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Aim: The aim of the present study was to isolate antifungal protein from seeds of Acacia senegal in order to develop a new, effective and environmental friendly biofungicide.Methodology: Antifungal protein from A. senegal seeds was extracted and purified through ammonium sulphate precipitation, dialysis, ion exchange and gel filtration chromatography. The novel antifungal was characterized employing SDS-PAGE, chitinase activity and antifungal efficacy. The purified protein was also characterized through MALDI-TOF MS/MS. Results: The yield of purified antifungal protein was estimated to be 0.96 mg 25 g-1 seeds and its molecular mass determined by SDS PAGE was 52.9 kDa. The purified protein exhibited antifungal activity against phytopathogenic fungi viz., Macrophomina phaseolina and Fusarium oxysporum also possessed chitinase activity. The purified protein was characterized through MALDI-TOF MS/MS and its spectra revealed 14 peptides with their specific amino acid sequences. Interpretation: The antifungal protein isolated from A. senegal seeds has broad-spectrum antifungal activity with chitinase activity against pathogenic fungi that can be exploited for management of fungal disease as biopesticide to promote sustainable agriculture
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Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.
Subject(s)
Aldose-Ketose Isomerases , Arabidopsis , Arabidopsis Proteins , Cloning, Molecular , Escherichia coli , Metabolism , Metals , Pentosephosphates , Recombinant ProteinsABSTRACT
Objective Pathogens from the nosocomial infection have been analyzed by MALDI‐TOF microbial identification system ,to evaluate mass spectrometry analysis advantage and explore the mass spectrometry method .Methods The pathogens have been analyzed by MALDI‐TOF microbial identification system ,by compared with the VITEK‐2 compact detection in the tes‐ting time ,detection rate and the amounts of identified strains .The homology differences have been analyzed by comparison calcula‐tion of common peaks from the fingerprint spectrums .Results Thirty‐one Escherichia coli strains ,28 Klebsiella pneumonia strains and 9 unusual pathogen strains have been identified by MALDI‐TOF MS for only 1 hours .It has more advantages than VITEK‐2 in the testing time and other aspects .Conclusion Nosocomial infection of pathogen shows a point source propagation mode centering on the department .MALDI‐TOF mass spectrometry is able to rapidly and correctly identify the pathogen .MALDI‐TOF microbial i‐dentification system is expected to be the major detecting technique in the field of the pathogen monitor and resistance monitoring a ‐nalysis .
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Objective To study the expression ot secreted proteins in aggregated dermal papilla cells (DPCs).Methods DPCs were isolated from human scalp tissue and subjected to primary culture and subculture.Aggregated and non-aggregated DPCs served as the subject of this study.Secreted proteins were prepared from these cells and subjected to two-dimensional polyacrylamide gel electrophoresis.Differentially expressed proteins were screened by the PDQuest image analysis software.Protein spots were digested and identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,and finally analyzed using the National Center for Biotechnology Information (NCBI) non-redundant (Nr) protein database.Results Two-dimensional electrophoresis maps with good repeatability and high resolution were established.Image analysis of 2-D gels revealed that the average number of detected protein spots was 1 134 ± 52 and 1 078 ± 36 in aggregated and nonaggregated DPCs respectively,and the majority of these protein spots were matched between aggregated and nonaggregated DPCs.Twenty-eight protein spots showed more than 5-fold difference between the two groups of cells,and 10 proteins were preliminarily identified as differentially expressed proteins by peptide-mass fingerprinting.Of these differentially expressed proteins,8 proteins including Rhogdi 1,filamin A,cystatin C,fibronectin,cyclophilin A,procollagen C proteinase enhancer 1,tissue inhibitor of metalloproteinase and tissue inhibitor of metalloproteinase-2 were up-regulated,and 2 proteins including neuropolypeptide h3 and matrix metalloproteinase-3/tissue inhibitor of metalloproteinase-1 complex were down-regulated in aggregated DPCs compared with non-aggregated DPCs.Conclusions Differentially expressed proteins between aggregated and non-aggregated DPCs are mainly implicated in cell signaling pathway,cellular proliferation and differentiation,extracellular matrix synthesis and degradation,and so on.
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BACKGROUND: Microbiological laboratories seek technologically innovative solutions to cope with large numbers of samples and limited personnel and financial resources. One platform that has recently become available is the Kiestra Total Laboratory Automation (TLA) system (BD Kiestra B.V., the Netherlands). This fully automated sample processing system, equipped with digital imaging technology, allows superior detection of microbial growth. Combining this approach with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) (Bruker Daltonik, Germany) is expected to enable more rapid identification of pathogens. METHODS: Early growth detection by digital imaging using Kiestra TLA combined with MS was compared to conventional methods (CM) of detection. Accuracy and time taken for microbial identification were evaluated for the two methods in 219 clinical blood culture isolates. The possible clinical impact of earlier microbial identification was assessed according to antibiotic treatment prescription. RESULTS: Pathogen identification using Kiestra TLA combined with MS resulted in a 30.6 hr time gain per isolate compared to CM. Pathogens were successfully identified in 98.4% (249/253) of all tested isolates. Early microbial identification without susceptibility testing led to an adjustment of antibiotic regimen in 12% (24/200) of patients. CONCLUSIONS: The requisite 24 hr incubation time for microbial pathogens to reach sufficient growth for susceptibility testing and identification would be shortened by the implementation of Kiestra TLA in combination with MS, compared to the use of CM. Not only can this method optimize workflow and reduce costs, but it can allow potentially life-saving switches in antibiotic regimen to be initiated sooner.