scholarly journals Cloning and secretory expression of functional diisopropyl-fluorophosphatase (DFPase) in Bacillus subtilis

2021 ◽  
Vol 23 (2) ◽  
pp. 87-92
Author(s):  
Ali Ahmadizad Firouzjaei ◽  
Shamsozzoha Abolmaali ◽  
Samaneh Khodi ◽  
Hossein Aghamollaei ◽  
Ali Mohammad Latifi ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Active Form ◽  
Protein Band ◽  
Secretory Expression ◽  
Diisopropyl Fluorophosphate ◽  
Pcr Product ◽  
Sds Page ◽  
Polymerase Chain ◽  
First Time ◽  
35 Kda Protein

Background and aims: Synthetic organophosphates (OPs) inhibit acetylcholinesterase resulting in the accumulation of acetylcholine, failure of organs, and eventually death. Diisopropyl-fluorophosphatase (DFPase) is one of the OPs degrading enzymes that has broad substrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order to accelerate the biodegradation rate of OPs. Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET28-inaV/N-dfpase plasmid. The PCR product was subcloned in the pWB980 plasmid. Competent B. subtilis WB600 were transformed with recombinant plasmid. SDS PAGE technique was used to study the expression of protein secreted in superrich medium. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Approximately, 35 kDa protein band was shown in culture medium after incubating at 35°C for 72 hours and 150 rpm. Measurement of enzyme’s activity was done by monitoring the release of fluoride from diisopropyl fluorophosphate (DFP), using ion-meter. Results showed that enzyme’s activity was 3333 U/L. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.

Characterization of cytoplasmic fibril structures found in gliding cells of Saprospira sp.

2005 ◽  
Vol 51 (10) ◽  
pp. 875-880 ◽  
Author(s):  
Gou Furusawa ◽  
Takeshi Yoshikawa ◽  
Yoshitaka Takano ◽  
Kazuyuki Mise ◽  
Iwao Furusawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Band ◽  
Amino Acid Sequences ◽  
Gliding Motility ◽  
Pcr Product ◽  
Nutrient Agar Medium ◽  
Sds Page ◽  
Tail Sheath ◽  
A Subunit ◽  
Triton X

The cytoplasmic fibril structures of Saprospira sp. strain SS98-5 grown on a low-nutrient agar medium were purified from cell lysates treated with Triton X-100 and were observed by electron microscopy to be about 7 nm in width and 200–300 nm in length. SDS–PAGE of the fibril structures exhibited a single protein band with a molecular mass of 61 kDa. A Saprospira cytoplasmic fibril protein (SCFP), which is a subunit of the fibril structures, was digested with trypsin to oligopeptides and analyzed for amino acid sequences. A partial nucleotide sequence of the SCFP gene was determined after PCR using primers designated from the amino acid sequences of the oligopeptides. SCFP gene including DNA fragments were detected by Southern hybridization using the PCR product for an SCFP gene as a probe and were cloned to determine whole nucleotide sequences. The SCFP gene indicated relatively higher similarity to conserved hypothetical phage tail sheath proteins. A Western immunoblotting analysis showed that SCFP was significantly expressed in gliding cells as compared with nongliding cells. The above findings with the previously reported results suggest that the cytoplasmic fibril structures are possibly related to the gliding motility of Saprospira sp. strain SS98-5.Key words: Saprospira, gliding motility, Saprospira cytoplasmic fibril protein (SCFP).

Cloning and expression of Ma duck interleukin-18 mature protein gene and biological activity detection

2007 ◽  
Vol 4 (3) ◽  
pp. 207-212
Author(s):  
Chen Hong-Ying ◽  
Cui Bao-An ◽  
Xia Ping-An ◽  
Li Xin-Sheng ◽  
Yang Ming-Fan ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Protein Gene ◽  
Haemagglutination Inhibition ◽  
Cloning And Expression ◽  
Mature Protein ◽  
Oil Emulsion ◽  
Pcr Product ◽  
Sds Page ◽  
Polymerase Chain

AbstractDuck interleukin (IL)-18 mature protein gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from Ma duck (Tadorna ferruginea) splenocytes. The PCR product was cloned into pGEM-T Easy vector for sequencing. The result revealed that the nucleotide sequence of duck IL-18 mature protein gene (mDuIL-18) consisted of a 513 bp band. A prokaryotic plasmid of mDuIL-18, pQE30-mDuIL18, was obtained by subcloning the encoding region of the DuIL-18 mature peptide into pQE30. pQE30-mDuIL18 transformed Escherichia coli M15. The expression of mDuIL-18 gene was identified by SDS-PAGE and Western blotting. Its molecular weight was 19.76 kDa, and could be specifically recognized by rabbit sera to chicken IL-18. The expressed products existed as inclusion bodies. After being degenerated, then renatured, the activities of the inclusion bodies were detected by methyl thiazolyl tetrazolium (MTT) assay. In ducks injected intramuscularly with mDuIL-18 protein (150 ng or 200 ng per duck) and Avian influenza virus (AIV) vaccine 2 weeks after immunization, the average titres of haemagglutination inhibition (HI) antibodies to AIV reached 7.5–7.7 log2, while those of HI antibody ranged between 6.3 and 6.6 log2 in ducks vaccinated with AIV vaccine only or with 100 ng mDuIL-18 and AIV vaccine. The results clearly showed that 150 ng mDuIL-18 per duck strengthened the in vivo immune responses induced by the inactivated oil emulsion AIV vaccine.

scholarly journals Antifreeze proteins in naturally cold acclimated leaves of Drimys angustifolia, Senecio icoglossus, and Eucalyptus ssp.

2016 ◽  
Vol 19 (0) ◽  
Author(s):  
João Gustavo Provesi ◽  
Pedro Alexandre Valentim Neto ◽  
Ana Carolina Maisonnave Arisi ◽  
Edna Regina Amante
Keyword(s):  
Potential Application ◽  
Protein Concentration ◽  
Antifreeze Proteins ◽  
Protein Band ◽  
Frozen Foods ◽  
Intense Band ◽  
Ice Recrystallization ◽  
Low Concentrations ◽  
Sds Page ◽  
First Time

Summary Antifreeze proteins (AFPs) present in plants may inhibit ice recrystallization even at low concentrations, and show potential application to many frozen foods. This study evaluated the presence of antifreeze proteins in naturally cold acclimated and non-acclimated leaves of Drimys angustifolia, Senecio icoglossus and Eucalyptus ssp. No proteins were detected in apoplastic extracts of Eucalyptus ssp. Extracts of cold acclimated and non-acclimated S. icoglossus showed protein concentrations of 42.89 and 17.76 µg mL-1, both with bands between 25 and 37 kDa in the SDS-PAGE. However, they did not inhibit recrystallization. The extract of cold acclimated D. angustifolia contained a protein concentration of 95.17 µg mL-1, almost five times higher than the extract of non-acclimated D. angustifolia. In the extract of cold acclimated D. angustifolia, there was presence of ice recrystallization inhibitors. This extract showed a protein band just below 37 kDa and another more intense band between 20 and 25 kDa. It is the first time that the presence of antifreeze proteins in this species is being described.

scholarly journals CS22, a Novel Human EnterotoxigenicEscherichia coli Adhesin, Is Related to CS15

2000 ◽  
Vol 68 (6) ◽  
pp. 3280-3285 ◽  
Author(s):  
Mariana Pichel ◽  
Norma Binsztein ◽  
Gloria Viboud
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Bacterial Surface ◽  
Pcr Product ◽  
Defective Mutant ◽  
Sds Page ◽  
Structural Subunit ◽  
Colonization Factors

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H− strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from thenfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.

An unusual case of spinal cord restricted mycobacteriosis in a European mink

2013 ◽  
Vol 41 (01) ◽  
pp. 63-66
Author(s):  
D. Schaudien ◽  
C. Flieshardt ◽  
I. Moser ◽  
H. Hotzel ◽  
A. Tipold ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Unusual Case ◽  
Histological Evaluation ◽  
Mustela Lutreola ◽  
Chain Reaction ◽  
European Mink ◽  
Pcr Product ◽  
Granulomatous Lesions ◽  
The Central Nervous System ◽  
Polymerase Chain

SummaryGranulomatous myelitis due to infection with Mycobacterium avium was diagnosed in a 4-year-old male neutered European mink (Mustela lutreola). The causative agent was detected by an acid-fast stain and further characterized by polymerase chain reaction and DNA sequencing of the PCR product. A thorough histological evaluation of the remaining organs revealed no granulomatous lesions or detectable acid-fast organisms. Although minks are generally highly susceptible for mycobacteria, localised infections, especially of the central nervous system, are unusual and may represent an atypical chronic form of the disease.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

scholarly journals Granulocytic anaplasmosis in captive ring-tailed lemur (Lemur catta) in Poland

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Łukasz Adaszek ◽  
Anna Wilczyńska ◽  
Jerzy Ziętek ◽  
Marcin Kalinowski ◽  
Oliwier Teodorowski ◽  
...  
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Polymerase Chain Reaction Test ◽  
Anaplasma Ovis ◽  
Case Presentation ◽  
Granulocytic Anaplasmosis ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Obligate Intracellular Bacteria ◽  
Chain Reaction Test ◽  
Anaplasma Bovis

Abstract Background Anaplasma are obligate intracellular bacteria and aetiological agents of tick-borne diseases of both veterinary and medical interest. The genus Anaplasma comprises six species: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma bovis and Anaplasma platys. They can infect humans, carnivores, ruminants, rodents, insectivores, birds and reptiles. The aim of this study was to present the first clinical case of granulocytic anaplasmosis in a captive ring-tailed lemur in Poland. Case presentation A 4-year-old female lemur presented anorexia, epistaxis and tick infestation. The microscopic examination of a blood smear revealed morulae in neutrophils. Polymerase chain reaction test and sequencing of obtained PCR product confirmed infection by the GU183908 Anaplasma phagocytophilum strain. Therapeutic protocol included doxycycline (2.5 mg/kg p.o., b.i.d.) for 3 weeks and the lemur recovered within 24 h. Conclusions This is the first report on granulocytic anaplasmosis in a ring-tailed lemur in Europe, indicating that A. phagocytophilum infection must also be considered in differential diagnosis in this animal species, especially in individuals with thrombocytopenia associated with Ixodes ricinus parasitism.

scholarly journals A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes

1989 ◽  
Vol 86 (17) ◽  
pp. 6691-6695 ◽  
Author(s):  
B Davies ◽  
S Feo ◽  
E Heard ◽  
M Fried
Keyword(s):  
Ribosomal Protein ◽  
Recombinant Dna ◽  
Single Gene ◽  
Ribosomal Protein Gene ◽  
Pcr Primers ◽  
Pcr Product ◽  
Dna Libraries ◽  
Processed Pseudogenes ◽  
Polymerase Chain ◽  
Genomic Dna Sequence

We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns.

scholarly journals Rickettsia typhi IN RODENTS AND R. felis IN FLEAS IN YUCATÁN AS A POSSIBLE CAUSAL AGENT OF UNDEFINED FEBRILE CASES

2015 ◽  
Vol 57 (2) ◽  
pp. 129-132 ◽  
Author(s):  
Gaspar PENICHE-LARA ◽  
Karla DZUL-ROSADO ◽  
Carlos PÉREZ-OSORIO ◽  
Jorge ZAVALA-CASTRO
Keyword(s):  
Rattus Rattus ◽  
Conventional Polymerase Chain Reaction ◽  
Causal Agent ◽  
Wild Rodents ◽  
Murine Typhus ◽  
Rickettsia Typhi ◽  
Rickettsia Felis ◽  
Vector Borne ◽  
Polymerase Chain ◽  
First Time

Rickettsia typhi is the causal agent of murine typhus; a worldwide zoonotic and vector-borne infectious disease, commonly associated with the presence of domestic and wild rodents. Human cases of murine typhus in the state of Yucatán are frequent. However, there is no evidence of the presence of Rickettsia typhi in mammals or vectors in Yucatán. The presence of Rickettsia in rodents and their ectoparasites was evaluated in a small municipality of Yucatán using the conventional polymerase chain reaction technique and sequencing. The study only identified the presence of Rickettsia typhi in blood samples obtained from Rattus rattus and it reported, for the first time, the presence of R. felis in the flea Polygenis odiosus collected from Ototylomys phyllotis rodent. Additionally, Rickettsia felis was detected in the ectoparasite Ctenocephalides felis fleas parasitizing the wild rodent Peromyscus yucatanicus. This study’s results contributed to a better knowledge of Rickettsia epidemiology in Yucatán.

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