Development of Immunomodulatory Properties L-Lysine-Α-Oxidase Under Irradiation of Animals

 

I. P. Smirnova1, S. P. Syatkin4, T. A. Lobaeva1, A. S. Skorik1, S. M. Chibisov2, M. L. Blagonravov2, G. I. Myandina3, V. I. Kuznetsov3

1T. T. Berezov Department of Biochemistry of PFUR. Moscow, Medical faculty

2Department of Pathological Physiology of PFUR. Moscow. Medical faculty

3Department of Biology and General Genetics of PFUR. Moscow, Medical faculty

4Peoples’ Friendship University of Russia (PFUR). Miklukho-Maklaya str., 8, Moscow, Russia

*Corresponding Author E-mail: russia@prescopus.com 

 

ABSTRACT:

We studied the immunomodulatory activity of L-lysine-α-oxidase (LO) from Trichoderma harzianum Rifai F-180 to model endogenous colony-forming cells surface of the spleen of the mice of line F/SVA С157В1. LO shows immunomodulatory activity on models in vivo and in vitro.

 

KEYWORDS: L-lysine-α-oxidase, x-ray, immunomodulatory activity.

 

 


INTRODUCTION:

At T. T. Berezov Department of Biochemistry of PFUR we conducted studies of the enzyme L-lysine-α-oxidase from Trichoderma harzianum Rifai F-180 for several years. The purpose of this work is to study the immunomodulatory properties of L-lysine-α-oxidase under irradiation f animals.

 

Methodology of the study:

Getting of l-lysine-α-oxidase. In the experiments we used the producer of L-lysine-α-oxidase (LO) Trichoderma harzianum Rifai F-180 grown on the previously described method [1].

 

Activity was determined by spectrophotometrically ontogenetically method, the selection and purification of the enzyme were performed according to previously developed methods [2]. In the experiment we used the LO with the activity of 70 ng/ml.

 

Evaluation of the influence of LO was conducted on models of endogenous colony-forming cells surface of the spleen of the mice of line F/SVA С157В1 after the introduction of the protein according to the method described in the monograph of Petrov R. V., Khaitov M.[3]. Protein dose of 0.5 mg. was twice intravenously injected to animals. On the third day the animals were irradiated with a dose of 600 R (roentgen) on apparatus RUM-17. After exposure, the drug was injected three more times at the same dose. In the experiment, the animals were divided into four groups:

1-irradiated animals not immunized with LO;

2 - irradiated animals immunized with LO;

3 - irradiated animals, to which the cells of the lymph nodes parental genotype;

4 - irradiated animals, to which the cells of the lymph nodes of parental genotype with the introduction of the LO were injected.

 

Lymphocytes dose of mice SVA, causing 50% inactivation of colony-forming cells (CFU) was 1X 106 cells. On the 9th day, the mice were killed, spleens were removed and after fixation the number of colonies on the surface of the body was counted. Cell line MT-4 (lymphoblastoid cell line of human cultured in vitro) was cultivated on medium RPM1-1640) containing 10% fetal calf serum and 100 u/ml of ampicillin. Counting of cells was performed by the camera of Goryaeva[4]. Determining of the number of dead cells was performed by staining cells with trypan blue. Rate of DNA synthesis was followed by incorporation into the acid-insoluble fraction of 3H-thymidine. The final concentration of label was 5 mu/ml After 1 hour of incubation with the labeled precursor to precipitate cells MT-4 (1X 106) were added to 5% trichloroacetic acid, cooled to a temperature of 45° C; the precipitate was washed 3 times with cold acid and then the precipitate was dissolved in 1 ml of 0.1 n NaOH. 0.1 of the sample was applied on filters for counting of radioactivity[5].

 

The radioactivity of the samples were recorded on spectrometer in toluene-based fluid (10 ml) for 1 min.

 

DISCUSSION OF OBTAINED RESULTS:

Table 1 presents the results of determination of immunomodulatory activity of LO. It is seen that the introduction of drug causes stimulation of colony-forming cells CFU by more than two times, indicating the presence of pronounced immunomodulatory properties of LO. It is also clear that the immunomodulatory activity of the enzyme is retained and in the experiment with the inhibition of endogenous colony-forming cells in the lymph nodes of the parent genotype. The difference between experience and control is 181%. The results indicate that LO has the immunomodulatory activity.

 


Table 1. Stimulation of L-lysine-α-oxidase for incensement the number of formation of endogenous colony-forming cells.

Analysis variants

The average number of endogenous colonies on the spleen of sublethal irradiated mice after injection of LO.

The average number of endogenous colonies of sublethal irradiated mice after injection of 1 mln of lymphocytes with parental genotype and LO

Р

1. Control (natural saline solution)

12,18±2,76

3,8±0,53

 >0,05

2. L-lysine-α-oxidase

30,9±4,6

6,9±1,22

 >0,05

 

Time in hours

 In the presence of the enzyme  Control

Fig. (1). The inclusion of marked thymidine at different stages of cell growth.

 


However, the results obtained by the study of LO stimulation of formation of endogenous colonies did not exclude the possibility that LO will not be have the endogenous stimulation of cells cultured in vitro. With this prose we made the analysis of the stimulation of the replicative enzyme of synthesis used in the lymphoblastoid cell line MT-4 that are known to have actually high replicative potential.

 

Results of study the rate of DNA synthesis by incorporation of marked thymidine incorporation at different stages of cell growth are presented in figure No. 1.

It is seen that after 2 days of culture growth LO slightly stimulates the replicative synthesis of the cells, which is explained by its own high rate of cell division. 3rd day is characterized by an already seen difference in the rate of DNA synthesis between experimental and control cultures. By the 7th day of growth in the presence of LO the intensity of biosynthesis of DNA is more than 2 times higher than the control level.

 

n subsequent experiments, it was interesting to find out whether enzyme is toxic in test concentrations as immunomodulator.

The results are presented in table No. 2.


Table No. 2. Determination of the cytotoxic effect of different concentrations of LO-immunomodulator on cells MT-4.

Concentration of LO (ng/ml)

700

70

7

0,7

Cytotoxicity of LO

Moderately toxic

 Non-toxic

Non-toxic

 Non-toxic

 


From table No. 2 we can see, LO from Trichoderma harzianum Rifai F-180 is non-toxic within a concentration of 0.7-70 ng/ml and can be used as substance of the drug with immunomodulatory activity.

 

CONCLUSIONS:

Thus, L-lysine-α-oxidase from Trichoderma harzianum Rifai F-180 shows immunomodulatory activity on models in vivo and in vitro.

 

CONFLICT OF INTEREST:

The author confirms that the submitted data does not contain conflict of interests.

 

REFERENCES:

1.        Smirnova I. P., Skinev V. M., Rudnev A. V., Shneider Yu. A., Kuzovnikov A. E., Technology of selecton and purification of L-lysine-α-Oxidase. Journal. Biotechnology, 2010 No. 6, p. 47-54.

2.        Smirnova I. P., Syatkin S. P., Berezov T. T. Spectrophotometric Micromethod of determination of L-Lysine-α-Oxidase. // Journal. Questions of medicinal chemistry, 1984, No. 1, p. 133-136.

3.        Petrov R. V., Khairov R. M. Control and regulation of the immune response. - L., - 1981 - p. 310-311.

4.        I. P. Smirnova, O. M. Kuznetsova, V. I. Ivanova-Radkevich, V. S. Orlova, V. M. Podboronov, A. A. Alexeev. Testing of an Antitumor Enzyme L-lysine-A-oxidase from Trichoderma Harzianum Rifai F-180. World Journal of Medical Sciences 2014. 11 (2): 233-236

5.        I. P. Smirnova, Yu. A. Shneider, and E. V. Karimova. Trichoderma L-Lysine-α-Oxidase Producer Strain Culture Fluid Inhibits Impatiens Necrotic Spot Virus. // Bulletin of Experimental Biology and Medicine Vol. 160, No. 3, January, 2016 Virology, P. 357-359.

 

 

 

 

 

Received on 22.01.2017             Modified on 15.02.2017

Accepted on 07.03.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(3): 674-676.

DOI: 10.5958/0974-360X.2017.00125.1