scholarly journals Biological Production of an Integrinαvβ3 Targeting Imaging Probe and Functional Verification

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Mi-Hye Hwang ◽  
Jung Eun Kim ◽  
Sang-Yeob Kim ◽  
Senthilkumar Kalimuthu ◽  
Shin Young Jeong ◽  
...  
Keyword(s):  
Low Cost ◽  
Functional Verification ◽  
Gaussia Luciferase ◽  
Imaging Probe ◽  
E Coli ◽  
His Tag ◽  
Bacterial Vector ◽  
Bacterial Clone

The aim of the present study is to establish a bacterial clone capable of secreting an integrinαvβ3 targeting probe with bioluminescent and fluorescent activities, and to verify its specific targeting and optical activities using molecular imaging. A bacterial vector expressing a fusion of secretory Gaussia luciferase (sGluc), mCherry, and RGD (sGluc-mCherry-RGDX3; GCR), and a control vector expressing a fusion of secretoryGaussia luciferaseand mCherry (sGluc-mCherry; GC) were constructed. The GCR and GC proteins were expressed inE. coliand secreted into the growth medium, which showed an approximately 10-fold higher luciferase activity than the bacterial lysate. Successful purification of GCR and GC was achieved using the 6X His-tag method. The GCR protein bound with higher affinity to U87MG cells than CHO cells in confocal microscopy and IVIS imaging, and also showed a high affinity for integrinαvβ3 expressing tumor xenografts in anin vivoanimal model. AnE. coliclone was established to secrete an integrinαvβ3 targeting imaging probe with bioluminescent and fluorescent activities. The probe was produced feasibly and at low cost, and has shown to be useful for the assessment of angiogenesisin vitroandin vivo.

scholarly journals In Vivo Expression of the β-Glucoside (bgl) Operon of Escherichia coli Occurs in Mouse Liver

1998 ◽  
Vol 180 (17) ◽  
pp. 4746-4749 ◽  
Author(s):  
M. Ayub Khan ◽  
Richard E. Isaacson
Keyword(s):  
Escherichia Coli ◽  
Infected Mouse ◽  
Mouse Liver ◽  
Transcription Terminator ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Bacterial Clone ◽  
Chloramphenicol Acetyltransferase Gene

ABSTRACT An Escherichia coli DNA fragment was identified that contained part of the β-glucoside (bgl) operon. This fragment was identified because it contained a promoter that was responsible for the expression of a reporter gene, the chloramphenicol acetyltransferase gene, in a mouse liver during bacterial infection but not when a bacterial clone was grown in vitro. This fragment contained a promoter and a rho-independent transcription terminator which were flanked by the 3′ end of bglG and the 5′ end ofbglF. Reverse transcription-PCR confirmed thatcat-specific mRNA was produced in infected mouse liver but not in vitro. mRNA encoding the positive regulator of thebgl operon, bglG, also was detected in mouse liver infected with an E. coli strain. These results demonstrated that expression of the bgl operon occurs in infected mouse liver and suggests a unique role for this operon in vivo.

scholarly journals The Disulfide Bond of the Peptide Thanatin Is Dispensible for Its Antimicrobial ActivityIn VivoandIn Vitro

2016 ◽  
Vol 60 (7) ◽  
pp. 4283-4289 ◽  
Author(s):  
Bo Ma ◽  
Chao Niu ◽  
Ying Zhou ◽  
Xiaoyan Xue ◽  
Jingru Meng ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Umbilical Vein ◽  
Low Cost ◽  
Survival Rates ◽  
Antimicrobial Activities ◽  
Bacterial Strains ◽  
Content Type ◽  
E Coli

ABSTRACTThanatin (THA) displays potent antibiotic activity, especially against extended-spectrum-β-lactamase (ESBL)-producingEscherichia colibothin vitroandin vivo, with minimal hemolytic toxicity and satisfactory stability in plasma. However, the high cost of thanatin significantly limits its development and clinical application. To reduce the cost of peptide synthesis, a formulation of cyclic thanatin (C-thanatin) called linear thanatin (L-thanatin) was synthesized and its activity was evaluatedin vivoandin vitro. Results showed that C-thanatin and L-thanatin MICs did not differ against eight Gram-negative and two Gram-positive bacterial strains. Furthermore, the survival rates of ESBL-producing-E. coli-infected mice were consistent after C-thanatin or L-thanatin treatment at 5 or 10 mg/kg of body weight. Neither C-thanatin nor L-thanatin showed toxicity for human red blood cells (hRBCs) and human umbilical vein endothelial cells (HUVECs) at a concentration as high as 256 μg/ml. Results of circular dichroism spectroscopy indicated that the secondary structure of L-thanatin is extremely similar to that of C-thanatin. Membrane permeabilization and depolarization assays showed that C-thanatin and L-thanatin have similar abilities to permeabilize the outer and inner membranes and to induce membrane depolarization in ESBL-producingE. coli. However, neither of them caused significant HUVEC membrane permeability. These findings indicate that the two peptides have similar effects on bacterial cell membranes and that the disulfide bond in thanatin is not essential for its antimicrobial activitiesin vivoandin vitro. L-thanatin is thus a promising low-cost peptide candidate for treating ESBL-producingE. coliinfections.

scholarly journals Direct purification of CRISPR/Cas ribonucleoprotein from E. coli in a single step

2018 ◽  
Author(s):  
Siyu Lin ◽  
Jie Qiao ◽  
Lixin Ma ◽  
Yi Liu
Keyword(s):  
Large Scale ◽  
Low Cost ◽  
Cost Effective ◽  
Nuclease Activity ◽  
Single Step ◽  
Simple Method ◽  
E Coli ◽  
Purification System

AbstractCRISPR/Cas ribonucleoprotein (RNP) complexes have been recently used as promising biological tools with plenty of applications, however, there are by far no efficient methods to prepare them at large scale and low cost. Here, we present a simple method to directly produce and purify Cas RNP, including the widely used Cas9 and Cas12a nuclease, from E.coli in a single step using an ultra-high-affinity CL7/Im7 purification system. The prepared Cas RNP shows high stability, solid nuclease activity in vitro, and profound genome editing efficiency in vivo. Our method is convenient, cost-effective, and applicable to prepare other CRISPR associated nucleases.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Effects of Bacterial CLPB Protein Fragments on Food Intake and PYY Secretion

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2223
Author(s):  
Manon Dominique ◽  
Nicolas Lucas ◽  
Romain Legrand ◽  
Illona-Marie Bouleté ◽  
Christine Bôle-Feysot ◽  
...  
Keyword(s):  
Food Intake ◽  
Peptide Yy ◽  
Molecular Mimicry ◽  
Potential Effect ◽  
In Vivo Studies ◽  
Sprague Dawley ◽  
E Coli ◽  
In Vivo Effects

CLPB (Caseinolytic peptidase B) protein is a conformational mimetic of α-MSH, an anorectic hormone. Previous in vivo studies have already shown the potential effect of CLPB protein on food intake and on the production of peptide YY (PYY) by injection of E. coli wild type (WT) or E. coli ΔClpB. However, until now, no study has shown its direct effect on food intake. Furthermore, this protein can fragment naturally. Therefore, the aim of this study was (i) to evaluate the in vitro effects of CLPB fragments on PYY production; and (ii) to test the in vivo effects of a CLPB fragment sharing molecular mimicry with α-MSH (CLPB25) compared to natural fragments of the CLPB protein (CLPB96). To do that, a primary culture of intestinal mucosal cells from male Sprague–Dawley rats was incubated with proteins extracted from E. coli WT and ΔCLPB after fragmentation with trypsin or after a heat treatment of the CLPB protein. PYY secretion was measured by ELISA. CLPB fragments were analyzed by Western Blot using anti-α-MSH antibodies. In vivo effects of the CLPB protein on food intake were evaluated by intraperitoneal injections in male C57Bl/6 and ob/ob mice using the BioDAQ® system. The natural CLPB96 fragmentation increased PYY production in vitro and significantly decreased cumulative food intake from 2 h in C57Bl/6 and ob/ob mice on the contrary to CLPB25. Therefore, the anorexigenic effect of CLPB is likely the consequence of enhanced PYY secretion.

scholarly journals Proanthocyanidins against Oxidative Stress: From Molecular Mechanisms to Clinical Applications

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Lingyu Yang ◽  
Dehai Xian ◽  
Xia Xiong ◽  
Rui Lai ◽  
Jing Song ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Metabolic Diseases ◽  
Low Cost ◽  
Natural Agents ◽  
Molecular Damage ◽  
And Control

Proanthocyanidins (PCs) are naturally occurring polyphenolic compounds abundant in many vegetables, plant skins (rind/bark), seeds, flowers, fruits, and nuts. Numerousin vitroandin vivostudies have demonstrated myriad effects potentially beneficial to human health, such as antioxidation, anti-inflammation, immunomodulation, DNA repair, and antitumor activity. Accumulation of prooxidants such as reactive oxygen species (ROS) exceeding cellular antioxidant capacity results in oxidative stress (OS), which can damage macromolecules (DNA, lipids, and proteins), organelles (membranes and mitochondria), and whole tissues. OS is implicated in the pathogenesis and exacerbation of many cardiovascular, neurodegenerative, dermatological, and metabolic diseases, both through direct molecular damage and secondary activation of stress-associated signaling pathways. PCs are promising natural agents to safely prevent acute damage and control chronic diseases at relatively low cost. In this review, we summarize the molecules and signaling pathways involved in OS and the corresponding therapeutic mechanisms of PCs.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

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