Therapeutic methods using bacterial strains which are capable of increasing adenosine levels
A technology for strains, adenosine, applied in the field of products containing such strains to address patient emotional and physical stress, death, inability to progress to proliferative and remodeling stages
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Embodiment 1
[0139] Method for identifying strains with genes encoding cell surface localized 5'-nucleotidases
[0140] Bacteria were grown on MRS plates for 16 h at 37 °C in an anaerobic atmosphere. Ten bacterial colonies were collected with a sterile plastic loop and suspended in 100 microliters of sterile water (PCR quality). (Alternative: prepare DNA from bacterial culture using a suitable method).
[0141]The presence of the 5'-nucleotidase gene can be checked by PCR, for example by using PuReTaq Ready ToGo PCR beads (GE HealthCare) and either of the primer pairs described below (0.4 mM each). The bacterial suspension or DNA prep (0.5 µl) should be added to the PCR mix and the PCR reaction should be performed by running the following program: 95°C, 5 min; 30x (95°C, 30 sec; 55°C, 30 sec ; 72°C, 30 seconds); 72°C, 10 minutes. PCR products can be separated and visualized by using standard agarose gel electrophoresis and sequenced by standard Sanger sequencing (using the forward prime...
Embodiment 2
[0158] Procedure for the analysis of 5'-nucleotidase activity
[0159] The Crystal Chem 5′-nucleotidase assay kit (Crystal Chem, Elk Grove Village, IL, USA) was used to determine the 5′-nucleotidase activity of bacterial cells and fermentation supernatants. Briefly, the procedure is as follows.
[0160]In two steps, reagents CC1 and CC2 were added to samples containing bacteria or supernatants. Reagent 1 contains AMP converted to adenosine by 5'-nucleotidase from bacteria. Adenosine is further hydrolyzed to inosine and hypoxanthine by the components in Reagent 1. In the second step, hypoxanthine is converted to uric acid and hydrogen peroxide, which are used to generate the quinone dye whose kinetics are measured in a spectrophotometer at 550 nm. Activity was determined by calculating the change in absorbance between 3 and 5 minutes and comparing to values from calibration samples.
Embodiment 3
[0162] 5'-Nucleotidase Activity in Lactobacillus reuteri Strains
[0163] Experimental data were generated using the method described in Example 2 above, which showed Lactobacillus reuteri DSM32846, DSM 32847, DSM 32848 and DSM 32849, and Lactobacillus reuteri DSM 17938 (published on January 30, 2006 5'-nucleotidase activity deposited in accordance with the Budapest Treaty at the DSMZ - German Collection of Microorganisms (Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH) (Mascheroder Weg 1b, D-38124 Braunschweig). Results are shown in figure 1 middle.
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