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Aim: To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes., Materials and Methods: Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet., Results: Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils., Conclusion: The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.

This review summarizes the main achievements of recent years in molecular organization research of yeast cell surface, i.e., the compartment that consists of the coordinately functioning plasma membrane, periplasmic space, and cell wall. There are data on vesicular transport to the external environment through the cell wall and the formation of channels in the wall, which indicate the possibility of dynamic rearrangements of the molecular structure of the yeast cell wall. There is an idea about the mosaic arrangement of the compartments of the plasma membrane. The hypothesis has been suggested on the heterogeneity of the molecular structure of the cell wall, which is usually considered as uniform except for the budding zones. The groups of proteins that form the molecular assembly of the yeast cell surface have been described. Special attention has been paid for proteins with amyloid properties, including Bgl2p glucanosyltransglycosylase, which is important for virulence in pathogenic yeast, and Gas1p, the first of the studied proteins of the cell surface, which is involved in the regulation of ribosomal DNA transcriptional silencing. The data on the structure of receptors localized on the cell surface and the "moonlight" proteins, involved in the cell stress response of yeasts, have been given.

TA characteristic feature of amyloid structures is polymorphism. The study of amyloid structures and their formation process was carried out for synthetic and recombinant Ab(1-40) and Ab(1-42) peptide preparations. In the study of these peptides, we recognized fibrils of different morphologies. We observed fibrillar formations in the form of single fibrils, ribbons, bundles, bunches, and clusters. Polymorphism of fibrils was observed not only when the environmental conditions changed, but under the same conditions and this was a common characteristics of all amyloid formations. Fibrils of Ab(1-40) peptides tended to form aggregates of fibrils in the form of ribbons, while Ab(1-42) peptide under the same conditions polymerized in the form of rough fibrils of different diameters and tends to branch. We assume that the formation of fibrils of Ab(1-40) and Ab(1-42) peptides occurs according to a simplified scheme: a destabilized monomer ® a ring oligomer ® a mature fibril consisting of ring oligomers. Proceeding from the proposition that the ring oligomer is the main building block of amyloid fibril (similar to the cell in the body), it is easy to explain fibril polymorphism, as well as fragmentation of mature fibrils under various external influences, branching and irregularity of diameter (surface roughness) of fibrils. One aspect of the study of amyloidogenesis is the determination of the regions of the protein chain forming the core of the amyloid fibril. We theoretically predicted amyloidogenic regions for two isoforms of Ab peptides capable of forming an amyloid structure: 16-21 and 32-36 residues. Using the method of tandem mass spectrometry, these regions were determined experimentally. It was shown that the regions of Ab(1-40) peptide from 16 to 22 and from 28 to 40 residues were resistant to the action of proteases, i.e. its formed the core of the amyloid fibril. For Ab(1-42) peptide the whole sequence is not available for the action of proteases, which indicates a different way of associating ring oligomers in the formation of fibrils. Based on electron microscopy and mass spectrometry data we proposed a molecular model of the fibril formed by Ab(1-40) and Ab(1-42) peptides.

The review considers various aspects of myocardium aging in normal and dilated cardiomyopathy (DC). There is a partial fibrosis of the heart tissue due to accumulation of collagen type 1, and accumulation of amyloid during normal aging. Men myocardial aging is accompanied by loss of cardiomyocytes and increased volume of the remaining cells, whereas in women this effect is absent. There is an expression of nuclear proteins decrease (lamin A and C), the accumulation of lipofuscin, increased content of reactive oxygen species, reduction of protein synthesis Sirt1 in cardiomyocytes with aging. Sirt1 has a cardioprotective effect, supports the balance of lipid metabolism, inhibits the development of inflammation and the formation of atherosclerotic plaques. Reduction of its expression not only indicates the accelerated pace of myocardial aging, but can be a predictor of the development of DC. In DC occurs stretching of the heart cavities and systolic dysfunction, predominantly in the left ventricle. DC is characterized by the development of inflammation in the myocardium. Its markers is increased expression of ICAM-1 adhesion molecule, MMP-2, MMP-9 matrix metalloproteinases, TIMP-1 tissue inhibitor of matrix metalloproteinases and decreased expression of MMP-1 matrix metalloproteinase.

The aim of the present study was to optimize the histochemical method of amyloid staining using Congo red. The study was performed on specimens of the myocardium of left ventricle of the heart obtained at autopsy from the patients with amyloidosis of myocardium diagnosed postmortem. It was shown that a positive impact on the quality of the staining of amyloid is provided by a procedure of pre-heating the slides in the liquid, especially at an acidic pH. The staining protocol was developed allowing to obtain preparations characterized by high-contrast staining of amyloid at light microscopic level and by high intensity of its fluorescence. The advantage of the protocol presented is also a significant reduction in the total duration of staining, an increase in stability of the specific staining of amyloid and the absence of nonspecific background staining.

The aim of the present study was to optimize the histochemical method of amyloid staining using Congo red. The study was performed on specimens of the myocardium of left ventricle of the heart obtained at autopsy from the patients with amyloidosis of myocardium diagnosed postmortem. It was shown that a positive impact on the quality of the staining of amyloid is provided by a procedure of pre-heating the slides in the liquid, especially at an acidic pH. The staining protocol was developed allowing to obtain preparations characterized by high-contrast staining of amyloid at light microscopic level and by high intensity of its fluorescence. The advantage of the protocol presented is also a significant reduction in the total duration of staining, an increase in stability of the specific staining of amyloid and the absence of nonspecific background staining.

The dystrophin-deficient mdx mouse is the most commonly used experimental model of Duchenne muscular dystrophy (DMD). Although the amyloid has been shown in the muscle biopsies of patients with different types of muscular dystrophies, there are no data on the amyloid accumulations in the biopsy of DMD patients or mdx mouse. Therefore, the aim of the present study was to testify the hypothesis of probable accumulation of amyloid in the visceral organs of mdx mouse. Specimens of myocardium, kidneys, and liver of male and female mdx mice aged from 2 months to 1.5 years (n = 9) were used in the study. The histochemical staining with Congo red demonstrated amyloid accumulations in the studied organs of the mdx mice. Morphology and localization of the found accumulations were described in details and analyzed. The mass-spectrometric study determined the vitronectin and apolipoprotein A-II as the most probable components of the amyloid accumulations in the mdx mouse.

Objective: to investigate the histopathology of amyloid in liver biopsy specimens. MATERIAL AND METHODS. A total of 46 liver biopsy specimens were investigated in patients with histologically verified amyloidosis in 2006 to 2009 from the Amyloid Registry of the Charité University Hospital (Berlin). The liver biopsy specimens were fixed in formalin and embedded in paraffin. The paraffin sections were stained with hematoxylin and eosin and with Congo red. Amyloid was immunohistochemically classified using antibodies against amyloid P component, AA amyloid, apolipoprotein Al, lysozyme, fibrinogen, transthyretin, and κ and λ light chains., Results: Amyloid deposits were diagnosed in the 46 liver biopsy specimens from 17 women and 29 men (mean age 60 years). Immunohistochemical subtyping was successful in 91% of the cases. AL amyloidosis was diagnosed in 87% of the biopsy specimens and further classified as AL lambda-light chain amyloidosis (57%) and AL kappa-light chain amyloidosis (30%). The 46 liver biopsy specimens showed one case of AA amyloidosis (2%) and one case of transthyretin amyloidosis (2%). The type of amyloid could not be classified in 9% of the biopsy specimens., Conclusion: The investigation revealed that the most common types of hepatic amyloidosis are AL lambda- and AL kappa-light chain amyloidosis associated with the signs of parenchymal atrophy and steatosis.

One hundred and thirteen oral mucosal biopsy specimens were analyzed in patients suspected of having systemic amyloidosis. Histological, immunohistochemical, and genetic studies and polarized light microscopy revealed oral amyloid deposits in 72.6% of cases, including 63.5% with metabolic syndrome and 36.5% with another etiology of chronic heart failure (coronary heart disease, cardiomyopathy). Systemic amyloidosis was found in 13.4% of cases (hereditary transthyretin, AL, and senile forms in 1.8, 10.5, and 1.1% cases, respectively). An anterior abdominal wall skin flap was a valid location to reveal the systemic forms of amyloidosis. Patients with metabolic syndrome and periodontal diseases may have local oral amyloidosis.

Brain protein BASP1 forms oligomers that resemble amyloid protein oligomers in some respects, including interaction with conformation-specific antibodies against amyloid oligomers. Aggregation-prone N-terminal myristoylated peptide myr-BASP1 (1-13) forms fibrillar aggregates of amyloid-like structure under physiological conditions in vitro, which is also the evidence of BASP1 similarity to amyloid proteins. Protein cross-linking by glutaraldehyde in situ demonstrated that BASP1 exists on the presynaptic membrane as lipid raft-associated oligomers. In addition, BASP1 is non-toxic to PC12 cells, when applied either as a monomer or oligomer. We conclude that BASP1 oligomer is a non-pathological physiologically relevant functional form of this protein.

Geriatric eye diseases (age-related macular regeneration, pseudoexfoliation syndrome, pseudoexfoliative glaucoma, senile cataract) are one of the most important problems of modern ophthalmology. Meanwhile, the treatment of these diseases continues to be primarily empirically based for lack of not only valid data on their etiology, but even consensus of opinion on their pathogenesis. This review gives the current views on the commonness of the etiopathogenetic and morphological manifestations of Alzheimer's disease and age-related eye diseases in the aspect of amyloid genesis. This approach is a promising attempt to specify the mechanisms responsible for the occurrence and development of neurodegenerative diseases, their markers, and new perspectives in their treatment.

Beta-amyloid peptide (betaAP) is a product of proteolytic cleavage of wide-spread transmembrane amyloid precursor protein. Betaap is physiological oligopeptide, which is present in biological fluids and tissues of healthy human organism at picomolar concentrations. However, excessive production, polymerization and accumulation of betaAP in tissues (first in neural tissue) underlie the pathogenesis of a number of neurodegenerative diseases (Alzheimer's disease and others). Although the presence of insoluble deposits of fibrillar betaAP (senile plaques) is a characteristic histopathological sign of Alzheimer's disease, soluble betaAP oligomers (dimers, trimers, etc.) possess most neurotoxicity. Soluble betaAP induces mitochondrial dysfunction, increased production of reactive oxygen species in neurons, disorganization of cell plasma membranes, disturbances of ion transport across cell membranes, impairment of synaptic transmission and long-term synaptic plasticity, etc. Recently a lot of attention is paid to possible peripheral effects of betaAP, related to its toxic influence on excitable structures of neuromuscular apparatus and cardiovascular system. At current review we represented state-of-art views on the processes of production and aggregation of betaAP in organism, cellular and molecular mechanisms of betaAP influence on physiological functions of excitable cells, the role of betaAP in pathogenesis of neurodegenerative diseases (Alzheimer's disease and others), as well as the results of our own studies in this field.

We have shown previously the presence of full length (50 kD) and truncated proteolytic form (45 kD) of pigment epithelium derived factor (PEDF) in the eye Tenon's capsule in progressive myopia. The full length PEDF is prevalent in myopia that correlates with breach in collagen fibrils forming. Immunohistochemical analysis of Tenon's capsule with polyclonal antibodies to PEDF revealed PEDF in control group being exclusively inside fibroblasts, whereas in myopia, PEDF was distributed extracellularly as halo around blasted fibroblasts. By means of atomic force microscopy and immunodot analysis with anti amyloid fibrils antibodies the ability was studied of recombinant PEDF fragments to form fibrils. Only full length PEDF was shown to form amyloid like fibril structures, but not the truncated form. Accumulation offibrils results in fibroblasts destruction and might be the cause of changes in biochemical and morphological structure of Tenon's capsule observed in myopia.

In-depth scholar literature analysis of Alzheimer's disease neurodegenerative features of amyloid beta protein neurochemistry modification and excessive phosphorylation of tau protein (and associated neuronal cytoskeleton rearrangements) are secondary phenomena. At early disease stage these neurobiochemical mechanisms are reversible and serve to heal an impairment of biophysical properties of neuronal membranes, neurotransmission, basic neuronal function and neuroplasticity, while preserving anatomical and functional brain fields. Abeta and tau could well serve to biochemically restore physico-chemical properties of neual membranes due to a role these proteins play in lipid metabolism. Under such scenario therapeutic block of aggregation and plaque formation of Abeta and inhibition of tau phosphorylation, as well as pharmaceutical modification of other secondary neurodegenerative features (such as a cascade of oxidative stress reactions) are unable to provide an effective cure of Alzheimer's disease and related pathologies of the Central and peripheral nervous systems, because they are not arraying primary pathagenetic cause. We review the role of Abeta in compensatory mechanisms of neuroplasticity restoration under normal physiological condition and Alzheimer's disease.

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.

Studies of neurodegenetrative disorders have become particularly actual attracting the attention of researchers from over the world because of the dissemination of Alzheimer's disease. The reason for such pathogenesis is the transition of a "healthy" molecule or peptide from the native conformation into a very stable "pathological" isoform. During this, molecules in the "pathological" conformation aggregate, forming amyloid fibrils that can increase without any control. Novel knowledge is required on sporadic isoforms of Alzheimer's disease, on the nature of triggering mechanisms of conformational transitions of beta-amyloid fragments from normally functioning proteins into new formations--nano-beta-amyloids--that spiral out of control of neurons and organism which leads to the loss of neurons. Herein we review studies devoted to the formation of amyloid fibrils and their role in pathogenesis of amyloid diseases.

Laser correlation spectroscopy has indicated that plasma C-reactive protein (CRP) is present as a monomer and a pentamer (pentraxin); the latter is similar in physicochemical properties to apolipoproteins, the proteins that transfer fatty acids as nonpolar triglycerides to the cells in the intercellular medium. Concurrently, sepharose column chromatography revealed an association of the bulk of plasma CRP with very low-density lipoproteins. In parallel with CRP, the content of secretory phospholipase A, as a component of lipoproteins is on the rise; the enzyme hydrolyzes phosphatidylcholine in the surface monolayer of lipoproteins to form lysophosphatidylcholine that the CRP-pentamere displays a high affinity binding to. Two different immunochemical assays for CRP suggest that the CRP-monomere and the CRP-pentamere show different functional activity: the CRP-monomere is a humoral immunomodulator while the CRP-pentamere also activates interstitial tissue cells, but differently, by enhancing their energy provision with substrates, namely with saturated and unsaturated fatty acids as triglycerides as components of very low-density lipoproteins via CRP/apoB-100-receptor endocytosis. The vector ligand redirects the flow offatty acids towards the cells that directly show a biological inflammatory response. For this, lipoprotein-associated phospholipase A, hydrolyzes phosphatidylcholine to produce lysophosphatidylcholine that the CRP-pentamere binds to; it superimposes a physiological apoE/B-100-ligand, becomes itself a pathophysiological CRP/B-100-ligand, and directs a flow of the energy substrates towards the interstitial cells that exhibit pathophysiological CRB/B-100-receptors on the membrane. In the arterial intima, the functionally triglyceride-overloaded resident macrophages form lipid spots that form in each systemic inflammatory response or with the elevated plasma levels of a CRP-pentamere and phospholipase A, and are directly unrelated to atheromatosis. It seems likely that we have identified a monomer with the low plasma values of CRP and its bulk (this is a pentamere) with its high plasma values.

Amyloid bound thioflavine T fluorescence was studied in the lysates of yeast strains carrying mutations in genes ADE1 or ADE2 and accumulating red pigment, a result of polymerization of aminoimidazoleribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in the case of cells grown in media containing high concentration of adenine (100 mg/l) that blocks accumulation of red pigment. Blocks at first stages of purine biosynthesis de novo also impede red pigment and lead to the same effect on thioflavine fluorescence. At the same time induction of mutations in genes ADE1 or ADE2 in originally white prototrophic strains leads to considerable drop of fluorescence. A fraction of protein polymers was studied by agarose gel electrophoresis and this permitted to conclude that lowering of fluorescence intensity is indeed connected with the decrease of amyloid amount in cells accumulating red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with Thioflavine T. 2D-electrophoretic comparison of pellet proteins of red and white isogenic strains, followed by MALDI, allowed identification of 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins, involved in glucose metabolism, closely corresponding to prion-dependent proteins characterized in our previous work. We suppose that, binding amyloid fibrils, red pigment hinders formation of prion aggregates and also, blocking fibril contact with chaperones, impedes prion propagation.

A short review of investigations along a new line: the antiamyloid action of fullerenes C60 and correction of disturbed cognitive processes is presented. The prospects for the development of drugs based on fullerenes acting on the key molecular mechanisms at the early stage of Alzheimer's disease are discussed.

Recently it became evident that proteins can perform their function not only in globular state but also in partially or completely disordered state. The majority of globular proteins are enzymes which function is strictly determined. Regulation and signaling proteins participating in interconnection with variety of partners must have much more lability, and macromolecules of such proteins are mainly in partially or completely disordered state. The aim of this work was to describe from the unified viewpoint in the frame of energy landscape model the existence of native globular, native partially or completely disordered proteins, formation of intermolecular complexes with various partners, formation of amorphous aggregates and amyloid fibrils. Compact globular proteins are formed if polypeptide chain provides strong intramolecular interconnections. The ability of polypeptide chain to fold in a compact globule depends on the relation of hydrophobic and charged aminoacids in its composition. Many partially or completely disordered proteins can form compact structure in complexes with their partners, which are composed by intermolecular interactions of polypeptide chains of protein and its partner. Intermolecular interaction of proteins can lead to formation associates, amorphous aggregates, amyloid and amyloid-like fibrils. The requisite condition of such contact formation is the availability of hydrophobic clusters of polypeptide chain exposed to the solution. That is why aggregation of partially or completely disordered proteins is more favorable in comparison with globular proteins.

An attempt was made at estimating the overall amyloid content of yeast cells by treating crude cellular lysates with thioflavin T, the agent specifically staining amyloid fibrils. We demonstrated that overproduction of the yeast chaperone Hsp104p, as well as GuHCI treatment of the [PSI+] cells led both to elimination of the [PSI+] factor and to a stable decrease of the overall amyloid content estimated by intensity of fluorescence (IF) of the thioflavin T. At the same time, overexpression of gene SUP35, coding the protein prionizable to [PSI+], led to generation of [PSI+] clones with higher IF of thioflavin T. Cytoduction in the crosses involving PSI factor leads to considerable enhancement of IF; cytoductants with the nucleus of the recipient [psi-] strain not only got [PSI+] factor from the donor strain but also increased their amyloid content. In these model experiments all treatments modifying one of the yeast prions, [PSI+] factor, led to a predictable shift of IF of thioflavin T that behaved like a cytoplasmic hereditary determinant. The data obtained show that IF of thioflavin T staining gives reliable estimates of cellular amyloid content and that mitotically stable shift of IF after a battery of treatments modifying cellular prion set provides quantitative estimate of the input of prionizable protein molecules to the amyloid pool. The combination of thioflavin staining and prionotropic treatments applied here can be possibly used for future attempts of checking yeast strains for cryptic prions.

Prion proteins are infective amyloids and cause several neurodegenerative diseases in humans and animals. In yeasts, prions are expressed as cytoplasmic heritable determinants of a protein nature. Yeast prion [PSI], which results from a conformational rearrangement and oligomerization of translation termination factor eRF3, is used as an example to consider the structural--functional relationships in a potentially prion molecule, specifics of its evolution, and interactions with other prions, which form so-called prion networks. In addition, the review considers the results of modeling mammalian prion diseases and other amyloidoses in yeast cells. A hypothesis of proteomic networks is proposed by analogy with prion networks, involving interactions of different amyloids in mammals.

Studying amyloid diseases (amyloidoses) has been especially actual and has attracted the attention of researchers all over the world in connection with the appearance of epidemic prion diseases such as Bovine Spongiform Encephalopathie in cattle and Creutzfeldt-Jakob in human. Amyloidoses are caused by transition of a "healthy" protein molecule or peptide from a native conformation to a very stable pathologic form: molecules in a pathologic conformation aggregate, that results in the amyloid fibrils which can grow infinitely. Investigation of molecular mechanisms of these diseases and the ability to find protein regions responsible for the appearance of the given diseases is a fundamental problem. Theoretical and experimental studies of amyloid fibril formation are considered in this review.

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to decrease in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The sizes of individual proteins (GroEL, GAPDH, and the recombinant prion), as well as aggregates formed after their mixing, were determined by the dynamic light scattering method. It was shown that at 25 degrees C the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the aggregate size to 70-80 nm. On the blocking of one of the chaperonin centers by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.

It was shown for the first time that skeletal muscle sarcomeric proteins of the titin family (X-, C- and H-proteins) are able to form in vitro amyloid aggregates of different types: granular aggregates, protofibrils, helically twisted ribbons, linear fibrils, and bundles of linear fibrils. Their amyloid nature was confirmed by electron, polarization, and fluorescence microscopy and by spectral methods. As opposed to other amyloidogenic proteins, X-, C-, and H-proteins easily form amyloids under mild conditions close to physiological ones (pH, ionic strength, temperature). Like amyloid fibrils of Abeta-peptide and tau protein in Alzheimer's disease, amyloid aggregates formed by X-, C-, and H-proteins are destroyed by the antibiotic tetracycline. Thus, new proteins-precursors of amyloids and possible participants of amyloidoses in muscles were discovered. Further study of in vitro amyloidogenesis of these proteins would help to find approaches to controlling this process in organs and tissues.

A conception on amyloidosis as a key factor of neuronal death in neurodegenerative diseases is stated. Experimental evidence is presented that amyloidosis is caused by alterations in the activity of a number of enzymes as well as conformational changes in pathogenic proteins. Arguments for amyloidosis as the universal biological mechanism of specific elimination of neurons showing changed metabolic and physiological status of cell differentiation are adduced. The final pattern of cell death seems to differ cardinally from that in both apoptosis and necrosis.

Using actin, alpha-lactalbumin and insulin as examples, it was shown that the formation of amorphous aggregates of proteins and amyloid fibrils leads to an increase in the rigidity of tryprophan and tyrosine residues micro-environment and, consequently, to the appearance of tryptophan (tyrosine) room temperature phosphorescence (RTP). RTP was used for examining a slow intramolecular mobility of native (G-, F-form) and inactivated (I) rabbit skeletal muscle actin during the process of GdnHCl induced protein unfolding. This method made it possible to confirm that an essentially unfolded intermediate precedes the formation of inactivated actin. It has been found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting a high lability of its structure. Symbate changes of integral intensity (relative quantum yield) and the mean lifetime of RTP during the U*-->I transition suggest a gradual increase of the number of monomers incorporated in the associate (U*-->11...-->In...-->I15), which is accompanied by an increase of protein structural rigidity. The rate of inactivated actin formation (I-->I15) is shown to increase with the increase of protein concentration. It is shown that, no matter what method of inactivation was employed (1--2 M GdnHCl or 3.0-3.5 M urea, Ca2+ removal, incubation at 70 degrees C, refolding from completely unfolded state by dialysis from 8 M urea or 6 M GdnHCl), actin transition to the inactivated state is accompanied by a significant increase in both integral intensity and the mean lifetime of RTP, suggesting the rigid structure of inactivated actin. It is shown that the lifetime of inactivated actin RTP does not depend on GdnHCl concentration within the limits from 0 to 4 M. On using insulin and alpha-lactalbumin as examples, it is shown that RTP can be used in studies of fibrillogenesis and properties of amyloid fibrils.

The present concepts of protein folding in vitro are reviewed. According to these concepts, amino acid sequence of protein, which has appeared a result of evolutionary selection, determines the native structure of protein, the pathway of protein folding, and the existence of free energy barrier between native and denatured states of protein. The latter means that protein macromolecule can exist in either native or denatured state. And all macromolecules in the native state are identical but for structural fluctuations due to Brownian motion of their atoms. Identity of all molecules in native state is of primary importance for their correct functioning. The dependence of protein stability, which is measured as the difference between free energy of protein in native and denatured states, on temperature and denaturant concentration is discussed. The modern approaches characterizing transition state and nucleation are regarded. The role of intermediate and misfolded states in amorphous aggregate and amyloid fibril formation is discussed.

Recent data on the use of yeast as a model for studying the molecular basis of prion infection are summarized. In contrast to mammalian prions, which are related to incurable neurodegenerative diseases, yeast prions determine the appearance of non-chromosomally inherited phenotypic traits. Prions of yeast are structurally similar to amyloids of mammals and their replication involves not only growth, but also fragmentation of prion amyloid-like fibrils. In mammals the fragmentation should lead to an increase in infectious titer. The use of yeast for study of the mechanisms of human amyloidoses, development of new anti-prion drugs and search for new proteins with prion properties is described.

We showed antisecretory effects of peptides of PGP, semax and a new pancreatic hormone, amylin, on the model of continuous stomach perfusion with the registration of pH, PCO2 as well as the determination of the amount of pepsinogen in the stomach perfusate. The intraperitoneal introduction of these peptides in the doses that proved to have an apparent anti-ulcer effect caused a decrease of both basal production of hydrochloric acid in the stomach and production caused by the irritation of the vagus nerve. Semax and amylin also decreased basal and stimulated secretion of pepsinogen. Amylin and PGP did not have any effect on basal bicarbonate secretion of the stomach, and semax caused its short-time increase 35 minutes after the introduction. We made a conclusion that the decrease of aggressive factors in gastric juices can be a mechanism of the anti-ulcer action of all three peptides under study.

17 thyroid medullary carcinomas (TMC) were studied electron microscopically (EM). Two cell groups were revealed regardless of their histological structure: EM differentiated (with signs of C-cell differentiation) and non-differentiated (without these signs). There is no correlation between histological structure of the tumor and the degree of its EM differentiation. In patients who lived 5 years maximum after the diagnosis there were different histological types but EM the tumors had relatively monomorphous structure with domination of non-differentiated cells (more than 70%), high content of dark cells (50% and more) and low content of desmosomes (less than 30%). The prognosis is more favourable the higher is content of EM differentiated cells, the better desmosomes are developed and the lower is content of dark cells. The scope of surgery and local metastases do not influence the prognosis while distant metastases (stage IV) deteriorate the prognosis.

Aim: To evaluate the duration of rheumatoid arthritis (RA) remission with respect to different drug treatments., Materials and Methods: Remission duration observed at 20-year follow-up of 442 RA patients living in the south of Estonia has been reviewed. Also, the data are provided on the disease onset, articular status, systemic lesions, RA activity and progression, the latest exacerbation and previous remission, standard laboratory indices, humoral immunity, examination of the biopsy of the articular tissues and subcutaneous fat for amyloid., Results: According to the retrospective analysis, slow-progressive RA course occurs primarily in remissions longer than 5 years and less frequently in remissions lasting from 1 to 5 years. No matter what the drug was used, 14% of the patients have failed the treatment. 3% of the patients were in remission longer than 5 years. Short-term remissions (1-3 months) were induced in 13%, stable ones (3 months-1 year) in 39% of the cases. These remissions were observed in early RA, more frequently in patients with initial arthritis of the small joints. Remissions from 1 to 5 years were registered in 31% of the patients., Conclusion: RA remissions up to 1 year represent temporary clinical improvement and do not inhibit progression of the rheumatoid process. Consideration of association between RA clinical course and remission duration helps to recognize groups of RA risk and to timely change treatment policy.

We studied cellular aspects of amyloid angiopathy of the iris upon decompensation of ocular pressure and the role of microcirculatory pathology in pathogenesis of the open angle glaucoma. A total of 115 iris biopsies were studied, which were taken during antiglaucoma operations and stained by Congo rot and thioflavine for amyloid and Toluidine blue for glycosaminoglycans. The biopsies from nine patients were studied at the ultrastructural level. Intravasal increase of the concentration of preamyloid proteins during spasms of the arterioles and capillaries should be considered as a provoking moment of the iris amyloidogenesis. Blockade of reticular structures of the microcirculatory basal membranes by preamyloid serum proteins, which provides for shedding of amyloid fibrils, dystrophic changes and detachment of the endothelial cells from the basal membrane, obliteration of lumens of the capillaries and arterioles, disturbed integrity of the microvascular barrier and accumulation of preamyloid and amyloid proteins in the extracellular matrix, ischemia and focal microinfarctions of the iris, and dystrophia of the melanocytes, are subsequent key factors. Amyloid angiopathy of the iris is a cause of ischemia of the anterior eye part, thus creating conditions for increased intraocular pressure and glaucoma.

Endocrine (isolated atrium amyloidosis and insular amyloidosis of the pancreas) and non-endocrine (aorta, eye, prostate and/or seminal vesicles, cerebral amyloidosis) forms should be distinguished among senile local amyloidosis. Senile polyorgan amyloidosis is also possible: combination of endocrine forms with aorta amyloidosis or that of insular amyloidosis with cerebral and eye amyloidosis. These data reject Schwartz tetrad as an obligatory manifestation of an old age. Peptide hormones, serum and specialized proteins are precursors of amyloid fibrillary protein; each of them is able to beta-cross-conformation and formation of the fibrillar amyloid protein. This implies that conformational protein disturbances in increasing senile tissue bradytrophia and extinction of the diffuse endocrine system function are a basis of amyloidogenesis in senile local amyloidosis. Clinical manifestations of senile amyloidosis are very variable, but local senile amyloidosis is not recognized clinically. Its diagnosis will probably become possible with clinical use of molecular biology achievements and, first of all, those of molecular genetics.

The mechanism of accumulation of prion amyloid in guinea pig CNS in experimental slow virus disease--amyotrophic leuco-spongiosis (AL) was studied. The complex histochemical, immuno-cytochemical and ultrastructural studies revealed specific amyloid deposits in a few brain capillaries and in most of pia matter vessels. Taking into account the high AL agent titer in spleen throughout the disease period, conclusion was drawn of entering AL agent in CNS through blood-liquor barrier and blood-brain barrier. It was supposed that primary immune system damaging took place in AL pathogenesis.

The reported and the authors' data are provided on the pathogenesis, morphofunctional characteristics and clinical manifestations of acute and chronic gastroenteropathies in patients suffering from periodic disease with and without amyloidosis. Under discussion is the significance of changes in the gastrointestinal tract for the diagnosis of periodic disease, its association with other diseases and early recognition of amyloidosis.

One should differentiate apudomas, two-component tumors (a combination of apudoma with a non-endocrine tumor), apudoma-like tumors and non-endocrine ones with admixture of APUD cells among the tumors with cells possessing the properties of APUD cells. Apudomas in their turn are represented by microapudomas and high- and low-differentiated macroapudomas. Routine light microscopy in most cases provides for apudoma verification, estimation of its differentiation and tissue origin, occasional identification of a secreted hormone.

Electron-microscopic investigation carried out following the intravenous administration of ferritin revealed an increase in permeability of the myocardium capillary wall in mice in experimental casein amyloidosis. Additionally, a complex electron-microscopic, immunohistochemical and histological investigation of the development of amyloidosis in the myocardium of mice was conducted. It was established that deposits of typical amyloid in the myocardium of mice consisted of two ultrastructural components--granular material and fibrils. It was shown that formation of amyloid with characteristic tinctorial properties was preceded by depositions of specific antigen fibrils of granular ultrastructure.