Biodegradation of 4-nitrophenol by a Rhodococcus species and a preliminary insight into its toxicoproteome based on mass spectrometry analysis

Aim: The objective of this study was to understand the possible fate of 4-NP through the molecular mechanism and to identify potential enzymes involved in 4-NP biodegradation by Rhodococcus sp. strain BUPNP1 Methodology: Biodegradation of 4-NP was detected spectrophotometrically at 400 nm and also confirmed by TLC and HPLC. Comparative study of proteomes was performed by 2-D gel electrophoresis followed by peptide mass fingerprinting and bioinformatic analysis to identify and/ or predict the possible functions of over-expressed proteins in 4-NP treated cells of BUNP1. Results: Utilization of 4-nitrophenol and its hydrolysis intermediate 4-nitrocatechol (4-NC) and 1,2,4-Benzenetriol as sole carbon source indicated the presence of genomic information encoding the enzyme necessary for the operation of 4-nitrophenol degradation pathway in the strain BUPNP1. It could transform 4-NP into 4-NC by monooxygenase whose major activity was detected during initial stage of degradation. The 4-NC further depleted in the medium to release nitrite ions. In order to investigate the molecular changes occurring during degradation, a comparative study of proteome profiles was carried out where; 4-nitrophenol treated cells were compared against cells grown on glucose as control. The comparative study indicated expression of several protein spots under 4-nitrophenol treated condition. Interpretation: This study showed the potential of BUPNP1 strain belonging to genus Rhodococcus towards induced expression of some unique proteins which might have possible role in 4-NP biodegradation process

Characterization of entamoeba histolytica antigens in circulating immune complexes in sera of patients with amoebiasis.

Isolated circulating immune complexes (CICs) from sera of patients with amoebiasis were characterized to determine Entamoeba histolytica antigens that participate in the disease process. In total, 116 serum samples were collected before starting anti-amoebic therapy, and their CICs were isolated by differential polyethylene glycol precipitation. The presence of amoeba-specific antigens in CICs was detected by antigen capture enzyme-linked immunosorbent assay (ELISA) and by immunoblot assay. Antigen capture ELISA showed significantly higher optical density (p < 0.001) in all patients with amoebiasis than in the normal healthy controls and patients of non-amoebic hepatic disorder. Immunoblot assay detected amoeba-specific CICs in all 18 patients (100%) with confirmed amoebic liver abscess, 28 (80%) of 35 patients with clinically-suspected amoebic liver abscess, and 18 (78.26%) of 23 patients with amoebic colitis. No patients with non-amoebic hepatic disorders and healthy control subjects had any detectable level of amoebic antigens in CICs. Immunoblot assay revealed E. histolytica antigens of relative molecular masses of 35, 56, 70, and 90 kDa present in CICs of 64 of 76 patients with amoebiasis. The 35-kDa polypeptide was observed in 52 patients (81.25%). The results of the study suggest that the 35-kDa polypeptide antigen can be a diagnostic marker in active amoebiasis.

Comparative evaluation of somatic & excretory-secretory antigens of Entamoeba histolytica in serodiagnosis of human amoebiasis by ELISA.

The excretory-secretory antigens collected from the axenic culture medium and conventional somatic antigen prepared from the whole trophozoites of E. histolytica were compared for their efficacy in serodiagnosis of amoebiasis. A total of 280 sera collected from different clinically proven cases of amoebiasis and healthy subjects were analysed against both the antigens in ELISA. Both antigens showed a 100 per cent correlation in amoebic liver abscess cases, patients infected with enteropathogens other than amoeba and healthy subjects. However, excretory-secretory antigens showed slightly higher detection rate in patients suffering from acute amoebic dysentery and asymptomatic cyst passers groups. These results clearly suggested the use of excretory-secretory antigens by ELISA for serodiagnosis of amoebiasis due to its better or equal sensitivity, specificity and easier preparation compare to conventional antigen. The use of excretory-secretory antigen in serodiagnosis will not only help in performing more tests utilizing the same chemicals, but also save the cost, time and troubles for importing the foreign chemicals required for cultivation of E. histolytica.