Aggregated proteins in malignant and benign neoplasms.

AIM: To evaluate the presence of the aggregated proteins in malignant and benign neoplasms for clarifying the role of impaired protein metabolism in the formation of the altered tissues. OBJECT AND METHODS: The histological specimens prepared from the operative materials of 196 patients with different forms of malignant and benign neoplasms were stained with Congo red and Thioflavin T and studied under the light and polarization microscope. RESULTS: The various forms of ß-stacked protein aggregates (ß-SPA) inclusions were detected in amyloids, keloid tissue, benign polyps, and several malignant tumors. CONCLUSION: The formation of non-functional protein aggregates proves the complex character of the impairment of protein metabolism resulting in local or systemic accumulation of secondary protein toxins results in ß-SPA formation as the self-sustaining complex of parametabolic processes. The ß-SPA formation is of considerable interest since their properties lead to the impairment of the normal physiological processes in adjacent tissues ensuring the chronic course of the pathology.

EPR spectroscopy studies of changes in erythrocyte membranes in patients with laryngeal cancer.

AIM: To evaluate microviscosity and sorption capacity of erythrocyte membranes (SCEM) from patients with laryngeal cancer (LC). MATERIALS AND METHODS: Samples from 35 patients with LC of stages II and III and 20 healthy volunteers were investigated by electron paramagnetic resonance with Bis(1-oxyl-2,2,6,6-tetramethylpiperidinyl-4)-ester of 5,7-dimethyladamantane-1,3-dicarbonic acid (AdTEMPO) probe. SCEM was evaluated by amount of unabsorbed methylene blue. RESULTS: Microviscosity of erythrocyte membranes was determined by the effective rotational diffusion correlation times (τeff) and a decrease in radical spectrum signal intensity per hour. The most apparent decrease in mobility of the AdTEMPO in erythrocytes was observed prior to washing of erythrocytes with 0.9% NaCl for 5 min after probe insertion. The deceleration after 60 min was observed only in stage II LC. τeff was at control values after washing of erythrocytes of stage II LC 5 min after probe insertion and was significantly reduced in stage III LC in comparison to control. Radical spectrum signal intensity per hour in samples of stage II and III patients prior to and after washing of erythrocytes was on average 1.5-fold higher than that of control. SCEM in samples of stage II and III LC was found in 40 and 33% cases, respectively and was on average significantly reduced in comparison to control. CONCLUSIONS: The initial interaction of AdTEMPO with erythrocyte membranes of stage II and III LC patients is accompanied by an increase in τeff, indicating deceleration of probe rotation. τeff of the probe in membranes remains unchanged in 60 min, indicating changes in the structural organization of lipid bilayer and its associated proteins in particular. The similarity of SCEM for both studied groups reflects the pathological changes in function of erythrocyte membranes.

Combined use of haemostatic system indices for evaluation of upper respiratory tract cancer progression.

AIM: To analyze whether comprehensive assessment of haemostatic system components, in particular, indices of coagulation and fibrinolytic systems along with functionally related proteins, could be indicative of upper respiratory tract (URT) cancer progression. MATERIALS AND METHODS: Indices of coagulation and fibrinolytic systems along with functionally related proteins, in particular, trypsin-like amidolytic activity, trypsin-like proteolytic activity, thrombin-like amidolytic activity, elastase-like amidolytic activity, fibrinolytic activity, potential amidolytic plasmin activity, content of fibrinogen, antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin, and prothrombin time were evaluated in blood plasma of patients with URT cancer of II (n = 10) and III (n = 25) stages with the use of routine biochemical methods. RESULTS: For both groups of patients with URT cancer there have been shown notable differences for the majority of the studied indices, especially the indexes of proteolytic activities, from these of healthy donors, and in the case of URT cancer of III stage they reached statistical significance. In contrary, the changes in the content of antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin were insignificant. In both groups of patients significant increase of fibrinogen content has been registered, while the content of soluble fibrinogen didn't change. Also, in both groups of patients there a significant increase of potential activity of plasminogen was documented, while clot lysis time was significantly increased only in patients with III stage URT cancer. Multifactorial analysis of haemostatic system indices evidenced for efficacy of their combined use for evaluation of URT cancer progression risk. CONCLUSION: Combined use of fibrinogen and α2-macroglobulin content and the level of amidolytic thrombin-like activity could serve as an indicator of URT cancer progression.

[Use of soybean trypsin inhibitor for modification of gold surface of the sensor chips in surface plasmon resonance spectrometer]. / Zastosuvannia soievoho inhibitora Trypsynu dlia modyfikatsiï poverkhni zolota sensornykh chipiv spektrometra poverkhnevoho plazmonnoho rezonansu.

Kunitz soybean trypsin inhibitor (STI) and glutaraldehyde are used to create an interlayer for proteins immobilization on the gold surface of the sensor chips of surface plasmon resonance spectrometer. Human serum albumin (HSA), goat Ig G and bovine eye lens alpha-crystallin are immobilized via the proposed interlayer. We studied the effects of the duration of storage of the sensor chips before use and pre-treatment by "piranha" solution on the STI adsorption by the gold surface. The influence of STI surface concentration, as well as the effect of the duration of storage of STI-modified sensor chips on the HSA immobilization are investigated. The binding of specific antibodies to the immobilized proteins and non-specific binding to the modified surfaces are studied. HSA immobilization on the bare gold surface is compared to that on the surfaces, modified by different methods.

[Prions and protein inhibitors of proteinases: structural analogies and their consequences. IV. The nature of the interspecies barrier of prion disease]. / Priony i belkovye ingibitory proteinaz: strukturnye analogii i ikh sledstviia. IV. O prirode mezhvidovogo bar'era prionovykh zabolevanii.

Declining of unproved supposition about the transformation of the cellular isoform of prion protein into pathogenic one allows the pathogenesis of prion diseases to be reduced to the series of interdependent processes caused by prion-mediated selective damage of the components of the cell chaperoning system with following membrane folding of the de novo synthesized prion protein. Dependence of the level of the cell chaperoning system damage on the similarity of the sequences of cellular and infectious prion proteins explains exhaustively the existence of interspecies barrier in prion pathogenesis as well as makes it natural and inevitable property of the latter. The structural ensurance of separate prion diseases strains, their transformation at repeated infectious passages commonly with known and supposed ways for prion-less initiation of spongiform encephalopathies are discussed.

[Ligand-induced structuring of polyreactive immunoglobulins]. / Ligandindutsirovannoe strukturirovanie polireaktivnykh immunoglobulinov.

The influence of several hydrophobic compounds with different structures on the binding of native and chaotropically modified bovine immunoglobulins to native and denatured nonspecific protein immobilized on the surface is researched. It is shown that the character of binding depends on the amount and mutual location of hydrophobic nuclei in the effectors structure. The authors observed moderate suppression of binding or absence of the expressed effect while using one-nucleus effector. The influence of compounds with two distanced benzene nuclei differs qualitatively from the influence of the compound with two condensate ones, described earlier: it was observed that the stimulation level of the binding depends on structural-functional condition of immunoglobulin and immobilized protein. In case of the effector with three spaced hydrophobic nuclei the stimulating effect is much more expressed (3-4 times higher). The concentration dependence of the ligand-induced effects is demonstrated. It is supposed, that stimulative influence of effectors with spaced hydrophobic nuclei is caused by two different processes. The first one is the formation of highly binding center as the result of ligand-induced structuring of immunoglobulins. The other one is the competition for the center formed between dissolved polynuclear effector and ligand groups of aminoacids hydrophobic residues statistically formed on the surface of immobilized protein.

[Interferonogenic activity of immobilized ribopolynucleotides in vitro]. / Interferonogennoe deistvie immobilizovannykh ribopolinukleotidov in vitro.

In vitro experiments the authors have studied a property of yeast RNA--tilorone hydrochloride complex covalently linked to spheron to induce the synthesis of interferons type I (alpha- and beta-interferons) in the culture of peripheral mononuclear human cells. Such a complex is shown to possess a marked interferonogenic activity. The data obtained appear to be a proof of the interferon induction to be realised by a mechanism needing at the first stage the contact between the inducer and the cell surface without its penetration into the cell.

[Orientation of proteins on the sensor surface and optimization of their immobilization by prior protection of the active center]. / Orientatsiia proteinov na poverkhnosti sensora i optimizatsiia ikh immobilizatsii putem predvaritel'noi zashchity aktivnogo tsentra.

When studying biospecific interactions with application of surface plasmon resonance, one of the main problems is reagent proper orientation to the sensor surface. Due to rather high chemical activity of molecular receptor sites, the interaction between these areas and surface may become predominant. Here we propose a technique for prevention of such orientation of bioreceptors using soybean trypsin inhibitor STI as an example. To obtain oriented STI immobilization on a modified gold surface its active site has been previously blocked through interaction with its specific partner trypsin. After conjugate immobilization on the sensor surface the components were separated using a glycine buffer (pH 2.2).

[Molecular mechanisms of systemic enzyme therapy]. / K voprosu o molekuliarnykh mekhanizmakh sistemnoi énzimoterapii.

This work deals with the molecular mechanisms of system enzymotherapy agents action--both administrated perorally and intratestially sorbed enzymatic complexes. The basic of functional composite part of SET agents are supposed to be trypsin degraded forms.

1-X-3 motif in inter-protein recognition: structures, widespreading and possible practical application.

Similarities in the discrete mode and size of contact areas of a wide range of protein complexes allows us to suggest the existence of a limited number of types of inter-protein interactions. Comparison of structures of bound determinants indicates that the double-module, 1-X-3 type of motif is widespread in recognition processes. Thus, in many cases, the sites of ligand recognition are formed by two significant amino acids and separated by insignificant ones. Typical examples of such motifs are the RGD sequence of some adhesive and haemostatic proteins, the primary sites for plasminogen sorption on the fibrin network, the reactive sites of protein inhibitors of serine proteinases, and the sites for activation of the hydrolysis of protein pro-forms and receptors. It is assumed that there is widespread double-module determinants in many inter-protein interactions.

Prions and protein inhibitors of proteinases: structural analogies and their consequences. III. Additive risk factor in transgenic technologies.

The article presents the risk factor connected with transgenous technologies. The probable risk is stipulated by a possibility to produce the proteins capable to initiate prion-conditioned neurodegenerative processes as a consequence of possible membrane folding of the recombinant protein portion.

Solid-state conjugation of proteins with hydrophobic compounds in non-denaturing conditions. I. Acylation of proteins by dansyl proline using a polymeric N-hydroxysuccinimide ester.

A method of conjugating protein molecules under native conditions with water-insoluble hydrophobic compounds is developed. It permits very water-insoluble acids to be gently coupled to the primary amines on proteins or other biopolymers. For this purpose we synthesized a polymer (co-polymer of N-hydroxymaleimide and N-vinylpyrrolidone cross-linked by benzidine) which swells equally well in water and in organic solvents. Hydrophobic substances are first activated by esterification to this polymer in organic solvent and then conjugated to protein by acylation in aqueous medium at pH 8.0-8.5. Thus, the contact of native protein with organic co-solvent may be completely avoided. The application of this approach is demonstrated by labeling trypsinogen and plasminogen with dansyl proline.

[Prions and proteinaceous proteinase inhibitors: structural analogs and their consequences. II. Dynamics of prion diseases]. / Priony i belkovye ingibitory proteinaz: strukturnye analogii i ikh sledstviia. II. O dinamike prionovykh zabolevanii.

The assumption about pathogenic prions as the proteins supplying the extracellular proteinases transport into intracellular space permits to bring the pathogenesis of prion diseases to order of the known and partially proved process regarding the case of prion diseases. We present the mathematical model of the dynamics of prion pathogenesis explaining the existence of the minimal infectious dose and small influence of its exceeding on the duration of long-term latent period of the disease. According to the model proposed the transformation of the neuronal cell into PrPSc breeder is the result of proteolytic damage of shaperoning system caused by accumulation in the cell of some crucial amount of proteinase-transporting prions. Such an accumulation is considered as the result of successive and centripheral lay-by-lay transformation of compact cellular locus from higher affinity to prions to normal one. The formation in the moveable frontier lays of the wave with high prion consisting and its closing into the locus center leads to dramatic splash of prion concentration even at moderate difference between higher and normal affinity levels. The final concentration of prions depends mainly on the correlation between these affinities whilst on exceeding of some value the dimension of the locus is of no importance.

[Structural regularities in activated cleavage sites of thrombin receptors]. / Strukturnye zakonomernosti uchastkov aktivatsionnykh rasshcheplenii trombinovykh retseptorov.

Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

[Prions and serpins: structural analogs and their consequences. I. Protease-transport hypothesis]. / Priony i serpiny: strukturnye analogii i ikh sledstviia. I. Proteinazo-transportnaia gipoteza.

The structural analogy between prions pathogenic form and serine proteinase inhibitors (serpins) was laid into the basis of explaining the prion diseases main peculiarities--nucleic-acid-free transfer of infection, neurodegenerative processes, existence of the minimal infective dose, long-term latent period and some others. The assumption about pathogenic prions as the proteins supplying the extracellular proteinases transport into the intracellular space permits to bring the pathogenesis of prion diseases to the known and partially proved processes order regarding the case of prion diseases.

On the role of effector site in serine proteinases hydrophobic chromatography.

Causes of differences between the calculated and experimentally obtained properties of affine sorbents with low-molecular ligands. The effectory character of the so-called hydrophobic chromatography of serine proteins and the role of a spacer in this process are supposed.

[Kinetic regularities of S2'-stimulation of alpha-chymotrypsin]. / Kineticheskie zakonomernosti S2'-stimuliatsii alpha-khimotripsina.

Data on the kinetics of S2'-stimulated alpha-chymotrypsin action have been presented. It is supposed that the increase of the catalytic action of S2'-stimulated chymotrypsin occurs at all three stages of the hydrolytic process-at the enzyme-substrate complex formation, its transformation to covalent acyl-enzyme and at the hydrolysis of the latter.

[Structural similarity of regions of activation cleavage of protein-precursors by reactive centers of serpins]. / O strukturnom podobii uchastkov aktivatsionnykh rasshcheplenii belkov-preshestvennikov reaktivym tsentram serpinov.

A comparative analysis of primary sequences of the region of "leaving group" of a number of splittings evoked by serine proteinases under the activation of protein-precursors has been carried out. A pronounced domination of amino acids of hydrophobic nature in P2'-position similar to that observed in case of the reactive centres of serpines has been noticed. Physiological role of S2'-stimulation of serine proteinases is discussed.

[Role of S'2-stimulation of serine proteases in regulation of proteolysis]. / O roli S'2-stimululiatsii serinovykh proteaz v reguliatsii proteoliza.

Data about ligand specificity and functionality of the nearest surrounding of hydrolytic centre of serine proteases are presented. A regulatory role of S'2-site predetermining highly-specific activation of proenzymes into the enzymes and formation of stable enzyme-serine complexes is discussed.