Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant kass=(2.3 ± 0.7) × 106 M-1s-1 and an SI of 4.7 ± 0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes.

In this study, we performed transcriptome profiling of oligodendrocyte culture of mice treated with the remyelinating therapeutic agent benztropine in the presence and absence of interferon gamma (IFNγ). The results of this work are important for understanding the expression profile of oligodendrocytes under conditions of systemic inflammation in the central nervous system in multiple sclerosis as well as the mechanisms of cellular response to benztropine in light of its possible use for the treatment of multiple sclerosis.

Protein B23/nucleophosmin is a polyfunctional protein existing in cells in numerous structural forms. In this work, for the immunochemical analysis of nucleophosmin we used the antibodies specific to different forms of nucleophosmin, namely, antibodies selectively revealing monomers of all the known forms of this protein and antibodies specific only to isoform B23.1. Homogenates of different rat tissues such as the brain, liver, kidney, lung and heart were used, as well as nuclei from liver and brain cells. For the first time, we show that the structural state of nucleophosmin in brain differs from its state in other tissues, including the liver that is enriched with nucleophosmin. It was revealed that on immunoblots of brain homogenates not only monomeric form of nucleophosmin but also unique SDS-resistant oligomeric forms were detected in the SDS-PAGE. Analysis of nucleophosmin in the cerebellum, cortex, amygdala, brainstem, and hippocampus showed that most enriched with nucleophosmin were hippocampus and cerebellum; on their immunoblots SDS-resistant oligomeric forms of nucleophosmin dominated. Using immunochemical analysis of the protein in primary cultures of cerebellum glial cells and neurons, significant structural differences of nucleophosmin in proliferating glial cells and non-proliferating neurons were revealed for the first time. It was found also that the nucleophosmin content in glial cells is much higher than in neurons and that the main forms of protein B23 in glial cells on immunoblots are the SDS-resistant oligomers, while a monomeric form was present in much smaller quantities. In contrast to glial cells, neurons did not contain such oligomers. In neurons, only trace amounts of a monomeric form of nucleophosmin were found, which were undetectable by the antibodies specific to isoform B23.1.

The results of 163 YAG-laser interventions for the elimination of the papillary block following the extraction of congenital, traumatic, complicated, and secondary cataracts are reported. Basal colomba was present in all treated eyes after YAG-laser iridectomy and iris bombe was eliminated in 93,9% of the children. The papillary block was eliminated with the complete restoration of normal anatomical features in 65,5% of the patients in the immediate postoperative period (within 3 months after surgery) and in 54,9% during the follow-up period from 6 months to 17 years in duration with the partial restoration of anatomical and morphological relationships in the affected region in one third of the children (32,5% and 35,3% respectively). The hypotensive effect was documented in 96,6% and 85,3% of the children in the early and late postoperative periods respectively. It was shown that indications for the elimination of the papillary block include any variants of clinical manifestations of this pathology, such as seclusion of the pupil, iris bombe ( both with ophthalmic hypertension (requiring emergency intervention) and without it (threatening iridocorneal adhesions)), and preangular block (planned treatment). The strategy for the choice of the laser-assisted treatment is recommended with special reference to various clinical manifestations of the papillary block in the children. The most persistent anatomical effect characterized by the reduced frequency of hemorrhagic complications at lower energy consumption was achieved with the use of the combined Ar-YAG-laser iridectomy (in comparison with YAG-laser iridectomy) in the case of laser colomba over 1 mm in diameter. The importance of maintaining the minimal energy regime is emphasized in conjunction with taking into consideration the degree of child’s communicativeness, the possibility of surgical intervention without narcosis (usually by performing surgery in several steps), and the reduction of the number of narcotization procedures. It is concluded that YAG-laser iridectomy provides an effective and safe tool for the elimination of the pupillary block as an alternative to surgical iridectomy; moreover, it does not require repeated capsulotomy and can not infrequently be performed possible without narcosis.

Protein B23/nucleophosmin is a multifunctional protein existing in cells in numerous structural fowls. In this work for immunochemical analysis of nucleophosmin we used the antibodies specific to different forms of nucleophosmin: antibodies selectively revealing monomers of all the known forms of protein B23 and antibodies specific only to isoform B23.1. Homogenates from different rat tissues such as brain, liver, kidney, lung and the heart were used, as well as nuclei from liver and brain cells. It was shomin for the first time that the structural state of nucleophosmin in the brain cells differs from its state in other tissues including liver enriched with nucleophosmin. Immunochemical analysis showed that in contrast to other tissues, immunoblots of brain cells contained not only monomeric form of nucleophosmin but also unique oligomeric forms detected in the SDS-PAGE. Analysis of nucleophosmin in the cerebellum, cortex, amygdala, stem, and hippocampus revealed that most enriched with nucleophosmin are hippocampus and cerebellum. The SDS-resistant oligomeric form of nucleophosmin dominated in their immunoblots. Immunochemical analysis of the protein in primary cultures of cerebellum glial cells indicated that nucleophosmin exists in these cells in different states and revealed structural differences of nucleophosmin in proliferating glial cells and non-proliferating neurons. It was found nucleophosmin content in glial cells is much higher than in neurons. The main forms of the protein in glial cells on immunoblots were SDS -resistant oligomers, while a monomeric form was present in much smaller amounts. In contrast, neurons did not contain such oligomers. In neurons only a trace amount of a monomeric form of nucleophosmin was found, which was not detectable by the antibodies against isoform B23.1.

Нуклеофозмин – полифункциональный белок, представленный в клетках разными структурными формами. В данной работе для анализа состояния нуклеофозмина в нормальных тканях иммунохимическим методом впервые помимо моноклональных антител были применены противопептидные антитела к разным формам нуклеофозмина: антитела, специфично выявляющие мономеры всех известных форм белка В23, и антитела, селективные только для мономеров и олигомеров изоформы В23.1. Объектами исследования служили гомогенаты головного мозга, печени, почек, легких и сердца крыс, а также ядра клеток, полученные из препаратов печени и мозга. Обнаружено, что структурное состояние нуклеофозмина в клетках мозга отличается о его состояния в клетках других тканей. Выявлено, что только на иммуноблотах гомогенатов мозга, помимо мономерной, детектируется уникальная олигомерная форма нуклеофозмина, устойчивая к обработке SDS и видимая в условиях проведения SDS-ПААГ-электрофореза. Анализ нуклеофозмина в мозжечке, коре, амигдале, стволе, гиппокампе позволил установить, что нуклеофозмином наиболее обогащены мозжечок и гиппокамп, причем на иммуноблотах обоих препаратов олигомерная форма белка была превалирующей. Состояние нуклеофозмина в пролиферирующих клетках глии и непролиферирующих нейронах мозга изучали с помощью иммунохимического анализа на препаратах первичных культур клеток глии и нейронов. Впервые обнаружено, что клетки глии и нейроны принципиально отличаются по содержанию и структурному состоянию нуклеофозмина. Содержание нуклеофозмина в клетках глии значительно больше, чем в нейронах. На иммуноблотах клеток глии нуклеофозмин детектируется в основном в виде SDS-устойчивых олигомеров, а мономерная форма присутствует в существенно меньшем количестве. На иммуноблотах нейронов видны лишь следовые количества мономерной формы нуклеофозмина.

Butyrylcholinesterase (BChE) is a serine hydrolase (EC 3.1.1.8) which can be found in most animal tissues. This enzyme has a broad spectrum of efficacy against organophosphorus compounds, which makes it a prime candidate for the role of stoichiometric bioscavenger. Development of a new-age DNA-encoded bioscavenger is a vival task. Several transgenic expression systems of human BChE were developed over the past 20 years; however, none of them has been shown to make economic sense or has been approved for administration to humans. In this study, a CHO-based expression system was redesigned, resulting in a significant increase in the production level of functional recombinant human butyrylcholinesterase as compared to the hitherto existing systems. The recombinant enzyme was characterized with Elman and ELISA methods.

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.

We have previously shown that, normally, two forms of the pigment epithelium-derived factor (PEDF) with molecular weights of 50 and 45 kDa are present in the Tenon's capsule in equal amounts. These forms represent the full-length protein and a product of restricted proteolysis of PEDF. In persons with myopia, the full-length uncleaved factor was predominantly detected, which correlated with the disturbance of collagen fiber formation. An immunohistochemical study of the Tenon's capsule using polyclonal antibodies to PEDF showed that, in the control group, the factor is localized solely inside fibroblasts, whereas in patients with myopia, PEDF is distributed outside the cell as a halo around destroyed fibroblasts. It was shown in the present work using atomic force microscopy and immunodot assay with antibodies specific to amyloid fibrils that only the full-length PEDF is capable of forming amyloid-like fibrillar structures. The accumulation of fibrils leads to the destruction of fibroblasts and is a cause of changes in the biochemical composition and morphological structure of the Tenon's capsule in myopia.

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer’s disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by injection of beta-amyloid peptide (25–35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the exploratory behavior of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of the rat hippocampal and cerebellar neurons under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer’s disease and in the medium of the culture of hippocampal neurons in the presence of the Aβ(25–35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer's disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by administration of the peptide corresponding to the 25-35 sequence of beta-amyloid peptide (25-35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the orientation-investigation activity of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of neurons of the hippocampus and cerebellum of rats under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer's disease and in the medium of the culture of hippocampus neurons in the presence of the Abeta(25-35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.

Показано нейропротекторное действие гексапептида HLDF-6 (Thr-Gly-Glu-Asn-His-Arg) - биологически активного фрагмента фактора дифференцировки лейкемических клеток человека HLDF-на моделях болезни Альцгеймера in vivo и in vitro. Синдромы данной патологии у самцов крыс индуцировались путем введения пептида (25-35) бета-амилоида и иботеновой кислоты в гиппокамп. Пептид HLDF-б предотвращал потерю долговременной памяти и снижение ориентировочно-исследовательской активности у таких животных, а также значительно уменьшал число пикнотических нейронов в области СА1 гиппокампа. Протекторный эффект данного пептида проявлялся также in vitro на первичных культурах нейронов мозжечка и гиппокампа крыс в условиях бета-амилоидной токсичности. Показано значительное увеличение содержания дигидротестостерона (DHT) в плазме крови крыс с синдромами болезни Альцгеймера, а также в культуральной среде нейронов гиппокампа, культивируемых в присутствии пептида (25-35) бета-амилоида. В обоих случаях это увеличение подавлялось пептидом HLDF-6. Предложен возможный механизм нейропротекторного эффекта пептида HLDF-б, предполагающий его влияние как на биосинтез, так и на метаболизм половых стероидных гормонов.

The mature differentiation factor HLDF, isolated from cultural medium, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48–50 and 100–102 (numbering according to the structure of S21), i.e., in the area preceding the cDNA fragment that encodes the N -terminal fragment of the mature protein. The mRNAs of HLDF and S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N -terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C -terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N -terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21.

Previously we identified a six-membered fragment 354TQVEHR359 of the C-terminal part of the PEDF (Pigment Epithelium-Derived Factor) differentiation factor molecule that shares homology with fragment 41TGENHR46 of the HLDF (Human Leukemia Differentiation Factor) differentiation factor molecule, which is responsible for its differentiation activity. HLDF has been isolated from the culture medium of human promyelocytic leukemia cell line HL-60. Hexapeptides HLDF-6 (TGENHR) and PEDF-6 (TQVEHR) corresponding to these HLDF and PEDF molecule fragments, which were previously shown to induce cell differentiation (Kostanyan et al. (2000) Russian Journal of Bioorganic Chemistry, 26, 505-511), also have neuroprotective properties. Both peptides prevent degeneration of Purkinje cells of rat cerebellar vermis upon chemical hypoxia induced by sodium azide in vivo; this effect is also observed on a behavioral level. Peptide HLDF-6 but not PEDF-6 promotes survival of HL-60 cells upon chemical hypoxia. Peptides HLDF-6 and PEDF-6 affect different second messenger biosynthesis systems in HL-60 cells. HLDF-6 diminishes cyclic AMP level in those cells due to adenylate cyclase inhibition, while PEDF-6 inhibits phosphatidylinositol-specific phospholipase C stimulated by aluminum tetrafluoride anions.