Expression and properties of lysyl oxidase from archeal halophile Haloterrigena turkmenica

Background: Lysyl oxidases (LOX) were studied mostly in mammals, whereas properties of recently found homologs in prokaryotic genomes remain enigmatic. Methods: LOX gene from Haloterrigena turkmenica has been cloned by PCR in a E. coli expression vector. Protein purification has been done using metal affinity chromatography under denaturing conditions followed by refolding. Catalytic activity has been fluorometrically a release of hydrogen peroxide coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. Results: H. turkmenica LOX (HTU-LOX) may be successfully expressed in E. coli with a high yield. However, full-length protein gives no catalytic activity. On the other hand, a deletion of putative signal peptide allows the protein to be refolded into an active enzyme. Further deletion until the boundary of the catalytic C-terminal domain greatly increases the activity. Refolding is optimal at pH around 6.0, with addition of Cu2+, and surprisingly does not respond to changing concentration s of NaCl. The active HTU-LOX is sensitive to the lysyl oxidase inhibitor β-aminopropionitrile. HTU-LOX is active towards usual substrates of mammalian LOX such as lysine-containing basic peptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed specificity of the former. HTU-LOX readily oxidizes various amines including such compounds as taurine and glycine. Moreover, it is active also towards aminoglycoside antibiotics. Benzyl amine is a poor substrate for HTU-LOX. H. turkmenica cells or culture medium do not contain any detectable amine oxidase activity. Polyclonal antibodies against HTU-LOX detect a band among H. turkmenica proteins with the molecular weight corresponding to the unprocessed enzyme. Conclusion: H. turkmenica contains a lysyl oxidase gene that may give active recombinant enzyme with important biochemical features conserved, for example, sensitivity to β-aminopropionitrile. However, its function in the host may be cryptic. Significance: This is the first report on some properties of lysyl oxidase that originated from a horizontal transfer event into Archea.

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Expression and properties of lysyl oxidase from archeal halophile Haloterrigena turkmenica

10.7287/peerj.preprints.2691 ◽

2017 ◽

Author(s):

Nikolay B Pestov ◽

Daniel V Kalinovsky ◽

Tatyana D Larionova ◽

Alia Z Zakirova ◽

Nikolai N Modyanov ◽

...

Keyword(s):

Catalytic Activity ◽

Amine Oxidase ◽

Lysyl Oxidase ◽

Polyclonal Antibodies ◽

Oxidase Inhibitor ◽

High Yield ◽

Transfer Event ◽

Oxidase Gene ◽

E Coli ◽

Haloterrigena Turkmenica

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Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica

PeerJ ◽

10.7717/peerj.6691 ◽

2019 ◽

Vol 7 ◽

pp. e6691

Author(s):

Nikolay B. Pestov ◽

Daniel V. Kalinovsky ◽

Tatyana D. Larionova ◽

Alia Z. Zakirova ◽

Nikolai N. Modyanov ◽

...

Keyword(s):

Western Blotting ◽

Oxidase Activity ◽

Amine Oxidase ◽

Lysyl Oxidase ◽

Polyclonal Antibodies ◽

Primary Amines ◽

Oxidase Gene ◽

E Coli ◽

Haloterrigena Turkmenica ◽

Amine Oxidase Activity

Background Lysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic. Methods LOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. Results Cultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells. Conclusion H. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic. Significance This is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.

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Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657308 ◽

1983 ◽

Vol 49 (01) ◽

pp. 024-027 ◽

Cited By ~ 20

Author(s):

David Vetterlein ◽

Gary J Calton

Keyword(s):

Monoclonal Antibody ◽

Light Chain ◽

Culture Media ◽

Biological Fluids ◽

Single Step ◽

High Yield ◽

Step Method ◽

Commercial Preparation ◽

E Coli ◽

Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

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Evaluation of germ plasm for resistant to Soybean mosaic virus (SMV)

JURNAL AGRI-TEK Jurnal Penelitian Ilmu-Ilmu Eksakta ◽

10.33319/agtek.v21i1.66 ◽

2020 ◽

Vol 21 (1) ◽

pp. 6-9

Author(s):

Wuye Ria Andayanie

Keyword(s):

Abiotic Stress ◽

Environmental Factors ◽

Mosaic Virus ◽

Polyclonal Antibody ◽

Symptom Severity ◽

Germ Plasm ◽

Polyclonal Antibodies ◽

Soybean Mosaic Virus ◽

High Yield ◽

High Yields

Soybean superior varieties with high yields and are resistant to abiotic stress have been largely released, although some varieties grown in the field are not resistant to SMV. In addition, the opportunity to obtain lines of hope as prospective varieties with high yield and resistance to SMV is very small. The method for evaluating soybean germplasm is based on serological observations of 98 accessions of leaf samples from SMV inoculation with T isolate. The evaluation results of 98 accessions based on visual observations showed 31 genotypes reacting very resistant or healthy to mild resistant category to SMV T isolate with a percentage of symptom severity of 0 −30 %. Among 31 genotypes there are 2 genotypes (PI 200485; M8Grb 44; Mlg 3288) with the category of visually very resistant and resistant, respectively and Mlg 3288 with the category of mild resistant. They have a good agronomic appearance with a weight of 100 seeds (˃10 g) and react negatively with polyclonal antibodies to SMV, except Mlg 3288 reaction is not consistent, despite the weight of 100 seeds (˃ 10 g). Leaf samples from 98 accessions revealed various symptoms of SMV infection in the field. This diversity of symptoms is caused by susceptibility to accession, when infection occurs, and environmental factors. Keywords—: soybean; genotipe; Soybean mosaic virus (SMV); disease severity; polyclonal antibody

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Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

International Journal of Bioassays ◽

10.21746/ijbio.2017.04.004 ◽

2017 ◽

Vol 6 (04) ◽

pp. 5347 ◽

Cited By ~ 3

Author(s):

Omar B. Ahmed* ◽

Anas S. Dablool

Keyword(s):

Dna Extraction ◽

Control Method ◽

Extraction Procedure ◽

Cost Effective ◽

High Yield ◽

Gram Negative Bacteria ◽

Bacterial Dna ◽

Gram Negative ◽

E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

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FRI-314-A novel semicarbazide-sensitive amine oxidase inhibitor, TERN-201, reduces NAS and fibrosis in rodent models of non-alcoholic steatohepatitis

Journal of Hepatology ◽

10.1016/s0618-8278(19)31057-6 ◽

2019 ◽

Vol 70 (1) ◽

pp. e534-e535

Author(s):

Kevin Klucher ◽

Yujin Wang ◽

Yingzi Xu ◽

Randall Halcomb ◽

Martijn Fenaux

Keyword(s):

Amine Oxidase ◽

Oxidase Inhibitor ◽

Rodent Models ◽

Alcoholic Steatohepatitis

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1671. Diagnosis and management of Escherichia coli bacteriuria at a Veterans Affairs hospital

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1849 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S821-S821

Author(s):

Niyati H Shah ◽

Brooke K Decker ◽

Gaetan Sgro ◽

Monique Y Boudreaux-Kelly ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Treatment ◽

High Yield ◽

Comorbid Conditions ◽

Provider Education ◽

Transmitted Infection ◽

Physician Practices ◽

E Coli ◽

Diagnosis And Management ◽

Tract Infections

Abstract Background The IDSA recommends against screening for and treating ASB in all patients except for those pregnant or undergoing urologic procedures. Nevertheless, antibiotic treatment of ASB is widespread. We conducted a retrospective analysis of physician practices in diagnosis and management of Escherichia coli (E. coli) ASB in a male Veteran population, and compared outcomes in ASB patients treated or not treated with antibiotics. Methods Patients with an E. coli positive urine culture during an ED visit or inpatient admission from 01/2017 to 12/2017 were screened. Patients admitted to the intensive care unit or diagnosed with a sexually transmitted infection, pyelonephritis, prostatitis, or epididymitis/orchitis were excluded. A total of 163 patients were included. Demographics, clinical comorbidities and severity of illness, and outcomes were compared in ASB patients managed with or without antibiotics. ANOVA and Chi-square or Fisher’s exact tests were utilized for comparing measurements. Results ASB was present in 92/163 patients. The majority (74%) of these patients were given antibiotics. Regardless of qSOFA score or alternate infection, there were no significant differences in outcomes between ASB patients treated or not treated with antibiotics: 3-month mortality (15% vs 21%; p = 0.53), emergence of newly resistant bacterial pathogens (7% vs 13%; p = 0.43), recurrent urinary tract infections (61% vs 50%; p = 0.72), clearance of urinary pathogens (75% vs 58%; p = 0.45), length of hospital stay (7 vs 6 days, p = 0.67). Factors that were predictive of physician treatment of ASB included patient comorbid conditions such as benign prostatic hyperplasia, pyuria, and the absence of hematuria. The incidence of adverse events with antibiotic treatment of ASB was low. Conclusion The rate of antibiotic treatment of E. coli ASB in male veterans is high. Outcomes do not differ among ASB patients managed with or without antibiotics. Future studies examining outcomes in patients prescribed antibiotics for multiple episodes of ASB may yield differences, particularly in emergence of resistant pathogens. Focusing on patients with comorbid conditions who are not critically ill would be a high yield target for provider education to reduce ASB treatment. Disclosures All Authors: No reported disclosures

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Potential Public Health Significance of Non-Escherichia coli Coliforms in Food

Journal of Food Protection ◽

10.4315/0362-028x-42.2.161 ◽

1979 ◽

Vol 42 (2) ◽

pp. 161-163 ◽

Cited By ~ 10

Author(s):

ROBERT M. TWEDT ◽

BRENDA K. BOUTIN

Keyword(s):

Public Health ◽

Escherichia Coli ◽

Diarrheal Disease ◽

Health Hazard ◽

High Yield ◽

Potential Health ◽

E Coli ◽

Potential Health Hazard ◽

Health Significance

Several coliform species other than Escherichia coli are often associated with and possibly responsible for acute and chronic diarrheal disease. Recent evidence suggests that non-Escherichia coli coliforms may be capable of colonizing the human intestine and producing enterotoxin(s) in high-yield. Whether these organisms are newly capable of causing disease because of infestation with extrachromosomal factors mediating pathogenicity or simply because of inherent pathogenic capabilities that have gone unrecognized, they pose a potential health hazard. Food, medical, and public health microbiologists should be aware that the non-E. coli coliforms contaminating foods may be potential enteropathogens. This possibility may make determination of their pathogenic capabilities even more important than identification of their taxonomic characteristics.

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