[Protective Activity of Prion Protein Fragments after Immunization of Annimals with Experimentally Induced Alzheimer's Disease].

The prion protein is considered as one of the membrane targets of neurotoxic beta-amyloid during Alzheimer's disease development. We have chosen and synthesized 17-33, 23-33, 95-110 and 101-115 prion fragments involved in beta-amyloid binding. The effect of immunization with the peptides on the features of Alzheimer's disease was investigated in animals with an experimentally induced form of the disease. It was shown that immunization either with peptide 17-33 or with protein conjugates of peptides 23-33 and 101-115 increases the level of brain beta-amyloid and improves morfofunctional state of the brain.

[The reduced level of antibodies to acetylcholine receptor alpha 7 fragment in the blood serum of patients with Alzheimer's disease].

OBJECTIVE: Determination of antibodies to neuronal membrane proteins in the blood serum of patients is of interest for diagnosis and optimization of treatment of Alzheimer's disease (AD). Authors studied the level of antibodies to acetylcholine receptor alpha 7 protein fragment (AChR), prion protein (РrР) and glycation end-products (RAGE) as well as to intracellular proteins nucleophosmin (Nuc) and survivin (Sur) in the serum of AD patients. MATERIAL AND METHODS: Serum samples of 26 patients with probable AD and 13 healthy people were studied. Exposed sections of each protein were used for the choice of peptides for antibody visualization. ELIZA was a main method in this study. RESULTS AND CONCLUSION: Antibodies to several proteins were identified but significant differences were found only for AChR-(173-193). The results demonstrated the involvement of AChR and AChR-antibodies in the development of AD. Determination of antibodies to AChR-(173-193) may be a marker of AD and a method for specifying the diagnosis of AD.

[Fragment of Receptor for Advanced Glycation End Products Improves Memory State in a Model of Alzheimer's Disease].

A number of synthetic peptides corresponding to potentially important regions in the sequence of the four membrane proteins known as beta-amyloid cell receptors have been investigated on their ability to improve memory state in experimental model of Alzheimer's disease. Nine fragments repeating all the exposed nonstructural regions of the RAGE protein according to X-ray data, have been synthesized. The activity of these peptides and synthesized earlier immunoprotective fragments of other three proteins (acetylcholine receptor alpha7-type, prion protein and neurotrophin receptor p75) has been investigated under intranasal administration, without immune response to the peptide. Only one fragment RAGE (60-76) was shown to have a therapeutic activity improving the memory state of bulbectomized mice and leads to decreasing in the level of brain beta-amyloid.

[Antibodies for detection of E/K amino acid substitution in 129 position of the survivin sequence].

Survivin is an oncofetal protein involved both in inhibiting of apoptosis and in cell cycle regulation. The functions of survivin are defined by its structural state. Due to nature polymorphism, survivin cancontain either E or K amino acid in 129 residue, and K129 is commonly acetylated. Only the protein having acetylated K129 tends to form dimeric structure. Thus, antibodies detecting the amino acid substitution can be a useful tool for structural and functional research of the protein. To obtain the antibodies specific to amino acid substitution E129/K129 peptide fragments overlapping 129 amino acid residue were synthesized, rabbits were immunized with the peptides and affinity purification of the antibodies on sepharose conjugated with the peptides was carried out. The data of ELISA and western blot showed that antibodies obtained were able to detect amino acid substitution E129/K129 in the recombinant and endogenous survivin.

[Immunization witha synthetic fragment 155-164 of neurotrophin receptor p75 prevents memory loss and decreases beta-amyloid level in mice with experimentally induced Alzheimer's disease].

Neurotoxic beta-amyloid peptide plays an important role in the pathology of Alzheimer's disease. In aggregated form it binds to several proteins on the surface of the brain cells leading to their death. p75 receptor in- volved in supporting of cell balance is one of the targets for toxic beta-amyloid. We proposed that induction of antibodies against potential binding sites of p75 with beta-amyloid can be a promising approach towards new drug development for Alzheimer's disease therapy. Four potentially immunoactive fragments of p75 were chosen and chemically synthesized. Investigation of immunoprotective effect of the peptide fragments carried out in mice with experimentally induced form of Alzheimer's disease helped to reveal two fragments effectively preserving murine memory from impairment. Results obtained by ELISA biochemical analysis showed that only immunization with fragment p75 155-164 led to significant decrease in beta-amyloid level in the brain of the experimental mice. Thus, immunization with both fragments of p75 receptor is believed to be an effective tool for the development of new drugs against Alzheimer's disease.

[Obtaining of the affinity purified antibodies against survivin for the structure functional study of the protein].

Tumor-associated protein survivin is the bifunctional protein which can participate either in cell division regulation or in apoptosis inhibition depending on its localization and structure state. The aim of this work was to obtain monospecific antibodies useful for investigation of protein structure and functional features. Six affinity purified antibodies directed to different protein regions were obtained. The ability of antibodies obtained to detect survivin in tumor cells and breast cancer tissues was studied. It was shown that antibodies to (1-22) and (95-105) survivin fragments have the highest specific activity. In western-blot antibodies to (1-22) region predominantly binds with survivin-containing complex, which may be the survivin dimer as we suppose, while antibodies to (95-105) region detects only monomeric form of the protein. Breast cancer tissues study demonstrated that survivin monomer presents only in the tumor core tissues, while survivin-containing complex is expressed both in tumor core and tumor periphery tissues. It was shown that antibodies to (1-22) fragment detect predominantly nuclear survivin, which participates in mitosis regulation, while antibodies to (95-105) fragment gave nucleoplasm and cytoplasm staining at all stages of cell cycle. Thereby antibodies obtained are the useful tool for structure-functional study of survivin.

[The apoptosis inhibitor survivin in transitional cell carcinoma of the urinary bladder].

Whether the expression of the apoptosis inhibitor survivin was correlated with the degree of differentiation and the stage of transitional cell carcinoma of the urinary bladder was studied. Sixty samples of surgical specimens from patients with urothelial carcinomas of various degrees of differentiation and different stages were examined. An immunohistochemical study using the monoclonal antibodies obtained by the authors was conducted. The high expression of survivin was shown to be correlated with the lower-grade differentiation of a tumor and its higher stage.

[Antibodies to synthetic peptides for the detection of survivin in tumor tissues].

Survivin, an endogenous protein, is a promising marker for the diagnosis of cancer. The aim of the present work was to obtain antibodies specific to survivin and capable of detecting this protein in tumor tissues. Four peptides corresponding to fragments (1-22), (54-74), (80-88)-(153-165), and (118-144) of the survivin-2B sequence were selected and synthesized. Rabbits were immunized with the synthetic peptides. It has been shown that all peptides in a free state, without conjugation with a high-molecular-weight carrier, stimulate the production of antibodies capable of binding with recombinant survivin. Antipeptide antibodies were isolated from sera and their performance in the immunohistochemical detection of survivin in human tumor tissues was studied. It was shown that only antibodies to the (80-88)-(153-165) peptide bind to the survivin present in breast and bladder tumors. The ability of antibodies to this peptide to detect survivin in tumor tissue lysates was demonstrated by immunoblotting. The part of the sequence targeted by the antibodies against the (80-88)-(153-165) peptide was localized using truncated peptide fragments.

[Antibodies to synthetic fragments of nucleophosmin for the specific detection of its monomeric and oligomeric forms].

Immunoactive fragments corresponding to the N-terminal (19-36) and C-terminal (283-294) regions of the NPM1.1 isoform of nucleophosmin and their shortened fragments were chosen and synthesized. Rabbits were immunized with free full-size peptides and their protein conjugates. Antibodies produced against the 19-36 and 283-294 peptides were purified by affinity chromatography on bromocyanogen-activated sepharose that was preliminary conjugated with the synthetic peptides. An analysis of immunoblots of lysates of the HeLa and Ramos cells demonstrated that the antibodies produced against the 19-36 peptide detected the monomeric form of nucleophosmin, whereas the antibodies against the 283-294 peptide predominantly revealed its oligomeric form. It was established by immunocytochemical analysis that the antibodies induced by the 19-36 peptide stained the nucleoplasm and perinuclear space of the cytoplasm of the HeLa and Ramos cells, but did not stain the nucleoli, while the antibodies against the 283-294 peptide stained only the nucleoli of the same cells. On the basis of these results, one could propose that the monomeric and oligomeric forms of nucleophosmin were located in the nucleoplasm and nucleoli of the examined cells, respectively. Thus, antibodies which can predominantly detect monomeric and oligomeric forms of nucleophosmin were produced for the first time. An analysis of the monomeric-oligomeric state and the location of the nucleophosmin in tumor cells could be performed using these antibodies.

[Production of monoclonal antibodies to the prion protein and their characterization].

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25-36 of PrP corresponds to one antibody, and epitopes located in region 222-229, to the other two. The antibodies to fragment 222-229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.