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1.  Serum activity of DPPIV and its expression on lymphocytes in patients with melanoma and in people with vitiligo 
BMC Immunology  2012;13:48.
Dipeptidyl peptidase IV, a multifunctional serine protease, is implicated in regulation of malignant transformation, promotion and further progression of cancer, exerting tumor-suppressing or even completely opposite - tumor-promoting activities.
The aim of present research was to determine the serum DPPIV activity, as well as the percentages of CD26+ lymphocytes, CD26+ overall white blood cells and the mean fluorescence intensity of CD26 expression on lymphocytes in patients with melanoma, people with vitiligo and in healthy controls.
The activity of DPPIV in serum was determined by colorimetric test. Expression of DPPIV (as CD26) on immunocompetent peripheral white blood cells was done using flow cytometry analysis.
Data from our study show for the first time statistically significant decrease: in the serum DPPIV activity, in the percentage of CD26+ overall white blood cells and in the percentage of lymphocytes in patients with melanoma in comparison to healthy control people. In addition, significantly lower serum DPPIV activity was found in the group of patients with melanoma in relation to people with vitiligo too.
This study indicates the need for exploring the cause and the importance of the disturbances in the serum DPPIV activity and in the CD26 expression on immunocompetent cells in complex molecular mechanisms underlying the development and progression of melanoma.
PMCID: PMC3464610  PMID: 22908963
CD26 expression; DPPIV serum activity; Melanoma; Vitiligo
2.  Immunity to melanin and to tyrosinase in melanoma patients, and in people with vitiligo 
The aim of this study was to determine the presence and the intensity of humoral immunity to melanoma-associated antigens: tyrosinase and melanin, in patients with melanoma, in persons with vitiligo and in control healthy people.
The study involved 63 patients with melanoma and 19 persons with vitiligo. Control group consisted up to 41 healthy volunteers. Mushroom tyrosinase and synthetic melanin were used as the antigens.
ELISA test showed significantly (p < 0.0000004 and p < 0.04) lower levels of IgM anti-tyrosinase autoantibodies, in melanoma and vitiligo patients respectively, compared to controls.
Although there was no significant difference between the levels of IgA anti-melanin autoantibodies in melanoma or vitiligo patients in comparison with controls, the enhanced concentrations of anti-melanin IgA autoantibodies were preferentially found in melanoma patients with metastatic disease. Significantly high percentage in the Fc alphaRI (CD89) positive cells was determined in melanoma patients (p < 0.002 and p < 0.008) in comparison to that found in healthy people or in patients with vitiligo, in the already mentioned order, pointing that IgA dependent cellular cytotoxicity is not important for the immune action against melanoma, even more that it is included in some immune suppression.
Levels of IgG autoantibodies to mentioned antigens in melanoma patients although low were not significantly lower from controls. These findings analyzed together with the statistically significant low percentage of FcgammaRIII, (CD16) positive immunocompetent cells (p < 0.0007 and p < 0.003), which was found in patients with melanoma compared with healthy or vitiligo people respectively, and statistically significant low percentage of (CD16 + CD56+) natural killer (NK) cells (p < 0.005) found in melanoma patients in comparison to healthy controls pointed to the low probability for anti-melanoma IgG mediated, antibody mediated cellular cytotoxicity, (ADCC) and NK cytotoxicity. Moreover the ratio of the percentages of granulocytes and percentage of lymphocytes was statistically higher in patients with melanoma in relation to healthy people as well as to people with vitiligo (p < 0.0007 and p < 0.05 respectively).
Autoantibodies to tyrosinase and to melanin which are found even in healthy people, point that consummation of edible mushrooms that carry the antigen tyrosinase and melanin, could influence the humoral anti-melanoma immune response.
Levels of different immunoglobulin classes of anti-melanin and anti-tyrosinase antibodies varied depending on the presence and the stage of studied diseases. Besides, the statistically enhanced ratio of the percentages of granulocytes and percentage of lymphocytes, together with statistically decreased percentage of NK cells is found in analyzed melanoma patients.
PMCID: PMC3457868  PMID: 22834951
Melanin; Tyrosinase; Melanoma; Vitiligo; Anti-tyrosinase antibodies; Anti-melanin antibodies; CD16+ CD56+; CD89+
3.  Self-guided Langevin dynamics study of regulatory interactions in NtrC 
Proteins  2009;76(4):1007-1019.
Multiple self-guided Langevin dynamics (SGLD) simulations were performed to examine structural and dynamical properties of the receiver domain of Nitrogen Regulatory Protein C (NtrCr). SGLD and MD simulations of the phosphorylated active form structure suggest a mostly stable but broad structural ensemble of this protein. The finite difference Poisson-Boltzmann calculations of the pKa values of the active site residues suggest an increase in the pKa of His-84 upon phosphorylation of Asp-54. In SGLD simulations of the phosphorylated active form with charged His-84 the average position of the regulatory helix α4 is found closer to the starting structure than in simulations with the neutral His-84. To model the transition pathway the phosphate group was removed from the simulations. After 7 ns of simulations, the regulatory helix α4 was found approximately halfway between positions in the NMR structures of the active and inactive forms. Removal of the phosphate group stimulated loss of helix α4, suggesting that the pathway of conformational transition may involve partial unfolding mechanism. The study illustrates the potential utility of the SGLD method in studies of the coupling between ligand binding and conformational transitions.
PMCID: PMC3373014  PMID: 19384996
Self-guided Langevin dynamics; allostery; conformational transitions; NtrC
4.  pH Replica-Exchange Method based on discrete protonation states 
Proteins  2011;79(12):3420-3436.
We propose a new algorithm for obtaining proton titration curves of ionizable residues. The algorithm is a pH replica-exchange method (PHREM) which is based on the constant pH algorithm of Mongan et al. [1]. In the original replica-exchange method, simulations of different replicas are performed at different temperature, and the temperatures are exchanged between the replicas. In our pH replica-exchange method, simulations of different replicas are performed at different pH values, and the pHs are exchanged between the replicas. The PHREM was applied to a blocked amino acid and to two protein systems (Snake Cardiotoxin and Turkey Ovomucoid Third Domain), in conjunction with a generalized Born implicit solvent. The performance and accuracy of this algorithm and the original constant pH method (PHMD) were compared. For a single set of simulations at different pHs, the use of PHREM yields more accurate Hill coefficients of titratable residues. By performing multiple sets of constant pH simulations started with different initial states the accuracy of predicted pKa values and Hill coefficients obtained with PHREM and PHMD methods becomes comparable. However, the PHREM algorithm exhibits better samplings of the protonation states of titratable residues and less scatter of the titration points and thus better precision of measured pKa values and Hill coefficients. In addition, PHREM exhibits faster convergence of individual simulations than the original constant pH algorithm.
PMCID: PMC3373023  PMID: 22002801
Generalized ensemble algorithm; molecular dynamics; Monte Carlo; pKa calculation; free energy
5.  Conformational relaxation and water penetration coupled to ionization of internal groups in proteins 
The Journal of Physical Chemistry. a  2011;115(16):4042-4053.
Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral, and moved towards crevices and the protein-water interface when they were charged. The only exceptions are Lys-36, Lys-62, Lys-92 and Lys-103, which remained buried even after charging. With the exception of Lys-38, the number of internal water molecules surrounding the ionizable group increased upon charging: the average number of water oxygen atoms within the first hydration shell increased by 1.7 for Lys residues, by 5.2 for Asp residues, and by 3.2 for Glu residues. The polarity of the micro environment of the ionizable group also increased when the groups were charged: the average number of polar atoms of any kind within the first hydration shell increased by 2.7 for Lys residues, by 4.8 for Asp residues, and by 4.0 for Glu residues. An unexpected linear relationship was observed between the absolute value of the shifts in pKa values measured experimentally, and structural relaxation as described in terms of the net difference in the polarity of the micro environment of the charged and neutral forms of the ionizable groups, and of the RMSD values of the charged side chains. The effects of ionization of internal groups on the conformation of the backbone were noticeable but mostly small and localized to the area immediately next to the internal ionizable moiety. Some variants did exhibit local unfolding.
PMCID: PMC3373309  PMID: 21428436

Results 1-5 (5)