**2.2.3 Results**

The studies disclosed significant decreased levels of apoptotic proteins (p53, Bax:Bcl-2, PARP, p<0.01; Bax, Bcl-2, p<0.001) in PD as compared to the controls (Table 4). Decreased level of apoptotic proteins in PD patients probably was result influence of α–synuclein on lower p53 protein expression and caspase-3 activity.


Table 4. Level of p53, Bax, Bcl-2, PARP proteins and of 85-kDa protein subunit in peripheral blood lymphocytes in PD patients and in the control group. Apoptotic proteins represent % of area of immunoreactivity bonds. Results are expressed as a means ± SD. The nonparametric test of Mann-Whitney was used. Differences significant at \*\*p<0.01; \*\*\*p<0.001 as compared to the controls.

Simultaneously, in PD patients treated with L-dopa preparations (Table 5) levels of p53, Bax, Bcl-2 proteins increased unsignificant as compared with untreatment patients. In the PD patients treated with L-dopa significant increased only the levels of PARP protein (p<0.001 as compared to patients not treated with L-dopa) and 85-kDa fragment (p<0.01 as compared to patients untreated with L-dopa).

protease inhibitor cocktail (Sigma) and homogenized in a mixture of RIPA with protease inhibitor cocktail (16:1) and 0.5 µl PSMF (Sigma) in isopropanol (10 mg/100 µl), centrifuged, and the obtained supernatant underwent further analysis (Ohnishi et al.,

*Western Blot.* The Bax and Bcl-2 proteins were analyzed in 12% and p53, and PARP proteins were analyzed in 7.5% polyacrylamide gel. Equivalent amounts of protein (30 µg protein/lane) were loaded to the wells. The gel-separated proteins were electrotransferred to nitrocellulose filter in a semidry Western Blot analysis apparatus (Apelex, France). To estimate the levels of the PARP protein, the filters were exposed first to an anti-PARP monoclonal antibody (G-2-10, IgG, 0.05 ml, Sigma, USA), diluted 1:2000, while the p53, Bax, Bcl-2 proteins were identified using anti-p53 (IgG-2a, 200 µg/1.0 ml; Santa Cruz, USA), anti-Bax (IgG-2b, 200 µg/1.0 ml; Santa Cruz, USA) and anti-Bcl-2 (IgG-1, 200 µg/1.0 ml; Santa

Subsequently, individual sheets of nitrocellulose filter were incubated with the second antibody, goad antimouse IgG-HRP (200 µg/0.5 ml; Santa Cruz, USA) at a dilution of 1:2000. To stain immunoreactive bands, peroxidase BMB was added (BM blue POD substrate precipitation; Roche, Germany). The surface area of the immunoreactive bands was registered using a densitometer (GS-710; Bio-Rad, Hercules, CA) in the Quantity One

The studies disclosed significant decreased levels of apoptotic proteins (p53, Bax:Bcl-2, PARP, p<0.01; Bax, Bcl-2, p<0.001) in PD as compared to the controls (Table 4). Decreased level of apoptotic proteins in PD patients probably was result influence of α–synuclein on

> **PD patients 41-81 years of age**

**35-73 years of age** 

of area of immunoreactivity bonds. Results are expressed as a means ± SD. The

p53 0.52 ± 0.37 0.25 ± 0.14\*\* Bax 0.60 ± 0.50 0.13 ± 0.07\*\*\* Bcl-2 1.20 ± 0.77 0.15 ± 0.08\*\*\* Bax/Bcl-2 1.46 ± 3.77 1.13 ± 0.83\*\* PARP 2.12 ± 0.83 1.61 ± 1.12\*\* 85-kDa 0.42 ± 0.80 0.41 ± 0.31 Table 4. Level of p53, Bax, Bcl-2, PARP proteins and of 85-kDa protein subunit in peripheral blood lymphocytes in PD patients and in the control group. Apoptotic proteins represent %

nonparametric test of Mann-Whitney was used. Differences significant at \*\*p<0.01; \*\*\*p<0.001

Simultaneously, in PD patients treated with L-dopa preparations (Table 5) levels of p53, Bax, Bcl-2 proteins increased unsignificant as compared with untreatment patients. In the PD patients treated with L-dopa significant increased only the levels of PARP protein (p<0.001 as compared to patients not treated with L-dopa) and 85-kDa fragment (p<0.01 as compared

Cruz, USA) mouse monoclonal antibody, respectively, diluted 1:500.

lower p53 protein expression and caspase-3 activity.

**Parameter Controls** 

1996).

System.

**2.2.3 Results** 

as compared to the controls.

to patients untreated with L-dopa).


Table 5. Level of p53, Bax, Bcl-2, PARP proteins and of 85-kDa protein subunit in peripheral blood lymphocytes in PD patients untreatment L-dopa (-) and treatment L-dopa (+), and in the control group. Apoptotic proteins represent % of area of immunoreactivity bonds. Results are expressed as a means ± SD. The nonparametric test of Kruskal-Wallis was used. Differences significant at \*p<0.05; \*\*p<0.01; \*\*\*p<0.001 as compared to the controls, and (\*\*)p<0.01; (\*\*\*)p<0.001 between PD patients untreatment L-dopa (-) and treatment L-dopa (+).

However, (Table 6) long-term (more than 5 years) therapy of L-dopa in PD patients probably leads to apoptosis, because elevated levels of Bax:Bcl-2 ratio (p<0.05 as compared to the controls) and 85-kDa fragment (p<0.05 as compared to the controls).


Table 6. Level of p53, Bax, Bcl-2, PARP proteins and of 85-kDa protein subunit in peripheral blood lymphocytes in PD patients treatment L-dopa less and more than 5 years, and in the control group. Apoptotic proteins represent % of area of immunoreactivity bonds. Results are expressed as a means ± SD. The nonparametric test of Kruskal-Wallis was used. Differences significant at \*p<0.05; \*\*p<0.01; \*\*\*p<0.001 as compared to the controls.

It seems that pharmacological treatment of PD patients with L-dopa has a major role in modulating of levels in lymphocytes of some apoptotic proteins, important for this process. Further investigation is thus requisite to analysis expression and mutations of genes encoding proteins important for effective repair and/or apoptosis in PD patients treatment with L-dopa.
