Characterization of phenoloxidases from larval cuticle of Sarcophaga bullata and a comparison with cuticular enzymes from other species

1987 ◽  
Vol 65 (5) ◽  
pp. 1158-1166 ◽  
Author(s):  
F. Michael Barrett
Keyword(s):  
Musca Domestica ◽  
Sodium Azide ◽  
Lucilia Sericata ◽  
Phormia Regina ◽  
Ph Optimum ◽  
Larval Cuticle ◽  
Sarcophaga Bullata ◽  
Tyrosinase Enzyme ◽  
Laccase Enzyme

A tyrosinase, enzyme A, and a laccase, enzyme B, have been partially purified from larval cuticle of the flesh fly Sarcophaga bullata. Enzyme A (EC 1.10.3.1, o-diphenol: O2 oxidoreductase) oxidizes o-diphenols but not p-diphenols, is strongly inhibited by phenylthiourea, and has a pH optimum around pH 6.5–7.0. Assays on intact cuticle suggest that it becomes maximally activated at pH between 8 and 9. Enzyme B (EC 1.10.3.2, p-diphenol: O2 oxidoreductase) oxidizes both o-diphenols and p-diphenols, is not inhibited by phenylthiourea but is inhibited by concentrations of sodium azide that have little effect on enzyme A, and has a pH optimum near pH 4.5. Enzyme A was identified in extracts of cuticle from nine other species representing five orders. Enzyme B was much less readily extractable but was partially purified from larval cuticle of Phormia regina, Musca domestica, and Lucilia sericata. A summary of all species studied to date makes possible the test of a hypothesis about the distribution of these cuticular phenoloxidases within the Insecta.

Characterization of the Volatiles’ Profiles of the Eggs of Forensically Relevant Lucilia sericata and Phormia regina (Diptera: Calliphoridae) Blow Flies by SPME-Facilitated GC-MS

2020 ◽  
Vol 57 (4) ◽  
pp. 994-1005
Author(s):  
Justine E Giffen-Lemieux ◽  
Koji Okuda ◽  
Jennifer Y Rosati ◽  
Rabi A Musah
Keyword(s):  
Solid Phase ◽  
Lucilia Sericata ◽  
Phormia Regina ◽  
Organosulfur Compounds ◽  
Blow Fly ◽  
Headspace Volatiles ◽  
Blow Flies ◽  
Wide Range ◽  
Published Research

Abstract The attraction of necrophagous insects, particularly blow flies, to corpses and carrion is of ecological, economic, and agricultural importance, although the mechanisms by which it occurs are not well understood. Much of the published research on blow fly attractants has focused on volatiles emitted from carrion surrogates, but little attention has been given to the possibility that blow fly eggs themselves may emit chemical cues that are responsible for conspecific and heterospecific insect attraction. In this study, the headspace volatiles emitted from eggs representing two aggregated oviposition events that were collected 1 mo apart from two species of the Calliphoridae family (Order: Diptera), Lucilia sericata (Meigen), and Phormia regina (Meigen) were analyzed via solid-phase microextraction-facilitated GC-MS. The volatiles’ profiles were found to be consistent between samples representing the same species, but unique between the two species. Over 100 molecules covering a wide range of compound classes that included alcohols, aldehydes, esters, amines, ketones, and organosulfur compounds were identified. The profile of volatiles emitted from the L. sericata eggs contained several alkanes and aldehydes, whereas salient features of the P. regina headspace included numerous esters and ketones. Between the two species, 42 compounds were shared, several of which were carboxylic acids. Little overlap between the range of compounds detected and those reported to be emitted from decomposing remains was observed.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals Impact of Comingled Heterospecific Assemblages on Developmentally Based Estimates of the Post-Mortem Interval—A Study with Lucilia sericata (Meigen), Phormia regina (Meigen) and Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae)

Insects ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 280
Author(s):  
Krystal R. Hans ◽  
Sherah L. Vanlaerhoven
Keyword(s):  
Growth Rate ◽  
Development Time ◽  
Lucilia Sericata ◽  
Phormia Regina ◽  
Calliphora Vicina ◽  
Immature Stages ◽  
Post Mortem ◽  
Blow Fly ◽  
Blow Flies ◽  
Post Mortem Interval

Estimates of the minimum post-mortem interval (mPMI) using the development rate of blow flies (Diptera: Calliphoridae) are common in modern forensic entomology casework. These estimates are based on single species developing in the absence of heterospecific interactions. Yet, in real-world situations, it is not uncommon to have 2 or more blow fly species developing on a body. Species interactions have the potential to change the acceptance of resources as suitable for oviposition, the timing of oviposition, growth rate, size and development time of immature stages, as well as impacting the survival of immature stages to reach adult. This study measured larval development and growth rate of the blow flies Lucilia sericata (Meigen, 1826), Phormia regina (Meigen, 1826) and Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) over five constant temperatures (15, 20, 25, 30, 35 °C), in the presence of conspecifics or two-species heterospecific assemblages. Temperature and species treatment interacted such that L. sericata larvae gained mass more rapidly when in the presence of P. regina at 20 and 30 °C, however only developed faster at first instar. At later stages, the presence of P. regina slowed development of L. sericata immatures. Development time of C. vicina immatures was not affected by the presence of P. regina, however larvae gained mass more slowly. Development time of P. regina immatures was faster in the presence of either L. sericata or C. vicina until third instar, at which point, the presence of L. sericata was neutral whereas C. vicina negatively impacted development time. Phormia regina larvae gained mass more rapidly in the presence of L. sericata at 20 °C but were negatively impacted at 25 °C by the presence of either L. sericata or C. vicina. The results of this study indicate that metrics such as development time or larval mass used for estimating mPMI with blow flies are impacted by the presence of comingled heterospecific blow fly assemblages. As the effects of heterospecific assemblages are not uniformly positive or negative between stages, temperatures or species combinations, more research into these effects is vital. Until then, caution should be used when estimating mPMI in cases with multiple blow fly species interacting on a body.

scholarly journals Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Ahmad R. Attia ◽  
Reyed M. Reyed ◽  
Nadia A. Soliman
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Maximum Activity ◽  
Sodium Oxalate ◽  
Partial Purification ◽  
Bromophenol Blue ◽  
Bromocresol Purple ◽  
Laccase Enzyme

Abstract Background Due to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO were studied in the present investigation. Results Alcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C. Some metal ions Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents [ethylenediamine tetraacetic acid (EDTA), sodium citrate, and sodium oxalate] caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine. Conclusion The promising properties of the newly studied laccase enzyme from Alcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.

scholarly journals Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines

1981 ◽  
Vol 197 (2) ◽  
pp. 523-526 ◽  
Author(s):  
Paul D. Wightman ◽  
Mary Ellen Dahlgren ◽  
James C. Hall ◽  
Philip Davies ◽  
Robert J. Bonney
Keyword(s):  
Phospholipase C ◽  
High Activity ◽  
Peritoneal Macrophages ◽  
Ph Optimum ◽  
Reaction Velocity ◽  
Neutral Ph ◽  
Maximum Reaction ◽  
Mouse Peritoneal Macrophages ◽  
Identification And Characterization

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca2+-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.

scholarly journals Molecular cloning and biochemical characterization of sialidases from zebrafish (Danio rerio)

2007 ◽  
Vol 408 (3) ◽  
pp. 395-406 ◽  
Author(s):  
Marta Manzoni ◽  
Paolo Colombi ◽  
Nadia Papini ◽  
Luana Rubaga ◽  
Natascia Tiso ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Biochemical Characterization ◽  
Chromosome 21 ◽  
Ph Optimum ◽  
Reverse Transcription Pcr ◽  
Genome Wide ◽  
Genome Wide Search ◽  
Putative Orthologues

Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT–PCR (reverse transcription–PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.

scholarly journals Characterization of Lassa Virus Cell Entry and Neutralization with Lassa Virus Pseudoparticles

2009 ◽  
Vol 83 (7) ◽  
pp. 3228-3237 ◽  
Author(s):  
François-Loic Cosset ◽  
Philippe Marianneau ◽  
Geraldine Verney ◽  
Fabrice Gallais ◽  
Noel Tordo ◽  
...  
Keyword(s):  
Immune Response ◽  
Humoral Immune Response ◽  
Neutralizing Antibodies ◽  
Cell Attachment ◽  
Low Ph ◽  
Cell Entry ◽  
Lassa Virus ◽  
Ph Optimum ◽  
Humoral Immune

ABSTRACT The cell entry and humoral immune response of the human pathogen Lassa virus (LV), a biosafety level 4 (BSL4) Old World arenavirus, are not well characterized. LV pseudoparticles (LVpp) are a surrogate model system that has been used to decipher factors and routes involved in LV cell entry under BSL2 conditions. Here, we describe LVpp, which are highly infectious, with titers approaching those obtained with pseudoparticles displaying G protein of vesicular stomatitis virus and their the use for the characterization of LV cell entry and neutralization. Upon cell attachment, LVpp utilize endocytic vesicles for cell entry as described for many pH-dependent viruses. However, the fusion of the LV glycoproteins is activated at unusually low pH values, with optimal fusion occurring between pH 4.5 and 3, a pH range at which fusion characteristics of viral glycoproteins have so far remained largely unexplored. Consistent with a shifted pH optimum for fusion activation, we found wild-type LV and LVpp to display a remarkable resistance to exposure to low pH. Finally, LVpp allow the fast and quantifiable detection of neutralizing antibodies in human and animal sera and will thus facilitate the study of the humoral immune response in LV infections.

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