Evaluation of the presence of reduced nicotinamide adenine dinucleotide phosphate in bacterial metabolites used as immunostimulators and its role in nitric oxide induction

Abstract

Bacterial metabolites that act as immunostimulators have aroused interest because of their therapeutic potential in several immune disorders. These metabolites are complex, heterogeneous, and comprise numerous immune-boosting biomolecules. To better understand their immune stimulatory properties, characterization of their components is essential. An ether extract of metabolites from nine bacterial species was analyzed for the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) or other fluorophores. This metabolite in combination with bile lipids is a licensed immune stimulatory drug. Excitation of the extract at 340 nm resulted in fluorescence with an emission maximum of around 410 nm, which is fairly specific for NADH and NADPH. Reverse-phase-HPLC and electro-spray ionization-mass analysis confirmed the presence of NADPH in the bacterial metabolites. Quantification by glutathione reductase assay indicated 11.90 AE 0.01 mM of NADPH in the metabolites. Further characterization of the individual bacterial extracts of the metabolite confirmed the presence of NADPH. Subsequently, studies were performed to evaluate the role/s of NADPH in immune-stimulatory drugs. NADPH is known to be involved in production of nitric oxide (NO), which has versatile roles in the immune system. The biological function of NADPH in NO induction by RAW 264.7 (mouse macrophage) cells was evaluated and it was found that bacterial NADPH has a significant role in inducing NO and that NADPH from individual bacterial extracts is capable of inducing NO. Investigation on the stability and biological potency of NADPH in bacterial metabolites is important because of NADPH's wide therapeutic applications, most of which are associated with its role in NO induction.

Cholesterol and Its Derivatives Reversibly Inhibit Proteinase K

“…The nonspecific immune stimulatory drug used in this study was manufactured and supplied by M/s Union Drugs Co. Ltd., Kolkata, India under the trade name “Sterodin.” It is constituted of an ether extract of nine bacterial metabolites in combination with solvent extracts of ox bile lipids. Composition of the final aqueous solution has been described earlier (Singh and Bhattacharyya, , , ).…”

Novel gene similar to nitrite reductase (NO forming) plays potentially important role in the latency of tuberculosis

“…Subsequently, extensive characterization of the cloned and purified MRA2164 protein under different experimental conditions has clearly showed that it is functionally very similar to NirK protein known to be present in other bacteria (Figs. 4 , 5 , S4 ) 36 – 38 . The Km of this enzyme was found to be ~ 17 mM, which justifies the level of NO produced at > 5 mM of nitrite in the medium (Figs.…”

Ultrasensitive detection of β-lactamase-associated drug-resistant bacteria using a novel mass-tagged probe-mediated cascaded signal amplification strategy

“…4C). [37][38][39][40] Additionally, no signicant signal was observed aer the MP-CMSA probe was incubated with non-BLA-expressing E. coli ATCC 25922, that is the Clinical and Laboratory Standards Institute (CLSI) recommended negative quality control strain. 41 Comparatively, a remarkable MS signal can be detected in three clinically prevalent BLA-expressing bacterial strains including K. pneumonia/KPC-3 ATCC BAA-2814, K. pneumonia/SHV-18 ATCC 700603 and E. coli/TEM-1 ATCC 35218 (Fig.…”