Investigating the Electrostatic Role of a Critical Arginine for the Catalysis of E. Coli ADP-Glucose Pyrophosphorylase

SummaryThe local Sanarelli-Shwartzman phenomenon (SSP-L) in the skin of 30 rats was induced by an intr a cutaneous sensitizing injection of leukaemic leucocytes isolated from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL), acute myeloblastic leukaemia (AL) and chronic granulocytic leukaemia (CGL) and challenged by an intravenous injection of 100(μ of E. coli endotoxin. SSP-L was observed in 7 rats after injection of CLL lymphocytes and in 6 and 2 rats after AL myeloblasts and the CGL granulocytes, respectively. The lesions in the skin after AL myeloblasts appeared in a shorter time and were of longer duration compared with those observed after CLL lymphocytes and CGL granulocytes. Histologically, the lesions consisted of areas of destruction in the superficial layers of the skin ; the demarcation line showed the presence of neutrophils, macrophages and erythrocytes. Haemorrhages and fibrin deposits near the demarcation line were larger after injection of CLL lymphocytes and AL myeloblasts than after CGL granulocytes. The possible role of leucocyte procoagulative substances in the differences observed have been discussed.

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Bioenhancing Effect of Cow Urine Distillate and Pepper Extract on Antibacterial Activity of Azadirachta Indica Leaves

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2016.9.2.8 ◽

1970 ◽

Vol 9 (2) ◽

pp. 3212-3214

Author(s):

Pramod Dhakal ◽

Ankit a Achary ◽

Vedamurthy Joshi

Keyword(s):

Antibacterial Activity ◽

Azadirachta Indica ◽

Pathogenic Bacteria ◽

Ethanol Extract ◽

Watery Diarrhea ◽

Diffusion Method ◽

E Coli ◽

Drug Molecules ◽

Cow Urine

Bioenhancers are drug facilitator which do not show the typical drug activity but in combination to enhance the activity of other molecule in several way including increase the bioavailability of drug across the membrane, potentiating the drug molecules by conformational interaction, acting as receptor for drug molecules and making target cell more receptive to drugs and promote and increase the bioactivity or bioavailability or the uptake of drugs in combination therapy. The objective of the present study was to evaluate the antibacterial and activity of combination in Azadirachta indica extract with cow urine distillate and pepper extract against common pathogenic bacteria, a causative agent of watery diarrhea. It has been found that Indian indigenous cow urine and its distillate also possess bioenhancing ability. Bioenhancing role of cow urine distillate (CUD) and pepper extract was investigated on antibacterial activity of ethanol extract of Azadirachta indica. Antibacterial activity of ethanol extract neem alone and in combination with CUD and pepper extract were determined the ATCC strains against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and E-coli by cup plate diffusion method. Ethanol extract of neem has showed more effect on P. aeruginosa, E-coli than S. aureus and K. pneumonia with combination of CUD and pepper extract. CUD and pepper did not show any inhibition of test bacteria in low concentration. The antibacterial effect of combination of extract and CUD was higher than the inhibition caused by extract alone and is suggestive of the bioenhancing role of cow urine distillate and pepper. Moreover, inhibition of test bacteria was observed with less concentration of extract on combining with CUD

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E. coli Enterotoxin LtB Enhances Vaccine-Induced Anti-H. pylori Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions

Cells ◽

10.3390/cells8090982 ◽

2019 ◽

Vol 8 (9) ◽

pp. 982

Author(s):

Xiaoyan Peng ◽

Rongguang Zhang ◽

Chen Wang ◽

Feiyan Yu ◽

Mingyang Yu ◽

...

Keyword(s):

Cellular Immunity ◽

Protective Efficacy ◽

Gastric Injury ◽

Oral Vaccines ◽

Gastric Mucus ◽

E Coli ◽

Non Invasive ◽

Considerable Impact ◽

H Pylori

Current studies indicate that the anti-H. pylori protective efficacy of oral vaccines to a large extent depends on using mucosal adjuvants like E. coli heat-lable enterotoxin B unit (LtB). However, the mechanism by which Th17/Th1-driven cellular immunity kills H. pylori and the role of LtB remains unclear. Here, two L. lactis strains, expressing H. pylori NapA and LtB, respectively, were orally administrated to mice. As observed, the administration of LtB significantly enhanced the fecal SIgA level and decreased gastric H. pylori colonization, but also markedly aggravated gastric inflammatory injury. Both NapA group and NapA+LtB group had elevated splenocyte production of IL-8, IL-10, IL-12, IL-17, IL-23 and INF-γ. Notably, gastric leukocytes’ migration or leakage into the mucus was observed more frequently in NapA+LtB group than in NapA group. This report is the first that discusses how LtB enhances vaccine-induced anti-H. pylori efficacy by aggravating gastric injury and leukocytes’ movement into the mucus layer. Significantly, it brings up a novel explanation for the mechanism underlying mucosal cellular immunity destroying the non-invasive pathogens. More importantly, the findings suggest the necessity to further evaluate LtB’s potential hazards to humans before extending its applications. Thus, this report can provide considerable impact on the fields of mucosal immunology and vaccinology.

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Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

International Journal of Molecular Sciences ◽

10.3390/ijms20184416 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4416 ◽

Cited By ~ 2

Author(s):

Lara Console ◽

Maria Tolomeo ◽

Matilde Colella ◽

Maria Barile ◽

Cesare Indiveri

Keyword(s):

Cardiovascular Disease ◽

Amino Acids ◽

Risk Factor ◽

Biologically Active ◽

Suitable Model ◽

Native Protein ◽

E Coli ◽

Few Data ◽

Riboflavin Transport

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

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The essential role of mRNA degradation in understanding and engineering E. coli metabolism

Biotechnology Advances ◽

10.1016/j.biotechadv.2021.107805 ◽

2021 ◽

pp. 107805

Author(s):

Charlotte Roux ◽

Thibault Etienne ◽

Eliane Hajnsdorf ◽

Delphine Ropers ◽

A.J. Carpousis ◽

...

Keyword(s):

Essential Role ◽

Mrna Degradation ◽

E Coli

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

Applied Sciences ◽

10.3390/app11156865 ◽

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.

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Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Scientific Reports ◽

10.1038/s41598-021-94528-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Krystyna Ślaska-Kiss ◽

Nikolett Zsibrita ◽

Mihály Koncz ◽

Pál Albert ◽

Ákos Csábrádi ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Binding Affinity ◽

Dna Methyltransferase ◽

Restriction Enzymes ◽

Dna Binding Protein ◽

E Coli ◽

Dna Binding Affinity ◽

Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

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Proteomic Analysis Reveals a Role of ADP-glucose Pyrophosphorylase in the Asynchronous Filling of Rice Superior and Inferior Spikelets

Protein Expression and Purification ◽

10.1016/j.pep.2021.105875 ◽

2021 ◽

pp. 105875

Author(s):

Hong Zhao ◽

Zhou Li ◽

Hira Amjad ◽

Guopei Zhong ◽

Muhammad Umar Khan ◽

...

Keyword(s):

Proteomic Analysis ◽

Inferior Spikelets ◽

Adp Glucose Pyrophosphorylase

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Chromosomal Lesion Suppression and Removal in Escherichia coli via Linear DNA Degradation

Genetics ◽

10.1093/genetics/163.4.1255 ◽

2003 ◽

Vol 163 (4) ◽

pp. 1255-1271 ◽

Cited By ~ 1

Author(s):

Anabel Miranda ◽

Andrei Kuzminov

Keyword(s):

Dna Degradation ◽

Dna Helicase ◽

Structural Element ◽

E Coli ◽

Linear Dna ◽

Replication Factory ◽

Low Copy Number ◽

Chromosomal Lesion ◽

Recbcd Enzyme

Abstract RecBCD is a DNA helicase/exonuclease implicated in degradation of foreign linear DNA and in RecA-dependent recombinational repair of chromosomal lesions in E. coli. The low viability of recA recBC mutants vs. recA mutants indicates the existence of RecA-independent roles for RecBCD. To distinguish among possible RecA-independent roles of the RecBCD enzyme in replication, repair, and DNA degradation, we introduced wild-type and mutant combinations of the recBCD chromosomal region on a low-copy-number plasmid into a ΔrecA ΔrecBCD mutant and determined the viability of resulting strains. Our results argue against ideas that RecBCD is a structural element in the replication factory or is involved in RecA-independent repair of chromosomal lesions. We found that RecBCD-catalyzed DNA degradation is the only activity important for the recA-independent viability, suggesting that degradation of linear tails of σ-replicating chromosomes could be one of the RecBCD’s roles. However, since the weaker DNA degradation capacity due a combination of the RecBC helicase and ssDNA-specific exonucleases restores viability of the ΔrecA ΔrecBCD mutant to a significant extent, we favor suppression of chromosomal lesions via linear DNA degradation at reversed replication forks as the major RecA-independent role of the RecBCD enzyme.

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Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa315 ◽

2020 ◽

Author(s):

Lina Y Alkaissi ◽

Martin E Winberg ◽

Stéphanie DS Heil ◽

Staffan Haapaniemi ◽

Pär Myrelid ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ex Vivo ◽

Ussing Chambers ◽

E Coli ◽

Follicle Associated Epithelium ◽

Vitro Model ◽

Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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