Preliminary characterization of vasocontractile activities in erythrocytes

1980 ◽  
Vol 53 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Shu T. Cheung ◽  
Michael P. McIlhany ◽  
Ramon Lim ◽  
Sean Mullan
Keyword(s):  
Biochemical Characterization ◽  
Contractile Activity ◽  
Clinical Situation ◽  
Enzymatic Digestion ◽  
Content Type ◽  
Exclusion Chromatography ◽  
Basilar Arteries ◽  
Peripheral Arterial

✓ The vasocontractile activities of washed red cell preparations hemolyzed by various methods were studied in vitro using isolated canine basilar arteries. Significant contractions were induced by each preparation. The maximum strength of contraction attained by the various preparations was similar. The contractile activity appeared to be dose-related, and molecular exclusion chromatography demonstrated that the activity migrated with the fraction of approximately 40,000 to 45,000 molecular weight. The vasocontractile effect of the active fraction was sustained in vitro when tested against basilar artery, but was inactive in peripheral arterial preparations. Preliminary biochemical characterization indicates that the contractile activity resides in a protein. Enzymatic digestion of the crude fraction appears to enhance the contractile activity significantly, and this observation suggests a possible mechanism for the delayed onset of ischemic symptoms encountered in the clinical situation.

scholarly journals Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Chao He ◽  
Ning Liu ◽  
Fudong Li ◽  
Xiaoyu Jia ◽  
Hui Peng ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Conformational Changes ◽  
Biochemical Characterization ◽  
Complex Structure ◽  
Elongation Factor ◽  
Titration Calorimetry ◽  
Content Type ◽  
Elongation Factor P

ABSTRACT A bacterial inverting glycosyltransferase EarP transfers rhamnose from dTDP-β-l-rhamnose (TDP-Rha) to Arg32 of translation elongation factor P (EF-P) to activate its function. We report here the structural and biochemical characterization of Pseudomonas aeruginosa EarP. In contrast to recently reported Neisseria meningitidis EarP, P. aeruginosa EarP exhibits differential conformational changes upon TDP-Rha and EF-P binding. Sugar donor binding enhances acceptor binding to EarP, as revealed by structural comparison between the apo-, TDP-Rha-, and TDP/EF-P-bound forms and isothermal titration calorimetry experiments. In vitro EF-P rhamnosylation combined with active-site geometry indicates that Asp16 corresponding to Asp20 of N. meningitidis EarP is the catalytic base, whereas Glu272 is another putative catalytic residue. Our study should provide the basis for EarP-targeted inhibitor design against infections from P. aeruginosa and other clinically relevant species. IMPORTANCE Posttranslational rhamnosylation of EF-P plays a key role in Pseudomonas aeruginosa, establishing virulence and antibiotic resistance, as well as survival. The detailed structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP from P. aeruginosa not only demonstrates that sugar donor TDP-Rha binding enhances acceptor EF-P binding to EarP but also should provide valuable information for the structure-guided development of its inhibitors against infections from P. aeruginosa and other EarP-containing pathogens.

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.

Prostacyclin and cerebral vessel relaxation

1982 ◽  
Vol 57 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Kamal S. Paul ◽  
Eric T. Whalley ◽  
Christine Forster ◽  
Richard Lye ◽  
John Dutton
Keyword(s):  
Angiotensin Ii ◽  
Arterial Spasm ◽  
Cerebral Vessel ◽  
Content Type ◽  
Wide Range ◽  
Potent Vasodilator ◽  
Dose Dependent Fashion ◽  
Basilar Arteries ◽  
High Concentrations

✓ The authors have studied the ability of prostacyclin to reverse contractions of human basilar arteries in vitro that were induced by a wide range of substances implicated in the etiology of cerebral arterial spasm. Prostacyclin (10−10 to 10−6M) caused a dose-related reversal of contractions induced by 5-hydroxytryptamine, noradrenaline, angiotensin II, prostaglandin (PG)F2α, and U-46619 (a thromboxane-A2 mimetic). These agents were tested at concentrations or volumes that produced almost maximum or maximum responses and those that produced approximately 50% of the maximum response. Contractions induced by maximum concentrations of angiotensin II and U-46619 were least affected by prostacyclin. In addition, contractions induced by thromboxane-A2 generated from guinea-pig lung were reversed in a dose-dependent fashion by prostacyclin. This ability of prostacyclin to physiologically antagonize contractions of the human basilar artery in vitro induced by high concentrations of various spasmogenic agents suggests that such a potent vasodilator agent or more stable analogue may be of value in the treatment of such disorders as cerebral arterial spasm following subarachnoid hemorrhage.

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.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

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

scholarly journals Biochemical Characterization of Cassiopea andromeda (Forsskål, 1775), Another Red Sea Jellyfish in the Western Mediterranean Sea

Marine Drugs ◽  
2021 ◽  
Vol 19 (9) ◽  
pp. 498
Author(s):  
Gianluca De De Rinaldis ◽  
Antonella Leone ◽  
Stefania De De Domenico ◽  
Mar Bosch-Belmar ◽  
Rasa Slizyte ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Biochemical Characterization ◽  
Biological Activities ◽  
Enzymatic Digestion ◽  
Western Mediterranean ◽  
Western Mediterranean Sea ◽  
Blue Growth ◽  
Jellyfish Species ◽  
Italian Coasts

Increasing frequency of native jellyfish proliferations and massive appearance of non-indigenous jellyfish species recently concur to impact Mediterranean coastal ecosystems and human activities at sea. Nonetheless, jellyfish biomass may represent an exploitable novel resource to coastal communities, with reference to its potential use in the pharmaceutical, nutritional, and nutraceutical Blue Growth sectors. The zooxanthellate jellyfish Cassiopea andromeda, Forsskål, 1775 (Cnidaria, Rhizostomeae) entered the Levant Sea through the Suez Canal and spread towards the Western Mediterranean to reach Malta, Tunisia, and recently also the Italian coasts. Here we report on the biochemical characterization and antioxidant activity of C. andromeda specimens with a discussion on their relative biological activities. The biochemical characterization of the aqueous (PBS) and hydroalcoholic (80% ethanol) soluble components of C. andromeda were performed for whole jellyfish, as well as separately for umbrella and oral arms. The insoluble components were hydrolyzed by sequential enzymatic digestion with pepsin and collagenase. The composition and antioxidant activity of the insoluble and enzymatically digestible fractions were not affected by the pre-extraction types, resulting into collagen- and non-collagen-derived peptides with antioxidant activity. Both soluble compounds and hydrolyzed fractions were characterized for the content of proteins, phenolic compounds, and lipids. The presence of compounds coming from the endosymbiont zooxanthellae was also detected. The notable yield and the considerable antioxidant activity detected make this species worthy of further study for its potential biotechnological sustainable exploitation.

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 The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

2015 ◽  
Vol 59 (8) ◽  
pp. 4446-4452 ◽  
Author(s):  
Vadim Makarov ◽  
João Neres ◽  
Ruben C. Hartkoorn ◽  
Olga B. Ryabova ◽  
Elena Kazakova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitro Group ◽  
Covalent Bond ◽  
Antimycobacterial Activity ◽  
Content Type ◽  
Sar Studies ◽  
Covalent Bond Formation

ABSTRACT8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity againstMycobacterium tuberculosisin vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml againstM. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorablein vitroabsorption-distribution-metabolism-excretion/toxicity (ADME/T) andin vivopharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

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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