Репозиторий Университета

© 2018 Izdatel'stvo Meditsina. All rights reserved. Results of investigation of the analgesic activity of the natural recombinant peptide PT1, which specifically binds to P2X3 receptors, are presented. The test for hypersensitivity provoked by complete Freund's adjuvant (CFA) showed evidence of the analgesic activity of PT1 peptide in CD-I mice after single intravenous administration in a dose range of 0.01-1 mg/kg.

© 2018 ABV-Press Publishing House. All rights reserved. The DNA analysis revealed a transthyretin amyloidosis with a rare Phe53Leu mutation in a woman of 62 y.o. with the late onset progressive generalized axonal sensomotor neuropathy, dry eye syndrome, and an episode of severe unintentionally weight loss. The same mutation was found in her healthy 34 y.o. son, but not in a 42 y.o. daughter. The Congo red staining were negative in nerve and salivar gland biopsies samples. The reassessment of the transthyretin amyloidosis “red flags” showed that the patient fulfilled the criteria of the disease and the absence of amyloid deposition was not the ground to reject the hereditary cause of the condition. The reasons of the misdiagnosis are discussed.

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway.

Marine sponges contain a variety of low-molecular-weight compounds including guanidine alkaloids possessing different biological activities. Monanchomycalin B and urupocidin A were isolated from the marine sponge Monanchora pulchra. We found that they act as inhibitors of the TRPV1, TRPV2, and TRPV3 channels, but are inactive against the TRPA1 receptor. Monanchomycalin B is the most active among all published marine alkaloids (EC50 6.02, 2.84, and 3.25 μM for TRPV1, TRPV2, and TRPV3, correspondingly). Moreover, monanchomycalin B and urupocidin A are the first samples of marine alkaloids affecting the TRPV2 receptor. Two semi-synthetic urupocidin A derivatives were also obtained and tested against TRP (Transient Receptor Potential) receptors that allowed us to collect some data concerning the structure-activity relationship in this series of compounds. We showed that the removal of one of three side chains or double bonds in the other side chains in urupocidin A led to a decrease of the inhibitory activities. New ligands specific to the TRPV subfamily may be useful for the design of medicines as in the study of TRP channels biology.

The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.

Обширное образование рубцовой ткани часто происходит после тяжелой ожоговой травмы, травмы или как одно из осложнений после хирургического вмешательства. Несмотря на значительные терапевтические успехи, все еще остается серьезной проблемой управлять формированием массивной рубцовой ткани, одновременно способствуя нормальному заживлению ран. Целью данного исследования было изучение терапевтического эффекта костных мезенхимальных стволовых клеток (BMSCs), которые были генетически модифицированы для сверхэкспрессии трансформирующего фактора роста-бета 3 (TGF-β 3).), ингибитор пролиферации миофибробластов и отложения коллагена I типа, на заживление кожных ран полной толщины на модели кролика. Двадцать четыре кролика с хирургически индуцированными кожными ранами полной толщины, созданными на внешнем ухе (1,5 × 1,5 см, две раны / ухо), были рандомизированы на четыре группы: (G1), раны без специальной обработки, но с обычной бессывороточной культурой средний как отрицательный контроль; (G2) местно применяемый рекомбинантный аденовирус, экспрессирующий TGF-β 3 / GFP; (G3), только для местного применения BMSC; (G4), местно применяемые BMSC, трансфицированные Ad-TGF-β 3 / GFP (BMSCs TGF-β3); и (G5), дополнительный нормальный контроль (n = 2) без раны или обработки кожи наружного уха. Размеры ран на тканях уха были тщательно исследованы, а глубина рубца и плотность ран были оценены гистологически через 21, 45 и 90 дней после создания хирургической раны. Наши результаты показали, что G4 значительно уменьшил глубину и плотность рубцов на ране по сравнению с G1 ~ 3. Количество клеток, экспрессирующих GFP, значительно увеличилось в G4 по сравнению с G2. Экспрессия белка TGF-β 3 и коллагена типа III в G4 значительно увеличилась, в то время как отношение типа I к коллагену типа III также значительно уменьшилось, что аналогично структуре ткани, обнаруженной в G5, по сравнению с другими группами лечения. В заключение трансплантация BMSCs TGF-β3 значительно улучшает заживление ран и уменьшает образование рубцовой ткани кожи на животной модели, что потенциально может предоставить альтернативу в лечении обширного образования рубцовой ткани после повреждения мягких тканей.

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community.

Acid-sensing ion channels (ASICs) play an important role in synaptic plasticity and learning, as well as in nociception and mechanosensation. ASICs are involved in pain and in neurological and psychiatric diseases, but their therapeutic potential is limited by the lack of ligands activating them at physiological pH.

EXPERIMENTAL APPROACH:

We extracted, purified and determined the structure of a bisbenzylisoquinoline alkaloid, lindoldhamine, (LIN) from laurel leaves. Its effect on ASIC3 channels were characterized, using two-electrode voltage-clamp electrophysiological recordings from Xenopus laevis oocytes.

KEY RESULTS:

At pH 7.4 or higher, LIN activated a sustained, proton-independent, current through rat and human ASIC3 channels, but not rat ASIC1a or ASIC2a channels. LIN also potentiated proton-induced transient currents and promoted recovery from desensitization in human, but not rat, ASIC3 channels.

CONCLUSIONS AND IMPLICATIONS:

We describe a novel ASIC subtype-specific agonist LIN, which induced proton-independent activation of human and rat ASIC3 channels at physiological pH. LIN also acts as a positive allosteric modulator of human, but not rat, ASIC3 channels. This unique, species-selective, ligand of ASIC3, opens new avenues in studies of ASIC structure and function, as well as providing new approaches to drug design.

Osteoarthritis (OA), the most common form of arthritis, is characterized by inflammation of joints and cartilage degradation leading to disability, discomfort, severe pain, inflammation, and stiffness of the joint. It has been shown that adenosine, a purine nucleoside composed of adenine attached to ribofuranose, is enzymatically produced by the human synovium. However, the functional significance of adenosine signaling in homeostasis and pathology of synovial joints remains unclear. Adenosine acts through four cell surface receptors, i.e., A1, A2A, A2B, and A3, and here, we have systematically analyzed mice with a deficiency for A3 receptor as well as pharmacological modulations of this receptor with specific analogs. The data show that adenosine receptor signaling plays an essential role in downregulating catabolic mechanisms resulting in prevention of cartilage degeneration. Ablation of A3 resulted in development of OA in aged mice. Mechanistically, A3 signaling inhibited cellular catabolic processes in chondrocytes including downregulation of Ca2+/calmodulin-dependent protein kinase (CaMKII), an enzyme that promotes matrix degradation and inflammation, as well as Runt-related transcription factor 2 (RUNX2). Additionally, selective A3 agonists protected chondrocytes from cell apoptosis caused by pro-inflammatory cytokines or hypo-osmotic stress. These novel data illuminate the protective role of A3, which is mediated via inhibition of intracellular CaMKII kinase and RUNX2 transcription factor, the two major pro-catabolic regulators in articular cartilage.

The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.

Обширное образование рубцовой ткани часто происходит после тяжелой ожоговой травмы, травмы или как одно из осложнений после хирургического вмешательства. Несмотря на значительные терапевтические успехи, все еще остается серьезной проблемой управлять формированием массивной рубцовой ткани, одновременно способствуя нормальному заживлению ран. Целью данного исследования было изучение терапевтического эффекта костных мезенхимальных стволовых клеток (BMSCs), которые были генетически модифицированы для сверхэкспрессии трансформирующего фактора роста-бета 3 (TGF-β 3).), ингибитор пролиферации миофибробластов и отложения коллагена I типа, на заживление кожных ран полной толщины на модели кролика. Двадцать четыре кролика с хирургически индуцированными кожными ранами полной толщины, созданными на внешнем ухе (1,5 × 1,5 см, две раны / ухо), были рандомизированы на четыре группы: (G1), раны без специальной обработки, но с обычной бессывороточной культурой средний как отрицательный контроль; (G2) местно применяемый рекомбинантный аденовирус, экспрессирующий TGF-β 3 / GFP; (G3), только для местного применения BMSC; (G4), местно применяемые BMSC, трансфицированные Ad-TGF-β 3 / GFP (BMSCs TGF-β3); и (G5), дополнительный нормальный контроль (n = 2) без раны или обработки кожи наружного уха. Размеры ран на тканях уха были тщательно исследованы, а глубина рубца и плотность ран были оценены гистологически через 21, 45 и 90 дней после создания хирургической раны. Наши результаты показали, что G4 значительно уменьшил глубину и плотность рубцов на ране по сравнению с G1 ~ 3. Количество клеток, экспрессирующих GFP, значительно увеличилось в G4 по сравнению с G2. Экспрессия белка TGF-β 3 и коллагена типа III в G4 значительно увеличилась, в то время как отношение типа I к коллагену типа III также значительно уменьшилось, что аналогично структуре ткани, обнаруженной в G5, по сравнению с другими группами лечения. В заключение трансплантация BMSCs TGF-β3 значительно улучшает заживление ран и уменьшает образование рубцовой ткани кожи на животной модели, что потенциально может предоставить альтернативу в лечении обширного образования рубцовой ткани после повреждения мягких тканей.

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community.

Acid-sensing ion channels (ASICs) play an important role in synaptic plasticity and learning, as well as in nociception and mechanosensation. ASICs are involved in pain and in neurological and psychiatric diseases, but their therapeutic potential is limited by the lack of ligands activating them at physiological pH.

EXPERIMENTAL APPROACH:

We extracted, purified and determined the structure of a bisbenzylisoquinoline alkaloid, lindoldhamine, (LIN) from laurel leaves. Its effect on ASIC3 channels were characterized, using two-electrode voltage-clamp electrophysiological recordings from Xenopus laevis oocytes.

KEY RESULTS:

At pH 7.4 or higher, LIN activated a sustained, proton-independent, current through rat and human ASIC3 channels, but not rat ASIC1a or ASIC2a channels. LIN also potentiated proton-induced transient currents and promoted recovery from desensitization in human, but not rat, ASIC3 channels.

CONCLUSIONS AND IMPLICATIONS:

We describe a novel ASIC subtype-specific agonist LIN, which induced proton-independent activation of human and rat ASIC3 channels at physiological pH. LIN also acts as a positive allosteric modulator of human, but not rat, ASIC3 channels. This unique, species-selective, ligand of ASIC3, opens new avenues in studies of ASIC structure and function, as well as providing new approaches to drug design.

Osteoarthritis (OA), the most common form of arthritis, is characterized by inflammation of joints and cartilage degradation leading to disability, discomfort, severe pain, inflammation, and stiffness of the joint. It has been shown that adenosine, a purine nucleoside composed of adenine attached to ribofuranose, is enzymatically produced by the human synovium. However, the functional significance of adenosine signaling in homeostasis and pathology of synovial joints remains unclear. Adenosine acts through four cell surface receptors, i.e., A1, A2A, A2B, and A3, and here, we have systematically analyzed mice with a deficiency for A3 receptor as well as pharmacological modulations of this receptor with specific analogs. The data show that adenosine receptor signaling plays an essential role in downregulating catabolic mechanisms resulting in prevention of cartilage degeneration. Ablation of A3 resulted in development of OA in aged mice. Mechanistically, A3 signaling inhibited cellular catabolic processes in chondrocytes including downregulation of Ca2+/calmodulin-dependent protein kinase (CaMKII), an enzyme that promotes matrix degradation and inflammation, as well as Runt-related transcription factor 2 (RUNX2). Additionally, selective A3 agonists protected chondrocytes from cell apoptosis caused by pro-inflammatory cytokines or hypo-osmotic stress. These novel data illuminate the protective role of A3, which is mediated via inhibition of intracellular CaMKII kinase and RUNX2 transcription factor, the two major pro-catabolic regulators in articular cartilage.

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway.

Marine sponges contain a variety of low-molecular-weight compounds including guanidine alkaloids possessing different biological activities. Monanchomycalin B and urupocidin A were isolated from the marine sponge Monanchora pulchra. We found that they act as inhibitors of the TRPV1, TRPV2, and TRPV3 channels, but are inactive against the TRPA1 receptor. Monanchomycalin B is the most active among all published marine alkaloids (EC50 6.02, 2.84, and 3.25 μM for TRPV1, TRPV2, and TRPV3, correspondingly). Moreover, monanchomycalin B and urupocidin A are the first samples of marine alkaloids affecting the TRPV2 receptor. Two semi-synthetic urupocidin A derivatives were also obtained and tested against TRP (Transient Receptor Potential) receptors that allowed us to collect some data concerning the structure-activity relationship in this series of compounds. We showed that the removal of one of three side chains or double bonds in the other side chains in urupocidin A led to a decrease of the inhibitory activities. New ligands specific to the TRPV subfamily may be useful for the design of medicines as in the study of TRP channels biology.