Up-regulation of amino acid transporter SLC6A19 activity and surface protein abundance by PKB/Akt and PIKfyve

Background: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. Methods: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive T308A/S473APKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant S318APIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. Results: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (Ile), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of T308A/S473APKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of S318APIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of Ile following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells

A New Method for Treating Burn Wounds Using Targeted Delivery of Medicinal Substances by Magnetic Nanocarrier (Experimental Part)

Проведено экспериментальное исследование на лабораторных животных по изучению эффективности адресной доставки мази левомеколь с помощью магнитных наночастиц и внешнего магнитного поля при термических ожогах. В исследовании принимало участие 20 крыс с двумя очагами ожога. Крысы были разделены на 4 группы: без лечения, терапия с использованием мази левомеколь, лечение с использованием наночастиц, мази левомеколь и внешнего магнитного поля и только магнитотерапии. При гистологическом исследовании на 14-е сутки во всех группах в зоне термического повреждения кожи были отмечены признаки глубокого ожога III и IV степени с распространением некроза на всю глубину дермы и на мышцы. В группе с наночастицами, мазью левомеколь и магнитным полем на фоне уменьшения воспаления отмечалось очаговое появление грануляционной ткани. Таким образом, гистологические исследования ожогового раневого процесса лабораторных животных показали, что использование инновационного биологически активного ранозаживляющего средства на основе наночастиц в сочетании с мазью левомеколь улучшает регенерацию тканей и приводит к ускорению эпителизации, что в целом повышает результаты лечения ожоговой раны. Использование внешнего магнитного поля способствует адресной доставке лечебного нанокомплекса и поддержанию оптимальной концентрации препарата в ранеExperimental studies have been carried out on laboratory animals to investigate the effectiveness of targeted delivery of levomekol ointment using magnetic nanoparticles and an external magnetic field for treatment of thermal burns. The study involved 20 rats, with two burns on each. The rats were divided into 4 groups: untreated; treated with levomekol ointment; treated with levomekol ointment associated with nanoparticles and an external magnetic field; and treated with magnetic field alone. Histological examination was conducted on Day 14, and in all groups, in the thermal burn zone of the skin there were signs of deep three- and four-degree burns with necrosis spread through the dermis, reaching the muscle. In the group with levomekol ointment associated with nanoparticles and magnetic field, inflammation was decreased, and focal granulation tissue formation was observed. Thus, histological studies of the burn wound process in laboratory animals showed that the use of an innovative biologically active wound healing agent based on nanoparticles in combination with the levomecol ointment improved tissue regeneration and accelerated epithelialization, which enhanced the effectiveness of burn wound treatment. The use of an external magnetic field facilitated targeted delivery of the therapeutic nanosystem and maintenance of the optimal concentration of the drug in the woun

Towards Better Understanding of Etiological Mechanisms at the Neuromuscular Junction

The neuromuscular junction (NMJ) serves as a model for understanding the mechanisms that determine communication between neurons and their target cells. Disorders of the NMJ can be either autoimmune or genetic (hereditary). The autoimmune disorder myasthenia gravis (MG) is caused by antibodies against the presynaptic nerve terminal or the postsynaptic muscle membrane, which make up the NMJ. The most common antibodies are directed against the acetylcholine receptor (AChR) or muscle specific tyrosine kinase (MuSK). An alternative to expand on preclinical in-vivo methods for studying mechanisms underlying diseases of neuromuscular transmission is to apply physiologic in-vitro models that would allow tissue-tissue as well as cell-cell interactions. A system that would allow cell-cell interactions in a biological fashion is the micro-electrode array (MEA) chip that allows co-culturing of motor neurons and muscle cells. The primary hypothesis is that the suggested MEA can be used in creating a reliable model for healthy and diseased NMJ, allowing for manipulations and treatment assays. The secondary hypothesis is that small non-coding RNA, so called microRNAs (miRNA) have a specific role in neuromuscular transmission and in MG. Study I demonstrated a method of long-term muscle cell culture on the MEA chips, which allows us to trace the development of muscle cells through the observation of their electrical activity at subcellular resolution. The maturation of skeletal muscle tissue was accompanied by a gradual increase in the amplitude and frequency of extracellular individual electrical spikes. The mature muscle tissue demonstrated the steady electrical activity with synchronized spike propagation in different directions across the chip. Study II showed a specific upregulated profile of miRNAs in the muscles of MuSK antibody seropositive MG mice. Transfection of these miRNAs, miR-1933 and miR-1930, promoted downregulation of several proteins and further confirmation with qPCR revealed a specific blocking of IMPA1 and MRPL27, which are involved in intracellular signal transduction and mitochondrial biogenesis in skeletal muscles. Study III revealed no correlation between the morphology of skeletal muscle cells and their electrical activity at an early developmental stage. However, the application of recombinant rat agrin increased the number of AChRs clusters in the culture of skeletal muscle and promoted a higher degree of spontaneous activity

Towards Better Understanding of Etiological Mechanisms at the Neuromuscular Junction

The neuromuscular junction (NMJ) serves as a model for understanding the mechanisms that determine communication between neurons and their target cells. Disorders of the NMJ can be either autoimmune or genetic (hereditary). The autoimmune disorder myasthenia gravis (MG) is caused by antibodies against the presynaptic nerve terminal or the postsynaptic muscle membrane, which make up the NMJ. The most common antibodies are directed against the acetylcholine receptor (AChR) or muscle specific tyrosine kinase (MuSK). An alternative to expand on preclinical in-vivo methods for studying mechanisms underlying diseases of neuromuscular transmission is to apply physiologic in-vitro models that would allow tissue-tissue as well as cell-cell interactions. A system that would allow cell-cell interactions in a biological fashion is the micro-electrode array (MEA) chip that allows co-culturing of motor neurons and muscle cells. The primary hypothesis is that the suggested MEA can be used in creating a reliable model for healthy and diseased NMJ, allowing for manipulations and treatment assays. The secondary hypothesis is that small non-coding RNA, so called microRNAs (miRNA) have a specific role in neuromuscular transmission and in MG. Study I demonstrated a method of long-term muscle cell culture on the MEA chips, which allows us to trace the development of muscle cells through the observation of their electrical activity at subcellular resolution. The maturation of skeletal muscle tissue was accompanied by a gradual increase in the amplitude and frequency of extracellular individual electrical spikes. The mature muscle tissue demonstrated the steady electrical activity with synchronized spike propagation in different directions across the chip. Study II showed a specific upregulated profile of miRNAs in the muscles of MuSK antibody seropositive MG mice. Transfection of these miRNAs, miR-1933 and miR-1930, promoted downregulation of several proteins and further confirmation with qPCR revealed a specific blocking of IMPA1 and MRPL27, which are involved in intracellular signal transduction and mitochondrial biogenesis in skeletal muscles. Study III revealed no correlation between the morphology of skeletal muscle cells and their electrical activity at an early developmental stage. However, the application of recombinant rat agrin increased the number of AChRs clusters in the culture of skeletal muscle and promoted a higher degree of spontaneous activity

Long-Term High-Density Extracellular Recordings Enable Studies of Muscle Cell Physiology

Skeletal (voluntary) muscle is the most abundant tissue in the body, thus making it an important biomedical research subject. Studies of neuromuscular transmission, including disorders of ion channels or receptors in autoimmune or genetic neuromuscular disorders, require high-spatial-resolution measurement techniques and an ability to acquire repeated recordings over time in order to track pharmacological interventions. Preclinical techniques for studying diseases of neuromuscular transmission can be enhanced by physiologic ex vivo models of tissue-tissue and cell-cell interactions. Here, we present a method, which allows tracking the development of primary skeletal muscle cells from myoblasts into mature contracting myotubes over more than 2 months. In contrast to most previous studies, the myotubes did not detach from the surface but instead formed functional networks between the myotubes, whose electrical signals were observed over the entire culturing period. Primary cultures of mouse myoblasts differentiated into contracting myotubes on a chip that contained an array of 26,400 platinum electrodes at a density of 3,265 electrodes per mm2. Our ability to track extracellular action potentials at subcellular resolution enabled study of skeletal muscle development and kinetics, modes of spiking and spatio-temporal relationships between muscles. The developed system in turn enables creation of a novel electrophysiological platform for establishing ex vivo disease models.ISSN:1664-042

Long-Term High-Density Extracellular Recordings Enable Studies of Muscle Cell Physiology

Skeletal (voluntary) muscle is the most abundant tissue in the body, thus making it an important biomedical research subject. Studies of neuromuscular transmission, including disorders of ion channels or receptors in autoimmune or genetic neuromuscular disorders, require high-spatial-resolution measurement techniques and an ability to acquire repeated recordings over time in order to track pharmacological interventions. Preclinical techniques for studying diseases of neuromuscular transmission can be enhanced by physiologic ex vivo models of tissue-tissue and cell-cell interactions. Here, we present a method, which allows tracking the development of primary skeletal muscle cells from myoblasts into mature contracting myotubes over more than 2 months. In contrast to most previous studies, the myotubes did not detach from the surface but instead formed functional networks between the myotubes, whose electrical signals were observed over the entire culturing period. Primary cultures of mouse myoblasts differentiated into contracting myotubes on a chip that contained an array of 26,400 platinum electrodes at a density of 3,265 electrodes per mm(2). Our ability to track extracellular action potentials at subcellular resolution enabled study of skeletal muscle development and kinetics, modes of spiking and spatio-temporal relationships between muscles. The developed system in turn enables creation of a novel electrophysiological platform for establishing ex vivo disease models

Stimulation of HERG Channel Activity by beta-catenin

The multifunctional protein ß-catenin governs as transcription factor the expression of a wide variety of genes relevant for cell proliferation and cell survival. In addition, ß-catenin is localized at the cell membrane and may influence the function of channels. The present study explored the possibility that ß-catenin participates in the regulation of the HERG K(+) channel. To this end, HERG was expressed in Xenopus oocytes with or without ß-catenin and the voltage-gated current determined utilizing the dual electrode voltage clamp. As a result, expression of ß-catenin markedly upregulated HERG channel activity, an effect not sensitive to inhibition of transcription with actinomycin D (10 µM). According to chemiluminescence, ß-catenin may increase HERG channel abundance within the oocyte cell membrane. Following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) the decay of current was similar in oocytes expressing HERG together with ß-catenin to oocytes expressing HERG alone. The experiments uncover a novel function of APC/ß-catenin, i.e. the regulation of HERG channels

The effect of ß-catenin on HERG currents is not modified by actinomycin D.

<p>Arithmetic means±SEM (n = 9–13) of the normalized outward tail current following a depolarization to +80 mV recorded in oocytes injected with cRNA encoding HERG (white bars) or with RNA encoding HERG and ß-catenin (black bars), incubated for 24 hours without (left bars) or with (right bars) 10 µM actinomycin D prior to the measurement. *indicates statistical significance (p<0.05) from the absence of ß-catenin cRNA.</p