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Physiology and Molecular Biology of... Apr 2011The Santalum peroxidase was extracted from the leaves and precipitated with double volume of chilled acetone. The optimum percent relative activity for the Santalum...
The Santalum peroxidase was extracted from the leaves and precipitated with double volume of chilled acetone. The optimum percent relative activity for the Santalum peroxidase was observed at pH 5.0 and 50 °C temperature. The Santalum peroxidase per cent relative activity was stimulated in the presence of phenolic compounds like ferrulic acid and caffeic acids; however, indole-3-acetic acid (IAA) and protocatechuic acid act as inhibitors. All divalent cations Fe(2+), Mn(2+), Mg(2+), Cu(2+) and Zn(2+) stimulate the relative activity of the Santalum peroxidase at concentration of 2.0 μM. Amino acids like L-alanine and L-valine activate the per cent relative activity, while L-proline and DL-methionine showed moderate inhibition for the Santalum peroxidase. However, a very low a concentration of cysteine acts as a strong inhibitor of Santalum peroxidase at the concentration of 0.4 mM. Native polyacrylamide gel electrophoresis (Native-PAGE) was performed for isoenzyme determination and two bands were observed. Km and Vmax values were calculated from Lineweaver-Burk graph. The apparent Vmax/Km value for O-dianisidine and H2O2 were 400 and 5.0 × 105 Units/min/mL respectively.
PubMed: 23573005
DOI: 10.1007/s12298-011-0054-x -
Haematologica Feb 2011The Meis1 protein represents an important cofactor for Hox and Pbx1 and is implicated in human and murine leukemias. Though much is known about the role of meis1 in...
BACKGROUND
The Meis1 protein represents an important cofactor for Hox and Pbx1 and is implicated in human and murine leukemias. Though much is known about the role of meis1 in leukemogenesis, its function in normal hematopoiesis remains largely unclear. Here we characterized the role of the proto-oncogene, meis1, during zebrafish primitive and definitive hematopoiesis.
DESIGN AND METHODS
Zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells and Sudan black to quantify neutrophils. The numbers of other cells (scl-, gata1- and alas2-positive cells) were also quantified by measuring the corresponding stained areas of the embryos. We used anti-Meis1 antibody and whole mount immunohistochemistry to determine the pattern of expression of Meis1 during zebrafish development and then analyzed the functional role of Meis1 by knocking-down the meis1 gene.
RESULTS
Using antisense morpholino oligomers to interrupt meis1 expression we found that, although primitive macrophage development could occur unhampered, posterior erythroid differentiation required meis1, and its absence resulted in a severe decrease in the number of mature erythrocytes. Furthermore a picture emerged that meis1 exerts important effects on later stages of erythrocyte maturation and that these effects are independent of gata1, but under the control of scl. In addition, meis1 morpholino knock-down led to dramatic single arteriovenous tube formation. We also found that knock-down of pbx1 resulted in a phenotype that was strikingly similar to that of meis1 knock-down zebrafish.
CONCLUSIONS
These results imply that meis1, jointly with pbx1, regulates primitive hematopoiesis as well as vascular development.
Topics: Animals; Cell Differentiation; Embryo, Nonmammalian; Erythrocytes; Gene Expression Regulation, Developmental; Hematopoiesis; Homeodomain Proteins; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Macrophages; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Neutrophils; Pre-B-Cell Leukemia Transcription Factor 1; Proto-Oncogene Mas; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 21048033
DOI: 10.3324/haematol.2010.027698 -
Comparative Biochemistry and... May 2010Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental...
Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats (Rattusnorvegicus), albino mice (Mus musculus), Cp-/- mice, and adult humans (Homo sapiens) to investigate the copper-iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp-/- mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp-/- mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60-90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency.
Topics: Amine Oxidase (Copper-Containing); Animal Feed; Animals; Ceruloplasmin; Copper; Deficiency Diseases; Disease Models, Animal; Female; Food, Formulated; Gene Expression; Humans; Male; Mice; Mice, Knockout; RNA, Messenger; Rats; Rats, Sprague-Dawley; Species Specificity
PubMed: 20170749
DOI: 10.1016/j.cbpc.2010.02.005 -
Protein Engineering, Design & Selection... Mar 2010Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient...
Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs.
Topics: Alcaligenes faecalis; Azurin; Chromogenic Compounds; Copper; Crystallography, X-Ray; Dianisidine; Directed Molecular Evolution; Electrochemistry; Electrons; Enzyme Assays; High-Throughput Screening Assays; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Nitrite Reductases; Oxidation-Reduction; Oxygen; Protein Conformation; Reducing Agents; Reproducibility of Results; Spectrum Analysis
PubMed: 20083495
DOI: 10.1093/protein/gzp084 -
BMC Gastroenterology Jan 2010Mutations in the gene ATP7B cause Wilson disease, a copper storage disorder with a high phenotypic and genetic heterogeneity. We aimed to evaluate whether 'severe'... (Comparative Study)
Comparative Study
BACKGROUND
Mutations in the gene ATP7B cause Wilson disease, a copper storage disorder with a high phenotypic and genetic heterogeneity. We aimed to evaluate whether 'severe' protein-truncating ATP7B mutations (SMs) are associated with low serum ceruloplasmin oxidase activities and an early age of onset when compared to missense mutations (MMs).
METHODS
The clinical phenotype of 59 genetically confirmed WD patients was analyzed retrospectively. Serum ceruloplasmin was measured by its oxidase activity with o-dianisidine dihydrochloride as substrate and immunologically.
RESULTS
Thirty-nine patients had two MMs, 15 had the genotype SM/MM, and 5 patients had two SMs on their ATP7B alleles. Enzymatic and immunologic serum ceruloplasmin levels differed significantly between the three groups (P < 0.001 and P < 0.01, respectively). The lowest levels were measured in patients with two SMs (0.0 U/L; IQR, 0.0-0.0 U/L and 0.02 g/L; IQR, 0.01-0.02 g/L, respectively) and the highest in patients with two MMs (17.8 U/L; IQR, 5.8-35.1 U/L and 0.11 g/L; IQR,0.10-0.17 g/L, respectively). The age of onset was also significantly different between the three patient groups (P < 0.05), with SM/SM patients showing the earliest onset (13 years; IQR, 9-13 years) and patients with two MMs showing the latest onset (22 years; IQR, 14-27 years). By ROC curve analysis a ceruloplasmin oxidase level
CONCLUSIONS
In our German study cohort truncating ATP7B mutations were associated with lower ceruloplasmin serum oxidase levels and an earlier age of onset when compared to MMs. Measurement of serum ceruloplasmin oxidase might help to predict presence of truncating ATP7B mutations and might facilitate the mutation analysis.
Topics: Adenosine Triphosphatases; Adolescent; Adult; Age of Onset; Cation Transport Proteins; Ceruloplasmin; Child; Cohort Studies; Copper-Transporting ATPases; Female; Genotype; Hepatolenticular Degeneration; Humans; Male; Mutation; Mutation, Missense; ROC Curve; Young Adult
PubMed: 20082719
DOI: 10.1186/1471-230X-10-8 -
Journal of the American Chemical Society Jan 2010Myeloperoxidase (MPO) is increasingly being recognized as an important factor in many inflammatory diseases, particularly cardiovascular and neurological diseases....
Myeloperoxidase (MPO) is increasingly being recognized as an important factor in many inflammatory diseases, particularly cardiovascular and neurological diseases. MPO-specific imaging agents would thus be highly useful to diagnose early disease, monitor disease progression, and quantify treatment effects. This study reports in vitro and in vivo characterizations of the mechanism of interaction between MPO and paramagnetic enzyme substrates based on physical and biological measurements. We show that these agents are activated through a radical mechanism, which can combine to form oligomers and, in the presence of tyrosine-containing peptide, bind to proteins. We further identified two new imaging agents, which represent the near extremes in either oligomerization (mono-5HT-DTPA-Gd) or protein-binding in their activation mechanism (bis-o-dianisidine-DTPA-Gd). On the other hand, we found that the agent bis-5HT-DTPA-Gd utilizes both mechanisms when activated. These properties yield distinct in vivo pharmacokinetics profiles for each of these agents that may be exploited for different applications. Specificity studies show that only MPO, but not eosinophil peroxidase, can highly activate these agents, and that MPO activity as low as 0.005 U/mg of tissue can be detected. Gd kinetic lability and cytotoxicity studies further confirm stability of the Gd ion and low toxicity for the 5HT-based agents, suggesting that these agents are suitable for translational in vivo studies.
Topics: Animals; Electron Spin Resonance Spectroscopy; Gadolinium; Kinetics; Magnetic Resonance Imaging; Mice; NIH 3T3 Cells; Organometallic Compounds; Oxidation-Reduction; Peroxidase; Proteins; Tyrosine
PubMed: 19968300
DOI: 10.1021/ja905274f -
Molecular Plant Sep 2009Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation...
Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically. To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Cross-linking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases (assayed on o-dianisidine) were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylan-cross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.
Topics: Cells, Cultured; Chromatography, Gel; Coumaric Acids; Horseradish Peroxidase; Oxidation-Reduction; Peroxidases; Plant Proteins; Xylans; Zea mays
PubMed: 19825665
DOI: 10.1093/mp/ssp044 -
The International Journal of... 2010The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However,...
The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.
Topics: Animals; Cardiovascular System; Dianisidine; Embryo, Nonmammalian; Embryonic Development; Female; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Nervous System; Receptor, IGF Type 1; Signal Transduction; Somatomedins; Zebrafish
PubMed: 19757379
DOI: 10.1387/ijdb.092922lh -
Indian Journal of Clinical Biochemistry... Jan 2009Serum ceruloplasmin is one of the most commonly used screening tests for Wilson's disease. However immunological assays for ceruloplasmin are not recommended for...
Serum ceruloplasmin is one of the most commonly used screening tests for Wilson's disease. However immunological assays for ceruloplasmin are not recommended for diagnosis and management of Wilson's disease through calculation of free copper index. Enzymatic methods using non-physiological substrates have toxicity and stability problems, making them difficult to automate. Ferroxidase assays may be a satisfactory alternative for measuring serum ceruloplasmin. The o-dianisidine hydrochloride manual method for estimation of serum ceruloplasmin enzyme activity was compared with an automated method using the ferroxidase activity of ceruloplasmin in measurement in a double blind study in 91 consecutive patients screened for Wilson's disease. The o-dianisidine and ferroxidase methods both successfully identified 7 patients with Wilson's disease. Values for these 7 patients in the o-dianisidine and ferroxidase methods were median 5.0 (range 0-16.0 U/L) and median 45.0 (range 4-166 U/L) respectively. There were 7 other positive values (<62 U/L) with the o-diansidine method and 2 (<200 U/L) with the ferroxidase method, where WD was not confirmed. ROC curves for both methods showed area under the curve of 0.998 for o-dianisidine and 0.997 for ferroxidase. Using literature cut off values of 62 U/L and 200 U/L respectively both methods had 100% sensitivity and specificity was 91.7% (o-dianisidine) and 97.6% (ferroxidase). For the o-dianisidine assay, specificity was improved to 98.8% using a cut off of 22.5 U/L. In the 84 persons (46 adults and 38 children) in whom the diagnosis of Wilson's disease was not established, the mean value for ceruloplasmin activity by the o-dianisidine and ferroxidase methods was 124.7 ± 48.7 U/L and 571.4 ± 168.1 U/L respectively. There were no significant differences between sex or age of patients (p > 0.29). In a subsequent evaluation with 372 specimens, the Pearson correlation coefficient between the assays was 0.908, p < 0.01, slope 4.06, intercept 265.8, with the manual assay as the x-axis. The ferroxidase assay is a suitable replacement for the o-dianisidine assay in detecting patients with Wilson's disease.
PubMed: 23105801
DOI: 10.1007/s12291-009-0003-4 -
Experimental Hematology Sep 2008Inherited or acquired mutations in the heme biosynthetic pathway leads to a debilitating class of diseases collectively known as porphyrias, with symptoms that can...
OBJECTIVE
Inherited or acquired mutations in the heme biosynthetic pathway leads to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant.
MATERIALS AND METHODS
Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with reverse transcriptase polymerase chain reaction was utilized to identify the genetic mutation, which was confirmed via allele-specific oligo hybridizations. Whole mount in situ hybridizations and o-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype.
RESULTS
Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hours post-fertilization are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox.
CONCLUSION
In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria.
Topics: Amino Acid Sequence; Anemia, Hypochromic; Animals; Codon, Nonsense; Conserved Sequence; DNA, Complementary; Disease Models, Animal; Embryo, Nonmammalian; Hemoglobins; Homozygote; Humans; Mice; Molecular Sequence Data; Phenotype; Porphyria, Variegate; Protoporphyrinogen Oxidase; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Zebrafish; Zebrafish Proteins
PubMed: 18550261
DOI: 10.1016/j.exphem.2008.04.008