. and Brevibacillus spp. The GenBank accession numbers of the 16S rRNA gene sequences determined in this study for the strains AK-P1, AK-P2, and AK-P3 are HM359120, HM359119, and HM359118, respectively.

  2. Gene cloning, expression and purification of human mito-chondrial tRNALeu(UUR) and its mutant

    Institute of Scientific and Technical Information of China (English)

    HAN; Weiguo

    2001-01-01

    [1]Anderson, S., Bankier, A. T., Barrell, B. G. et al., Sequence and organization of the human mitochondrial gene, Nature, 1981, 290(9): 457.[2]Wang, X. M., Yang, Y. S., The mutation of human mitochondrial tRNA and diseases, Chemistry of Life, 1999, 19(4): 171.[3]Goto, Y., Nonaka, I., Horai, S., A mutation in the tRNAleu(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1990, 348(13): 651.[4]Schapira, A. H. V., Mitochondrial disorders, Current Opinion in Genetics and Development, 1993, 3: 457.[5]Moraes, C. T., Ricci, E., Bonilla, W. et al., The mitochondrial tRNAleu(UUR) mutation in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS): genetic, biochemical and morphological correlations in skeletal muscle, Am. J. Hum. Genet., 1992, 50: 934.[6]Chen, L., Jin, Y. X., Wang, D. B. et al., Species-specific identity elements of tRNATrp, Prog. Natl. Sci., 2000, 10(3): 192.[7]Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 556-558.[8]Merri, C. R., Goldman, D., Keuren, M. L. V., Gel protein stains: silver stain, Methods Enzymol, 1984, 104: 441.[9]Li, Y., Wang, E., Wang, Y., Overproduction and purification of Escherichia coli tRNAleu, Science in China, Ser. C, 1998, 41(3): 225.[10]Hess, J. F., Parisi, M. A., Bennett Clayton, D. A., Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1991, 352: 236.[11]Yousufzai, A. Y. K., Bradford, M. W., Shrago, E. et al., Characterization of the adenine nucleotide translocase of pancreatic islet mitochondria, FEBS Lett., 1982, 137: 201.[12]Welsh, N., Paabo, S., Welsh, M., Decreased mitochondrial gene expression in isolated islet of rats injected neonatally with streptozotocin, Diabetologia, 1991, 34: 626.[13] Chen, L., Jin

  3. Preparation and characterization of Pd/Al{sub 2}O{sub 3} and Pd nanoparticles as standardized test material for chemical and biochemical studies of traffic related emissions

    Energy Technology Data Exchange (ETDEWEB)

    Leopold, K. [Technische Universitaet Muenchen, Arbeitsgruppe fuer Analytische Chemie, Lichtenbergstrasse 4, D-85747 Garching (Germany)], E-mail: kerstin.leopold@lrz.tum.de; Maier, M.; Schuster, M. [Technische Universitaet Muenchen, Arbeitsgruppe fuer Analytische Chemie, Lichtenbergstrasse 4, D-85747 Garching (Germany)

    2008-05-01

    Palladium model particles similar to those emitted from catalytic car exhaust converters were prepared and characterized with the intention of providing a standardized material for investigations of the chemical behavior and bioavailability of traffic related Pd emissions. Two series of Pd particles were prepared and characterized in detail: Pd nanoparticles (2-4 nm) dispersed on aluminum oxide particles of a diameter range between 0.1 to 30 {mu}m and 'Pd-only' nanoparticles of 5-10 nm in diameter. The Pd/{alpha}-Al{sub 2}O{sub 3} particles are very similar to particles emitted from catalytic converters by mechanical abrasion. The Pd-only particles are useful e.g. for exposure studies in which the presence of aluminum could lead to interferences when studying biological and biochemical effects. The sample preparation procedure of both series was optimized in order to achieve elemental particles with proper sizes and a narrow size distribution. The obtained particles were characterized by transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), selective area diffraction (SAD), laser granulometry and graphite furnace atomic absorption spectrometry (GFAAS) for the measurement of Pd concentrations.

  4. Heinrich Wieland--prize lecture. Transport of proteins across mitochondrial membranes.

    Science.gov (United States)

    Neupert, W

    1994-03-01

    The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of pre-proteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments--the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix--are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c1 heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c1 preproteins, and the

  5. Betaine is a positive regulator of mitochondrial respiration

    International Nuclear Information System (INIS)

    Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect

  6. Betaine is a positive regulator of mitochondrial respiration

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Icksoo, E-mail: icksoolee@dankook.ac.kr

    2015-01-09

    Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.

  7. Mitochondrial DNA Variants of Respiratory Complex I that Uniquely Characterize Haplogroup T2 Are Associated with Increased Risk of Age-Related Macular Degeneration

    OpenAIRE

    SanGiovanni, John Paul; Arking, Dan E.; Sudha K. Iyengar; Elashoff, Michael; Clemons, Traci E.; Reed, George F.; Henning, Alice K.; Sivakumaran, Theru A; Xu, Xuming; DeWan, Andrew; Agrón, Elvira; Rochtchina, Elena; Carolyn M Sue; Wang, Jie Jin; Mitchell, Paul

    2009-01-01

    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 ...

  8. Mitochondrial restriction fragment length polymorphism (RFLP) and sequence variation among closely related avian species and the genetic characterization of hybrid Dendroica warblers.

    Science.gov (United States)

    Lovette, I J; Bermingham, E; Rohwer, S; Wood, C

    1999-09-01

    To address several interconnected goals, we used mitochondrial DNA (mtDNA) sequences to explore evolutionary relationships among four potentially hybridizing taxa in a North American avian superspecies (Dendroica occidentalis, D. townsendi, D. virens, and D. nigrescens). We first compared the results of a previous restriction fragment length polymorphism (RFLP)-based study with 1453 nucleotides from the mitochondrial cytochrome oxidase subunit I (COI), ATP-synthase 6 (ATPase 6), and ATP-synthase 8 (ATPase 8) genes. Separate phylogenetic analyses of the RFLP and sequence data provided identical and well-supported hierarchical species-level reconstructions that grouped occidentalis and townsendi as sister taxa. We then explored several general features of mitochondrial evolution via a comparison of the RFLP and sequence data sets. Qualitative rate differences that seemed evident in highly autocorrelated comparisons of RFLP vs. sequence pairwise distances were not supported when autocorrelation was removed. We also noted a high variance in corresponding RFLP and sequence distances after the removal of autocorrelation effects. This variance suggests that caution should be used when combining RFLP and sequence-based data in studies that require the large-scale synthesis of divergence estimates drawn from sources employing different molecular techniques. Finally, we used our parallel RFLP and sequence data to design and validate a rapid and inexpensive polymerase chain reaction-RFLP (PCR-RFLP) protocol for determining species-specific mitochondrial haplotypes. This PCR-RFLP technique will be applied in ongoing studies of the occidentalis/townsendi hybrid zone, where the historic and geographical complexity of the interbreeding populations necessitates the genotyping of thousands of individual warblers. PMID:10564448

  9. Strokes in mitochondrial diseases

    Directory of Open Access Journals (Sweden)

    N V Pizova

    2012-06-01

    Full Text Available It is suggested that mitochondrial diseases might be identified in 22—33% of cryptogenic stroke cases in young subjects. The incidence of mitochondrial disorders in patients with stroke is unknown; it is 0.8 to 7.2% according to the data of some authors. The paper gives data on the prevalence, pathogenesis, and clinical manifestations of mitochondrial diseases, such as mitochondrial encephalopathy, lactic acidosis, and stroke-like syndrome (MELAS and insulin-like episodes; myoclonic epilepsy and ragged-red fibers (MERRF syndrome, and Kearns-Sayre syndrome (sporadic multisystem mitochondrial pathology.

  10. Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae.

    Science.gov (United States)

    Alizadeh, Ali; Talebi-Jahromi, Khalil; Hosseininaveh, Vahid; Ghadamyari, Mohammad

    2014-01-01

    The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively

  11. Biochemical characterization of laccase from hairy root culture of Brassica juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes.

    Science.gov (United States)

    Telke, Amar A; Kagalkar, Anuradha N; Jagtap, Umesh B; Desai, Neetin S; Bapat, Vishwas A; Govindwar, Sanjay P

    2011-12-01

    In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg(-1) of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg(-1) specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0-6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and L: -cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots. PMID:21735196

  12. Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities.

    Directory of Open Access Journals (Sweden)

    Nadia Zaraî Jaouadi

    Full Text Available Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD, biological oxygen demand (BOD, and total suspended solid (TSS loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca(2+. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF and diiodopropyl fluorophosphates (DFP, which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional

  13. Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities.

    Science.gov (United States)

    Jaouadi, Nadia Zaraî; Rekik, Hatem; Badis, Abdelmalek; Trabelsi, Sahar; Belhoul, Mouna; Yahiaoui, Amina Benkiar; Ben Aicha, Houda; Toumi, Abdessatar; Bejar, Samir; Jaouadi, Bassem

    2013-01-01

    Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca(2+). The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals

  14. Structural and biochemical characterization reveals LysGH15 as an unprecedented "EF-hand-like" calcium-binding phage lysin.

    Directory of Open Access Journals (Sweden)

    Jingmin Gu

    2014-05-01

    Full Text Available The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA. Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408 in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.

  15. Metabolic alterations produced by 3-nitropropionic acid in rat striata and cultured astrocytes: quantitative in vitro 1H nuclear magnetic resonance spectroscopy and biochemical characterization

    International Nuclear Information System (INIS)

    Quantitative high resolution in vitro 1H nuclear magnetic resonance spectroscopy was employed to study the metabolic effects of 3-nitropropionic acid associated with aging from perchloric acid extracts of rat striata. Systemic injection of 3-nitropropionic acid in rats at a dose of 10 mg/kg/day for seven consecutive days significantly impaired energy metabolism in rats one, four and eight months of age, as evidenced by a marked elevation of succinate and lactate levels. However, a significant decrease in N-acetyl-l-aspartate level, a neuronal marker, was observed in four- and eight-month-old rats but not in one-month-old rats. This would indicate that rats at four to eight months are more susceptible to 3-nitropropionic acid than those at one month. A significant decrease in GABA level was observed in four-month-old 3-nitropropionic acid-treated rats, which is consistent with the literature that GABAergic neurons are particularly vulnerable to 3-nitropropionic acid treatment. In addition, glutamine and glutamate levels were markedly decreased at four and eight months in 3-nitropropionic acid-treated rats. Since glutamine is synthesized predominantly in glia, the observation above suggests that 3-nitropropionic acid intoxication may involve perturbation of energy metabolism, glial injury and consequent neuronal damage. Astrocytes which are essential in the metabolism of glutamate and glutamine were used to further assess 3-nitropropionic acid-induced toxicity. Glial proliferation, mitochondrial metabolism and glutamine synthetase activity were all reduced by 3-nitropropionic acid treatment with a concomitant increase, in a dose-dependent manner, of lactate levels, suggesting that 3-nitropropionic acid is also detrimental to astrocytes in vivo and thus may affect metabolic interaction between neurons and glia.These results not only imply that 3-nitropropionic acid blocks energy metabolism prior to exerting neurotoxic damage but also demonstrate that the degree of

  16. Keshan disease and mitochondrial cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    YANG Fuyu

    2006-01-01

    Keshan disease (KD) is a potentially fatal form of cardiomyopathy (disease of the heart muscle) endemic in certain areas of China. From 1984 to 1986, a national comprehensive scientific investigation on KD in Chuxiong region of Yunnan Province in the southwest China was conducted. The investigation team was composed of epidemiologists, clinic doctors, pathologists, biochemists, biophysicists and specialists in ecological environment. Results of pathological, biochemical and biophysical as well as clinical studies showed: an obvious increase of enlarged and swollen mitochondria with distended crista membranes in myocardium from patients with KD; significant reductions in the activity of oxidative phosphorylation (succinate dehydrogenase, cytochrome oxidase, succinate oxidase, H+-ATPase) of affected mitochondria; decrease in CoQ, cardiolipin, Se and GSHPx activity, while obvious increase in the Ca2+ content. So, it was suggested that mitochondria are the predominant target of the pathogenic factors of KD. Before Chuxiong KD survey only a few cases of mitochondrial cardiomyopathy were studied. During the multidisciplinary scientific investigation on KD in Chuxiong a large amount of samples from KD cases and the positive controls were examined. On the basis of the results obtained it was suggested that KD might be classified as a "Mitochondrial Cardiomyopathy" endemic in China. This is one of the achievements in the three years' survey in Chuxiong and is valuable not only to the deeper understanding of pathogenic mechanism of KD but also to the study of mitochondrial cardiomyopathy in general.Keshan disease is not a genetic disease, but is closely related to the malnutrition (especially microelement Se deficiency). KD occurs along a low Se belt, and Se supplementation has been effective in prevention of such disease. The incidence of KD has sharply decreased along with the steady raise of living standard and realization of preventive measures. At present, patients of

  17. Control of gene expression and mitochondrial biogenesis in the muscular adaption to endurance exercise

    DEFF Research Database (Denmark)

    Joseph, A. M.; Pilegaard, H.; Leick, L.;

    2006-01-01

    adaptations is an increase in mitochondrial content, which confers a greater resistance to muscle fatigue. This essay reviews current knowledge on the regulation of exercise-induced mitochondrial biogenesis at the molecular level. The major steps involved include, (i) transcriptional regulation of nuclear......-encoded genes encoding mitochondrial proteins by the coactivator peroxisome-proliferatoractivated receptor coactivator-1, (ii) control of mitochondrial DNA gene 1To whom correspondence should be addressed (email dhood@yorku.ca). 13 © 2006 The Biochemical Society Ch-02_essbiochem_hood.indd Page 13 11/13/06 10......:27:15 PM elhi /Volumes/ju108/POIN001/essbiochem_indd%0/Chapter 2 © 2006 The Biochemical Society 14 Essays in Biochemistry volume 42 2006 expression by the transcription factor Tfam, (iii) mitochondrial fi ssion and fusion mechanisms, and (iv) import of nuclear-derived gene products into the mitochondrion...

  18. Biochemical Analysis of Microbial Rhodopsins.

    Science.gov (United States)

    Maresca, Julia A; Keffer, Jessica L; Miller, Kelsey J

    2016-01-01

    Ion-pumping rhodopsins transfer ions across the microbial cell membrane in a light-dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro analysis of their light-absorbing properties and in vivo analysis of ion pumping will remain critical to characterizing these proteins. As we learn more about the variety of physiological roles performed by microbial rhodopsins in different cell types and environments, observing the localization patterns of the rhodopsins and/or quantifying the number of rhodopsin-bearing cells in natural environments will become more important. Here, we provide protocols for purification of rhodopsin-containing membranes, detection of ion pumping, and observation of functional rhodopsins in laboratory and environmental samples using total internal reflection fluorescence microscopy. © 2016 by John Wiley & Sons, Inc. PMID:27153387

  19. Dynamics of mitochondrial transport in axons

    Directory of Open Access Journals (Sweden)

    Robert Francis Niescier

    2016-05-01

    Full Text Available The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons.

  20. Dynamics of Mitochondrial Transport in Axons.

    Science.gov (United States)

    Niescier, Robert F; Kwak, Sang Kyu; Joo, Se Hun; Chang, Karen T; Min, Kyung-Tai

    2016-01-01

    The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons. PMID:27242435

  1. Biochemical and Structural Characterization of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation Between Distant Phylogenetic Trees of Life

    Energy Technology Data Exchange (ETDEWEB)

    Hobdey, Sarah E.; Knott, Brandon C.; Momeni, Majid Haddad; Taylor, Larry E., II; Borisova, Anna S.; Podkaminer, Kara K.; VanderWall, Todd A.; Himmel, Michael E.; Decker, Stephen R.; Beckham, Gregg T.; Stahlberg, Jerry

    2016-06-01

    Glycoside Hydrolase Family 7 (GH7) cellobiohydrolases (CBHs) are commonly employed enzymes in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions remain regarding structure-activity relationships in these important natural and commercial enzymes. Here, we present crystal structures and biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum and Dictyostelium purpureum (DdiCel7A and DpuCel7A, respectively). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures, resolved to 2.1 A (DdiCel7A), and 2.7 A (DpuCel7A), are homologous to other GH7 CBHs with an enclosed active site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH from Trichoderma reesei Cel7A (TreCel7A) occur near the hydrolytic active site and the product binding sites. To compare the activity of these enzymes with TreCel7A, the Family 1 TreCel7A CBM and linker was added to the C-terminus of the Dictyostelium enzymes, DdiCel7ACBM and DpuCel7ACBM, which were recombinantly expressed in T. reesei. DdiCel7ACBM and DpuCel7ACBM hydrolyze Avicel, pretreated corn stover, and phosphoric acid swollen cellulose as efficiently as TreCel7A when compared at their temperature optima. The Ki of cellobiose is significantly higher for DdiCel7ACBM and DpuCel7ACBM than for TreCel7A: 205, 130, and 29 uM, respectively. Taken together, the present study highlights the remarkable conservation in the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.

  2. Surface‐enhanced Raman spectral biomarkers correlate with Ankle Brachial Index and characterize leg muscle biochemical composition of patients with peripheral arterial disease

    OpenAIRE

    Cluff, Kim; Kelly, Abby M.; Koutakis, Panagiotis; He, Xiang N.; Huang, Xi; Lu, Yong Feng; Pipinos, Iraklis I; Casale, George P.; Subbiah, Jeyamkondan

    2014-01-01

    Abstract Peripheral arterial disease (PAD) is characterized by atherosclerotic blockages of the arteries supplying the lower extremities, which cause a progressive accumulation of ischemic injury to the skeletal muscles of the lower limbs. This injury includes altered metabolic processes, damaged organelles, and compromised bioenergetics in the affected muscles. The objective of this study was to explore the association of Raman spectral signatures of muscle biochemistry with the severity of ...

  3. Biochemical Properties of Pectate Lyases Produced by Three Different Bacillus Strains Isolated from Fermenting Cocoa Beans and Characterization of Their Cloned Genes▿ †

    OpenAIRE

    Ouattara, Honoré G.; Reverchon, Sylvie; Niamke, Sébastien L.; Nasser, William

    2010-01-01

    Pectinolytic enzymes play an important role in cocoa fermentation. In this study, we characterized three extracellular pectate lyases (Pels) produced by bacilli isolated from fermenting cocoa beans. These enzymes, named Pel-22, Pel-66, and Pel-90, were synthesized by Bacillus pumilus BS22, Bacillus subtilis BS66, and Bacillus fusiformis BS90, respectively. The three Pels were produced under their natural conditions and purified from the supernatants using a one-step chromatography method. The...

  4. Mitochondrial pathway mediates the antileukemic effects of Hemidesmus indicus, a promising botanical drug.

    Directory of Open Access Journals (Sweden)

    Carmela Fimognari

    Full Text Available BACKGROUND: Although cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation. METHODOLOGY/PRINCIPAL FINDINGS: A variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca(2+](i raise through the mobilization of intracellular Ca(2+ stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine. A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients. CONCLUSIONS/SIGNIFICANCE: These results indicate the molecular basis of

  5. Genetic and Biochemical Characterization of 4-Carboxy-2-Hydroxymuconate-6-Semialdehyde Dehydrogenase and Its Role in the Protocatechuate 4,5-Cleavage Pathway in Sphingomonas paucimobilis SYK-6

    OpenAIRE

    Masai, Eiji; Momose, Kiyotaka; Hara, Hirofumi; Nishikawa, Seiji; Katayama, Yoshihiro; Fukuda, Masao

    2000-01-01

    Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that ligC was located 295 bp downstream of ligB, which encodes the large subun...

  6. Human liver mitochondrial carnitine palmitoyltransferase I: characterization of its cDNA and chromosomal localization and partial analysis of the gene.

    OpenAIRE

    Britton, C H; Schultz, R.A.; Zhang, B; Esser, V; Foster, D W; McGarry, J D

    1995-01-01

    Using the cDNA for rat liver mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) as a probe, we isolated its counterpart as three overlapping clones from a human liver cDNA library. Both the nucleotide sequence of the human cDNA and the predicted primary structure of the protein (773 aa) proved to be very similar to those of the rat enzyme (82% and 88% identity, respectively). The CPT I mRNA size was also found to be the same (approximately 4.7 kb) in both species. Screening o...

  7. Identification and characterization of a highly variable region in mitochondrial genomes of fusarium species and analysis of power generation from microbial fuel cells

    Science.gov (United States)

    Hamzah, Haider Mousa

    In the microbial fuel cell (MFC) project, power generation from Shewanella oneidensis MR-1 was analyzed looking for a novel system for both energy generation and sustainability. The results suggest the possibility of generating electricity from different organic substances, which include agricultural and industrial by-products. Shewanella oneidensis MR-1 generates usable electrons at 30°C using both submerged and solid state cultures. In the MFC biocathode experiment, most of the CO2 generated at the anodic chamber was converted into bicarbonate due the activity of carbonic anhydrase (CA) of the Gluconobacter sp.33 strain. These findings demonstrate the possibility of generation of electricity while at the same time allowing the biomimetic sequestration of CO2 using bacterial CA. In the mitochondrial genomes project, the filamentous fungal species Fusarium oxysporum was used as a model. This species causes wilt of several important agricultural crops. A previous study revealed that a highly variable region (HVR) in the mitochondrial DNA (mtDNA) of three species of Fusarium contained a large, variable unidentified open reading frame (LV-uORF). Using specific primers for two regions of the LV-uORF, six strains were found to contain the ORF by PCR and database searches identified 18 other strains outside of the Fusarium oxysporum species complex. The LV-uORF was also identified in three isolates of the F. oxysporum species complex. Interestingly, several F. oxysporum isolates lack the LV-uORF and instead contain 13 ORFs in the HVR, nine of which are unidentified. The high GC content and codon usage of the LV-uORF indicate that it did not co-evolve with other mt genes and was horizontally acquired and was introduced to the Fusarium lineage prior to speciation. The nonsynonymous/synonymous (dN/dS) ratio of the LV-uORFs (0.43) suggests it is under purifying selection and the putative polypeptide is predicted to be located in the mitochondrial membrane. Growth assays

  8. Biochemical Characterization of Liver Oil of Echinorhinus brucus (Bramble Shark) and Its Cytotoxic Evaluation on Neuroblastoma Cell Lines (SHSY-5Y)

    Science.gov (United States)

    Venugopal, Vishnu; Kumaran, Ajeeshkumar Kizhakkepurath; Sekhar Chatterjee, Niladri; Kumar, Suvanish; Kavilakath, Shyni; Nair, Jayarani Ramachandran; Mathew, Suseela

    2016-01-01

    The objective of the present study was to characterize the liver oil extracted from the deep sea shark, Echinorhinus brucus, caught from Central Indian Ocean and to evaluate its cytotoxic effect on neuroblastoma cell line (SHSY-5Y). Characterization of liver oil of Echinorhinus brucus revealed the presence of palmitic acid (15%), oleic acid (12%), stearic acid (8%), docosahexaenoic acid (DHA) (18%), and eicosapentaenoic acid (EPA) (16%). It was also found to be a good source of squalene (38.5%) and fat soluble vitamins such as A, D, and K (vitamin A: 17.08 mg/100 g of oil, vitamin D: 15.04 mg/100 g oil, and vitamin K: 11.45 mg/100 g oil). Since it was found to be rich in essential fatty acids, fat soluble vitamins, and squalene, it can be considered as better dietary supplement. The oil of Echinorhinus brucus also showed high in vitro cytotoxic effect against the human neuroblastoma cell line (SHSY-5Y) and the IC50 value laid between 35 and 45 ng. PMID:27340593

  9. Biochemical Characterization of Liver Oil of Echinorhinus brucus (Bramble Shark and Its Cytotoxic Evaluation on Neuroblastoma Cell Lines (SHSY-5Y

    Directory of Open Access Journals (Sweden)

    Vishnu Venugopal

    2016-01-01

    Full Text Available The objective of the present study was to characterize the liver oil extracted from the deep sea shark, Echinorhinus brucus, caught from Central Indian Ocean and to evaluate its cytotoxic effect on neuroblastoma cell line (SHSY-5Y. Characterization of liver oil of Echinorhinus brucus revealed the presence of palmitic acid (15%, oleic acid (12%, stearic acid (8%, docosahexaenoic acid (DHA (18%, and eicosapentaenoic acid (EPA (16%. It was also found to be a good source of squalene (38.5% and fat soluble vitamins such as A, D, and K (vitamin A: 17.08 mg/100 g of oil, vitamin D: 15.04 mg/100 g oil, and vitamin K: 11.45 mg/100 g oil. Since it was found to be rich in essential fatty acids, fat soluble vitamins, and squalene, it can be considered as better dietary supplement. The oil of Echinorhinus brucus also showed high in vitro cytotoxic effect against the human neuroblastoma cell line (SHSY-5Y and the IC50 value laid between 35 and 45 ng.

  10. Biochemical Characterization of Liver Oil of Echinorhinus brucus (Bramble Shark) and Its Cytotoxic Evaluation on Neuroblastoma Cell Lines (SHSY-5Y).

    Science.gov (United States)

    Venugopal, Vishnu; Kumaran, Ajeeshkumar Kizhakkepurath; Sekhar Chatterjee, Niladri; Kumar, Suvanish; Kavilakath, Shyni; Nair, Jayarani Ramachandran; Mathew, Suseela

    2016-01-01

    The objective of the present study was to characterize the liver oil extracted from the deep sea shark, Echinorhinus brucus, caught from Central Indian Ocean and to evaluate its cytotoxic effect on neuroblastoma cell line (SHSY-5Y). Characterization of liver oil of Echinorhinus brucus revealed the presence of palmitic acid (15%), oleic acid (12%), stearic acid (8%), docosahexaenoic acid (DHA) (18%), and eicosapentaenoic acid (EPA) (16%). It was also found to be a good source of squalene (38.5%) and fat soluble vitamins such as A, D, and K (vitamin A: 17.08 mg/100 g of oil, vitamin D: 15.04 mg/100 g oil, and vitamin K: 11.45 mg/100 g oil). Since it was found to be rich in essential fatty acids, fat soluble vitamins, and squalene, it can be considered as better dietary supplement. The oil of Echinorhinus brucus also showed high in vitro cytotoxic effect against the human neuroblastoma cell line (SHSY-5Y) and the IC50 value laid between 35 and 45 ng. PMID:27340593

  11. Oxygen Glucose Deprivation in Rat Hippocampal Slice Cultures Results in Alterations in Carnitine Homeostasis and Mitochondrial Dysfunction

    OpenAIRE

    Thomas F. Rau; Qing Lu; Shruti Sharma; Xutong Sun; Gregory Leary; Beckman, Matthew L.; Yali Hou; Wainwright, Mark S; Michael Kavanaugh; Poulsen, David J.; Black, Stephen M.

    2012-01-01

    Mitochondrial dysfunction characterized by depolarization of mitochondrial membranes and the initiation of mitochondrial-mediated apoptosis are pathological responses to hypoxia-ischemia (HI) in the neonatal brain. Carnitine metabolism directly supports mitochondrial metabolism by shuttling long chain fatty acids across the inner mitochondrial membrane for beta-oxidation. Our previous studies have shown that HI disrupts carnitine homeostasis in neonatal rats and that L-carnitine can be neurop...

  12. Nutritional and biochemical therapies for neurodegenerative diseases

    OpenAIRE

    Barreto, George E.

    2015-01-01

    Neurodegenerative diseases such as Parkinson’s disease, Huntington and Alzheimer’s disease are characterized by neuronal death and loss in different areas of the brain. Downstream signaling mechanisms associated to cellular death/survival are altered, where mitochondrial damage and inflammation, dysfunctional autophagy process, and accumulation of toxins proteins play a central role in the pathogenesis of these diseases. The disabling effects of these diseases on health system are high and gr...

  13. Proteomic Dissection of the Mitochondrial DNA Metabolism Apparatus in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    SAlly A. Mackenzie

    2004-01-06

    This study involves the investigation of nuclear genetic components that regulate mitochondrial genome behavior in higher plants. The approach utilizes the advanced plant model system of Arabidopsis thaliana to identify and functionally characterize multiple components of the mitochondrial DNA replication, recombination and mismatch repair system and their interaction partners. The rationale for the research stems from the central importance of mitochondria to overall cellular metabolism and the essential nature of the mitochondrial genome to mitochondrial function. Relatively little is understood about mitochondrial DNA maintenance and transmission in higher eukaryotes, and the higher plant mitochondrial genome displays unique properties and behavior. This investigation has revealed at least three important properties of plant mitochondrial DNA metabolism components. (1) Many are dual targeted to mitochondrial and chloroplasts by novel mechanisms, suggesting that the mitochondria a nd chloroplast share their genome maintenance apparatus. (2)The MSH1 gene, originating as a component of mismatch repair, has evolved uniquely in plants to participate in differential replication of the mitochondrial genome. (3) This mitochondrial differential replication process, termed substoichiometric shifting and also involving a RecA-related gene, appears to represent an adaptive mechanism to expand plant reproductive capacity and is likely present throughout the plant kingdom.

  14. Biochemical and pharmacological characterization of irradiated crotamine by gamma rays of {sup 60}Co; Caracterizacao bioquimica e farmacologica da crotamina irradiada por raios gama de {sup 60}Co

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Karina Corleto

    2014-07-01

    The serum production in Brazil, the only effective treatment in cases of snakebites, uses horses that although large size, have reduced l lifespan compared with horses not immunized. Ionizing radiation has been shown as an excellent tool in reducing the toxicity of venoms and toxins isolated, and promote the achievement of better immunogens for serum production, and contributing to the welfare of serum-producing animals. It is known, however, that the effects of ionizing radiation on protein are characterized by various chemical modifications, such as fragmentation, cross-linking due to aggregation and oxidation products generated by water radiolysis. However, the action of gamma radiation on toxins is not yet fully understood structurally and pharmacologically, a fact that prevents the application of this methodology in the serum production process. So we proposed in this paper the characterization of crotamine, an important protein from the venom of Crotalus durissus terrificus species, irradiated with {sup 60}Co gamma rays. After isolating the toxin by chromatographic techniques and testing to prove the obtaining of pure crotamine, it was irradiated with gamma rays and subjected to structural analysis, Fluorescence and Circular Dichroism. Using high hydrostatic pressure tests were also conducted in order to verify that the conformational changes caused by radiation suffer modifications under high pressures. From the pharmacological point of view, muscle contraction tests were conducted with the objective of limiting the action of crotamine in smooth muscle as well as the change in the action of toxin caused structural changes to the front. Analysis of Circular Dichroism and Fluorescence showed changes in structural conformation of crotamine when subjected to gamma radiation and that such changes possibly occurring in the secondary and tertiary structure of the protein. The observed in pharmacological tests showed that the irradiated crotamine was less effective

  15. Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

    Institute of Scientific and Technical Information of China (English)

    MA Lifang; ZHANG Shicui; ZHUANG Zhimeng; LIU Zhenhui; LI Hongyan; XIA Jianjun

    2005-01-01

    In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.

  16. Prevalence and clinical characterization of Japanese diabetes mellitus with an A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA{sup Leu (UUR)} gene

    Energy Technology Data Exchange (ETDEWEB)

    Odawara, Masato; Sasaki, Kayoko; Yamashita, Kamejiro [Univ. of Tsukuba (Japan)

    1995-04-01

    An A-to-G mutation at nucleotide position 3243 of the mitochondrial genome has been associated with insulin-dependent diabetes mellitus (IDDM) and with noninsulin-dependent diabetes mellitus (NIDDM) with deafness. We investigated the prevalence of this mutation in Japanese patients with IDDM, NIDDM, and impaired glucose tolerance (IGT) and in nondiabetic control individuals, and we identified it in 3 of 300 patients with NIDDM or IGT (1.0%). None of these individuals had significant sensorineural hearing loss. None of the 94 IDDM or the 115 nondiabetic control subjects was positive for this mutation. Oral glucose tolerance test revealed that a 57-yr-old male with this mutation was rather hyperinsulinemic in the fasting state. The insulin secretion in this patient decreased with age; he did not complain of any hearing disorder, although audiometry revealed a slight elevation of hearing threshold at high frequencies. In conclusion, we found that a mitochondrial gene mutation at nucleotide position 3243 was present in about 1% of NIDDM patients including those patients with IGT. The subtype of diabetes mellitus with this mutation may have a clinical profile similar to that found in patients with NIDDM commonly seen in outpatient clinics. 25 refs., 2 figs., 1 tab.

  17. Measures of Biochemical Sociology

    Science.gov (United States)

    Snell, Joel; Marsh, Mitchell

    2008-01-01

    In a previous article, the authors introduced a new sub field in sociology that we labeled "biochemical sociology." We introduced the definition of a sociology that encompasses sociological measures, psychological measures, and biological indicators Snell & Marsh (2003). In this article, we want to demonstrate a research strategy that would assess…

  18. Biochemical Education in Brazil.

    Science.gov (United States)

    Vella, F.

    1988-01-01

    Described are discussions held concerning the problems of biochemical education in Brazil at a meeting of the Sociedade Brazileira de Bioquimica in April 1988. Also discussed are other visits that were made to universities in Brazil. Three major recommendations to improve the state of biochemistry education in Brazil are presented. (CW)

  19. Multiple Targets for Drug-Induced Mitochondrial Toxicity.

    Science.gov (United States)

    Wallace, Kendall B

    2015-01-01

    Mitochondrial toxicity is rapidly gaining the interest of researchers and practitioners as a prominent liability in drug discovery and development, accounting for a growing proportion of preclinical drug attrition and post-market withdrawals or black box warnings by the U.S. FDA. To date, the focus of registries of drugs that elicit mitochondrial toxicity has been largely restricted to those that either inhibit the mitochondrial electron transport chain (ETC) or uncouple mitochondrial oxidative phosphorylation. Less appreciated are the toxicities that are secondary to the drug affecting either the molecular regulation, assembly or incorporation of the ETC into the inner mitochondrial membrane or those that limit substrate availability. The current article describes the complexities of molecular events and biochemical pathways required to sustain mitochondrial fidelity and substrate homeostasis with examples of drugs that interfere which the various pathways. The principal objective of this review is to shed light on the broader scope of drug-induced mitochondrial toxicities and how these secondary targets may account for a large portion of drug failures. PMID:25973981

  20. Development and characterization of a polyclonal antibody against rat liver mitochondrial overt carnitine palmitoyltransferase (CPT I). Distinction of CPT I from CPT II and of isoforms of CPT I in different tissues.

    Science.gov (United States)

    Kolodziej, M P; Crilly, P J; Corstorphine, C G; Zammit, V A

    1992-01-01

    The [3H]tetradecylglycidyl-CoA (TDG-CoA)-binding protein (Mr approx. 88,000) of purified outer membranes from rat liver mitochondria was identified by SDS/PAGE. The region in which it migrated was shown to contain another protein which stained strongly with periodic acid-Schiff reagent and could be removed from membrane extracts by incubation with Sepharose-concanavalin A. Amounts of TDG-CoA-binding protein were prepared from lectin-treated extracts using preparative SDS/PAGE and used to raise a polyclonal antibody in a sheep. The IgG fraction purified from this anti-serum reacted strongly with a protein of Mr approximately 88,000 on Western blots, and much more weakly with two other proteins of Mr approximately 76,000 and Mr approximately 53,000 in extracts of rat liver mitochondrial outer membranes. The crude IgG fraction and immunopurified IgG both removed carnitine palmitoyltransferase (CPT) I activity from very pure outer membrane extracts, suggesting that the TDG-CoA-binding protein against which the antiserum was raised also expresses CPT I activity. This was confirmed by the demonstration of a strong positive correlation between CPT I activity and the amount of immunoreactive protein of Mr approximately 88,000 in mitochondria prepared from rats in different physiological states. By contrast, the antibody did not react with CPT II either in mitochondria or in purified form. Similarly, an anti-(CPT II) antibody did not cross-react with CPT I on Western blots, proving conclusively that CPT I and CPT II are immunologically distinct proteins, as well as being of different functional molecular sizes [Zammit, Corstophine & Kelliher (1988) Biochem. J. 250, 415-420]. Immunoblots of mitochondrial proteins obtained from different tissues indicated that, of the rat tissues tested, only kidney cortex mitochondria contain the same isoform of CPT I as that in liver. Heart, skeletal muscle and brown adipose tissue mitochondria contain a slightly smaller isoform which was

  1. Mitochondrial oxidative stress causes hyperphosphorylation of tau.

    Directory of Open Access Journals (Sweden)

    Simon Melov

    Full Text Available Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD: tau phosphorylation, and beta-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2 die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576 with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.

  2. Study of biochemical characterization and tolerance to nickel and cadmium of 30 bacteria isolated from the microbasin of the Rio Purires (Cartago, Costa Rica)

    International Nuclear Information System (INIS)

    Heavy metal pollution has been a worldwide problem; Costa Rica, a developing country, receives more and more industries, increasing at an accelerated pollution. A study was performed, from water contaminated with heavy metals, in the microbasin of the Rio Purires to isolate and characterize the cultivable bacteria with nickel and cadmium tolerance. The bacterial growth was determined by measuring of changes in the optical density of a medium. A curve of growth and biomass production experiment was made to the bacteria more tolerant to metals. Pseudomonas synxantha proved to be more tolerant to nickel with a minimum inhibitory concentration of 75 ppm and was chosen to make the curve of growth. The bacterium more tolerant to cadmium was Pseudomonas spp with a minimum inhibitory concentration of 22 ppm. A time of 450 minutes was given for the change in logarithmic phase to stationary phase in the curve of number of bacteria versus time, where the yield of biomass production was 1.73% wet weight biomass. The bacterium Pseudomonas synxantha was established as a good alternative to go forward to conduct trials of biosorption and bioremediation. (author)

  3. Biochemical and physiological characterization of a new Na(+)-channel specific peptide from the venom of the Argentinean scorpion Tityus trivittatus.

    Science.gov (United States)

    Coronas, Fredy I V; Diego-García, Elia; Restano-Cassulini, Rita; de Roodt, Adolfo R; Possani, Lourival D

    2015-06-01

    A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na(+)-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the β-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent. PMID:24862827

  4. Crystal structure and biochemical characterization of Chlamydomonas FDX2 reveal two residues that, when mutated, partially confer FDX2 the redox potential and catalytic properties of FDX1.

    Science.gov (United States)

    Boehm, Marko; Alahuhta, Markus; Mulder, David W; Peden, Erin A; Long, Hai; Brunecky, Roman; Lunin, Vladimir V; King, Paul W; Ghirardi, Maria L; Dubini, Alexandra

    2016-04-01

    The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2 protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized. PMID:26526668

  5. X-ray crystallographic and biochemical characterizations of a mutant photosystem II complex from Thermosynechococcus vulcanus with the psbTc gene inactivated by an insertion mutation

    International Nuclear Information System (INIS)

    The characterization of a PsbTc-truncated mutant photosystem II complex is described. The crystal structure of a photosystem II (PSII) dimer from Thermosynechococcus vulcanus with its psbTc gene inactivated by insertion mutation of an antibiotic cassette in a site in the C-terminal region was analyzed at 3.8 Å resolution. In the crystal structure of the mutant PSII, the transmembrane helix of PsbTc remains, whereas the C-terminal loop of PsbTc has disappeared. In addition, the PsbM subunit, which seemed to be lost in a PsbTc-deletion mutant PSII of T. elongatus, is still present. The deletion of the C-terminal loop of PsbTc in the mutant PSII was verified by mass spectrometry. Thus, the insertion mutation of psbTc eliminated only the C-terminal loop of this subunit. Nevertheless, some features of the mutant PSII, namely a destabilization of the dimeric form and a slight decrease of the oxygen-evolving activity, were observed in the mutant, indicating that the C-terminal loop of PsbTc functions to maintain the stability of the PSII dimer and the activity of oxygen evolution

  6. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Rebecca Dovega

    Full Text Available Protein Phosphatase 2A (PP2A is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A subunit, a catalytic (C subunit and various regulatory (B subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  7. Expression and biochemical characterization of recombinant α-l-rhamnosidase r-Rha1 from Aspergillus niger JMU-TS528.

    Science.gov (United States)

    Li, Lijun; Yu, Yue; Zhang, Xia; Jiang, Zedong; Zhu, Yanbing; Xiao, Anfeng; Ni, Hui; Chen, Feng

    2016-04-01

    A putative cDNA of α-l-rhamnosidase was PCR-cloned from Aspergillus niger JMU-TS528 and further extracellular over-expressed in Pichia pastoris GS115. The activity of the recombinant α-l-rhamnosidase r-Rha1 was 711.9U/mL, eightfold higher than the native α-l-rhamnosidase from A. niger JMU-TS528. r-Rha1 is a N-glycosylated protein of 90kDa and possesses broad substrate specificities by hydrolyzing α-1,2, α-1,3 α-1,4, and α-1,6 linkages to β-d-glucosides. This is the first report presenting that α-l-rhamnosidase showed activity on four kinds of glucosidic linkages. Compared with other previously characterized α-l-rhamnosidases, r-Rha1 showed a good thermostability and wide range of pH-stability with the optimum pH of 5.0 and temperature of 60°C. r-Rha1 activity was not greatly affected by representative metal ions and other detected effectors and showed excellent tolerance abilities against glucose and ethanol. These beneficial characteristics of r-Rha1 suggest that r-Rha1 should be considered a potential new biocatalyst for food and drug industrial applications. PMID:26769090

  8. Mitochondrial morphology and cardiovascular disease

    OpenAIRE

    Ong, Sang-Bing; Hausenloy, Derek J

    2010-01-01

    Mitochondria are dynamic and are able to interchange their morphology between elongated interconnected mitochondrial networks and a fragmented disconnected arrangement by the processes of mitochondrial fusion and fission, respectively. Changes in mitochondrial morphology are regulated by the mitochondrial fusion proteins (mitofusins 1 and 2, and optic atrophy 1) and the mitochondrial fission proteins (dynamin-related peptide 1 and mitochondrial fission protein 1) and have been implicated in a...

  9. A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy.

    Science.gov (United States)

    Jonckheere, An I; Hogeveen, Marije; Nijtmans, Leo; van den Brand, Mariel; Janssen, Antoon; Diepstra, Heleen; van den Brandt, Frans; van den Heuvel, Bert; Hol, Frans; Hofste, Tom; Kapusta, Livia; Dillmann, U; Shamdeen, M; Smeitink, J; Smeitink, J; Rodenburg, Richard

    2009-01-01

    To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder.Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing.A homoplasmic nonsense mutation m.8529G→A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient's fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNAWe describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme. PMID:21686774

  10. The morphological changes of cardiomyocytes and mitochondrial dysfunction in spontaneous hypertensive rats with experimental diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Kolesnyk M.Yu.

    2013-01-01

    Full Text Available The conception of energetic deficiency in the pathogenesis of arterial hypertension and diabetes mellitus presents new perspectives in the understanding of molecular and biochemical mechanisms of these diseases. It was performed the comparison between morphological changes and mitochondrial dysfunction in spontaneous hypertensive rats with experimental diabetes mellitus. The mitochondrial state was assessed by investigation of the permeability of the giant mitochondrial pore. It was found that the permeability of mitochondrial pore is increased in spontaneous hypertensive rats. It was registrated the significant increasing of mitochondrial membrane permeability in case of diabetes. It was observed the increased area of cardiomyocytes’ nuclei and decreased nuclear cytoplasmic ratio in diabetic animals. It was demonstrated that nucleic and cytoplasmic RNA concentration is decreased in comparison with the intact spontaneous hypertensive rats. The RNA biosynthesis abnormalities are associated with the degree of mitochondrial dysfunction in the myocardium of spontaneous hypertensive rats with experimental diabetes mellitus.

  11. A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine

    OpenAIRE

    McCulloch, Vicki; Seidel-Rogol, Bonnie L.; Shadel, Gerald S.

    2002-01-01

    A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication. Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA. However, based on studies in model systems, the existence of a second human mito...

  12. Cytonuclear Interactions in the Evolution of Animal Mitochondrial tRNA Metabolism

    OpenAIRE

    Pett, Walker; Lavrov, Dennis V.

    2015-01-01

    The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-prote...

  13. Autism in the Son of a Woman with Mitochondrial Myopathy and Dysautonomia: A Case Report

    OpenAIRE

    Brown, Bradley D; Rais, Theodore

    2015-01-01

    The relationship between autism spectrum disorders and mitochondrial dysfunction, including mitochondrial myopathies and other mitochondrial diseases, is an area of ongoing research. All autism spectrum disorders are known to be heritable, via genetic and/or epigenetic mechanisms, but specific modes of inheritance are not well characterized. Nevertheless, autism spectrum disorders have been linked to many specific genes associated with mitochondrial function, especially to genes involved in m...

  14. Composição elementar e caracterização da fração lipídica de seis cultivares de caupi Biochemical composition and characterization of lipid fraction of six cowpea cultivars

    Directory of Open Access Journals (Sweden)

    Rolando E. R. Castellón

    2003-04-01

    Full Text Available A composição bioquímica elementar de sementes de seis cultivares de feijão de corda (Vigna unguiculata foi determinada e a fração lipídica de cada cultivar, caracterizada. Os resultados sugerem que todas as farinhas de semente compartilham composição bioquímica similar, embora as cultivares Br-14 e CE-315 difiram das Br-9 e Br-17 no teor de umidade. As frações lipídicas purificadas foram caracterizadas quanto ao teor e tipos de ácidos graxos presentes. A cultivar Br-17 possui alto conteúdo de ácido palmítico (58,2% e o menor conteúdo de ácido linoléico (6,4% enquanto a Vita 7 apresenta alto conteúdo de ácido linoléico (21,8% e relativamente baixo conteúdo de ácido palmítico (41% em uma base comparativa. Os ácidos graxos pentacosanóico e eicosanóico representam a fração lipídica menor e o primeiro não ocorre em CE-315. Os resultados sugerem que diferentes cultivares obtidas por melhoramento genético podem apresentar diferenças quantitativas e qualitativas na composição bioquímica; assim, diferenças na capacidade germinativa, resistência a predadores no campo de plantio e no armazenamento, poderiam estar relacionadas à inibição ou estímulo da expressão de genes codificadores da síntese de moléculas relevantes, que refletem diferenças de composição, como demonstrado neste trabalho.The biochemical composition of the seeds of six bean - Vigna unguiculata (Walp cultivars was determined and the lipid fraction of each of these cultivars was characterized. The results suggest that all analyzed seed flours share similar biochemical composition, although the cultivars Br-14 and CE-315 differ in relation to Br-9 and Br-17 in water content. The purified fatty acid fractions were characterized according to their lipid content. The cultivar Br-17 possesses high content of palmitic acid (58.2% and low content of linoleic acid (6.4% while Vita 7 possesses high contents of linoleic acid (21.8% and relatively low

  15. Caracterização bioquímica de linhagens de soja com alto teor de proteína Biochemical characterization of high protein soybean lines

    Directory of Open Access Journals (Sweden)

    Rita Maria Alves de Moraes

    2006-05-01

    Full Text Available O objetivo deste trabalho foi caracterizar bioquimicamente duas isolinhas de soja com alto teor de proteína. O aumento do teor de proteína nas isolinhas foi acompanhado por redução no teor de óleo e de carboidratos totais. Em relação à composição aminoacídica, o aumento do teor de proteína promoveu acréscimo em todos os aminoácidos, exceto glicina, alanina, metionina, cisteína e tirosina, mantendo a relação enxofre/nitrogênio. A quantificação dos polipeptídios mostrou que o aumento do teor de proteína manteve inalterado o teor das proteínas 7S, promoveu aumento no teor das proteínas 11S e, conseqüentemente, da relação 11S/7S. Pode haver melhoria na qualidade do farelo de soja das isolinhas, uma vez que as proteínas 11S têm melhor qualidade nutricional do que as proteínas 7S.The objective of this work was to characterize high protein soybean near isogenic lines. The increasing of protein was followed by reducing of oil and carbohydrate. In respect to aminoacid composition, increasing of protein promoted a rising in all aminoacids, except for glycine, alanine, methionine, cysteine and tyrosine, although the ratio S/N has been kept. The measure of polypeptides showed that the increasing of protein did not alter the quantity of 7S proteins, provided increasing of 11S proteins and 11S/7S ratio. An improvement of meal quality in these lines can occur once the 11S proteins have a better nutritional quality than 7S proteins.

  16. Calcium enhances the proteolytic activity of BACE1: An in vitro biophysical and biochemical characterization of the BACE1-calcium interaction.

    Science.gov (United States)

    Hayley, Michael; Perspicace, Samantha; Schulthess, Therese; Seelig, Joachim

    2009-09-01

    BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the beta-secretase, BACE1, is the first step in the production of the Abeta peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca(2+) binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca(2+). Isothermal titration calorimetry was then used to characterize the Ca(2+)-BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding (k(d) = 2.0 x microM) between Ca(2+) and BACE1 and that the binding process was exothermic (DeltaH= -3.5 kcal/mol). We also could demonstrate that low concentrations of Ca(2+) (microM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca(2+) and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca(2+). PMID:19486882

  17. Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Zhu, Chenguang; Fan, Qianlan; Wang, Wei; Shen, Chunlei; Meng, Xiangzong; Tang, Yuanping; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2014-02-25

    The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%-48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%-43%), total GS activity (39%-45%) and soluble protein concentration (23%-24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency. PMID:24334123

  18. Human liver mitochondrial carnitine palmitoyltransferase I: characterization of its cDNA and chromosomal localization and partial analysis of the gene.

    Science.gov (United States)

    Britton, C H; Schultz, R A; Zhang, B; Esser, V; Foster, D W; McGarry, J D

    1995-01-01

    Using the cDNA for rat liver mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) as a probe, we isolated its counterpart as three overlapping clones from a human liver cDNA library. Both the nucleotide sequence of the human cDNA and the predicted primary structure of the protein (773 aa) proved to be very similar to those of the rat enzyme (82% and 88% identity, respectively). The CPT I mRNA size was also found to be the same (approximately 4.7 kb) in both species. Screening of a human genomic library with the newly obtained cDNA yielded a positive clone of approximately 6.5 kb which, upon partial analysis, was found to contain at least two complete exons linked by a 2.3-kb intron. Oligonucleotide primers specific to upstream and downstream regions of one of the exon/intron junctions were tested in PCRs with DNA from a panel of somatic cell hybrids, each containing a single human chromosome. The results allowed unambiguous assignment of the human liver CPT I gene to the q (long) arm of chromosome 11. Additional experiments established that liver and fibroblasts express the same isoform of mitochondrial CPT I, legitimizing the use of fibroblast assays in the differential diagnosis of the "muscle" and "hepatic" forms of CPT deficiency. The data provide insights into the structure of a human CPT I isoform and its corresponding gene and establish unequivocally that CPT I and CPT II are distinct gene products. Availability of the human CPT I cDNA should open the way to an understanding of the genetic basis of inherited CPT I deficiency syndromes, how the liver CPT I gene is regulated, and which tissues other than liver express this particular variant of the enzyme. Images Fig. 4 Fig. 5 PMID:7892212

  19. Mitochondrial Dynamics in Diabetes

    OpenAIRE

    Yoon, Yisang; Galloway, Chad A.; Jhun, Bong Sook; Yu, Tianzheng

    2011-01-01

    Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emergin...

  20. Multiplexing oscillatory biochemical signals.

    Science.gov (United States)

    de Ronde, Wiet; ten Wolde, Pieter Rein

    2014-04-01

    In recent years it has been increasingly recognized that biochemical signals are not necessarily constant in time and that the temporal dynamics of a signal can be the information carrier. Moreover, it is now well established that the protein signaling network of living cells has a bow-tie structure and that components are often shared between different signaling pathways. Here we show by mathematical modeling that living cells can multiplex a constant and an oscillatory signal: they can transmit these two signals simultaneously through a common signaling pathway, and yet respond to them specifically and reliably. We find that information transmission is reduced not only by noise arising from the intrinsic stochasticity of biochemical reactions, but also by crosstalk between the different channels. Yet, under biologically relevant conditions more than 2 bits of information can be transmitted per channel, even when the two signals are transmitted simultaneously. These observations suggest that oscillatory signals are ideal for multiplexing signals. PMID:24685537

  1. Involvement of the mitochondrial compartment in human NCL fibroblasts

    International Nuclear Information System (INIS)

    Highlights: ► Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. ► Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. ► Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  2. Involvement of the mitochondrial compartment in human NCL fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Pezzini, Francesco; Gismondi, Floriana [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Tessa, Alessandra [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Tonin, Paola [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Carrozzo, Rosalba [IRCCS Bambino Gesu Hospital-Molecular Medicine Unit, Roma (Italy); Mole, Sara E. [MRC Laboratory for Molecular Cell Biology, Molecular Medicines Unit, UCL Institute of Child Health and Department of Genetics, Evolution and Environment, University College London (United Kingdom); Santorelli, Filippo M. [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Simonati, Alessandro, E-mail: alessandro.simonati@univr.it [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. Black-Right-Pointing-Pointer Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. Black-Right-Pointing-Pointer Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  3. Effects of mitochondrial dysfunction on the immunological properties of microglia

    Directory of Open Access Journals (Sweden)

    Ferger Annette I

    2010-08-01

    Full Text Available Abstract Background Neurodegenerative diseases are characterized by both mitochondrial dysfunction and activation of microglia, the macrophages of the brain. Here, we investigate the effects of mitochondrial dysfunction on the activation profile of microglial cells. Methods We incubated primary mouse microglia with the mitochondrial toxins 3-nitropropionic acid (3-NP or rotenone. These mitochondrial toxins are known to induce neurodegeneration in humans and in experimental animals. We characterized lipopolysaccharide- (LPS- induced microglial activation and the alternative, interleukin-4- (IL-4- induced microglial activation in these mitochondrial toxin-treated microglial cells. Results We found that, while mitochondrial toxins did not affect LPS-induced activation, as measured by release of tumor necrosis factor α (TNF-α, interleukin-6 (IL-6 and interleukin-1β (IL-1β, they did inhibit part of the IL-4-induced alternative activation, as measured by arginase activity and expression, induction of insulin-like growth factor 1 (IGF-1 and the counteraction of the LPS induced cytokine release. Conclusions Mitochondrial dysfunction in microglial cells inhibits part of the IL-4-induced alternative response. Because this alternative activation is considered to be associated with wound healing and an attenuation of inflammation, mitochondrial dysfunction in microglial cells might contribute to the detrimental effects of neuroinflammation seen in neurodegenerative diseases.

  4. Nd(III)-Induced Rice Mitochondrial Dysfunction Investigated by Spectroscopic and Microscopic Methods

    OpenAIRE

    Xia, Cai-Fen; Lv, Long; Chen, Xin-You; Fu, Bo-Qiao; Lei, Ke-Lin; Qin, Cai-Qin; Liu, Yi

    2015-01-01

    The production capacity and yield of neodymium (Nd) in China have ranked the first in the world. Because of its unique biophysical and biochemical properties, Nd compounds have entered into the agricultural environment greatly to promote plant growth. Mitochondria play a crucial role in respiration and metabolism during the growth of plants. However, little is known about the mechanism by which Nd act at the mitochondrial level in plant cells. In this study, rice mitochondrial swelling, colla...

  5. Noninvasive diagnostics of mitochondrial disorders in isolated lymphocytes with high resolution respirometry

    OpenAIRE

    Petr Pecina; Hana Houšťková; Tomáš Mráček; Alena Pecinová; Hana Nůsková; Markéta Tesařová; Hana Hansíková; Jan Janota; Jiří Zeman; Josef Houštěk

    2014-01-01

    Background: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. Methods: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-p...

  6. Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

    DEFF Research Database (Denmark)

    Larsen, Steen; Nielsen, Joachim; Neigaard Nielsen, Christina; Nielsen, Lars Bo; Wibrand, Flemming; Stride, Nis; Schroder, Henrik Daa; Boushel, Robert; Helge, Jørn W; Dela, Flemming; Hey-Mogensen, Martin

    closely associated these commonly used biochemical measures are to muscle mitochondrial content and muscle oxidative capacity (OXPHOS).Sixteen young healthy male subjects were recruited for this study. Subjects completed a graded exercise test to determine maximal oxygen uptake (VO(2peak)) and muscle...... biopsies were obtained from the vastus lateralis. Mitochondrial content was determined using transmission electron microscopy imaging and OXPHOS was determined as the maximal coupled respiration in permeabilized fibers. Biomarkers of interest were citrate synthase (CS) activity, cardiolipin content...

  7. Mitochondrial gene mutations and type 2 diabetes in Chinese families

    Institute of Scientific and Technical Information of China (English)

    LI Ming-zhen; YU De-min; YU Pei; LIU De-min; WANG Kun; TANG Xin-zhi

    2008-01-01

    Background Numerous mitochondrial DNA mutations are significantly correlated with development of diabetes. This study investigated mitochondrial gene, point mutations in patients with type 2 diabetes and their families. Methods Unrelated patients with type 2 diabetes(n=826)were randomly recruited; unrelated and nondiabetic subjects (n=637)served as controls. The clinical and biochemical data of the participants were collected. Total genome was extracted from peripheral leucocytes. Polymerase chain reaction, restriction fragment length polymorphism (PCR-RFLP)and clonig techniques were used to screen mitochondrial genes including np3316,np3394 and np3426 in the ND1 region and np3243 in the tRNALeu (UUR). Results In 39 diabetics with one or more mitochondrial gene point mutations, the prevalence(4.7%,39/826)of mtDNA mutations was higher than that(0.7%,5/637)in the controls. The identical mutation was found in 23 of 43 tested members from three pedigrees. Affected family members presented with variable clinical features ranging from normal glucose tolerance to impaired glucose tolerance (IGT)(n=2),impaired fasting glucose(IFG)(n=1)to type 2 diabetes (n=13)with 3 family members suffering from hearing loss. Conclusions Type 2 diabetes in China is associated with several mitochondrial gene mutations. Aged patients with diabetic family history had a higher prevalence of mutation and various clinical pictures. Mitochondrial gene mutation might be one of the genetic factors contributing to diabetic familial clustering.

  8. Molecular characterization of a mitochondrial DNA segment from the Japanese scallop (Patinopecten yessoensis): demonstration of a region showing sequence polymorphism in the population.

    Science.gov (United States)

    Sato, M; Nagashima, K

    2001-07-01

    A 1.3-kb mitochondrial DNA segment from the Japanese scallop Patinopecten yessoensis was cloned and sequenced. This segment contained the transfer RNA(Met) gene and partial sequences of 2 ribosomal RNA genes, together with 2 separate noncoding regions (designated NcR1 and NcR2). The NcR regions derived from 78 individuals cultured in Lake Saroma or Matsu Bay, were sequenced, and we found 15 loci with sequence alterations including 13 substitutions, 1 deletion, and 1 insertion (1 locus in NcR1, 14 loci in NcR2), and 17 haplotypes. Of the 17 haplotypes, 10 were found in the Saroma population only, 3 in the Mutsu population only, and 4 in both populations. The gene diversity and nucleotide diversity values were, respectively, 0.87 and 0.0069 for the Saroma population, 0.63 and 0.0040 for the Mutsu population, and 0.83 and 0.0203 overall. Thus the NcR segment was considered to have sufficient sequence variation for population genetic studies. The 16 variants of the NcR2 sequence were separated successfully by denaturing gradient gel electrophoresis, confirming the sequence variation in NcR2. PMID:14961353

  9. Glycolytic Coupling to Mitochondrial Energy Production Ensures Survival in an Oxygen Rich Environment

    Science.gov (United States)

    Stefano, George B.; Kream, Richard M.

    2016-01-01

    The mitochondrion exhibits biochemical and functional variations that emerged by random chance as an evolutionary survival strategy, which include enhanced energy production driven by anaerobic respiratory mechanisms. In invertebrates, this mitochondrial anaerobic respiration permits survival at a lower energy state suited for this type of environment while yielding more ATP than by glycolysis alone. This ability provides a protective existential advantage in naturally occurring hypoxic environments via diminished free radical generation. In the blue mussel Mytilus edulis and other marine organisms, a functionally active mitochondrial anaerobic respiratory mechanism tailored to hypoxic conditions reflects an evolutionary adaptation/reworking of ancient metabolic pathways. Components of these pathways were also discovered and characterized as metabolic intermediates in plant parasites, specifically crown gall tumors. Mechanistic similarities between anaerobically functioning mitochondria in M. edulis and crown gall tissues and metabolic processes in human tumors are known to occur, demonstrating commonalities in diverse life energy processes. Furthermore, cytoplasmic glycolytic processes are now shown also to exhibit a dynamic capacity for enhanced energy generation by increasing its efficiency in hypoxic environments, making it equally dynamic in meeting its cellular survival goal. PMID:27439008

  10. Mitochondrial diseases and epilepsy.

    Science.gov (United States)

    Bindoff, Laurence A; Engelsen, Bernt A

    2012-09-01

    The mitochondrial respiratory chain is the final common pathway for energy production. Defects affecting this pathway can give rise to disease that presents at any age and affects any tissue. However, irrespective of genetic defect, epilepsy is common and there is a significant risk of status epilepticus. This review summarizes our current understanding of the epilepsy that occurs in mitochondrial disease, focusing on three of the most common disorders: mitochondrial myopathy encephalopathy, lactic acidosis and stroke-like episodes (MELAS), myoclonus epilepsy and ragged-red fibers (MERRF), and polymerase gamma (POLG) related disease. In addition, we review the pathogenesis and possible treatment of these disorders. PMID:22946726

  11. Cerebral energy metabolism during mitochondrial dysfunction induced by cyanide in piglets

    DEFF Research Database (Denmark)

    Nielsen, Troels Halfeld; Olsen, N.V.; Toft, P; Nordström, C H

    2013-01-01

    variables related to energy metabolism. METHODS: Mitochondrial dysfunction was induced in piglets and evaluated by monitoring brain tissue oxygen tension (PbtO2 ) and cerebral levels of glucose, lactate, pyruvate, glutamate, and glycerol bilaterally. The biochemical variables were obtained by microdialysis...... insufficient energy metabolism and degradation of cellular membranes, respectively. CONCLUSION: Mitochondrial dysfunction is characterised by an increased LP ratio signifying a shift in cytoplasmatic redox state at normal or elevated PbtO2 . The condition is biochemically characterised by a marked increase in...

  12. Cytonuclear Interactions in the Evolution of Animal Mitochondrial tRNA Metabolism.

    Science.gov (United States)

    Pett, Walker; Lavrov, Dennis V

    2015-08-01

    The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-proteobacterial ancestor. However, variation in the complement of mitochondrial-encoded molecules involved in translation, including transfer RNAs (tRNAs), provides evidence for the ongoing evolution of mitochondrial protein synthesis. Here, we investigate the evolution of the mitochondrial translational machinery using recent genomic and transcriptomic data from animals that have experienced the loss of mt-tRNAs, including phyla Cnidaria and Ctenophora, as well as some representatives of all four classes of Porifera. We focus on four sets of mitochondrial enzymes that directly interact with tRNAs: Aminoacyl-tRNA synthetases, glutamyl-tRNA amidotransferase, tRNA(Ile) lysidine synthetase, and RNase P. Our results support the observation that the fate of nuclear-encoded mitochondrial proteins is influenced by the evolution of molecules encoded in mitochondrial DNA, but in a more complex manner than appreciated previously. The data also suggest that relaxed selection on mitochondrial translation rather than coevolution between mitochondrial and nuclear subunits is responsible for elevated rates of evolution in mitochondrial translational proteins. PMID:26116918

  13. Biochemical characterization of three mycobacterial ribosomal fractions.

    Science.gov (United States)

    Portelance, V; Beaudet, R

    1983-02-01

    The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity. PMID:6189570

  14. Babesia bovis clones: biochemical and enzymatic characterization

    International Nuclear Information System (INIS)

    Studies were undertaken to generate additional knowledge of the biochemistry of Babesia bovis. A modified in vitro culture technique used for cloning B. bovis. This technique included a low oxygen concentration atmosphere (2%, O2, 5% CO2, 93% N2) and 4 mm fluid level. Cultures initiated with one infected erythrocyte were maintained until parasitemias of positive wells reached 2% parasitemia. Primary clones were obtained and from these, nine clones were recloned twice and used for subsequent studies. A procedure was developed to concentrate and separate B. bovis merozoites and infected erythrocytes by Percoll density gradients. Merozoites separated at 1.087 g/ml specific density, whereas infected erythrocytes separated at 1.121 g/ml. Viability of purified parasites was not affected. Agarose gel electrophoresis was used to identify metabolic enzyme in B. bovis and B. bigemina. The enzymes LDH, GDH, GPI and HK were detected in both species. Molecular analysis by one and two-dimensional gel electrophoresis of proteins metabolically labeled with 35S-methionine indicated that two clones, derived from the same field strain, were similar but not identical to the parent. Fewer proteins were observed in the parental strain. Growth of two 60-Co irradiated B. bovis clones indicated a dose-effect relationship. Growth of parasites exposed for the longest period was initially retarded but returned to normal growth after two or three subcultures. Cultures exposed for shorter periods were unaffected with respect to the rate of growth. Analysis of electrophoretic mobility of metabolic enzyme showed a change in migration pattern

  15. Babesia bovis clones: biochemical and enzymatic characterization

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez Camarillo, S.D.

    1985-01-01

    Studies were undertaken to generate additional knowledge of the biochemistry of Babesia bovis. A modified in vitro culture technique used for cloning B. bovis. This technique included a low oxygen concentration atmosphere (2%, O/sub 2/, 5% CO/sub 2/, 93% N/sub 2/) and 4 mm fluid level. Cultures initiated with one infected erythrocyte were maintained until parasitemias of positive wells reached 2% parasitemia. Primary clones were obtained and from these, nine clones were recloned twice and used for subsequent studies. A procedure was developed to concentrate and separate B. bovis merozoites and infected erythrocytes by Percoll density gradients. Merozoites separated at 1.087 g/ml specific density, whereas infected erythrocytes separated at 1.121 g/ml. Viability of purified parasites was not affected. Agarose gel electrophoresis was used to identify metabolic enzyme in B. bovis and B. bigemina. The enzymes LDH, GDH, GPI and HK were detected in both species. Molecular analysis by one and two-dimensional gel electrophoresis of proteins metabolically labeled with /sup 35/S-methionine indicated that two clones, derived from the same field strain, were similar but not identical to the parent. Fewer proteins were observed in the parental strain. Growth of two 60-Co irradiated B. bovis clones indicated a dose-effect relationship. Growth of parasites exposed for the longest period was initially retarded but returned to normal growth after two or three subcultures. Cultures exposed for shorter periods were unaffected with respect to the rate of growth. Analysis of electrophoretic mobility of metabolic enzyme showed a change in migration pattern.

  16. Mitochondrial uncoupling protein is required for efficient photosynthesis

    OpenAIRE

    Sweetlove, Lee J.; Lytovchenko, Anna; Morgan, Megan; Nunes-Nesi, Adriano; Taylor, Nicolas L.; Baxter, Charles J.; Eickmeier, Ira; Fernie, Alisdair R.

    2006-01-01

    Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally used for ATP synthesis. Although the catalytic function and regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidopsis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results i...

  17. Mitochondrial functions on oocytes and preimplantation embryos

    Institute of Scientific and Technical Information of China (English)

    Li-ya WANG; Da-hui WANG; Xiang-yang ZOU; Chen-ming XU

    2009-01-01

    Oocyte quality has long been considered as a main limiting factor for in vitro fertilization (IVF). In the past decade,extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA (mtDNA) damages, chromosomal aneuploidies,the incidence of apoptosis, and changes in mitochondrial gene expression. All these dysfunctions may cause a high level of developmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species (ROS) that is closely associated with the oxidative energy production or calcium overload,which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.

  18. Biochemical Hypermedia: Galactose Metabolism.

    Directory of Open Access Journals (Sweden)

    J.K. Sugai

    2013-05-01

    Full Text Available Introduction: Animations of biochemical processes and virtual laboratory environments lead to true molecular simulations. The use of interactive software’s in education can improve cognitive capacity, better learning and, mainly, it makes information acquisition easier. Material and Methods: This work presents the development of a biochemical hypermedia to understanding of the galactose metabolism. It was developed with the help of concept maps, ISIS Draw, ADOBE Photoshop and FLASH MX Program. Results and Discussion: A step by step animation process shows the enzymatic reactions of galactose conversion to glucose-1-phosphate (to glycogen synthesis, glucose-6-phosphate (glycolysis intermediary, UDP-galactose (substrate to mucopolysaccharides synthesis and collagen’s glycosylation. There are navigation guide that allow scrolling the mouse over the names of the components of enzymatic reactions of via the metabolism of galactose. Thus, explanatory text box, chemical structures and animation of the actions of enzymes appear to navigator. Upon completion of the module, the user’s response to the proposed exercise can be checked immediately through text box with interactive content of the answer. Conclusion: This hypermedia was presented for undergraduate students (UFSC who revealed that it was extremely effective in promoting the understanding of the theme.

  19. A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNAVal mutation

    International Nuclear Information System (INIS)

    Highlights: → We report a young Tunisian patient with clinical features of MELAS syndrome. → Reported mitochondrial mutations were absent after a mutational screening of the whole mtDNA. → We described a novel m.1640A>G mutation in the tRNAVal gene which was absent in 150 controls. → Mitochondrial deletions and POLG1 gene mutations were absent. → The m.1640A>G mutation could be associated to MELAS syndrome. -- Abstract: Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNAVal. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.

  20. Neonatal mitochondrial hepatoencephalopathy caused by novel GFM1 mutations

    Directory of Open Access Journals (Sweden)

    Kirstine Ravn

    2015-06-01

    Full Text Available Disorders caused by defects in the mitochondrial translation system are clinically and genetically heterogeneous. The elongation phase of mitochondrial protein synthesis requires, among many other components, three nuclear-encoded elongation factors: EFTu (TUFM; 602389, EFTs (TSFM; 604723, and EFG1 (GFM1; 606639. Mutations have been identified in the genes encoding all three elongation factors, and they result in combined respiratory chain deficiencies and severe phenotypes with an early fatal outcome. So far, only eleven patients have been reported with mutations in GFM1. Here we describe an additional three patients with novel GFM1 mutations. Our results confirm the tissue-specific effect of GFM1 mutations, since we found only slightly decreased respiratory chain enzyme activities in muscle and fibroblasts, but a severe deficiency in the liver. Hence, a thorough biochemical evaluation is important to guide genetic investigation in patients suspected for a mitochondrial disorder.

  1. Neonatal mitochondrial hepatoencephalopathy caused by novel GFM1 mutations.

    Science.gov (United States)

    Ravn, Kirstine; Schönewolf-Greulich, Bitten; Hansen, Rikke M; Bohr, Anna-Helene; Duno, Morten; Wibrand, Flemming; Ostergaard, Elsebet

    2015-06-01

    Disorders caused by defects in the mitochondrial translation system are clinically and genetically heterogeneous. The elongation phase of mitochondrial protein synthesis requires, among many other components, three nuclear-encoded elongation factors: EFTu (TUFM; 602389), EFTs (TSFM; 604723), and EFG1 (GFM1; 606639). Mutations have been identified in the genes encoding all three elongation factors, and they result in combined respiratory chain deficiencies and severe phenotypes with an early fatal outcome. So far, only eleven patients have been reported with mutations in GFM1. Here we describe an additional three patients with novel GFM1 mutations. Our results confirm the tissue-specific effect of GFM1 mutations, since we found only slightly decreased respiratory chain enzyme activities in muscle and fibroblasts, but a severe deficiency in the liver. Hence, a thorough biochemical evaluation is important to guide genetic investigation in patients suspected for a mitochondrial disorder. PMID:26937387

  2. Mitochondrial dynamics and apoptosis

    OpenAIRE

    Suen, Der-Fen; Norris, Kristi L.; Youle, Richard J.

    2008-01-01

    In healthy cells, mitochondria continually divide and fuse to form a dynamic interconnecting network. The molecular machinery that mediates this organelle fission and fusion is necessary to maintain mitochondrial integrity, perhaps by facilitating DNA or protein quality control. This network disintegrates during apoptosis at the time of cytochrome c release and prior to caspase activation, yielding more numerous and smaller mitochondria. Recent work shows that proteins involved in mitochondri...

  3. The plant mitochondrial proteome

    DEFF Research Database (Denmark)

    Millar, A.H.; Heazlewood, J.L.; Kristensen, B.K.;

    2005-01-01

    The plant mitochondrial proteome might contain as many as 2000-3000 different gene products, each of which might undergo post-translational modification. Recent studies using analytical methods, such as one-, two- and three-dimensional gel electrophoresis and one- and two-dimensional liquid...... context to be defined for them. There are indications that some of these proteins add novel activities to mitochondrial protein complexes in plants....

  4. Mitochondrial metabolism and diabetes

    OpenAIRE

    Kwak, Soo Heon; Park, Kyong Soo; Lee, Ki‐Up; Lee, Hong Kyu

    2010-01-01

    Abstract The oversupply of calories and sedentary lifestyle has resulted in a rapid increase of diabetes prevalence worldwide. During the past two decades, lines of evidence suggest that mitochondrial dysfunction plays a key role in the pathophysiology of diabetes. Mitochondria are vital to most of the eukaryotic cells as they provide energy in the form of adenosine triphosphate by oxidative phosphorylation. In addition, mitochondrial function is an integral part of glucose‐stimulated insulin...

  5. Neurodegenerative and Fatiguing Illnesses, Infections and Mitochondrial Dysfunction: Use of Natural Supplements to Improve Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2014-01-01

    Full Text Available Background: Many chronic diseases and illnesses are associated with one or more chronic infections, dysfunction of mitochondria and reduced production of ATP. This results in fatigue and other symptoms that occur in most if not all chronic conditions and diseases. Methods: This is a review of the published literature on chronic infections in neurodegenerative diseases and fatiguing illnesses that are also typified by mitochondrial dysfunction. This contribution also reviews the use of natural supplements to enhance mitochondrial function and reduce the effects of chronic infections to improve overall function in various chronic illnesses. Results: Mitochondrial function can be enhanced by the use of various natural supplements, notably Lipid Replacement Therapy (LRT using glyerolphospholipids and other mitochondrial supplements. In various chronic illnesses that are characterized by the presence of chronic infections, such as intracellular bacteria (Mycoplasma, Borrelia, Chlamydia and other infections and viruses, LRT has proven useful in multiple clinical trials. For example, in clinical studies on chronic fatigue syndrome, fibromyalgia syndrome and other chronic fatiguing illnesses where a large majority of patients have chronic infections, LRT significantly reduced fatigue by 35-43% in different clinical trials and increased mitochondrial function. In clinical trials on patients with multiple intracellular bacterial infections and intractable fatigue LRT plus other mitochondrial supplements significantly decreased fatigue and improved mood and cognition. Conclusions: LRT formulations designed to improve mitochondrial function appear to be useful as non-toxic dietary supplements for reducing fatigue and restoring mitochondrial and other cellular membrane functions in patients with chronic illnesses and multiple chronic infections.

  6. Mitochondrial DNA disease—molecular insights and potential routes to a cure

    Energy Technology Data Exchange (ETDEWEB)

    Russell, Oliver; Turnbull, Doug, E-mail: doug.turnbull@newcastle.ac.uk

    2014-07-01

    Mitochondrial DNA diseases are common neurological conditions caused by mutations in the mitochondrial genome or nuclear genes responsible for its maintenance. Current treatments for these disorders are focussed on the management of the symptoms, rather than the correction of biochemical defects caused by the mutation. This review focuses on the molecular effects of mutations, the symptoms they cause and current work focusing on the development of targeted treatments for mitochondrial DNA disease. - Highlights: • We discuss several common disease causing mtDNA mutations. • We highlight recent work linking pathogenicity to deletion size and heteroplasmy. • We discuss recent advances in the development of targeted mtDNA disease treatments.

  7. Mitochondrial diseases: an overview of genetics, pathogenesis, clinical features and an approach to diagnosis and treatment.

    Directory of Open Access Journals (Sweden)

    Singhal N

    2000-07-01

    Full Text Available Defects in structures or functions of mitochondria, mainly involving the oxidative phosphorylation, mitochondrial biogenesis and other metabolic pathways have been shown to be associated with a wide spectrum of clinical phenotypes. The ubiquitous nature of mitochondria and their unique genetic features contribute to the clinical, biochemical and genetic heterogenecity of mitochondrial diseases. This article focuses on the recent advances in the field of mitochondrial disorders with respect to the consequences for an advanced clinical and genetic diagnostics. In addition, an overview on recently identified genetic defects and their pathogenic molecular mechanisms are given.

  8. Inherited mitochondrial neuropathies.

    Science.gov (United States)

    Finsterer, Josef

    2011-05-15

    Mitochondrial disorders (MIDs) occasionally manifest as polyneuropathy either as the dominant feature or as one of many other manifestations (inherited mitochondrial neuropathy). MIDs in which polyneuropathy is the dominant feature, include NARP syndrome due to the transition m.8993T>, CMT2A due to MFN2 mutations, CMT2K and CMT4A due to GDAP1 mutations, and axonal/demyelinating neuropathy with external ophthalmoplegia due to POLG1 mutations. MIDs in which polyneuropathy is an inconstant feature among others is the MELAS syndrome, MERRF syndrome, LHON, Mendelian PEO, KSS, Leigh syndrome, MNGIE, SANDO; MIRAS, MEMSA, AHS, MDS (hepato-cerebral form), IOSCA, and ADOA syndrome. In the majority of the cases polyneuropathy presents in a multiplex neuropathy distribution. Nerve conduction studies may reveal either axonal or demyelinated or mixed types of neuropathies. If a hereditary neuropathy is due to mitochondrial dysfunction, the management of these patients is at variance from non-mitochondrial hereditary neuropathies. Patients with mitochondrial hereditary neuropathy need to be carefully investigated for clinical or subclinical involvement of other organs or systems. Supportive treatment with co-factors, antioxidants, alternative energy sources, or lactate lowering agents can be tried. Involvement of other organs may require specific treatment. Mitochondrial neuropathies should be included in the differential diagnosis of hereditary neuropathies. PMID:21402391

  9. Mutations in APOPT1, Encoding a Mitochondrial Protein, Cause Cavitating Leukoencephalopathy with Cytochrome c Oxidase Deficiency

    Science.gov (United States)

    Melchionda, Laura; Haack, Tobias B.; Hardy, Steven; Abbink, Truus E.M.; Fernandez-Vizarra, Erika; Lamantea, Eleonora; Marchet, Silvia; Morandi, Lucia; Moggio, Maurizio; Carrozzo, Rosalba; Torraco, Alessandra; Diodato, Daria; Strom, Tim M.; Meitinger, Thomas; Tekturk, Pinar; Yapici, Zuhal; Al-Murshedi, Fathiya; Stevens, René; Rodenburg, Richard J.; Lamperti, Costanza; Ardissone, Anna; Moroni, Isabella; Uziel, Graziella; Prokisch, Holger; Taylor, Robert W.; Bertini, Enrico; van der Knaap, Marjo S.; Ghezzi, Daniele; Zeviani, Massimo

    2014-01-01

    Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation. PMID:25175347

  10. Molecular Details of the Yeast Frataxin-Isu1 Interaction during Mitochondrial Fe-S Cluster Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Cook, J.; Kondapalli, K; Rawat, S; Childs, W; Murugesan, Y; Dancis, A; Stemmler, T

    2010-01-01

    Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

  11. The mitochondrial transcription factor A functions in mitochondrial base excision repair

    DEFF Research Database (Denmark)

    Canugovi, Chandrika; Maynard, Scott; Bayne, Anne-Cécile V;

    2010-01-01

    mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles. The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated. However, a function for TFAM in BER has not yet been...

  12. Optical Biochemical Platforms for Nanoparticles Detection

    CERN Document Server

    Campanella, Clarissa Martina

    2014-01-01

    In the biochemical sensing field, a fervent research activity related to the development of real time, low cost, compact and high throughput devices for the detection and characterization of natural or synthetic nanoparticles NPs actually exists. In this research scenario, different platforms for biosensing purposes have been developed according to the huge amount of physical effects involved in the transduction of the biochemical-signal into a measurable output signal. In the present work two different optical platforms for NP detection have been investigated, one based on integrated optics and the other based on microscopy. Both the approaches rely on the study of the interaction of an electromagnetic wave with a small particle in the hypothesis of dealing with a Rayleigh scatterer, i.e. a nanoparticle having a size really smaller than the one of the wavelength of the incident light and scattering light elastically.

  13. Advancement in biochemical assays in andrology

    Institute of Scientific and Technical Information of China (English)

    Wolf-BernhardSchill; RaftHenkel

    1999-01-01

    Determination of maikers of sperm function, accessory sex gland secretion and silent male genital tract inflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemical tests into the analysis of male factor has the advantage that standardized assays with a coefficient of variafion characteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability .Biochemical parameters may be used in clinical practice to evaluate the sperm fertitizing capacity (acrosin, aniline blue,ROS), to characterize male accessory sex gland secretinns (fructose, a-glucosidase, PSA), and to identify men with silent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS). (As/an J Androl 1999 Jun; 1: 45-51)

  14. Mitochondrial emitted electromagnetic signals mediate retrograde signaling.

    Science.gov (United States)

    Bagkos, Georgios; Koufopoulos, Kostas; Piperi, Christina

    2015-12-01

    Recent evidence shows that mitochondria regulate nuclear transcriptional activity both in normal and cell stress conditions, known as retrograde signaling. Under normal mitochondrial function, retrograde signaling is associated with mitochondrial biogenesis, normal cell phenotype and metabolic profile. In contrast, mitochondrial dysfunction leads to abnormal (oncogenic) cell phenotype and altered bio-energetic profile (nucleus reprogramming). Despite intense research efforts, a concrete mechanism through which mitochondria determine the group of genes expressed by the nucleus is still missing. The present paper proposes a novel hypothesis regarding retrograde signaling. More specifically, it reveals the mitochondrial membrane potential (MMP) and the accompanied strong electromagnetic field (EF) as key regulatory factors of nuclear activity. Mitochondrial emitted EFs extend in long distance and affect the function of nuclear membrane receptors. Depending on their frequencies, EFs can directly activate or deactivate different groups of nuclear receptors and so determine nuclear gene expression. One of the key features of the above hypothesis is that nuclear membrane receptors, besides their own endogenous or chemical ligands (hormones, lipids, etc.), can also be activated by electromagnetic signals. Moreover, normal MMP values (about -140 mV) are associated with the production of high ATP quantities and small levels of reactive oxygen species (ROS) while the hyperpolarization observed in all cancer cell types leads to a dramatic fall in ATP production and an analogous increase in ROS. The diminished ATP and increased ROS production negatively affect the function of all cellular systems including nucleus. Restoration of mitochondrial function, which is characterized by the fluctuation of MMP and EF values within a certain (normal) range, is proposed as a necessary condition for normal nuclear function and cancer therapy. PMID:26474928

  15. Biochemical synthesis with stable isotopes

    International Nuclear Information System (INIS)

    Descriptions of the biochemical synthesis of glucose-13C6 from Agmenellum quadruplication; the biochemical labelling of [13C, 15N] Chlorella and [13C] E. coli, [15N] E. coli, and the production of lactic-13C3 acid utilizing Lactobacillus casei are discussed

  16. Neurological mitochondrial cytopathies.

    Directory of Open Access Journals (Sweden)

    Mehndiratta M

    2002-04-01

    Full Text Available The mitochondrial cytopathies are genetically and phenotypically heterogeneous group of disorders caused by structural and functional abnormalities in mitochondria. To the best of our knowledge, there are very few studies published from India till date. Selected and confirmed fourteen cases of neurological mitochondrial cytopathies with different clinical syndromes admitted between 1997 and 2000 are being reported. There were 8 male and 6 female patients. The mean age was 24.42+/-11.18 years (range 4-40 years. Twelve patients could be categorized into well-defined syndromes, while two belonged to undefined group. In the defined syndrome categories, three patients had MELAS (mitochondrial encephalopathy, lactic acidosis and stroke like episodes, three had MERRF (myoclonic epilepsy and ragged red fibre myopathy, three cases had KSS (Kearns-Sayre Syndrome and three were diagnosed to be suffering from mitochondrial myopathy. In the uncategorized group, one case presented with paroxysmal kinesogenic dystonia and the other manifested with generalized chorea alone. Serum lactic acid level was significantly increased in all the patients (fasting 28.96+/-4.59 mg%, post exercise 41.02+/-4.93 mg%. Muscle biopsy was done in all cases. Succinic dehydrogenase staining of muscle tissue showed subsarcolemmal accumulation of mitochondria in 12 cases. Mitochondrial DNA study could be performed in one case only and it did not reveal any mutation at nucleotides 3243 and 8344. MRI brain showed multiple infarcts in MELAS, hyperintensities in putaminal areas in chorea and bilateral cerebellar atrophy in MERRF.

  17. Caracterização química e bioquímica de sementes de Bauhinia variegata L. Chemical and biochemical characterization of Bauhinia variegata L. seeds

    Directory of Open Access Journals (Sweden)

    Luciano S. Pinto

    2005-09-01

    Full Text Available Sementes quiescentes de Bauhinia variegata foram submetidas a caracterização bioquímica, por meio de análise elementar, fracionamento de proteínas e atividade hemaglutinante. A análise elementar mostrou grande quantidade de proteína total e de lipídeos, com 29,41 e 14,89%, respectivamente. O ácido linoléico foi o mais encontrado na constituição lipídica das sementes e a composição mineral ficou dentro de níveis aceitáveis para o consumo humano. As diferentes frações protéicas (albuminas, globulinas, prolaminas, glutelinas ácidas e básicas apresentaram atividade hemaglutinante contra hemácias tratadas e não-tratadas com enzimas proteolíticas, mas a maior atividade hemaglutinante específica foi evidenciada na fração globulínica; já nas frações glutelinas ácidas e albuminas, esta atividade é maior quando se utilizam hemácias de coelho previamente tratadas com tripsina e papaína, respectivamente. Assim, por apresentarem alto valor energético, as sementes de Bauhinia variegata são uma possível fonte opcional na alimentação.Seeds of Bauhinia variegata were submitted to biochemical characterization through elementary analysis, protein fractioning and hemaglutinanting activity. The elementary analysis of seed showed high protein and lipid contents, with 29.41 and 14.89%, respectively. The linolenic acid was found in the lipidic constitution of the seeds and the mineral composition remained with in acceptable levels for the human consumption. The specific hemaglutinanting activity from different fractions of proteins (albumin, globulin, prolamin, glutelin acid and alkaline demonstrated hemaglutinanting activity against native and enzyme treated rabbit erythrocytes. However, the fraction globulins showed the largest specific hemaglutinanting activity. Acid glutelin and albumin had a larger specific hemaglutinanting activity against tripisin-treated and papain-treated rabbit erythrocytes, respectively. Thus, for

  18. Mitochondrial fusion and inheritance of the mitochondrial genome.

    Science.gov (United States)

    Takano, Hiroyoshi; Onoue, Kenta; Kawano, Shigeyuki

    2010-03-01

    Although maternal or uniparental inheritance of mitochondrial genomes is a general rule, biparental inheritance is sometimes observed in protists and fungi,including yeasts. In yeast, recombination occurs between the mitochondrial genomes inherited from both parents.Mitochondrial fusion observed in yeast zygotes is thought to set up a space for DNA recombination. In the last decade,a universal mitochondrial fusion mechanism has been uncovered, using yeast as a model. On the other hand, an alternative mitochondrial fusion mechanism has been identified in the true slime mold Physarum polycephalum.A specific mitochondrial plasmid, mF, has been detected as the genetic material that causes mitochondrial fusion in P. polycephalum. Without mF, fusion of the mitochondria is not observed throughout the life cycle, suggesting that Physarum has no constitutive mitochondrial fusion mechanism.Conversely, mitochondria fuse in zygotes and during sporulation with mF. The complete mF sequence suggests that one gene, ORF640, encodes a fusogen for Physarum mitochondria. Although in general, mitochondria are inherited uniparentally, biparental inheritance occurs with specific sexual crossing in P. polycephalum.An analysis of the transmission of mitochondrial genomes has shown that recombinations between two parental mitochondrial genomes require mitochondrial fusion,mediated by mF. Physarum is a unique organism for studying mitochondrial fusion. PMID:20196232

  19. Mitochondrial function in normal and diabetic beta-cells

    OpenAIRE

    Maechler, Pierre; Wollheim, Claes

    2001-01-01

    The aetiology of type 2, or non-insulin-dependent, diabetes mellitus has been characterized in only a limited number of cases. Among these, mitochondrial diabetes, a rare subform of the disease, is the consequence of pancreatic beta-cell dysfunction caused by mutations in mitochondrial DNA, which is distinct from the nuclear genome. The impact of such mutations on beta-cell function reflects the importance of mitochondria in the control of insulin secretion. The beta-cell mitochondria serve a...

  20. Mitochondrial myopathy presenting as fibromyalgia: a case report

    Directory of Open Access Journals (Sweden)

    Abdullah Mishal

    2012-02-01

    Full Text Available Abstract Introduction To the best of our knowledge, we describe for the first time the case of a woman who met the diagnostic criteria for fibromyalgia, did not respond to therapy for that disorder, and was subsequently diagnosed by biochemical and genetic studies with a mitochondrial myopathy. Treatment of the mitochondrial myopathy resulted in resolution of symptoms. This case demonstrates that mitochondrial myopathy may present in an adult with a symptom complex consistent with fibromyalgia. Case presentation Our patient was a 41-year-old Caucasian woman with symptoms of fatigue, exercise intolerance, headache, and multiple trigger points. Treatment for fibromyalgia with a wide spectrum of medications including non-steroidal anti-inflammatory drugs, antidepressants, gabapentin and pregabalin had no impact on her symptoms. A six-minute walk study demonstrated an elevated lactic acid level (5 mmol/L; normal Conclusions This case demonstrates that adults diagnosed with fibromyalgia may have their symptom complex related to an adult onset mitochondrial myopathy. This is an important finding since treatment of mitochondrial myopathy resulted in resolution of symptoms.