scholarly journals Antibodies Raised against Bcvir15, an Extrachromosomal Double-Stranded RNA-Encoded Protein from Babesia canis, Inhibit the In Vitro Growth of the Parasite

2003 ◽  
Vol 71 (3) ◽  
pp. 1056-1067 ◽  
Author(s):  
P. Drakulovski ◽  
B. Carcy ◽  
K. Moubri ◽  
C. Carret ◽  
D. Depoix ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Protozoan Parasite ◽  
Inhibitory Effect ◽  
Open Reading Frames ◽  
Babesia Canis ◽  
In Vitro Growth ◽  
Viral Origin ◽  
Double Stranded Rna

ABSTRACT As part of a search for homologous members of the Plasmodium falciparum Pf60 multigene family in the intraerythrocytic protozoan parasite Babesia canis, we report here the characterization of a cDNA of 1,115 bp, which was designated Bcvir for its potential viral origin. The Bcvir cDNA contained two overlapping open reading frames (ORFs) (ORF1 from nucleotide [nt] 61 to 486 and ORF2 from nt 417 to 919), where Bcvir15, the deduced ORF1 peptide (M1 to I141), is the main expressed product. The Bcvir cDNA was derived from an extrachromosomal dsRNA element of 1.2 kbp that was always found associated with a double-stranded RNA (dsRNA) of 2.8 kbp by hybridization, and no copy of this cDNA sequence was found in B. canis genomic DNA. Biochemical characterization of Bcvir15, by using polyclonal rabbit sera directed against recombinant proteins, indicated that it is a soluble protein which remained associated with the cytoplasm of the B. canis merozoite. Interestingly, purified immunoglobulins from the anti-glutathione S-transferase-Bcvir15 (at a concentration of 160 μg/ml) induced 50% inhibition of the in vitro growth of B. canis, and the inhibitory effect was associated with morphological damage of the parasite. Our data suggest that the extrachromosomal dsRNA-encoded Bcvir15 protein might interfere with the intracellular growth of the parasite rather than with the process of invasion of the host cell by the merozoite. Epitope mapping of Bcvir15 identified three epitopes that might be essential for the function of the protein.

Purification and characterization of glucose-6-phosphate dehydrogenase from Eisenia fetida and effects of some pesticides and metal ions

2020 ◽  
Vol 45 (4) ◽  
pp. 373-380
Author(s):  
Naciye Kayhan ◽  
Veysel Çomaklı ◽  
Sevki Adem ◽  
Caglar Güler
Keyword(s):  
Metal Ions ◽  
Eisenia Fetida ◽  
Biochemical Characterization ◽  
Inhibitory Effect ◽  
Biochemical Properties ◽  
Affinity Column ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

AbstractObjectivesEarthworms have a large impact on the soil ecosystem. They are quite sensitive to pollutants. Purification and biochemical characterization of glucose-6-phosphate dehydrogenases (G6PD) from the earthworm species Eisenia fetida were aimed. The determination of the toxicity potentials of some soil pollutants on G6PD activity was intended.MethodsG6PD was isolated using 2′,5′-ADP-Sepharose 4B affinity column. Enzyme purity and molecular mass were determined by SDS-PAGE. Its biochemical properties investigated. The effects of some soil pollutants on the enzyme were studied in vitro.ResultsEnzyme was purified with 28% yields and 232 fold. Optimum pH and buffer concentration, optimal and stable temperature was determined as pH: 8.5, 60 mM, 25 °C and 20 °C. Its molecular weight estimated as 36 kDa. The Ni2+, Hg2+, Pb2+, Cr2+, and Fe2+ ions with IC50 values in the range of 56 ± 06−120 ± 20 μM and the diniconazole, metalaxyl, methomyl, carboxyl, and oxamyl with IC50 values in the range of 7.6 ± 1.2−77 ± 12 μM exhibited an inhibitory effect on G6PD.ConclusionsG6PD was isolated and characterized from E. fetida. Its catalytic activity decreased with very low concentration by pesticides and metal ions. The results indicated that the inhibition of G6PD may be important in the toxicity mechanism of pollutants on this earthworm.

Biochemical characterization of some raptor trypanosomes. II. Enzyme studies, with a description of Trypanosoma bennetti n. sp.

1986 ◽  
Vol 64 (1) ◽  
pp. 195-203 ◽  
Author(s):  
Carl E. Kirkpatrick ◽  
Cynthia A. Terway-Thompson ◽  
M. Raja Iyengar
Keyword(s):  
Numerical Taxonomy ◽  
Biochemical Characterization ◽  
Growth Behavior ◽  
In Vitro Growth ◽  
Mobility Patterns ◽  
Leishmania Chagasi ◽  
Electrophoretic Data ◽  
A New Species

Comparing electrophoretic mobility patterns of 14 enzymes of nine strains of cultured avian trypanosomes and two strains of cultured mammalian trypanosomatids revealed marked differences between the strains isolated from Falco sparverius and those isolated from other host raptors as well as among the avian trypanosomatids and the mammalian ones. These results supported the differentiation of two species of raptor trypanosomes based on other criteria: size, form, in vitro growth behavior, surface carbohydrate expression, lectin-mediated agglutination, and qualitative activity profiles of 19 enzymes. The results indicate that biochemical characteristics, particularly isoenzyme electrophoretic data, are useful and objective additions to the traditional criteria used to type and classify Trypanosoma spp. of birds. Trypanosoma bennetti n. sp. from F. sparverius is described as a new species, and the other avian trypanosome strains are identified as T. avium Danilewsky, 1885. A phylogenetic tree, constructed by methods of numerical taxonomy, revealed that T. bennetti n. sp. is distantly related to T. avium as well as to T. rhodesiense and Leishmania chagasi.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

scholarly journals Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

1985 ◽  
Vol 101 (2) ◽  
pp. 427-440 ◽  
Author(s):  
E Bartnik ◽  
M Osborn ◽  
K Weber
Keyword(s):  
Positive Reaction ◽  
Intermediate Filaments ◽  
Biochemical Characterization ◽  
Helix Pomatia ◽  
Molar Ratio ◽  
Negative Stain ◽  
Epithelial Morphology ◽  
Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins

2016 ◽  
pp. 151-179 ◽  
Author(s):  
Dennis Zimmermann ◽  
Alisha N. Morganthaler ◽  
David R. Kovar ◽  
Cristian Suarez
Keyword(s):  
Binding Proteins ◽  
Biochemical Characterization ◽  
Actin Binding ◽  
Actin Binding Proteins

The In Vitro Growth and Characterization of Small Cell Lung Cancer

1983 ◽  
pp. 1-24
Author(s):  
Desmond N. Carney ◽  
Adi F. Gazdar ◽  
Herbert K. Oie ◽  
Francis Cuttitta ◽  
John D. Minna
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Small Cell ◽  
In Vitro Growth ◽  
Small Cell Lung

Biochemical characterization of HIV-1 Rev as a potent activator of casein kinase II in vitro

FEBS Letters ◽  
1998 ◽  
Vol 428 (3) ◽  
pp. 235-240 ◽  
Author(s):  
Kenzo Ohtsuki ◽  
Toshiro Maekawa ◽  
Shigeyoshi Harada ◽  
Atsushi Karino ◽  
Yuko Morikawa ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Hiv 1

scholarly journals Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

2019 ◽  
Vol 166 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Jan M Klenk ◽  
Max-Philipp Fischer ◽  
Paulina Dubiel ◽  
Mahima Sharma ◽  
Benjamin Rowlinson ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Metabolic Pathway ◽  
Biochemical Characterization ◽  
Gene Clusters ◽  
Cytochrome P450 Monooxygenases ◽  
Dihydroxyphenylacetic Acid ◽  
Meta Position ◽  
Redox Partners

AbstractCytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

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