Macrovipera lebetina obtusa Snake Venom as a Modulator of Antitumor Effect in S-180 Sarcoma Mouse Model

Molecular Biology - Macrovipera lebetina obtusa (MLO) is a venomous snake endemic to Middle East. Here we describe the therapeutic potential of the MLO snake venom. In S-180 sarcoma-bearing mouse...     

Interaction of dipeptydil peptidase IV with amyloid peptides

The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (K_m = 37.5 μM, k_cat/K_m = 1.7 × 10³ M⁻¹sec⁻¹) and Aβ42 (K_m = 138.4 μM, k_cat/K_m = 1.90 × 10² M⁻¹sec⁻¹). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.     

Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response.     

Effects of cathepsins B and L inhibition on postischemic protein alterations in the brain

The effects of selective inhibition of cathepsins B and L on postischemic protein alterations in the brain were investigated in a rat model of middle cerebral artery occlusion (MCAO). Cathepsin B activity increased predominantly in the subcortical region of the ischemic hemisphere where the levels of collapsing mediator response protein 2, heat shock cognate 70 kDa protein, 60 kDa heat shock protein, protein disulfide isomerase A3 and albumin, were found to be significantly elevated. Postischemic treatment with Cbz-Phe-Ser(OBzl)-CHN₂, cysteine protease inhibitor 1 (CP-1), reduced infarct volume, neurological deficits and cathepsin B activity as well as the amount of heat shock proteins and albumin found in the brain. Our data strongly suggests that the decrease in heat shock protein levels and the significant reduction of serum albumin leakage into the brain following acute treatment with CP-1 is indicative of less secondary ischemic damage, which ultimately, is related to less cerebral tissue loss and improved neurological recovery of the animals.     

Studying protein isoforms of the adaptor SETA/CIN85/Ruk with monoclonal antibodies

SETA/CIN85/Ruk is a multifunctional adaptor protein involved in signal transduction and attenuation downstream of receptor tyrosine kinases. It has a modular structure, and various isoforms that combine different protein-protein interaction domains have been proposed based on cDNA analysis. As a first step towards understanding SETA/CIN85/Ruk isoforms at the protein level, we have characterized 5 monoclonal antibodies against this protein. Three of these were used to study lysates fractionated on a pH gradient, leading to the identification of various SETA/CIN85/Ruk proteins on the basis of pI and apparent molecular weight. While good correspondence with proteins predicted from cDNA analysis was found for two isoforms, in most cases it was not possible to make an unequivocal assignment. We conclude that additional splice variants remain to be described, and that a deeper understanding of SETA/CIN85/Ruk post-translational processing and modification is necessary to gain further understanding of this complex gene product.