Nervous system growth factors promote axonal growth following acute spinal cord injury. In the present experiment, we examined whether delivery of neurotrophic factors after chronic spinal cord ...injury would also promote axonal growth and influence functional outcomes. Adult Fischer 344 rats underwent mid-thoracic spinal cord dorsal hemisection lesions. Three months later, primary fibroblasts genetically modified to express human neurotrophin-3 (NT-3) were placed in, and distal to, the lesion cavity. Upon sacrifice 3 months later (6 months following the initial lesion), NT-3-grafted animals exhibited significant growth of corticospinal axons up to 15 mm distal to the lesion site and showed a modest but significant 1.5-point improvement in locomotor scores (
P < 0.05) on the BBB scale, compared to control-grafted animals. Thus, growth factor gene delivery can elicit growth of corticospinal axons in chronic stages of injury and improves functional outcomes compared to non-growth-factor-treated animals.
Specific GABAA circuits for visual cortical plasticity FAGIOLINI, Michela; FRITSCHY, Jean-Marc; LÖW, Karin ...
Science (American Association for the Advancement of Science),
03/2004, Letnik:
303, Številka:
5664
Journal Article
Recenzirano
Weak inhibition within visual cortex early in life prevents experience-dependent plasticity. Loss of responsiveness to an eye deprived of vision can be initiated prematurely by enhancing ...gamma-aminobutyric acid (GABA)-mediated transmission with benzodiazepines. Here, we use a mouse "knockin" mutation to alpha subunits that renders individual GABA type A (GABA(A)) receptors insensitive to diazepam to show that a particular inhibitory network controls expression of the critical period. Only alpha1-containing circuits were found to drive cortical plasticity, whereas alpha2-enriched connections separately regulated neuronal firing. This dissociation carries implications for models of brain development and the safe design of benzodiazepines for use in infants.
The emerging viruses SARS‐CoV‐2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues ...requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS‐CoV‐2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme‐1/site‐1 protease (SKI‐1/S1P), respectively. We report improved luciferase‐based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high‐throughput manner. We identified 23 FDA‐approved small molecules, among them monensin which displayed broad activity against furin and SKI‐1/S1P. Monensin inhibited arenaviruses and SARS‐CoV‐2 in a dose‐dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS‐CoV‐2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS‐CoV‐2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad‐spectrum antivirals with therapeutic potential to combat current and future emerging viruses.
Recombinant adeno-associated viral (AAV) vectors of serotypes 6, 8, and 9 were characterized as tools for gene delivery to dopaminergic neurons in the substantia nigra for future gene therapeutic ...applications in Parkinson's disease. While vectors of all three serotypes transduced nigral dopaminergic neurons with equal efficiency when directly injected to the substantia nigra, AAV6 was clearly superior to AAV8 and AAV9 for retrograde transduction of nigral neurons after striatal delivery. For sequential transduction of nigral dopaminergic neurons, the combination of AAV9 with AAV6 proved to be more powerful than AAV8 with AAV6 or repeated AAV6 administration. Surprisingly, single-stranded viral genomes persisted in nigral dopaminergic neurons within cell bodies and axon terminals in the striatum, and intact assembled AAV capsid was enriched in nuclei of nigral neurons, 4 weeks after virus injections to the substantia nigra. 6-Hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons in the substantia nigra reduced the number of viral genomes in the striatum, in line with viral genome persistence in axon terminals. However, 6-OHDA-induced axonal degeneration did not induce any transsynaptic spread of AAV infection in the striatum. Therefore, the potential presence of viral particles in axons may not represent an important safety issue for AAV gene therapy applications in neurodegenerative diseases.
The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to ...overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides that may not accurately recapitulate the complex cellular context of the basic PC–substrate interaction. Herein we report basic PC sensor (BPCS), a novel cell‐based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. BPCS consists of Gaussia luciferase linked to a sortilin‐1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in the same membrane. Screening of selected candidate peptidomimetic inhibitors revealed that BPCS can readily distinguish between general and selective PC inhibitors in a high‐throughput screening format. The robust and cost‐effective assay format of BPCS makes it suitable to identify novel specific small‐molecule inhibitors against basic PCs for therapeutic application. Its cell‐based nature will allow screening for drug targets in addition to the catalytically active mature enzyme, including maturation, transport, and cellular factors that modulate the enzyme's activity. This broadened ‘target range’ will enhance the likelihood to identify novel small‐molecule compounds that inhibit basic PCs in a direct or indirect manner and represents a conceptual advantage.
The basic proprotein convertases (PCs) are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Here, we report a novel cell‐based basic PC sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells and is suitable for high‐throughput screening.
Specific GABA A Circuits for Visual Cortical Plasticity Fagiolini, Michela; Fritschy, Jean-Marc; Löw, Karin ...
Science (American Association for the Advancement of Science),
03/2004, Letnik:
303, Številka:
5664
Journal Article
Recenzirano
Weak inhibition within visual cortex early in life prevents experience-dependent plasticity. Loss of responsiveness to an eye deprived of vision can be initiated prematurely by enhancing ...γ-aminobutyric acid (GABA)–mediated transmission with benzodiazepines. Here, we use a mouse “knockin” mutation to α subunits that renders individual GABA type A (GABA
A
) receptors insensitive to diazepam to show that a particular inhibitory network controls expression of the critical period. Only α1-containing circuits were found to drive cortical plasticity, whereas α2-enriched connections separately regulated neuronal firing. This dissociation carries implications for models of brain development and the safe design of benzodiazepines for use in infants.
The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC ...possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.
Issue Title: Special Section: Pacific Rim Ceramic Societies Symposia - Biomaterials The use of an "over 1000-nm near-infrared (NIR) in vivo fluorescence bioimaging" system based on lanthanide ...containing inorganic nanostructures emitting in the visible and NIR range under 980-nm excitation is proposed. It may overcome problems of currently used biomarkers including color fading, phototoxicity and scattering. Gd^sub 2^O3:Er^sup 3+^,Yb^sup 3+^ nanoparticles and nanorods showing upconversion and NIR emission are synthesized and their cytotoxic behavior is investigated by incubation with B-cell hybridomas and macrophages. Surface modification with PEG-b-PAAc provides the necessary chemical durability reducing the release of toxic Gd^sup 3+^ ions. NIR fluorescence microscopy is used to investigate the suitability of the nanostructures as NIR-NIR biomarkers. The in vitro uptake of bare and modified nanostructures by macrophages is investigated by confocal laser scanning microscopy. In vivo investigations revealed nanostructures in liver, lung, kidneys and spleen a few hours after injection into mice, while most of the nanostructures have been removed from the body after 24 h.PUBLICATION ABSTRACT
Amino acids in the α‐ and γ‐subunits contribute to the benzodiazepine binding site of GABAA‐receptors. We show that the mutation of a conserved histidine residue in the N‐terminal extracellular ...segment (α1H101R, α2H101R, α3H126R, and α5H105R) results not only in diazepam‐insensitivity of the respective αxβ2,3γ2‐receptors but also in an increased potentiation of the GABA‐induced currents by the partial agonist bretazenil. Furthermore, Ro 15‐4513, an inverse agonist at wild‐type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABAA‐receptor subtypes in vivo.
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