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The article analyzes the conditions for the formation of recreation for children in the structure of an urbanized environment. In the conditions of large cities and metropolitan areas, specialists are faced with the acute issue of creating and shaping a comfortable and safe recreational environment for children. The article explores the main factors that have a decisive influence on the formation of a comfortable environment for organizing children's leisure. The problems of creating recreational areas for children in an urbanized environment are constantly considered by many specialists, including urban planners, psychologists, teachers and others. The article discusses domestic and foreign experience in the creation, operation and development of recreational areas for children in different periods of the development of society, the main tasks of children's leisure and recreation in general. The modern system of children's recreation, the typology of its objects and elements are analyzed, considering the current standards. In European countries, as well as in Ukraine, the organization of children's recreation is supported by the state and the legislative framework, but the conditions for its formation are milder in comparison with Ukraine. When designing children's recreation in Europe, the state of the urban environment, the unity of space, the overall design and safety issues are necessarily considered, and the child is taught to be careful and competently assess the current situation. In Ukraine, they adhere to the principle – the more sites, the better.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, e.g., soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests against three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs more experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

The role of small secreted peptides in plant defense responses to viruses has seldom been investigated. Here, we report a role for potato (Solanum tuberosum) PIP1, a gene predicted to encode a member of the pathogen-associated molecular pattern (PAMP)-induced peptide (PIP) family, in the response of potato to Potato virus Y (PVY) infection. We show that exogenous application of synthetic StPIP1 to potato leaves and nodes increased the production of reactive oxygen species and the expression of plant defense-related genes, revealing that StPIP1 triggers early defense responses. In support of this hypothesis, transgenic potato plants that constitutively overexpress StPIP1 had higher levels of leaf callose deposition and, based on measurements of viral RNA titers, were less susceptible to infection by a compatible PVY strain. Interestingly, systemic infection of StPIP1-overexpressing lines with PVY resulted in clear rugose mosaic symptoms that were absent or very mild in infected non-transgenic plants. A transcriptomics analysis revealed that marker genes associated with both pattern-triggered immunity and effector-triggered immunity were induced in infected StPIP1 overexpressors but not in non-transgenic plants. Together, our results reveal a role for StPIP1 in eliciting plant defense responses and in regulating plant antiviral immunity.

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1‐Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three‐dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin‐binding sites of tropomodulin and generated a three‐dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self‐consistent three‐dimensional model of tropomodulin assembly at the pointed end. The model of the pointed‐end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.

The article studies the creation and functioning of industrial parks. The potential for intensifying industrial development is achieved through the mechanism of industrial parks. The placement of industrial parks in areas free from building or already in existing industrial areas require special attention. The article examines the experience of the creation and functioning of industrial parks on the example of the countries of the world. A distinctive feature of the development and creation of industrial parks in Europe and the USA is their creation in already existing clusters and industrial zones with a clearly defined specialization or in the territories of special economic zones. The classification of industrial parks has been investigated. Their features and characteristics have been determined, which must be taken into account when initiating their creation and further development. The legislative basis for the creation and functioning of industrial parks is considered. The processes of formation of industrial parks in Ukraine, despite the adoption of the relevant law and development concept, are only at the initial stage and face a number of problems that require detailed consideration and improvement, namely: the lack of a competitive market for management companies; developed infrastructure; a truly preferential mode of their functioning is not provided; low investment attractiveness of the state as a whole, etc., Розглянуто досвід створення та функціонування індустріальних парків у світі та в Україні. Досліджено ознаки, характеристики та класифікації індустріальних парків, які необхідно враховувати при ініціюванні їх створення та подальшому розвитку.

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Plant recognition of invading organisms occurs through identification of foreign molecules associated with attackers and of self-derived, damage-associated molecules. Perception of these molecules activates signaling processes including dynamic changes in ion balance, production of second messengers such as reactive oxygen species and nitric oxide, increased levels of plant hormones, and map kinase cascade activation. Together these signaling events stimulate transcriptional changes to initiate plant defense responses. Among the earliest detectable signaling events is a rapid increase in apoplastic pH, i.e., extracellular alkalinization. Here, an assay for quantification of this alkalinization response using suspension-cultured cell lines for Arabidopsis, potato, and maize is described. This assay is an inexpensive, fast, simple, and reproducible method to quantify defense signaling output, providing a powerful tool for evaluating early plant responses to elicitors and pathogens. Results from the alkalinization assay are comparable to other more costly and time-consuming methods for assessing defense signaling, such as measurement of the oxidative burst, calcium influx, and marker gene expression. This bioassay is a quantitative and robust method for evaluation of plant defense output. © 2017 by John Wiley & Sons, Inc.

The first layer of plant immunity is deployed by recognition of pathogen-associated molecule patterns (PAMPs) and induction of early stress responses. Flagellin is the major protein component of the flagellum. Flagellin-derived peptide fragments such as Flg22, a short active peptide derived from the highly conserved part of the N-terminal region, are recognized as PAMPs by a specific perception system present in most higher plants. Some bacteria evade the plant recognition system by altering the Flg22 region in the flagellin. Instead, a small subset of plants (i.e., solanaceous plants) can sense these bacteria by recognizing a second region, termed FlgII-28. The function of FlgII-28 has been well-documented in tomato but not in potato plants. Here, we investigated the effect of FlgII-28 on several defense responses in potato. Cytosolic calcium (Ca2+) elevation is an early defense response upon pathogenic infection. We generated transgenic potato plants expressing aequorin, a nontoxic Ca2+-activated photoprotein. The results showed that FlgII-28 induced strong cytosolic Ca2+ elevation in a dose-dependent manner, whereas the response was attenuated when a Ca2+ channel blocker was added. In addition, the FlgII-28-triggered cytosolic Ca2+ elevation was shown to subsequently promote extracellular alkalinization, reactive oxygen species production, mitogen-activated protein kinase phosphorylation, and transcriptional reprogramming of defense-related genes in potato. Interestingly, all tested defense responses caused by FlgII-28 were significantly stronger than those caused by Flg22, suggesting that FlgII-28 acts as a primary flagellar PAMP to elicit multiple defense responses in potato.

The precise arrangement of the N-terminal actin- and tropomyosin-binding sites within leiomodin-2 is determined. A novel model is given of the roles of leiomodin-2 at thin filament pointed ends., Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2–knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43–90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124–201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly­merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities.

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein-protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7-41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.

Hypoxia inducible factor (HIF) prolyl hydroxylases (PHD) belong to α-ketoglutarate-dependent non-heme iron dioxygenases catalyzing hydroxylation of HIF prolines. The catalytic domains of all three enzyme isoforms (PHD1–3) were expressed in E.coli with the highest expression level being observed for the PHD2 isoform. In all cases, the recombinant portion of protein was mainly expressed as insoluble inclusion bodies. PHD was reactivated by refolding from inclusion bodies. To optimize the refolding procedure, a novel continuous assay based on measurement of ferrocyanide oxidation activity in the presence of HIF peptide or protein was developed. The comparison between the purified soluble enzyme samples and the PHD2 samples reactivated from inclusion bodies showed a 4–5-fold higher specific activity of the latter (5 mol min−1 vol) in the α-ketoglutarate consumption determined by fluorescence detection of ketoglutarate—o-phenylene diamine adduct. The PHD2 isoform is highly hydrophobic, has to be handled in high-molarity buffer solutions to prevent aggregation and precipitation, and is inactivated rapidly in the absence of dithiothreitol (DTT).

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we propose a fast and reliable alkalinization assay to monitor plant defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists (Phytophthora infestans and Spongospora subterranea) and fungi (Verticillium dahliae and Colletotrichum coccodes). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization assay is an effective tool to study early stages of defense response in potatoes.

Small molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1 alpha oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a "branching motif" immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best "hits" stabilize HIF1 alpha, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.

The development of some familial dilated cardiomyopathies (DCM ) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Whereas the biological consequence of mutations affecting protein folding, stability and levels of intracellular expression is more apparent, understanding of harmful effects of mutations interfering with yet uncharacterized protein-protein interactions might be elusive. We used nuclear magnetic resonance spectroscopy to precisely localize the region of striated muscle α-tropomyosin that interacts with leiomodin-2 (Lmod2), a member of tropomodulin family of actin-binding proteins. The K15N mutation known to be associated with familial DCM is located within the newly identified Lmod2 binding site of tropomyosin. We studied the effect of this mutation on binding Lmod2 and Tmod1, cardiac isoforms responsible for correct lengths of the thin filaments. The mutation reduced binding affinity for both Lmod2 and Tmod1, however the decrease was more pronounced for binding Lmod2. The reduced affinity and the uneven effect of the K15N mutation on TM binding to Lmod2 and Tmod1 provide a molecular rationale for the development of familial DCM.

Actin dynamics is fundamental for neurite development; monomer depolymerization from pointed ends is rate-limiting in actin treadmilling. Tropomodulins (Tmod) make up a family of actin pointed end-capping proteins. Of the four known isoforms, Tmod1–Tmod3 are expressed in brain cells. We investigated the role of Tmod’s C-terminal (LRR) domain in the formation of neurite-like processes by overexpressing Tmod1 and Tmod2 with deleted or mutated LRR domains in PC12 cells, a model system used to study neuritogenesis. Tmod1 overexpression results in a normal quantity and a normal length of processes, while Tmod2 overexpression reduces both measures. The Tmod2 overexpression phenotype is mimicked by overexpression of Tmod1 with the LRR domain removed or with three point mutations in the LRR domain that disrupt exposed clusters of conserved residues. Removal of Tmod2’s LRR domain does not significantly alter the outgrowth of neurite-like processes compared to that of Tmod2. Overexpression of chimeras with the N-terminal and C-terminal domains switched between Tmod1 and Tmod2 reinforces the idea that Tmod1’s LRR domain counteracts the reductive effect of the Tmod N-terminal domain upon formation of processes while Tmod2’s LRR domain does not. We suggest that the TM-dependent actin capping ability of both Tmods inhibits the formation of processes, but in Tmod1, this inhibition can be controlled via its LRR domain. Circular dichroism, limited proteolysis, and molecular dynamics demonstrate structural differences in the C-terminal region of the LRR domains of Tmod1, Tmod2, and the Tmod1 mutant.

Flagella are extracellular organelles that propel bacteria. Each flagellum consists of a basal body, a hook, and a filament. The major protein of the filament is flagellin. Induction of flagellin gene expression coincides with secretion of FlgM. The role of FlgM is to inhibit FliA (σ 28 ), a flagellum-specific RNA polymerase responsible for flagellin transcription. To prevent premature polymerization of newly synthesized flagellin molecules, FliS, the flagellin-specific chaperone, binds flagellin and facilitates its export. In this study, the interaction between FlgM and FliS from Salmonella enterica serovar Typhimurium was characterized using gel shift, intrinsic tryptophan fluorescence, circular dichroism, limited proteolysis, and cross-linking. We have demonstrated that (i) FliS and FlgM interact specifically, forming a 1:1 complex, (ii) the FliS binding site on FlgM is proximal to or even overlaps the binding site for FliA, and (iii) FliA competes with FliS for FlgM binding.

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer-by-layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility.

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Actin filaments are major components of the cytoskeleton in eukaryotic cells and are involved in vital cellular functions such as cell motility and muscle contraction. Tropomyosin is an alpha-helical, coiled coil protein that covers the grooves of actin filaments and stabilizes them. Actin filament length is optimized by tropomodulin, which caps the slow growing (pointed end) of thin filaments to inhibit polymerization or depolymerization. Tropomodulin consists of two structurally distinct regions: the N-terminal and the C-terminal domains. The N-terminal domain contains two tropomyosin-binding sites and one tropomyosin-dependent actin-binding site, whereas the C-terminal domain contains a tropomyosin-independent actin-binding site. Tropomodulin binds to two tropomyosin molecules and at least one actin molecule during capping. The interaction of tropomodulin with tropomyosin is a key regulatory factor for actin filament organization. The binding efficacy of tropomodulin to tropomyosin is isoform-dependent. The affinities of tropomodulin/tropomyosin binding influence the proper localization and capping efficiency of tropomodulin at the pointed end of actin filaments in cells. Tropomodulin and tropomyosin are crucial constituents of the actin filament network, making their presence indispensable in living cells. Here we describe how a small difference in the sequence of the tropomyosin-binding sites of tropomodulin may result in dramatic change in localization of Tmod in muscle cells or morphology of non-muscle cells. We also suggest most promising directions to study and elucidate the role of Tmod-TM interaction in formation and maintenance of sarcomeric and cytoskeletal structure.

New types of potent aprotinin-containing medicinal polymer films are elaborated for topical applications. The higher the temperature of drying, the lower the steam absorption, the film swelling, and the velocity of aprotinin in vitro release. The presence of antimicrobials having the basic functional groups contributed to compacting the structure of the films and retention of aprotinin in them. The velocity of aprotinin release from the films is regulated by adding different polymers. Inclusion of polyvinylpirrolidone in the film resulted in acceleration and increase of aprotinin release. This was probably because of increasing the film swelling by a factor of 1.7. Additional retention of the inhibitor in films was achieved by inclusion of sodium alginate and cellulose powder capable of binding aprotinin. Soluble bioadhesive films derived from a copolymer of acrylamide,N-vinyl-pirrolidone, ethyl acrylate (M r 30,000-600,000) and aprotinin were obtained and analyzed. Kinetics of aprotinin release from biosoluble films was studied under various conditions. The duration of aprotinin release was comparable with duration of gradual dissolution of the matrix. Bioavailability of aprotinin from soluble films was linear with time.

Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1–4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity.

Assembly of the actin cytoskeleton is an important part of formation of neurites in developing neurons. Tropomodulin, a tropomyosin-dependent capping protein for the pointed end of the actin filament, is one of the key players in this process. Tropomodulin binds tropomyosin in two binding sites. Tmod1 and Tmod2, tropomodulin isoforms found in neurons, were overexpressed in PC12 cells, a model system for neuronal differentiation. Tmod1 did not affect neuronal differentiation; while cells expressing Tmod2 showed a significant reduction in the number and the length of neurites. Both tropomodulins bind short α-, γ- and δ-tropomyosin isoforms. Mutations in one of the tropomyosin-binding sites of Tmod1, which increased its affinity to short γ- and δ-tropomyosin isoforms, caused a decrease in binding short α-tropomyosin isoforms along with a 2-fold decrease in the length of neurites. Our data demonstrate that Tmod1 is involved in neuronal differentiation for proper neurite formation and outgrowth, and that Tmod2 inhibits these processes. The mutations in the tropomyosin-binding site of Tmod1 impair neurite outgrowth, suggesting that the integrity of this binding site is critical for the proper function of Tmod1 during neuronal differentiation.

Regulation of actin filament assembly is essential for efficient contractile activity in striated muscle. Leiomodin is an actin-binding protein and homolog of the pointed-end capping protein, tropomodulin. These proteins are structurally similar, sharing a common domain organization that includes two actin-binding sites. Leiomodin also contains a unique C-terminal extension that has a third actin-binding WH2 domain. Recently, the striated-muscle-specific isoform of leiomodin (Lmod2) was reported to be an actin nucleator in cardiomyocytes. Here, we have identified a function of Lmod2 in the regulation of thin filament lengths. We show that Lmod2 localizes to the pointed ends of thin filaments, where it competes for binding with tropomodulin-1 (Tmod1). Overexpression of Lmod2 results in loss of Tmod1 assembly and elongation of the thin filaments from their pointed ends. The Lmod2 WH2 domain is required for lengthening because its removal results in a molecule that caps the pointed ends similarly to Tmod1. Furthermore, Lmod2 transcripts are first detected in the heart after it has begun to beat, suggesting that the primary function of Lmod2 is to maintain thin filament lengths in the mature heart. Thus, Lmod2 antagonizes the function of Tmod1, and together, these molecules might fine-tune thin filament lengths.

To enhance the antitumor potential of soybean Bowman-Birk inhibitor (BBI), the conjugate of BBI with an antibody via a macromolecular carrier was prepared. Clinical dextran (D) was used as a biocompatible biodegradable carrier for co-immobilization of BBI and antibody. A model immunoglobulin isolated from sheep serum (sIgG), raised against human IgM was utilized to develop the procedure of immunoconjugate synthesis. The molar ratio of the ingredients in the conjugate was the following BBI:D:sIgG=9:1:1. Comparison of the dose response curves for the native sIgG and the BBI-D-sIgG conjugate indicated that sIgG completely retained its specific activity (90%) after modification with dextran. The determination of the Ki values for chymotrypsin interaction with the native BBI and the BBI-D-sIgG conjugate indicated high anti-chymotrypsin activity. In the next step, the monoclonal antibody (ICO 25 MAb) against the mucin-like human epithelial membrane antigen was used for conjugation as it is the most universal vector for targeting different agents to human tumors of epithelial origin. The influence of conjugation on the specificity of the Mab reaction with its antigen was studied. The conjugated MAb reacted with tumor cells of different epithelial genesis (breast, lung, gastric, ovarian and uterus tumors), but did not react with tumor cells of non-epithelial origin. It was shown that BBI-D-ICO 25 MAb conjugate has almost the same immunohistochemical activity as non-conjugated MAb. These results demonstrated the feasibility of exploiting the activities of covalently bound BBI and ICO 25 MAb for anticarcinogenic agent targeting.