scholarly journals A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase

2021 ◽  
Vol 22 (11) ◽  
pp. 6148
Author(s):  
Matteo Miceli ◽  
Silvana Casati ◽  
Pietro Allevi ◽  
Silvia Berra ◽  
Roberta Ottria ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Kinetic Constants ◽  
New Method ◽  
Monoacylglycerol Lipase ◽  
Enzymatic Cleavage ◽  
Highly Sensitive ◽  
Bioluminescence Assay ◽  
Identification And Characterization ◽  
In Cells

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

scholarly journals Identification of a New Site of Sumoylation on Tel (ETV6) Uncovers a PIAS-Dependent Mode of Regulating Tel Function

2008 ◽  
Vol 28 (7) ◽  
pp. 2342-2357 ◽  
Author(s):  
M. Guy Roukens ◽  
Mariam Alloul-Ramdhani ◽  
Alfred C. O. Vertegaal ◽  
Zeinab Anvarian ◽  
Crina I. A. Balog ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Control Of Gene Expression ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Spatiotemporal Control ◽  
Identification And Characterization ◽  
In Cells

ABSTRACT Cell proliferation and differentiation are governed by a finely controlled balance between repression and activation of gene expression. The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue Yan, play pivotal roles in cell fate determination although the precise mechanisms by which repression of gene expression by these factors is achieved are not clearly defined. Here, we report the identification and characterization of the primary site of sumoylation of Tel, lysine 11 (K11), which is highly conserved in vertebrates (except Danio rerio). We demonstrate that in cells PIAS3 binds to Tel and stimulates sumoylation of K11 in the nucleus. Both Tel monomers and oligomers are efficiently sumoylated on K11 in vitro; but in cells only Tel oligomers are found conjugated with SUMO, whereas sumoylation of Tel monomers is transitory and appears to sensitize them for proteasomal degradation. Mechanistically, sumoylation of K11 inhibits repression of gene expression by full-length Tel. In accordance with this observation, we found that sumoylation impedes Tel association with DNA. By contrast, a Tel isoform lacking K11 (TelM43) is strongly repressive. This isoform results from translation from an alternative initiation codon (M43) that is common to all Tel proteins that also contain the K11 sumoylation consensus site. We find that PIAS3 may have a dual, context-dependent influence on Tel; it mediates Tel sumoylation, but it also augments Tel's repressive function in a sumoylation-independent fashion. Our data support a model that suggests that PIAS-mediated sumoylation of K11 and the emergence of TelM43 in early vertebrates are linked and that this serves to refine spatiotemporal control of gene expression by Tel by establishing a pool of Tel molecules that are available either to be recycled to reinforce repression of gene expression or are degraded in a regulated fashion.

scholarly journals Identification and Characterization of Escherichia coli from Bronchial Plug of Broiler Chicken with Respiratory Distress

2021 ◽  
Vol 10 (10) ◽  
pp. 221-228
Author(s):  
M. H. Chhatrola D. T. Fefar ◽  
A. R. Bhadaniya B. J. Trangadia ◽  
V. A. Kalaria S. N. Ghodasara ◽  
B. B. Javia J. M. Chauhan
Keyword(s):  
Respiratory Distress ◽  
Broiler Chicken ◽  
Pcr Assay ◽  
E Coli ◽  
Methane Sulphonate ◽  
Highly Sensitive ◽  
High Prevalence ◽  
Identification And Characterization ◽  
Pooled Sample

A Twenty-five bronchial plug samples from dead broiler chicken with signs of respiratory distress collected for identification of avian pathogenic E. coli (APEC). Samples comprised of one pooled sample each from 25 poultry farms located in and around Junagadh city. Isolation, culture and colony characteristics used for primary detection followed by PCR assay targeting four virulence genes (iss, papC, tsh and vat)for confirmatory diagnosis. All bronchial plugs were found to be positive for E. coli infection by isolation. Out of 25 flock sampled, the highest number of E. coli isolates were found positive for the presence of iss gene (22) followed by tsh gene (15), vat gene (13) and papC gene (8)by PCR. On antibiogram, overall E. coli was highly sensitive to colistin (Methane sulphonate) and meropenem(100%) followed by levofloxacin (76%), ceftriaxone (64%), gentamicin (60%), amikacin (60%), co-trimoxazole (40%), amoxycilin-clavunic acid (8%) and imepenem (8%). These results indicated the high prevalence of the E. coli with variable antibiotic resistance in broiler chicken.

scholarly journals Identification and characterization of genes upregulated in cells transformed by v-Jun

Oncogene ◽  
2000 ◽  
Vol 19 (31) ◽  
pp. 3537-3545 ◽  
Author(s):  
Shu-ling Fu ◽  
Anke Waha ◽  
Peter K Vogt
Keyword(s):  
Identification And Characterization ◽  
In Cells

scholarly journals Identification and Characterization of theErwinia amylovora rpoS Gene: RpoS Is Not Involved in Induction of Fireblight Disease Symptoms

1998 ◽  
Vol 180 (24) ◽  
pp. 6789-6792 ◽  
Author(s):  
M. Anderson ◽  
C. E. Pollitt ◽  
I. S. Roberts ◽  
J. A. Eastgate
Keyword(s):  
Sigma Factor ◽  
Erwinia Amylovora ◽  
Environmental Stresses ◽  
Alternative Sigma Factor ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Highly Sensitive ◽  
Identification And Characterization ◽  
Sigma Factor Rpos

ABSTRACT The Erwinia amylovora rpoS gene, encoding the alternative sigma factor RpoS, has been cloned and characterized. Though highly sensitive to a number of environmental stresses, anE. amylovora rpoS mutant was not compromised in its ability to grow or cause disease symptoms within apple seedlings or in an overwintering model.

scholarly journals Identification and Characterization of Four Azole-Resistant erg3 Mutants of Candida albicans

2010 ◽  
Vol 54 (11) ◽  
pp. 4527-4533 ◽  
Author(s):  
Claire M. Martel ◽  
Josie E. Parker ◽  
Oliver Bader ◽  
Michael Weig ◽  
Uwe Gross ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Total Sterol ◽  
Sterol Fraction ◽  
Missense Mutations ◽  
Multidrug Efflux ◽  
Highly Sensitive ◽  
Multiple Amino Acid ◽  
Identification And Characterization ◽  
Assay Conditions

ABSTRACT Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol Δ5,6-desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ≥256, 16, 16, 8, and 1 μg ml−1, respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 μg ml−1); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 μg ml−1). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.

Characterization of protein kinase C in early Xenopus embryogenesis

Development ◽  
1990 ◽  
Vol 110 (2) ◽  
pp. 461-470 ◽  
Author(s):  
A.P. Otte ◽  
I.M. Kramer ◽  
M. Mannesse ◽  
C. Lambrechts ◽  
A.J. Durston
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Inositol Phosphates ◽  
Kinase C ◽  
Low Concentrations ◽  
Highly Sensitive ◽  
Pkc Isozymes ◽  
Endodermal Cells

Recently, we presented evidence that protein kinase C (PKC) is involved in mediating the endogenous signals that induced competent Xenopus ectoderm to differentiate to neural tissue. We report here that PKC is already strongly activated in neural-induced ectoderm from midgastrula embryos and that this activation runs parallel with an increase in the level of inositol phosphates. We further identify several proteins that are phosphorylated, both in natural neural-induced ectoderm and in TPA-treated ectoderm, suggesting that they are phosphorylated through the PKC route. We found no major changes in PKC activity among different pregastrula stages, including the unfertilized egg. However, PKC isolated from animal, ectodermal cells is highly sensitive to Ca2+ and can be activated by low concentrations, (6–25 microM) of arachidonic acid, while PKC isolated from vegetal, endodermal cells is more insensitive to Ca2+ and cannot be activated by arachidonic acid. These results suggest that different PKC isozymes are present in animal and vegetal cells.

A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes

PROTEOMICS ◽  
2001 ◽  
Vol 1 (2) ◽  
pp. 239-247 ◽  
Author(s):  
Naoyuki Taniguchi ◽  
Atsuko Ekuni ◽  
Jeong Heon Ko ◽  
Eiji Miyoshi ◽  
Yoshitaka Ikeda ◽  
...  
Keyword(s):  
Identification And Characterization ◽  
In Cells

scholarly journals ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF SALMONELLA SEROVARS FROM DIARRHOEIC STOOL SAMPLES OF HUMAN

2012 ◽  
Vol 9 (1) ◽  
pp. 85-93 ◽  
Author(s):  
MK Nesa ◽  
MSR Khan ◽  
M Alam
Keyword(s):  
First Line ◽  
Isolation And Identification ◽  
First Line Therapy ◽  
Antimicrobial Sensitivity ◽  
Highly Sensitive ◽  
Stool Samples ◽  
Salmonella Serovars ◽  
Identification And Characterization ◽  
Human Stool

    The present study aimed at isolation and identification of Salmonella serovars from human stool and characterization of the isolated serovars using biochemical, serological, molecular and antimicrobial sensitivity techniques. A total of 25 samples were collected of which 16% were positive to Salmonella serovars. All the culturally positive isolates fermented dextrose, maltose and mannitol with the peoduction of acid and gas but not fermented sucrose and lactose. The same isolates showed Indole and V-P tests negative but M-R test positive. All the culturally and biochemically positive Salmonella serovars showed agglutination with poly ‘O’ and poly ‘H’ antisera. The antimicrobial susceptibility testing showed that the isolated Salmonella serovars were highly sensitive to ciprofloxacin and moderately sensitive to chloramphenicol, kanamycin, cotrimoxazol and nalidixic acid. However, the positive isolates were resistant to erythromycin. The present study indicates that ciprofloxacin can be used as a first line therapy for the treatment of Salmonella gastroenteritis.DOI = http://dx.doi.org/10.3329/bjvm.v9i1.11218Bangl. J. Vet. Med. (2011). 9(1): 85-93

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