scholarly journals A comparative study of GT27 Family N-acetylgalactosaminyltransferases

2021 ◽  
Author(s):  
Biprajit Sanyal
Keyword(s):  
Half Life ◽  
Homo Sapiens ◽  
Effective Means ◽  
Therapeutic Proteins ◽  
In Vitro Assays ◽  
Future Research ◽  
E Coli ◽  
Glycosylation Sites

Therapeutic proteins face short half lives in vivo. Their high costs and associated toxicity effects of increasing dosage warrant exploration of methods to increase serum half-life. These proteins can be produced with native or engineered glycosylation sites, which has been shown to be an effective means of prolonging serum half-life. Engineered E. coli represents an economical route of production. I have been able to produce, purify and test the activity of three N-acetylgalactosaminyltransferase isoform 2 in Escherichia coli and show glycosylation on peptides derived from Interleukin 29. I followed the activity of these enzymes on three candidate therapeutic proteins via lectin blotting. Data suggest the Homo sapiens orthologue of GalNAcT2 is the most efficient enzyme in the in vitro assays with all candidate therapeutic protein substrates displaying the Tn-antigen. Future research should investigate continuous assays for precise results as well as assaying native peptides as opposed to non-native ones.

scholarly journals A comparative study of GT27 Family N-acetylgalactosaminyltransferases

2021 ◽  
Author(s):  
Biprajit Sanyal
Keyword(s):  
Half Life ◽  
Homo Sapiens ◽  
Effective Means ◽  
Therapeutic Proteins ◽  
In Vitro Assays ◽  
Future Research ◽  
E Coli ◽  
Glycosylation Sites

Therapeutic proteins face short half lives in vivo. Their high costs and associated toxicity effects of increasing dosage warrant exploration of methods to increase serum half-life. These proteins can be produced with native or engineered glycosylation sites, which has been shown to be an effective means of prolonging serum half-life. Engineered E. coli represents an economical route of production. I have been able to produce, purify and test the activity of three N-acetylgalactosaminyltransferase isoform 2 in Escherichia coli and show glycosylation on peptides derived from Interleukin 29. I followed the activity of these enzymes on three candidate therapeutic proteins via lectin blotting. Data suggest the Homo sapiens orthologue of GalNAcT2 is the most efficient enzyme in the in vitro assays with all candidate therapeutic protein substrates displaying the Tn-antigen. Future research should investigate continuous assays for precise results as well as assaying native peptides as opposed to non-native ones.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

scholarly journals Systematic identification of metabolites controlling gene expression in E. coli

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Martin Lempp ◽  
Niklas Farke ◽  
Michelle Kuntz ◽  
Sven Andreas Freibert ◽  
Roland Lill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
In Vitro Assays ◽  
E Coli ◽  
A Genome ◽  
Concentration Changes ◽  
Systematic Identification ◽  
Genome Scale

Abstract Metabolism controls gene expression through allosteric interactions between metabolites and transcription factors. These interactions are usually measured with in vitro assays, but there are no methods to identify them at a genome-scale in vivo. Here we show that dynamic transcriptome and metabolome data identify metabolites that control transcription factors in E. coli. By switching an E. coli culture between starvation and growth, we induce strong metabolite concentration changes and gene expression changes. Using Network Component Analysis we calculate the activities of 209 transcriptional regulators and correlate them with metabolites. This approach captures, for instance, the in vivo kinetics of CRP regulation by cyclic-AMP. By testing correlations between all pairs of transcription factors and metabolites, we predict putative effectors of 71 transcription factors, and validate five interactions in vitro. These results show that combining transcriptomics and metabolomics generates hypotheses about metabolism-transcription interactions that drive transitions between physiological states.

scholarly journals Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli

2022 ◽  
Vol 10 (1) ◽  
pp. 119
Author(s):  
Bram Van den Bergh
Keyword(s):  
Drosophila Melanogaster ◽  
In Vitro Assays ◽  
Antibiotic Tolerance ◽  
In Vivo Models ◽  
Antibiotic Development ◽  
E Coli ◽  
Germ Free ◽  
Flexible System

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics’ efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

Natural Products as Potential Agents Against SARS-CoV and SARS-CoV-2

2021 ◽  
Vol 28 ◽  
Author(s):  
Joanda Paolla Raimundo e Silva ◽  
Chonny Alexander Herrera Acevedo ◽  
Thalisson Amorim de Souza ◽  
Renata Priscila Barros de Menezes ◽  
Zoe L. Sessions ◽  
...  
Keyword(s):  
Natural Products ◽  
In Silico ◽  
Principal Component ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Future Research ◽  
Natural Substances ◽  
Effective Drugs

Background: Natural products are useful agents for the discovery of new lead-compounds and effective drugs to combat coronaviruses (CoV). Objective: The present work provides an overview of natural substances, plant extracts, and essential oils as potential antiSARS-CoV agents. In addition, this work evaluates their drug-like properties which are essential in the selection of compounds in order to accelerate the drug development process. Methods: The search was carried out using PubMed, ScienceDirect and SciFinder. Articles addressing plant-based natural products as potential SARS-CoV or SARS-CoV-2 agents within the last seventeen years were analyzed and selected. The descriptors for Chemometrics analyzes were obtained in alvaDesc and the principal component analyzes (PCA) were carried out in SIMCA version 13.0. Results: Based on in vitro assays and computational analyzes, this review covers twenty nine medicinal plant species and more than 300 isolated substances as potential anti-coronavirus agents. Among them, flavonoids and terpenes were the most promising compound classes. In silico analyses of drug-like properties corroborate these findings and indicate promising candidates for in vitro and in vivo studies to validate their activity. Conclusion: This paper highlights the role of ethnopharmacology in drug discovery and simulates the use of integrative (in silico/ in vitro) and chemocentric approaches to strengthen current studies and guide future research in the field of antivirals agents.

scholarly journals DL-α-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase

1982 ◽  
Vol 204 (3) ◽  
pp. 771-775 ◽  
Author(s):  
A Kallio ◽  
P P McCann ◽  
P Bey
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Arginine Decarboxylase ◽  
Biosynthetic Enzyme ◽  
Irreversible Inhibitor ◽  
E Coli

DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased.

scholarly journals Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1201
Author(s):  
Márcia R. Couto ◽  
Joana L. Rodrigues ◽  
Lígia R. Rodrigues
Keyword(s):  
Glucose Dehydrogenase ◽  
Cell Extract ◽  
Capra Hircus ◽  
In Vitro Assays ◽  
Uridine Diphosphate ◽  
Phylogenetic Groups ◽  
Biotechnological Production ◽  
E Coli

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.

scholarly journals Mesenchymal Stromal Cell Characteristics and Regenerative Potential in Cardiovascular Disease

2018 ◽  
Vol 27 (5) ◽  
pp. 765-785 ◽  
Author(s):  
F. C. C. van Rhijn-Brouwer ◽  
H. Gremmels ◽  
J. O. Fledderus ◽  
M. C. Verhaar
Keyword(s):  
Cardiovascular Disease ◽  
Clinical Success ◽  
Predictive Ability ◽  
In Vitro Assays ◽  
Future Research ◽  
Promising Strategy

Administration of mesenchymal stromal cells (MSCs) is a promising strategy to treat cardiovascular disease (CVD). As progenitor cells may be negatively affected by both age and comorbidity, characterization of MSC function is important to guide decisions regarding use of allogeneic or autologous cells. Definitive answers on which factors affect MSC function can also aid in selecting which MSC donors would yield the most therapeutically efficacious MSCs. Here we provide a narrative review of MSC function in CVD based on a systematic search. A total of 41 studies examining CVD-related MSC (dys)function were identified. These data show that MSC characteristics and regenerative potential are often affected by CVD. However, studies presented conflicting results, and directed assessment of MSC parameters relevant to regenerative medicine applications was lacking in many studies. The predictive ability of in vitro assays for in vivo efficacy was rarely assessed. There was no correlation between quality of study reporting and study findings. Age mismatch was also not associated with study findings or effect size. Future research should focus on assays that assess regenerative potential in MSCs and parameters that relate to clinical success.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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