Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Emergence of non-stationary regimes in one- and two-dimensional models with internal rotators.

In the present paper, we give a selective review of some very recent works concerning the non-stationary regimes emerging in various one- and two-dimensional models incorporating internal rotators. In one-dimensional models, these regimes are characterized by the intense energy transfer from the outer element, subjected to initial or harmonic excitation, to the internal rotator. As for the two-dimensional models (incorporating internal rotators), we will mainly focus on the two special dynamical states, namely a state of the near-complete energy transfer from longitudinal to lateral vibrations of the outer element as well as the state of a permanent, unidirectional energy locking with mild, spatial energy exchanges. In this review, we will discuss the recent theoretical and experimental advancements in the study of essentially nonlinear mechanisms governing the formation and bifurcations of the regimes of intense energy transfer. The present review is composed of two parts. The first part will be mainly devoted to the emergence of resonant energy transfer states in one-dimensional models incorporating internal rotators, while the second part will be mainly concerned with the manifestation of various energy transfer states in two-dimensional ones.This article is part of the theme issue 'Nonlinear energy transfer in dynamical and acoustical systems'.

Consecutive transitions from localized to delocalized transport states in the anharmonic chain of three coupled oscillators.

In the present paper, we study the mechanism of formation and bifurcations of highly nonstationary regimes manifested by different energy transport intensities, emerging in an anharmonic trimer model. The basic model under investigation comprises a chain of three coupled anharmonic oscillators subject to localized excitation, where the initial energy is imparted to the first oscillator only. We report the formation of three basic nonstationary transport states traversed by locally excited regimes. These states differ by spatial energy distribution, as well as by the intensity of energy transport along the chain. In the current study, we focus on numerical and analytical investigation of the intricate resonant mechanism governing the inter-state transitions of locally excited regimes. Results of the analytical study are in good agreement with the numerical simulations of the trimer model.

[EEG frequency and regional properties in patients with paranoid schizophrenia: effects of positive and negative symptomatology prevalence].

OBJECTIVE: EEG changes in schizophrenic patients are caused by a multitude of factors related to clinical heterogeneity of the disease, current state of patients, and conducted therapy. EEG spectral analysis remains an actual methodical approach for the investigation of the neurophysiological mechanisms of the disease. The goal of the investigation was the study of frequency and regional EEG correlating with the intensity of productive and negative disorders. MATERIAL AND METHODS: Models of summary prevalence of positive/negative disorders and evidence of concrete clinical indices of the PANSS scale were used. Spectral characteristics of background EEG in the frequency range of 1-60 Hz were studied in 35 patients with paranoid schizophrenia free from psychoactive medication and in 19 healthy volunteers. RESULTS: It was established that the main index of negative symptomatology in summary assessment was diffuse increase of spectral power of gamma and delta ranges. Deficient states with the predominance of volitional disorders were characterized by a lateralized increase of spectral power of beta-gamma ranges in the left hemisphere, and of delta range - in frontal areas of this hemisphere. Positive symptomatology was noticeably less reflected in EEG changes than negative ones. CONCLUSION: An analysis of psychopathological symptom complexes revealed the significance of spatially structured EEG patterns in the beta range: for the delusion disturbances with psychic automatism phenomena - in frontal areas of the left hemisphere, and for the paranoid syndrome with primary interpretative delusion - in cortical areas of the right hemisphere.

[The application of proteomic technologies for the analysis of muscle proteins of farm animals used in the meat industry (review)].

The review briefly summarizes the data on the development of proteomic technologies that became actively used in studies of the muscular proteins of farm animals used in the meat industry in 2006-2013. It has been noted that the main research trends are connected with the detection of changes in muscle proteins during post-mortem autolysis and the search for species-specific and other protein biomarkers. Particular publications regarding the development of methods based on proteomic technologies for monitoring the state of muscle proteins are considered. According to the analyzed data, we can conclude that the field is promising for the solution of a number of pressing problems in.applied biochemistry.

[Effect of a complex preparation of sea urchin shells on blood glucose level and oxidative stress parameters in type II diabetes model].

The experimental preclinical study on mice showed that a complex of polyhydroxylated naphthoquinone pigments and minerals from shells of sea urchins decreases the concentration of glucose, stimulates the synthesis of phospholipids in liver, and has antioxidant properties. On the model of streptozotocin-nicotinamide-induced type II diabetes, this complex of polyhydroxylated naphthoquinone pigments and minerals from sea urchin shells exhibited activity after administration for 10 days in a dose of 1.8 mg/kg.

AGR2, ERp57/GRP58, and some other human protein disulfide isomerases.

This review considers the major features of human proteins AGR2 and ERp57/GRP58 and of other members of the protein disulfide isomerase (PDI) family. The ability of both AGR2 and ERp57/GRP58 to catalyze the formation of disulfide bonds in proteins is the parameter most important for assigning them to a PDI family. Moreover, these proteins and also other members of the PDI family have specific structural features (thioredoxin-like domains, special C-terminal motifs characteristic for proteins localized in the endoplasmic reticulum, etc.) that are necessary for their assignment to a PDI family. Data demonstrating the role of these two proteins in carcinogenesis are analyzed. Special attention is given to data indicating the presence of biomarker features in AGR2 and ERp57/GRP58. It is now thought that there is sufficient reason for studies of AGR2 and ERp57/GRP58 for possible use of these proteins in diagnosis of tumors. There are also prospects for studies on AGR2 and ERp57/GRP58 leading to developments in chemotherapy. Thus, we suppose that further studies on different members of the PDI family using modern postgenomic technologies will broaden current concepts about functions of these proteins, and this will be helpful for solution of urgent biomedical problems.

[Study of Dj-1 protein in tissue specimens, cultured cells and serum of prostate cancer patients].

Two isoforms of Dj-1 protein were identified using a proteomic study in tissue specimens from two groups of patients with confirmed benign prostate hyperplasia (BPH) and prostate cancer (PCa). Dj-1 was also found in the cell lines PC-3, DU-145, LNCaP, BPH-1, and the lowest level of Dj-1 was found in BPH-1. Immunochemical study (ELISA) of serum levels of Dj-1, Bcl-2, IGF-1 and IGFBP-3 proteins revealed statistically significant distinctions between two groups of patients (p=0,004, Mann-Whitney test) only for Dj-1. Taken together, these data suggest that Dj-1 protein is a perspective biomarker candidate for PCa.

[The use of a program of therapeutic physical exercises for the correction of the functional state of the cardiorespiratory system of adolescents residing in a megapolis].

The objective of this work was to study the influence of corrective and health-improving exercises on the functional characteristics of the cardiorespiratory system of adolescents residing in a megapolis before they developed any pathology. The health status of the children (n = 1760) was monitored using the "Health Navigator" technology; special attention was given to the initial state of the cardiorespiratory system and its dependence on the environmental factor. The proposed program of special corrective respiratory exercises in combination with physical endurance exercises was shown to improve the adaptive responsiveness of the cardiorespiratory system and correct the initial level vasoregulation.

[Is it possible to "cancel" aging process of cell cultures under optimal conditions for cultivation?].

The characteristics of the cells epigenotypes Dunaliella viridis Teod. in the process of chronological and replicative aging were investigated. By 40th day of accumulative cultivation (which coincided with the stationary growth phase) DNA content in the cells of Dunaliella viridis increased 2 times, triacylglycerides 3 times, beta-carotene and carbonyl proteins 2 times, RNA content decreased in comparison with cells in exponential growth phase, i. e., the 40th day of growth of culture forms the age-related epigenotype. 4 received subcultures were being transplanted during 2 years in mid-logarithmic growth phase (subculture-10), early stationary phase of growth (subculture-20), in the mid-stationary growth phase (subculture-30), and late stationary growth phase (subculture-40). It is shown that epigenotype of subculture-10 remained unchanged over 2 years of cultivation, i. e., it does not manifest replicative aging. At the same time, the subculture-20, although long enough (at least 40 passages), maintained epigenotype characteristic of young cultures, and showed age-related changes. Pronounced age-dependent changes of epigenotype in the course of cultivation were identified for subculture-30, and subculture-40 was characterized by unstable epigenotype. Thus, cultivation conditions determine the intensity of replicative aging in Dunaliella viridis.

Induction of permeability of the inner membrane of yeast mitochondria.

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a nonspecific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca2+-dependent pores, neither the classical Ca2+/P(i)-dependent, cyclosporin A-sensitive pore even under de-energization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca2+ concentrations (in the presence of the Ca2+ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca2+ uptake and is differently regulated compared to the mPTP of animal mitochondria.

[A study of protein profile changes in differentiating human myoblasts].

The changes in the protein profile in cultured human myoblasts after induction of differentiation was studied by proteomic techniques (a combination of O'Farrell two-dimensional electrophoresis and subsequent protein identification by MALDI-TOF MS and MS/MS analyses). Forty-one proteins have been identified, 25 of which were present in both proliferating and differentiating myoblasts, which allows them to be considered as myoblast housekeeping proteins. The changes in the distribution of some isoforms of tropomyosins, S100 proteins, cofilin, etc. have been revealed. The possible role of these changes in the cell protein profile in the realization of the program of skeletal muscle cell differentiation is discussed.

[Clinicobiochemical study of diagnostic value of the panel including 10 potential protein markers of prostatic cancer].

We tested diagnostic value of the panel of 10 specially selected proteins--potential markers of prostatic cancer. A double blind method and proteomic technologies were used in complex clinicobiochemical examination of 20 patients with benign and malignant tumors. The same diagnosis were obtained by clinicomorphological criteria and protein markers in 13 (65%) cases. The highest diagnostic efficacy was achieved in prostatic cancer--11 cases (79%) vs 14 by clinicomorphological criteria.

[Determination of "amino acids conflicts" and amino acids substitutions in primary structures of 41 human proteins by use the proteomic technologies].

Proteomic studies of some human tissues and organs (skeletal muscles, myometrium, motor zone of the brain, prostate), and also cultivated myoblasts revealed 41 of 300 identified proteins, in which the present of certain variants of amino acids ("conflicts") was recognized at several positions. Among the 93 registered amino acids "conflicts", seven cases represented the results of the protein polymorphisms caused by corresponding substitution of individual amino acid. Moreover, among prostate proteins the proteomic analysis revealed two isoforms of prostate-specific antigen, formed due to alternative splicing. Thus, our results have shown, that proteomic technologies allow to specify effectively the features of primary structures and to characterize various kinds of polymorphism in many human proteins.

[Age-related changes of albumin and actin of human myocardium].

In the human myocardium tissue, proteomic technologies revealed the products of 2 genes (alpha-actin and albumin), existing as fragments; their appearance and increased contents correlated with age. The age-related variants differ from the mature forms by the absence of N-terminal fragments of the amino acid sequences. In the chronic ischemic heart disease (CIHD), these age-dependent proteins were found in 50% of cases (in the age group 31-40 years), while in the control group such combination was detected only in 10% of the examined individuals. Subsequent studies in this field would probably reveal molecular mechanisms responsible for impairment and/or ageing of the cardiac muscle and also of the adaptation-dysadaptations mechanisms in the CIHD.

[Creatine as a metabolic controller of skeletal muscles structure and function in strength exercises in humans. The cellular mechanisms].

The effects of creatine oral supplementation combined with a 10-week resistive training of morphometric, contractile and molecular characteristics of human vast lateral muscle fibers were studied. 2 groups consisting of 9 young healthy men each were involved in resistive training of knee extensors for 10 weeks. Volunteers of the first group received per os 20 g of creatine for the 1st week of training and 5 g for the rest of the experimental training period. We found a significant increase of slow and fast-twitch fiber size in both trained groups and a significant increase of Ca-sensitivity of skinned single fiber contractility in creatine-supplemented group. The serum creatine phosphokinase activity in blood samples taken 24 hours after exercise session increased in all stages of the experimental training in both groups. At the same time, the adaptive decrease of the after-exercise CK concentration was observed in the placebo but not in the creatine-supplemented group. The altered integrity of the subsarcolemmal dystrophin layer was revealed in both groups after training.

[New approaches to molecular diagnosis of prostatic cancer].

We compared prostatic proteins in patients operated for adenoma or cancer. 630 protein fractions were obtained from each tissue sample after fractionation by two-dimentional electrophoresis according to O'Farrell. Comparison of the samples from adenoma and cancer showed their difference by 7 proteins among which were isoforms of glyceraldehydes-3-phosphate-dehydrogenase, alpha-collagen and several little known proteins. Most of the cancer patients had the protein with molecular mass 19 kDa and isoelectric point 9.0. By the results of mass-spectrometry this protein was identified as androgen-induced secreted protein AGR2. This protein is considered a potential oncomarker. Prospects of some postgenome technologies for detection of new diagnostic markers of prostatic cancer are discussed.

Factors affecting survival of reconstructed mouse embryos after nuclear transfer.

Reconstruction of mouse embryos was performed by injection of donor genetic material from differentiated cells of various types (cumulus cells, cardiomyocytes, and epithelial cells) into recipient cells (mature oocytes and zygotes). A medium for microsurgery was selected, which enhanced survival of both embryonic and somatic cells during the reconstructive manipulations. Special preparation of somatic cells to transplantation was carried out, which employed factors synchronizing the cells in a certain phase of the cell cycle in order to enhance their capacity to maintain the development of reconstructed embryos. The processes of nucleus reprogramming in specialized cells under the action of cytoplasmic factors of oocytes and zygotes were examined. During in vitro culturing of reconstructed embryos, the most successful development was observed in embryos implanted with donor material from cumulus cells. Mouse embryos reconstructed with a certain genome and subsequent production and use of stem cells are considered as the model system for developing the basic principles of replacement therapy.

Spectral study of interactions of 4,8,4'-trimethylpsoralen and 4,4'-dimethylangelicin dyes with DNA.

Absorption and fluorescence spectra for six new synthetic dyes of 4,8,4'-trimethylpsoralen and 4,4'-dimethylangelicin derivatives containing various terminal substituents at 5'-position have been investigated in different environments using a wide range of the DNA/ligand concentrations. Various spectral and binding characteristics of the DNA-ligand systems have been determined. General principles characterizing mechanisms responsible for changes in the fluorescent properties of nucleotide-specific dyes have been proposed; they take into consideration chemical structure of the dyes, properties of the environment, and degree of sorption on substrate.

[Gelatinase activity of protein fractions obtained by electrophoresis in polyacrylamide gel]. / Izuchenie zhelatinaznoi aktivnosti v belkovykh fraktsiiakh, poluchennykh élektroforezom v poliakrilamidnom gele.

The modification of a method of determination gelatinolytic activity in protein fractions obtained by one and two-dimensional polyacrylamide gel electrophoresis is presented. The presence of a fraction 115-125 kD among human proteins of cortex matter and glomerulus of kidney and also in myocardium having gelatinolytic activity is demonstrated.